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ARTICLE OPEN ACCESS Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis Delphine Sterlin, PharmD, PhD, Martin Larsen, PhD, Jehane Fadlallah, MD, PhD, Christophe Parizot, MS, Marina Vignes, PharmD, Ga¨ elle Autaa, MS, Karim Dorgham, PhD, Catherine Juste, PhD, Patricia Lepage, PhD, Jennifer Aboab, MD, Savine Vicart, MD, Elisabeth Maillart, MD, Olivier Gout, MD, Catherine Lubetzki, MD, PhD, Romain Deschamps, MD, Caroline Papeix, MD, and Guy Gorochov, MD, PhD Neurol Neuroimmunol Neuroinamm 2021;8:e997. doi:10.1212/NXI.0000000000000997 Correspondence Prof. Gorochov guy.gorochov@ sorbonne-universite.fr Abstract Objective Based on animal models and human studies, there is now strong suspicion that host/microbiota mutualism in the context of gut microbial dysbiosis could inuence immunity and multiple sclerosis (MS) evolution. Our goal was to seek evidence of deregulated microbiota-induced systemic immune responses in patients with MS. Methods We investigated gut and systemic commensal-specic antibody responses in healthy controls (n = 32), patients with relapsing-remitting MS (n = 30), and individuals with clinically isolated syndromes (CISs) (n = 15). Gut microbiota composition and diversity were compared between controls and patients by analysis of 16S ribosomal ribonucleic acid (rRNA) sequencing. Au- tologous microbiota and cultivable bacterial strains were used in bacterial ow cytometry assays to quantify autologous serum IgG and secretory IgA responses to microbiota. IgG-bound bacteria were sorted by ow cytometry and identied using 16S rRNA sequencing. Results We show that commensal-specic gut IgA responses are drastically reduced in patients with severe MS, disease severity being correlated with the IgA-coated fecal microbiota fraction (r = -0.647, p < 0.0001). At the same time, IgA-unbound bacteria elicit qualitatively broad and quantitatively increased serum IgG responses in patients with MS and CIS compared with controls (4.1% and 2.5% vs 1.9%, respectively, p < 0.001). Conclusions Gut and systemic microbiota/immune homeostasis are perturbed in MS. Our results argue that defective IgA responses in MS are linked to a breakdown of systemic tolerance to gut micro- biota leading to an enhanced triggering of systemic IgG immunity against gut commensals occurring early in MS. From the Sorbonne Universit´ e (D.S., M.L., J.F., C.P., M.V., G.A., K.D., G.G.), Inserm, Centre dImmunologie et des Maladies Infectieuses (CIMI-Paris), AP-HP Hˆ opital Piti´ e-Salpˆ etri` ere, France; Universit´ e Paris-Saclay (C.J., P.L.), INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France; Hˆ opital Ophtalmologique Adolphe de Rothschild (J.A., O.G., R.D.), D´ epartement de Neurologie, Paris, France; and Sorbonne Universit´ e (S.V., E.M., C.L., C.P.), D´ epartement de Neurologie, AP-HP Hˆ opital Piti´ e-Salpˆ etri` ere, Paris, France. Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by the AP-HP. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1
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Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

Feb 15, 2022

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Page 1: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

ARTICLE OPEN ACCESS

Perturbed MicrobiotaImmune Homeostasis inMultiple SclerosisDelphine Sterlin PharmD PhD Martin Larsen PhD Jehane Fadlallah MD PhD Christophe Parizot MS

Marina Vignes PharmD Gaelle Autaa MS Karim Dorgham PhD Catherine Juste PhD Patricia Lepage PhD

Jennifer Aboab MD Savine Vicart MD ElisabethMaillart MD Olivier Gout MD Catherine Lubetzki MD PhD

Romain Deschamps MD Caroline Papeix MD and Guy Gorochov MD PhD

Neurol Neuroimmunol Neuroinflamm 20218e997 doi101212NXI0000000000000997

Correspondence

Prof Gorochov

guygorochov

sorbonne-universitefr

AbstractObjectiveBased on animal models and human studies there is now strong suspicion that hostmicrobiotamutualism in the context of gut microbial dysbiosis could influence immunity and multiplesclerosis (MS) evolution Our goal was to seek evidence of deregulated microbiota-inducedsystemic immune responses in patients with MS

MethodsWe investigated gut and systemic commensal-specific antibody responses in healthy controls(n = 32) patients with relapsing-remitting MS (n = 30) and individuals with clinically isolatedsyndromes (CISs) (n = 15) Gutmicrobiota composition and diversity were compared betweencontrols and patients by analysis of 16S ribosomal ribonucleic acid (rRNA) sequencing Au-tologous microbiota and cultivable bacterial strains were used in bacterial flow cytometry assaysto quantify autologous serum IgG and secretory IgA responses to microbiota IgG-boundbacteria were sorted by flow cytometry and identified using 16S rRNA sequencing

ResultsWe show that commensal-specific gut IgA responses are drastically reduced in patients withsevere MS disease severity being correlated with the IgA-coated fecal microbiota fraction (r =minus0647 p lt 00001) At the same time IgA-unbound bacteria elicit qualitatively broad andquantitatively increased serum IgG responses in patients with MS and CIS compared withcontrols (41 and 25 vs 19 respectively p lt 0001)

ConclusionsGut and systemic microbiotaimmune homeostasis are perturbed in MS Our results argue thatdefective IgA responses in MS are linked to a breakdown of systemic tolerance to gut micro-biota leading to an enhanced triggering of systemic IgG immunity against gut commensalsoccurring early in MS

From the Sorbonne Universite (DS ML JF CP MV GA KD GG) Inserm Centre drsquoImmunologie et des Maladies Infectieuses (CIMI-Paris) AP-HP Hopital Pitie-SalpetriereFrance Universite Paris-Saclay (CJ PL) INRAE AgroParisTech Micalis Institute Jouy-en-Josas France Hopital Ophtalmologique Adolphe de Rothschild (JA OG RD) Departementde Neurologie Paris France and Sorbonne Universite (SV EM CL CP) Departement de Neurologie AP-HP Hopital Pitie-Salpetriere Paris France

Go to NeurologyorgNN for full disclosures Funding information is provided at the end of the article

The Article Processing Charge was funded by the AP-HP

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2021 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Over the years many microbes have been proposed as potentialmultiple sclerosis (MS) triggers but firm evidence for causalityhas never been provided A seminal murine study howeverexploiting germ-free mice engineered to develop spontaneousexperimental autoimmune encephalomyelitis (EAE) suggestedthat commensal gut bacteria contribute to the disease process1

Microbiota transfer from patients with MS into mice was thenshown to induce andor exacerbate symptoms using the samespontaneous model1 or a more classic model in which EAE isactively induced following colonization2

In patients with MS increased fecal abundances of Akker-mansia Blautia Ruminococcus and Bifidobacterium generahave been described whereas Faecalibacterium Para-bacteroides Prevotella and Lactobacilluswould have a relativelylow abundance compared with healthy controls345 Moststudies supported based on in vitro assays and murinemodels that MSmicrobiota composition affects host immuneresponses driving an impaired T-cell differentiation to CD25+

FoxP3+ regulatory T cells and inclined differentiation towardproinflammatory Th1 and Th17 cells26 or proinflammatorychanges in monocytes5 The effect of immunomodulatory(IMD) therapy was examined in multiple studies indicatingthat treatment might normalize the abundance of some mi-crobial taxa such as Prevotella and Sutterella5 yet affect othermicrobial taxa such as Methanobrevibacter578 Akkermansiamuciniphila57 Roseburia and Clostridium cluster IV89 Wehypothesized that such gut microbiota alterations may affecthost humoral responses

Original events leading to a breakdown of tolerance and gen-eration of autoimmune response are still poorly understood inMS The molecular mimicry theory argues in favor of homolo-gies between microbial components and human proteins likelytaking part in the development of systemic andor CNS auto-immune diseases Both commensal and pathogenic strains couldlead to autoimmune responses through molecular mimicry Inneuromyelitis optica patients develop autoantibodies againstaquaporin 4 the predominant astrocyte water channel whichcross-react with an adenosine triphosphate-binding cassettetransporter of Clostridium perfringens1011 Likewise Guillain-Barre syndrome (GBS) has been associated with Campylobacterjejuni a leading agent of bacterial gastroenteritis12 GBS patho-genesis would be related in some instances to the induction ofantibodies cross-reacting with C jejuni surface polysaccharidesand similar human myelin-associated ganglioside structures13

However systemic immune responses to autologous microbiotawere never extensively explored in patients with MS

Here we investigated gut and systemic antibody anti-microbiota responses in patients with MS We report thatintestinal IgA interacts with a reduced proportion of com-mensal bacteria in patients with severe MS Aberrant anti-microbiota IgG responses associated with symptoms ofbacterial translocation are also observed

MethodsPatientsIn the present study 18 patients with clinically isolated syn-dromes (CISs) (feces and serum were together available foronly 12 of them) 32 patients with relapsing-remitting (RR)-MS and 30 age- and sex-matched healthy individuals havebeen enrolled Patients with CIS and RR-MS were recruitedeither at the Fondation Ophtalmologique Adolphe deRothschild (Paris France) or at the Department of Neurologyof Pitie-Salpetriere Hospital (Paris France) Patients with RR-MS fulfilled McDonald diagnostic criteria for MS14 Patientdisability was assessed using the Expanded Disability StatusScale (EDSS)15 which ranges from 0 (no disability) to 10(death) with 05 unit increments EDSS of included patientsranged between 0 and 65 CIS was defined as a first CNSinflammatory event that lasts at least 24 hours1617 IMDtreatments including glatiramer acetate and IFN-β werereported in the table Blood and stool collection occurredbefore steroid therapy in patients with CIS and RR-MS Theinclusion criteria specified no use of corticosteroids antibi-otics or laxative drugs in the last 3 months prior the studyDetailed patient information is summarized in the table Freshstool and blood samples were collected simultaneously at asingle time point

Stool Processing Microbiota Purificationand ProcessingStool samples were collected in a container including a reagentfor the generation of an O2-depleted and CO2-enriched at-mosphere (Anaerocult band Mikrobiologie) aliquoted in ananaerobic atmosphere and stored atminus80degC Fecal bacteria werepurified by gradient purification as previously described1819

Bacterial extracts were suspended in 1xPBS (phosphate buffersaline)ndash10 glycerol immediately frozen in liquid nitrogenand then stored at minus80degC Genomic DNA was extracted fromwhole stool samples as previously described20 Briefly 200 mgof fecal sample was lysed chemically (guanidine thiocyanateand N-lauroyl sarcosine) and mechanically (glass beads) fol-lowed by elimination of cell debris by centrifugation and pre-cipitation of genomic DNA Finally genomic DNA was RNase

GlossaryCFU = colony-forming unit CIS = clinically isolated syndrome EAE = experimental autoimmune encephalomyelitis EDSS =Extended Disability Status Scale EI = enrichment indexGBS = Guillain-Barre syndrome IMD = immunomodulatoryMBP =myelin basic protein MS = multiple sclerosis OTU = operational taxonomic unit PBS = phosphate buffer saline PCR =polymerase chain reaction rRNA = ribosomal ribonucleic acid RR = relapsing-remitting TNF = tumor necrosis factor

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

treated DNA concentration was estimated by Nanodrop(Thermo Scientific)

Bacterial Strains and Culture ConditionsPrevotella oris and Fusobacterium nucleatum were isolated fromhuman samples and identified by matrix-assisted laser desorptionionizationndashtime of flight mass spectrometry (Microbiology De-partment Pitie-Salpetriere Hospital Paris) Both bacterial strainswere cultured on sheep red blood agar plates at 37degC underanaerobic conditions for 48 hours Ruminococcus obeum (VPI B3-21) Bacteroides caccae (VPI 3452A) Bifidobacterium longum(E194v variant A) and Akkermansia muciniphila (DSM-22959)were characterized at the Institut National de Recherche Agron-omique (Jouy-en-Josas France) A muciniphila was cultured inL-YHBHI4 medium (Liquid-Yeast extract Hemin Brain HeartInfusion) with mucin whereas R obeum was cultured in PYGmedium (peptonendashyeast extractndashdextrose) at 37degC under anaer-obic conditions Bacterial cultures were suspended in 1xPBSndash10glycerol (109 colony-forming units [CFUs]mL) and frozen atminus80degC Quantification of CFUs was performed by addingcounting beads (Beckman Coulter) to bacterial suspensions andthen analyzed by flow cytometry (FACSCanto II BD)

Bacterial Flow CytometrySystemic IgG and secretory IgA binding to microbiota wasassessed by bacterial flow cytometry as previously described21

Briefly thawed microbiota or bacterial strains (107 bacteriaconditions) were fixed in a solution of 4 paraformaldehyde andstained with Cell Proliferation Dye eFluor 450 (eBioscience)After washing with 1xPBS (10 minutes 4000g 4degC) cells weresuspended in 1xPBS 2 bovine serum albumin (Sigma) and002 sodium azide (Sigma) and incubated in a 96-V bottomwell plate with a 10 μgmL IgG solution (from either humanserum or pooled human IgG HizentrandashCSL Behring France orhuman anti-tumor necrosis factor (TNF) RemicadendashMSDFrance) per condition All buffers were passed through sterile022-μm filters before use After washing secondary conjugatedantibodies (goat anti-human IgA-FITC and goat anti-humanIgG-A647) or isotype controls (both from Jackson ImmunoR-esearch Laboratories West Grove) were added for 20 minutes at4degC Then bacteria were suspended in sterile PBS Thirtythousand bacterial events were acquired on a FACSCanto II flowcytometer (Becton Dickinson) Analysis was performed withFlowJo software (Treestar) Frequencies of Ig-bound microbiotawere expressed as percentages median minimum andmaximumvalues throughout the article Medians of fluorescence were usedto measure IgG-binding levels for pure bacterial strains

16S Ribosomal RNA Phylogenetic Analysis ofTotal Gut Microbiota and IgG-Coated FractionsPurified microbiota (108condition) was washed in 1xPBSand stained with secondary conjugated antibodies (goat anti-human IgA-FITC and goat anti-human IgG-A647) or isotypecontrols (both from Jackson ImmunoResearch LaboratoriesWest Grove) After washing sorting was performed using amicrobiota-dedicated single laser S3 cell sorter (Bio-RadLaboratories CA) 105 bacteria per fraction was collected in1xPBS centrifuged and immediately stored as dry pellets atminus80degC Purity for both fractions was systematically verifiedafter sorting DNA was extracted and the V3ndashV4 region ofthe 16S ribosomal ribonucleic acid (rRNA) gene was ampli-fied by seminested polymerase chain reaction (PCR) PrimersV3fwd (+357) 59 TACGGRAGGCAGCAG 39 and V4rev(+857) 59 ATCTTACCAGGGTATCTAATCCT 39 wereused during the first round of PCR (10 cycles) PrimersV3fwd and X926_Rev (+926) 59 CCGTCAATTCMTT-TRAGT 39 were used in the second PCR round (40 cycles)PCR amplicon libraries were sequenced using a MiSeq Illu-mina platform (Genotoul Toulouse France) The resultingsequences from whole microbiota were analyzed using theopen source software package Quantitative Insights IntoMicrobial Ecology22 as previously described23 Demultiplexedreads from IgG-coated bacteria were processed using MG-RAST analysis pipeline Sequencing artifacts host DNAcontamination and sequences less than 200 bp in length wereremoved Insufficient quality reads were discarded (lt5 oftotal reads) Sequences were then clustered into operationaltaxonomic units (OTUs) with a 97 homology usingGreengenes database OTUs containing only a single se-quence were discarded OTUs detected at gt01 relative

Table Demographic and Clinical Features of the Cohort

Healthydonors RR-MS CIS-MS

Sex (HF) 1220 1119 513

BMI 218(187ndash319)

226(164ndash327)

229(184ndash391)

Age 329(236ndash613)

384(189ndash606)

318(181ndash548)

Age at onset NA 298(123ndash532)

315(18ndash547)

Duration of thedisease

NA 7 (05ndash26) NA

EDSS score

lt3 NA 18 14

3ndash5 NA 10 4

gt5 NA 2 0

Treatment

IFN-β 0 8 NA

Glatirameracetate

0 6 NA

Mitoxantrone 0 1 NA

Solumedrol 0 1 NA

Natalizumab 0 1 NA

wo (or ND) 10 (3) NA

Abbreviations BMI = bodymass index IFN = interferon NA = not applicablewo = withoutNumbers for ages and duration of the disease refer to years

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 3

abundance in at least 2 samples were finally conserved Thisprocess reduced the total OTU count from 297 down to 102OTU table was rarefied to the minimum samplersquos depth(18727 reads) Shannon index was calculated according tothe following equation Shannon index = minusΣpiln(pi) where pi isthe relative abundance of the ith OTU in the data set Incalculating the enrichment index (EI) we scored a pseudo-relative abundance equal to 00001 which was the lower limitof detection if a taxon was not detected in a given fractionIgAminusIgG+ EI refers to

log10

IgA minus IgG+ taxon abundanceIgA minus IgGminus taxon abundance

and IgA+IgG+ EI to log10

IgA + IgG+ taxon abundanceIgA minus IgGminus taxon abundance

Statistical AnalysisStatistical analysis was performed using Graphpad Prism v6The Wilcoxon paired rank test was used when comparingpaired groups whereas the Mann-Whitney test was usedwhen comparing 2 independent groups For multiple com-parisons the Kruskall-Wallis test with post hoc Dunn test wasconducted Significant p values are indicated on plots (p lt005 p lt 001 lt0001)

Standard Protocol Approvals Registrationsand Patient ConsentsThe study had been approved by the local ethics committee ofPitie-Salpetriere Hospital (CPP Ile de France VI) A priorwritten consent was obtained from all the patients and con-trols before inclusion in the study

Data AvailabilityAll relevant data are available within the article Anonymizeddata are available and will be shared on request from anyqualified investigator

ResultsCollapsed IgA Interactions With FecalMicrobiota in Patients With Severe MSWe postulated that not only microbiota composition345 butalso the immunemicrobiota interface could be perturbed inMS We first compared IgA-bound fecal microbiota levels inhealthy donors and patients with MS In accordance withprevious studies19 we observed in the control group used forthe present study that IgA binds a median percentage of 76(08ndash188 n = 30) of the whole fecal microbiota (figure 1 Aand B) The proportion of IgA-bound fecal microbiota is notsignificantly decreased in patients with MS (median [minndashmax] 51 [02ndash272] n = 32 figure 1 A and B) comparedwith controls (p = 033) We then evaluated whether the IgAmicrobiota interface could be more perturbed in severely af-fected patients Disease severity at the time of serum andmicrobiota sampling was evaluated using the Extended Dis-ability Status Scale (EDSS)24 We observed that disabled

patients with RR-MS (EDSS score gt4) had significantly reducedproportions of IgA-bound fecal bacteria compared with patientswith RR-MS without disability (EDSS score lt2) (102[024ndash602] vs 123 [149ndash2723] respectively p lt 0001figure 1 C) Strikingly disease severity is inversely correlated withthe IgA-coated fecalmicrobiota fraction at the individual level (r=minus0647 p lt 00001 figure 1 D) Because the majority of severepatients were on immunosuppressive treatment we sought toexplore whether IMD therapies might alter IgA responses Wefound no difference in IgA-coated proportions of fecal microbiotabetween untreated and treated patients (figure e-1 A linkslwwcomNXIA468) Moreover interferon- and glatiramer-treatedpatients exhibited similar IgA-bound fecal microbiota levelscompared with untreated patients with MS (figure e-1 A linkslwwcomNXIA468) These results argue for a defective surveyof the gut microbiota by IgA in disabled patients with MS

MS-Associated Gut Microbiota DysbiosisWe reasoned that defective IgA responses in disabled patientswith MS might be related to differences in gut microbiotacomposition We therefore performed 16S rRNA sequencingand identified bacterial taxa at the genus level We first examinedthe microbial diversity by calculating the Shannon index Wefound that alpha-diversity ie the number of different specieswithin a sample was not different between healthy donors andpatients with MS (median [minndashmax] 051 [030ndash059] vs 053[039ndash061] p = 019 figure e-2 A linkslwwcomNXIA469)However neither disease severity nor treatments appeared toaffect bacterial diversity (figure e-2 A linkslwwcomNXIA469) We next investigated the frequency of prevalent genera(gt1 in any sample group) Consistent with previous findings8

the relative abundance of Clostridium cluster IV was increased inpatients withMS compared with healthy donors (51 times 10minus3 [54times 10minus4ndash003] vs 002 [15 times 10minus3 times 011] p = 002 figure e-2 BlinkslwwcomNXIA469) This difference was mainly drivenby a drastic increase ofClostridium cluster IV in disabled patients(0022 [31 times 10minus3ndash011] in patients with EDSS score gt2 p =00011 figure e-2 C linkslwwcomNXIA469) As previouslydescribed5 we found that Prevotella tended to be un-derrepresented in patients with MS (15 times 10minus3 [0ndash036] vs 004[0ndash06] p = 009 figure e-2 B linkslwwcomNXIA469) Wealso observed a decrease in the genus Coprococcus (001 [2 times10minus4ndash007] vs 0017 [0ndash008] p = 012 figure e-2 A linkslwwcomNXIA469) In both cases these alterations tended to bemore profound in severe cases (figure e-2 C linkslwwcomNXIA469) Previous reports described effects of IMD therapieson gut microbiota composition589 We therefore comparedmost prevalent genera in untreated interferon- and glatiramer-treated patients and healthy donors Although unclassified Clos-tridiales tended to be increased in interferon-treated patients(008 [17 times 10minus4ndash014] vs 0009 [93 times 10minus5ndash006] in healthydonors p = 006 figure e-3 linkslwwcomNXIA470) we didnot observe statistically significant differences between groupswhich is likely explained by the limited number of patients ineach subgroup Taken together these results indicate subtledifferences in the gutmicrobial communities in patients withMSthat might modulate gut IgA responses

4 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

Exacerbated Antimicrobiota IgG Response inPatients With MSWe then postulated that the perturbed gut IgAmicrobiota in-terface observed above could translate into abnormal systemicanticommensal immune responses in patients Indeed the hu-moral response to microbiota is not gut confined as high anti-commensal IgGs are detected in healthy donorrsquos serum232526

We used a previously described flow cytometric assay21 to con-comitantly detect secretory IgA and serum IgG binding to au-tologous fecal microbiota (figure 2 A) Of note IgG is about 5times more abundant in serum than IgA consequently thisprotocol allows us to detect serum IgG but not serum IgAbinding to fecal microbiota as shown in figure e-4 A linkslwwcomNXIA471 Moreover IgG is not transferred in the gutlumen leading to a negligible IgG-bound bacteria fraction inhuman feces (figure e-4 B linkslwwcomNXIA471)23 Alto-gether this assay measured specifically secretory IgA preboundlocally in the gut and serum IgG binding without interference ofserum IgA nor intestinal IgG We found both quantitative andqualitative antimicrobiota IgG alterations in patients with MSregardless of disease severity As shown MS serum IgG interactswith a broader proportion of fecal microbiota compared withcontrols (67 [05ndash171] n = 32 vs 11 [02ndash32] n = 30 p lt00001 figure 2 B)

To investigate whether IgG binding to fecal bacteria is ratherFc or Fab dependent we tested the reactivity of an irrelevant

human IgG (chimeric anti-human TNF) against MS fecalmicrobiota As shown irrelevant human IgG binding to MSmicrobiota is significantly reduced compared with autologousserum IgG (06 [0ndash22] vs 67 [05ndash171] p lt 00001figure 2 C) indicating that serum IgG targets fecal microbiotain a mostly Fab-mediated manner

We then askedwhether disease duration or treatments could skewantimicrobiota IgG response As shown recently diagnosed anduntreated patients with CIS also exhibit an enhanced anti-microbiota IgG response comparedwith controls (55 [34ndash84]n = 12 vs 11 [02ndash32] n = 30 p lt 00001 figure 2 B) FinallyIgG+ proportions of autologous microbiota did not significantlydiffer between treated and untreated patients with RR-MS (72[05ndash171] n = 16 vs untreated patients 54 [05ndash85] n = 10 p= 041 figure e-1 B linkslwwcomNXIA468) Taken togetherthese data reveal an enhanced triggering of systemic IgG immunityagainst gut microbiota occurring early in MS

Loss of Antimicrobiota IgAIgG Convergencein MSWe previously observed that serum antimicrobiota IgG con-verges with secretory IgA to target the same bacterial cells inhealthy individuals23 As expected serum IgG exclusivelycolabels secretory IgA-bound fecal bacteria in healthy donors23

but preferentially targets IgA-unbound bacteria in patients withMS (19 [02ndash69] in healthy donors (n = 30) vs 41

Figure 1 IgA-Coated Bacteria Are Reduced in Patients With Severe MS

(A) Representative Flow Cytometry Dot Plot Showing Endogenous Secretory IgA Coating on Indicated Fractions of Fecal Microbiota From aHealthy Donor or aPatientWith RR-MS (B) Flow cytometry analysis of the fraction of fecalmicrobiota bound by secretory IgA in healthy donors (n = 32) and patients with RR-MS (n= 32) Median values are indicated and groups were compared with a nonparametric Mann-Whitney test (ns not significant) (C) Flow cytometry analysis ofthe fraction of fecal microbiota bound by secretory IgA in healthy donors (n = 32) and patients with RR-MS classified according to disease severity measuredby the EDSS Median values are indicated Error bars represent minimum and maximum values p Values were calculated using the nonparametric Mann-Whitney test (p lt 005 p lt 0001) (D) Disease severity evaluated by the EDSS correlatedwith the percentage of secretory IgA-boundmicrobiota in patientswith MS-RR Spearman coefficient (r) and p values are indicated MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 5

Figure 2 Enhanced IgG Antimicrobiota Response in Patients With MS

(A) Procedure (top) and representative flow cytometry detection (bottom) of endogenous secretory IgA and autologous systemic IgG (10 μgmL) coating onhealthy or RR-MS fecal microbiota is presented (middle panels) (B) Proportions of IgG+IgAplusmn (left) and IgG+IgAminus (right) coated bacteria are compared in healthydonors (n = 30) patients with RR-MS (n = 32) or patients with CIS (n = 12) Median values are indicated p Values were calculated using the nonparametricMann-Whitney test (p lt 0001) (C) Representative flow cytometry dot plot (left) and analysis (right) of irrelevant IgG (anti-TNFα 10 μgmL) binding to RR-MSand CIS fecalmicrobiota (n = 44) (D) Procedure (top) representative flow cytometry dot plot (middle panel) and analysis (bottom) of autologous IgG or pooledhealthy IgG binding to RR-MS fecal microbiota (n = 32) Proportions of IgG+IgAplusmn coated bacteria (bottom left) IgG+IgA+ and IgG+IgAminus coated bacteria (bottomright) are shown p Value was calculated by using the Wilcoxon paired test (p lt 00001 ns = nonsignificant) CIS = clinically isolated syndrome MS =multiple sclerosis RR = relapsing-remitting

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

[01ndash164] and 25 [13ndash54] in patients with RR-MS (n =32) and CIS (n = 12) respectively p lt 0001 figure 2 B)

We then asked whether this observed loss of convergence wasrelated to IgG responses only found in MS or to an increasedMS microbiota reactivity to IgG in general To address thisissue we compared MS and pooled healthy IgG reactivity toMS microbiota As shown in figure 2 D although pooledhealthy IgG bound a smaller fraction of RR-MS microbiotacompared with autologous RR-MS IgG (19 [0ndash10] vs 67[05ndash171] n = 32 p lt 00001 figure 2 D) pooled healthyIgG targets not only in vivo IgA-bound but also IgA-free RR-MS microbiota (07 [0ndash64] vs 06 [0ndash62] p = 079figure 2 D) suggesting that MSmicrobiota are more prone tointeract with IgG regardless of their origin

From this part we conclude that the IgAIgG convergencerule does not apply to MS A previously IgA-ignored fractionof MS microbiota is able to interact with IgG

Antimicrobiota IgG Spreading in MSIt could be deduced from above that IgG binding to MSmicrobiota might extend beyond the usual bacterial repertoireof IgA targets that we and others previously defined2327 Todecipher antimicrobiota IgG specificities in patients with MSwith higher resolution we next isolated IgG-bound bacteria byflow cytometry sorting and identified their taxonomy using 16SrRNA sequencing (figure 3 A) To avoid confounding factorsfrom IMD therapies we sorted IgG-bound bacteria from un-treated patients We first examined microbial diversity by cal-culating the Shannon index and found a similar diversity inIgG-only and Ig-negative bacteria (206 [114ndash27] vs 212[18ndash232] n = 5 figure 3 B) In contrast dually coated bacteriatended to exhibit a decreased microbial diversity in comparisonwith the previous fractions (074 [053ndash135] n = 5 p = 0062figure 3 B) suggesting a state of restricted antimicrobiota se-cretory IgA diversity in MS We observed that individualIgA+IgG+ and IgAminusIgG+ bacterial repertoires were remarkablydistinct in all tested patients (figure 3 C for back-to-back

Figure 3 MS IgG Binds a Diverse Repertoire of Commensals

(A) Sorting strategy of IgG+IgA+ and IgG+IgAminus-coated bacteria in 5 patients with RR-MS Composition of sorted fractions was next analyzed by using 16S rRNAsequencing (B) Genera diversity in IgG+IgA+ and IgG+IgAminus-sorted fractions calculated by using the Shannon index (C) Median relative abundance of the 30most frequent genera in sorted fractions from 1 representative RR-MS patient MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 7

taxonomic analysis of fecal IgA+IgG+ IgAminusIgG+ and IgAminusIgG-

fractions in a representative patient) supporting an IgG re-sponse spread beyond IgA-surveyed taxa To quantitativelycompare IgAminusIgG+ and IgA+IgG+microbial compositions in thestudied population we thereafter narrowed the analysis down tothe 30 most frequent genera that were present in at least 80 ofthe Ig-sorted fractions Three genera derived from FirmicutesBlautia Clostridium and Eubacterium appeared preferentiallytargeted by MS serum IgG alone as deduced from their com-pared frequencies in IgAminusIgG+ bacteria and IgA+IgG+ sortedfractions (006 [002ndash02] vs 0004 [0002ndash0033] 007[0055ndash045] vs 0003 [0009ndash0041] and 0023 [0003ndash004] vs0002 [0001ndash0003] respectively p lt 005 figure 4 AndashC) Wesought to validate these observations by calculating a log-basedEI that is not influenced by genera representation in the studiedsamples We confirmed that both Clostridium and Eubacteriumwere predominant in IgAminusIgG+- compared with IgA+IgG+-

sorted fractions (EI minus06 [minus083 to 022] vs minus137 [minus166 to064] and minus018 [minus039 to 105] vs minus093 [minus174 to 019] forClostridium and Eubacterium respectively p lt 005 figure 4 B)

Taken together these results demonstrate thatMS is associatedwith a broad antimicrobiota systemic IgG response with avariety of bacterial genera being preferentially targeted by MSIgG compared with MS IgA Because systemic IgG could re-flect silent gut microbiota translocation episodes232829 theseobservations could support an enhanced bacterial translocationfrom the gut lumen to systemic compartments in patientswith MS

Increased MS IgG Antibacterial ResponsesConfirmed at the Strain LevelBacterial identification remains at the genus level using 16SrRNA sequencing Using bacterial flow cytometry we sought

Figure 4 IgG-Bound Commensal Shift in Patients With MS

(A) Relative abundance of the 30 most frequent genera in IgG+IgA+ and IgG+IgAminus-sorted fractions in blue and red respectively (B) EI of the 30 most frequentgenera in IgG+IgA+ and IgG+IgAminus-sorted fractions compared in blue and red respectively (C) Blautia Clostridium and Eubacterium frequencies in IgG+IgA+ andIgG+IgAminus fractions For all box plots each dot represents 1 donor errors bars represent maximum andminimum values andmedians are indicated p Valueswere calculated by using the nonparametric Mann-Whitney test (p lt 005 p lt 001) EI = enrichment index MS = multiple sclerosis

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

to determine whether increased antimicrobiota IgG reactiv-ities could also be measured at the species level in patientswith MS We therefore selected representative bacterialstrains that were previously described as secretory IgA targetsin humans (A muciniphila B longum and R obeum) and 3others that are not preferentially bound by IgA in humans (Bcaccae F nucleatum and P oris)30-33 A majority of strainstested (4 of 6) tended to be more brightly stained by MS (RRandor CIS) IgG that by control serum IgG (figure 5 AndashC)Preferred MS IgG targets are not systematically preferredcontrol IgA targets as neither B longum nor B caccae werepreferentially bound by MS IgG compared with control IgG(figure e-5 linkslwwcomNXIA472) Of interest we ob-served that F nucleatum is particularly well opsonized by CISserum compared with healthy control serum but also com-pared with RR serum (IgG median fluorescence intensity 518[146ndash5505] vs 126 [407ndash414] and 156 [421ndash3350] in CIShealthy control and RR serums respectively figure 5 AndashB)We conclude from this part thatMS serum contains high titersof antimicrobiota IgG that can be measured against specificbacterial strains

DiscussionWe report an imbalance between systemic and secretoryantimicrobiota antibody responses in patients with MS

Antimicrobiota secretory IgA responses are impaired whereas atthe same time systemic IgG responses directed against autolo-gous microbiota are increased compared with healthy controlsImportantly although we found MS-associated gut microbiotaalterations the systemic IgG responses observed were not di-rected to taxa overrepresented in MS microbiota Furthermorelow proportions of IgA-coated bacteria among fecal microbiotaare associated with disease severity Consistently with the latterobservation Rojas et al34 have recently reported that proportionsof IgA-coated bacteria among fecal microbiota are decreased inpatients with relapsing MS compared with inactive patients Re-duced proportions of fecal IgA+ bacteria might result from re-duced secretory IgA levels in relation with IgA-secreting cellsegress out of the gutmucosa Previous works indeed shed light onthe dynamic trafficking of intraepithelial CD4T cells between gutand an inflamed CNS35 and also of IgA-producing cells betweengut and bone marrow36 In the EAE model commensal-reactiveIgA-producing cells regulate inflammation via the production IL-10 after their migration in the CNS Probstel et al recentlydemonstrated that gut microbiota IgA-producing cells infiltratethe CNS in patients with MS leading to an increase in in situ IgAproduction during MS relapses However commensal-specificIgA does not cross-react with brain tissue37 Further studies arerequired to assess the role of gut-derived IgA-producing cells inMS pathophysiology Defective IgA binding to gut microbiota indisabled patients with MS might be also related to microbialcommunity alterations

Figure 5 MS Antimicrobiota IgG Signature Measured on Cultivable Bacterial Strains

(A) Representative flow cytometry analysis ofserum IgG binding to F nucleatum Gray histo-gram represents isotype control blue red andred dotted lines represent a healthy controlpatient with RR-MS and patient with CIS re-spectively (B) Flow cytometry analysis of serumIgG binding to IgA-unbound bacteria (from leftto right F nucleatum and P oris) in healthy do-nors (n = 30) patients with RR-MS (=32) andpatients with CIS (n = 12) (C) Flow cytometryanalysis of serum IgG binding to IgA-boundbacteria (from left to right A muciniphila and Robeum) in healthy donors (n = 30) patients withRR-MS (=32) and patients with CIS (n = 12) Darkbars represent medians p Values were calcu-lated by using the nonparametric Mann-Whit-ney test (p lt 005 p lt 001 p lt 0001)p lt 00001) MS = multiple sclerosis RR =relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 9

Impaired IgA-mediated microbiota survey might also favorbacterial translocation and induction of systemic IgG re-sponses as we previously described in patients with IgA de-ficiency23 Numerous studies underline that IgG typicallypresent in the CSF of the patients might play a role in MSpathogenesis38-40 IgG purified from patients withMS increasesmouse CNS demyelination and exacerbates EAE41 The mainclinical evidence for a potential pathogenic role for antibodiesin MS is the benefit of antibody removal in a subset of patientswith acute neurologic symptoms4243 However what triggersoligoclonal antibody responses observed in MS CSF remains amystery38-40 Antimicrobiota antibodies are frequently cross-reactive with self-antigens44-46 Cross-reactivity is probably anessential antimicrobial antibody feature enabling control of alarge diversity of gut commensals and invading pathogens4748

but is also associated with a theoretical risk of autoimmunity Ithas indeed been very recently proposed that some pathogenicimmune responses associated with lupus4950 or myocarditis51

can be triggered by commensal bacteria through a molecularmimicry mechanism Of interest more than 20 bacterial spe-cies well represented in human gut microbiota express pro-teins that contain potentially encephalitogenic peptides iehighly homologous to myelin basic protein (MBP) or myelinoligodendrocyte protein In particular 4 proteins expressed byF nucleatum have been identified as potentially encephalito-genic52 Moreover 3 proteins expressed by members of theClostridium genera were described to be homologous toMBP52 We report that RR-MS and even more so CIS IgGvery brightly stain F nucleatum We also report that the Clos-tridium genus is significantly more targeted by systemicMS IgGthan by IgA These observations open new avenues for ex-ploring the hypothesis that immune responses induced bycommensal antigens might play a role in MS pathogenesis

Enhanced induction of antimicrobiota IgG could also be aconsequence of intestinal barrier dysfunction in MS Indeednot only blood-brain barrier defects have been reported inMSbut also a state of increased intestinal permeability charac-terized by higher lactulosemannitol urinary ratios increasedlipopolysaccharide plasma levels and associated with in-creased plasma zonulin a regulator of epithelial and endo-thelial barrier functions53-55 In the EAE model suchalterations preceded the onset of neurologic symptoms56 It istherefore conceivable that bacterial translocation could occurat early stages of the disease and account for the increasedantimicrobiota IgG responses we observe as early as the CISstage Systemic antimicrobiota IgG responses are likely tomirror microbiota composition at the individual level Wetherefore tested some of the bacterial species differentiallyidentified between MS and healthy donor gut microbiota245

We observed that patients with RR-MS and CISharbor ele-vated serum antindashA muciniphila IgG reactivities comparedwith controls These results are in line with the higher levels ofantindashA muciniphila antibodies detected in CSF of patientswith MS57 Of interest a greater A muciniphila prevalence inMS gut microbiota has been shown in previous reports25 Apositive correlation between A muciniphila abundance and

gene expression in T cells and monocytes involved in keypathways previously implicated in MS pathogenesis was alsoreported5 Located in close vicinity with the intestinal cells Amuciniphila has also been reported to promoteTh1 lymphocyte differentiation a T-cell subset classicallyassociated with MS pathogenesis258 Altogether althoughonly correlative these observations suggest a role for Amuciniphila in driving exacerbated host immune responsein MS

As mentioned above signs of increased intestinal permeabilitypreceded the onset of neurologic symptoms in the EAEmodel56 It would now remain to determine to which extentantimicrobiota IgG monitoring could represent a tool topredict MS progression Not all patients with CIS progress tofull blown MS Baseline lesions age at onset and short timedelay between the first 2 relapses provide some predictivevalue for disease progression yet remain flawed5960 Whethermicrobialhost signatures including immune responses tocommensals could help to distinguish between stable casesand future progressive cases should be now tested in largerstudies

In summary we have found quantitative and qualitative al-terations of the antimicrobiota antibody response in patientswith MS involving peculiar bacterial taxa We propose thatsuch peculiar IgG responses associated with MS could rep-resent a lead in the quest for antigenic drivers possibly im-plicated in MS pathogenesis through a molecular mimicrymechanism We also propose that antimicrobiota fingerprintsshould be more widely used in further studies to seek forcorrelates with disease status Finally microbiota manipula-tion is now a recognized therapeutic option in the clinic61

How our results could translate into treatments shouldtherefore be tested in murine MS models

AcknowledgmentThe authors thank the patients who agreed to participate inthis study and doctors and nurses from the NeurologyDepartment (Hopital Pitie-Salpetriere 75013 Paris Franceand Hopital Fondation Adolphe de Rotschild 75019 ParisFrance) who participated in this study

Study FundingThe study was supported by a grant from Fondation pourlrsquoAide a la Recherche sur la Sclerose en Plaques (ARSEP) andby a proof of concept grant (CoPoc) from Inserm TransfertParis France

DisclosureG Gorochov is a scientific advisor for Luxia Scientific FranceThe other authors report no disclosures relevant to themanuscript Go to NeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 7 2020 Accepted in final form March 9 2021

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

This information is current as of May 11 2021

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is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Page 2: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

Over the years many microbes have been proposed as potentialmultiple sclerosis (MS) triggers but firm evidence for causalityhas never been provided A seminal murine study howeverexploiting germ-free mice engineered to develop spontaneousexperimental autoimmune encephalomyelitis (EAE) suggestedthat commensal gut bacteria contribute to the disease process1

Microbiota transfer from patients with MS into mice was thenshown to induce andor exacerbate symptoms using the samespontaneous model1 or a more classic model in which EAE isactively induced following colonization2

In patients with MS increased fecal abundances of Akker-mansia Blautia Ruminococcus and Bifidobacterium generahave been described whereas Faecalibacterium Para-bacteroides Prevotella and Lactobacilluswould have a relativelylow abundance compared with healthy controls345 Moststudies supported based on in vitro assays and murinemodels that MSmicrobiota composition affects host immuneresponses driving an impaired T-cell differentiation to CD25+

FoxP3+ regulatory T cells and inclined differentiation towardproinflammatory Th1 and Th17 cells26 or proinflammatorychanges in monocytes5 The effect of immunomodulatory(IMD) therapy was examined in multiple studies indicatingthat treatment might normalize the abundance of some mi-crobial taxa such as Prevotella and Sutterella5 yet affect othermicrobial taxa such as Methanobrevibacter578 Akkermansiamuciniphila57 Roseburia and Clostridium cluster IV89 Wehypothesized that such gut microbiota alterations may affecthost humoral responses

Original events leading to a breakdown of tolerance and gen-eration of autoimmune response are still poorly understood inMS The molecular mimicry theory argues in favor of homolo-gies between microbial components and human proteins likelytaking part in the development of systemic andor CNS auto-immune diseases Both commensal and pathogenic strains couldlead to autoimmune responses through molecular mimicry Inneuromyelitis optica patients develop autoantibodies againstaquaporin 4 the predominant astrocyte water channel whichcross-react with an adenosine triphosphate-binding cassettetransporter of Clostridium perfringens1011 Likewise Guillain-Barre syndrome (GBS) has been associated with Campylobacterjejuni a leading agent of bacterial gastroenteritis12 GBS patho-genesis would be related in some instances to the induction ofantibodies cross-reacting with C jejuni surface polysaccharidesand similar human myelin-associated ganglioside structures13

However systemic immune responses to autologous microbiotawere never extensively explored in patients with MS

Here we investigated gut and systemic antibody anti-microbiota responses in patients with MS We report thatintestinal IgA interacts with a reduced proportion of com-mensal bacteria in patients with severe MS Aberrant anti-microbiota IgG responses associated with symptoms ofbacterial translocation are also observed

MethodsPatientsIn the present study 18 patients with clinically isolated syn-dromes (CISs) (feces and serum were together available foronly 12 of them) 32 patients with relapsing-remitting (RR)-MS and 30 age- and sex-matched healthy individuals havebeen enrolled Patients with CIS and RR-MS were recruitedeither at the Fondation Ophtalmologique Adolphe deRothschild (Paris France) or at the Department of Neurologyof Pitie-Salpetriere Hospital (Paris France) Patients with RR-MS fulfilled McDonald diagnostic criteria for MS14 Patientdisability was assessed using the Expanded Disability StatusScale (EDSS)15 which ranges from 0 (no disability) to 10(death) with 05 unit increments EDSS of included patientsranged between 0 and 65 CIS was defined as a first CNSinflammatory event that lasts at least 24 hours1617 IMDtreatments including glatiramer acetate and IFN-β werereported in the table Blood and stool collection occurredbefore steroid therapy in patients with CIS and RR-MS Theinclusion criteria specified no use of corticosteroids antibi-otics or laxative drugs in the last 3 months prior the studyDetailed patient information is summarized in the table Freshstool and blood samples were collected simultaneously at asingle time point

Stool Processing Microbiota Purificationand ProcessingStool samples were collected in a container including a reagentfor the generation of an O2-depleted and CO2-enriched at-mosphere (Anaerocult band Mikrobiologie) aliquoted in ananaerobic atmosphere and stored atminus80degC Fecal bacteria werepurified by gradient purification as previously described1819

Bacterial extracts were suspended in 1xPBS (phosphate buffersaline)ndash10 glycerol immediately frozen in liquid nitrogenand then stored at minus80degC Genomic DNA was extracted fromwhole stool samples as previously described20 Briefly 200 mgof fecal sample was lysed chemically (guanidine thiocyanateand N-lauroyl sarcosine) and mechanically (glass beads) fol-lowed by elimination of cell debris by centrifugation and pre-cipitation of genomic DNA Finally genomic DNA was RNase

GlossaryCFU = colony-forming unit CIS = clinically isolated syndrome EAE = experimental autoimmune encephalomyelitis EDSS =Extended Disability Status Scale EI = enrichment indexGBS = Guillain-Barre syndrome IMD = immunomodulatoryMBP =myelin basic protein MS = multiple sclerosis OTU = operational taxonomic unit PBS = phosphate buffer saline PCR =polymerase chain reaction rRNA = ribosomal ribonucleic acid RR = relapsing-remitting TNF = tumor necrosis factor

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

treated DNA concentration was estimated by Nanodrop(Thermo Scientific)

Bacterial Strains and Culture ConditionsPrevotella oris and Fusobacterium nucleatum were isolated fromhuman samples and identified by matrix-assisted laser desorptionionizationndashtime of flight mass spectrometry (Microbiology De-partment Pitie-Salpetriere Hospital Paris) Both bacterial strainswere cultured on sheep red blood agar plates at 37degC underanaerobic conditions for 48 hours Ruminococcus obeum (VPI B3-21) Bacteroides caccae (VPI 3452A) Bifidobacterium longum(E194v variant A) and Akkermansia muciniphila (DSM-22959)were characterized at the Institut National de Recherche Agron-omique (Jouy-en-Josas France) A muciniphila was cultured inL-YHBHI4 medium (Liquid-Yeast extract Hemin Brain HeartInfusion) with mucin whereas R obeum was cultured in PYGmedium (peptonendashyeast extractndashdextrose) at 37degC under anaer-obic conditions Bacterial cultures were suspended in 1xPBSndash10glycerol (109 colony-forming units [CFUs]mL) and frozen atminus80degC Quantification of CFUs was performed by addingcounting beads (Beckman Coulter) to bacterial suspensions andthen analyzed by flow cytometry (FACSCanto II BD)

Bacterial Flow CytometrySystemic IgG and secretory IgA binding to microbiota wasassessed by bacterial flow cytometry as previously described21

Briefly thawed microbiota or bacterial strains (107 bacteriaconditions) were fixed in a solution of 4 paraformaldehyde andstained with Cell Proliferation Dye eFluor 450 (eBioscience)After washing with 1xPBS (10 minutes 4000g 4degC) cells weresuspended in 1xPBS 2 bovine serum albumin (Sigma) and002 sodium azide (Sigma) and incubated in a 96-V bottomwell plate with a 10 μgmL IgG solution (from either humanserum or pooled human IgG HizentrandashCSL Behring France orhuman anti-tumor necrosis factor (TNF) RemicadendashMSDFrance) per condition All buffers were passed through sterile022-μm filters before use After washing secondary conjugatedantibodies (goat anti-human IgA-FITC and goat anti-humanIgG-A647) or isotype controls (both from Jackson ImmunoR-esearch Laboratories West Grove) were added for 20 minutes at4degC Then bacteria were suspended in sterile PBS Thirtythousand bacterial events were acquired on a FACSCanto II flowcytometer (Becton Dickinson) Analysis was performed withFlowJo software (Treestar) Frequencies of Ig-bound microbiotawere expressed as percentages median minimum andmaximumvalues throughout the article Medians of fluorescence were usedto measure IgG-binding levels for pure bacterial strains

16S Ribosomal RNA Phylogenetic Analysis ofTotal Gut Microbiota and IgG-Coated FractionsPurified microbiota (108condition) was washed in 1xPBSand stained with secondary conjugated antibodies (goat anti-human IgA-FITC and goat anti-human IgG-A647) or isotypecontrols (both from Jackson ImmunoResearch LaboratoriesWest Grove) After washing sorting was performed using amicrobiota-dedicated single laser S3 cell sorter (Bio-RadLaboratories CA) 105 bacteria per fraction was collected in1xPBS centrifuged and immediately stored as dry pellets atminus80degC Purity for both fractions was systematically verifiedafter sorting DNA was extracted and the V3ndashV4 region ofthe 16S ribosomal ribonucleic acid (rRNA) gene was ampli-fied by seminested polymerase chain reaction (PCR) PrimersV3fwd (+357) 59 TACGGRAGGCAGCAG 39 and V4rev(+857) 59 ATCTTACCAGGGTATCTAATCCT 39 wereused during the first round of PCR (10 cycles) PrimersV3fwd and X926_Rev (+926) 59 CCGTCAATTCMTT-TRAGT 39 were used in the second PCR round (40 cycles)PCR amplicon libraries were sequenced using a MiSeq Illu-mina platform (Genotoul Toulouse France) The resultingsequences from whole microbiota were analyzed using theopen source software package Quantitative Insights IntoMicrobial Ecology22 as previously described23 Demultiplexedreads from IgG-coated bacteria were processed using MG-RAST analysis pipeline Sequencing artifacts host DNAcontamination and sequences less than 200 bp in length wereremoved Insufficient quality reads were discarded (lt5 oftotal reads) Sequences were then clustered into operationaltaxonomic units (OTUs) with a 97 homology usingGreengenes database OTUs containing only a single se-quence were discarded OTUs detected at gt01 relative

Table Demographic and Clinical Features of the Cohort

Healthydonors RR-MS CIS-MS

Sex (HF) 1220 1119 513

BMI 218(187ndash319)

226(164ndash327)

229(184ndash391)

Age 329(236ndash613)

384(189ndash606)

318(181ndash548)

Age at onset NA 298(123ndash532)

315(18ndash547)

Duration of thedisease

NA 7 (05ndash26) NA

EDSS score

lt3 NA 18 14

3ndash5 NA 10 4

gt5 NA 2 0

Treatment

IFN-β 0 8 NA

Glatirameracetate

0 6 NA

Mitoxantrone 0 1 NA

Solumedrol 0 1 NA

Natalizumab 0 1 NA

wo (or ND) 10 (3) NA

Abbreviations BMI = bodymass index IFN = interferon NA = not applicablewo = withoutNumbers for ages and duration of the disease refer to years

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 3

abundance in at least 2 samples were finally conserved Thisprocess reduced the total OTU count from 297 down to 102OTU table was rarefied to the minimum samplersquos depth(18727 reads) Shannon index was calculated according tothe following equation Shannon index = minusΣpiln(pi) where pi isthe relative abundance of the ith OTU in the data set Incalculating the enrichment index (EI) we scored a pseudo-relative abundance equal to 00001 which was the lower limitof detection if a taxon was not detected in a given fractionIgAminusIgG+ EI refers to

log10

IgA minus IgG+ taxon abundanceIgA minus IgGminus taxon abundance

and IgA+IgG+ EI to log10

IgA + IgG+ taxon abundanceIgA minus IgGminus taxon abundance

Statistical AnalysisStatistical analysis was performed using Graphpad Prism v6The Wilcoxon paired rank test was used when comparingpaired groups whereas the Mann-Whitney test was usedwhen comparing 2 independent groups For multiple com-parisons the Kruskall-Wallis test with post hoc Dunn test wasconducted Significant p values are indicated on plots (p lt005 p lt 001 lt0001)

Standard Protocol Approvals Registrationsand Patient ConsentsThe study had been approved by the local ethics committee ofPitie-Salpetriere Hospital (CPP Ile de France VI) A priorwritten consent was obtained from all the patients and con-trols before inclusion in the study

Data AvailabilityAll relevant data are available within the article Anonymizeddata are available and will be shared on request from anyqualified investigator

ResultsCollapsed IgA Interactions With FecalMicrobiota in Patients With Severe MSWe postulated that not only microbiota composition345 butalso the immunemicrobiota interface could be perturbed inMS We first compared IgA-bound fecal microbiota levels inhealthy donors and patients with MS In accordance withprevious studies19 we observed in the control group used forthe present study that IgA binds a median percentage of 76(08ndash188 n = 30) of the whole fecal microbiota (figure 1 Aand B) The proportion of IgA-bound fecal microbiota is notsignificantly decreased in patients with MS (median [minndashmax] 51 [02ndash272] n = 32 figure 1 A and B) comparedwith controls (p = 033) We then evaluated whether the IgAmicrobiota interface could be more perturbed in severely af-fected patients Disease severity at the time of serum andmicrobiota sampling was evaluated using the Extended Dis-ability Status Scale (EDSS)24 We observed that disabled

patients with RR-MS (EDSS score gt4) had significantly reducedproportions of IgA-bound fecal bacteria compared with patientswith RR-MS without disability (EDSS score lt2) (102[024ndash602] vs 123 [149ndash2723] respectively p lt 0001figure 1 C) Strikingly disease severity is inversely correlated withthe IgA-coated fecalmicrobiota fraction at the individual level (r=minus0647 p lt 00001 figure 1 D) Because the majority of severepatients were on immunosuppressive treatment we sought toexplore whether IMD therapies might alter IgA responses Wefound no difference in IgA-coated proportions of fecal microbiotabetween untreated and treated patients (figure e-1 A linkslwwcomNXIA468) Moreover interferon- and glatiramer-treatedpatients exhibited similar IgA-bound fecal microbiota levelscompared with untreated patients with MS (figure e-1 A linkslwwcomNXIA468) These results argue for a defective surveyof the gut microbiota by IgA in disabled patients with MS

MS-Associated Gut Microbiota DysbiosisWe reasoned that defective IgA responses in disabled patientswith MS might be related to differences in gut microbiotacomposition We therefore performed 16S rRNA sequencingand identified bacterial taxa at the genus level We first examinedthe microbial diversity by calculating the Shannon index Wefound that alpha-diversity ie the number of different specieswithin a sample was not different between healthy donors andpatients with MS (median [minndashmax] 051 [030ndash059] vs 053[039ndash061] p = 019 figure e-2 A linkslwwcomNXIA469)However neither disease severity nor treatments appeared toaffect bacterial diversity (figure e-2 A linkslwwcomNXIA469) We next investigated the frequency of prevalent genera(gt1 in any sample group) Consistent with previous findings8

the relative abundance of Clostridium cluster IV was increased inpatients withMS compared with healthy donors (51 times 10minus3 [54times 10minus4ndash003] vs 002 [15 times 10minus3 times 011] p = 002 figure e-2 BlinkslwwcomNXIA469) This difference was mainly drivenby a drastic increase ofClostridium cluster IV in disabled patients(0022 [31 times 10minus3ndash011] in patients with EDSS score gt2 p =00011 figure e-2 C linkslwwcomNXIA469) As previouslydescribed5 we found that Prevotella tended to be un-derrepresented in patients with MS (15 times 10minus3 [0ndash036] vs 004[0ndash06] p = 009 figure e-2 B linkslwwcomNXIA469) Wealso observed a decrease in the genus Coprococcus (001 [2 times10minus4ndash007] vs 0017 [0ndash008] p = 012 figure e-2 A linkslwwcomNXIA469) In both cases these alterations tended to bemore profound in severe cases (figure e-2 C linkslwwcomNXIA469) Previous reports described effects of IMD therapieson gut microbiota composition589 We therefore comparedmost prevalent genera in untreated interferon- and glatiramer-treated patients and healthy donors Although unclassified Clos-tridiales tended to be increased in interferon-treated patients(008 [17 times 10minus4ndash014] vs 0009 [93 times 10minus5ndash006] in healthydonors p = 006 figure e-3 linkslwwcomNXIA470) we didnot observe statistically significant differences between groupswhich is likely explained by the limited number of patients ineach subgroup Taken together these results indicate subtledifferences in the gutmicrobial communities in patients withMSthat might modulate gut IgA responses

4 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

Exacerbated Antimicrobiota IgG Response inPatients With MSWe then postulated that the perturbed gut IgAmicrobiota in-terface observed above could translate into abnormal systemicanticommensal immune responses in patients Indeed the hu-moral response to microbiota is not gut confined as high anti-commensal IgGs are detected in healthy donorrsquos serum232526

We used a previously described flow cytometric assay21 to con-comitantly detect secretory IgA and serum IgG binding to au-tologous fecal microbiota (figure 2 A) Of note IgG is about 5times more abundant in serum than IgA consequently thisprotocol allows us to detect serum IgG but not serum IgAbinding to fecal microbiota as shown in figure e-4 A linkslwwcomNXIA471 Moreover IgG is not transferred in the gutlumen leading to a negligible IgG-bound bacteria fraction inhuman feces (figure e-4 B linkslwwcomNXIA471)23 Alto-gether this assay measured specifically secretory IgA preboundlocally in the gut and serum IgG binding without interference ofserum IgA nor intestinal IgG We found both quantitative andqualitative antimicrobiota IgG alterations in patients with MSregardless of disease severity As shown MS serum IgG interactswith a broader proportion of fecal microbiota compared withcontrols (67 [05ndash171] n = 32 vs 11 [02ndash32] n = 30 p lt00001 figure 2 B)

To investigate whether IgG binding to fecal bacteria is ratherFc or Fab dependent we tested the reactivity of an irrelevant

human IgG (chimeric anti-human TNF) against MS fecalmicrobiota As shown irrelevant human IgG binding to MSmicrobiota is significantly reduced compared with autologousserum IgG (06 [0ndash22] vs 67 [05ndash171] p lt 00001figure 2 C) indicating that serum IgG targets fecal microbiotain a mostly Fab-mediated manner

We then askedwhether disease duration or treatments could skewantimicrobiota IgG response As shown recently diagnosed anduntreated patients with CIS also exhibit an enhanced anti-microbiota IgG response comparedwith controls (55 [34ndash84]n = 12 vs 11 [02ndash32] n = 30 p lt 00001 figure 2 B) FinallyIgG+ proportions of autologous microbiota did not significantlydiffer between treated and untreated patients with RR-MS (72[05ndash171] n = 16 vs untreated patients 54 [05ndash85] n = 10 p= 041 figure e-1 B linkslwwcomNXIA468) Taken togetherthese data reveal an enhanced triggering of systemic IgG immunityagainst gut microbiota occurring early in MS

Loss of Antimicrobiota IgAIgG Convergencein MSWe previously observed that serum antimicrobiota IgG con-verges with secretory IgA to target the same bacterial cells inhealthy individuals23 As expected serum IgG exclusivelycolabels secretory IgA-bound fecal bacteria in healthy donors23

but preferentially targets IgA-unbound bacteria in patients withMS (19 [02ndash69] in healthy donors (n = 30) vs 41

Figure 1 IgA-Coated Bacteria Are Reduced in Patients With Severe MS

(A) Representative Flow Cytometry Dot Plot Showing Endogenous Secretory IgA Coating on Indicated Fractions of Fecal Microbiota From aHealthy Donor or aPatientWith RR-MS (B) Flow cytometry analysis of the fraction of fecalmicrobiota bound by secretory IgA in healthy donors (n = 32) and patients with RR-MS (n= 32) Median values are indicated and groups were compared with a nonparametric Mann-Whitney test (ns not significant) (C) Flow cytometry analysis ofthe fraction of fecal microbiota bound by secretory IgA in healthy donors (n = 32) and patients with RR-MS classified according to disease severity measuredby the EDSS Median values are indicated Error bars represent minimum and maximum values p Values were calculated using the nonparametric Mann-Whitney test (p lt 005 p lt 0001) (D) Disease severity evaluated by the EDSS correlatedwith the percentage of secretory IgA-boundmicrobiota in patientswith MS-RR Spearman coefficient (r) and p values are indicated MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 5

Figure 2 Enhanced IgG Antimicrobiota Response in Patients With MS

(A) Procedure (top) and representative flow cytometry detection (bottom) of endogenous secretory IgA and autologous systemic IgG (10 μgmL) coating onhealthy or RR-MS fecal microbiota is presented (middle panels) (B) Proportions of IgG+IgAplusmn (left) and IgG+IgAminus (right) coated bacteria are compared in healthydonors (n = 30) patients with RR-MS (n = 32) or patients with CIS (n = 12) Median values are indicated p Values were calculated using the nonparametricMann-Whitney test (p lt 0001) (C) Representative flow cytometry dot plot (left) and analysis (right) of irrelevant IgG (anti-TNFα 10 μgmL) binding to RR-MSand CIS fecalmicrobiota (n = 44) (D) Procedure (top) representative flow cytometry dot plot (middle panel) and analysis (bottom) of autologous IgG or pooledhealthy IgG binding to RR-MS fecal microbiota (n = 32) Proportions of IgG+IgAplusmn coated bacteria (bottom left) IgG+IgA+ and IgG+IgAminus coated bacteria (bottomright) are shown p Value was calculated by using the Wilcoxon paired test (p lt 00001 ns = nonsignificant) CIS = clinically isolated syndrome MS =multiple sclerosis RR = relapsing-remitting

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

[01ndash164] and 25 [13ndash54] in patients with RR-MS (n =32) and CIS (n = 12) respectively p lt 0001 figure 2 B)

We then asked whether this observed loss of convergence wasrelated to IgG responses only found in MS or to an increasedMS microbiota reactivity to IgG in general To address thisissue we compared MS and pooled healthy IgG reactivity toMS microbiota As shown in figure 2 D although pooledhealthy IgG bound a smaller fraction of RR-MS microbiotacompared with autologous RR-MS IgG (19 [0ndash10] vs 67[05ndash171] n = 32 p lt 00001 figure 2 D) pooled healthyIgG targets not only in vivo IgA-bound but also IgA-free RR-MS microbiota (07 [0ndash64] vs 06 [0ndash62] p = 079figure 2 D) suggesting that MSmicrobiota are more prone tointeract with IgG regardless of their origin

From this part we conclude that the IgAIgG convergencerule does not apply to MS A previously IgA-ignored fractionof MS microbiota is able to interact with IgG

Antimicrobiota IgG Spreading in MSIt could be deduced from above that IgG binding to MSmicrobiota might extend beyond the usual bacterial repertoireof IgA targets that we and others previously defined2327 Todecipher antimicrobiota IgG specificities in patients with MSwith higher resolution we next isolated IgG-bound bacteria byflow cytometry sorting and identified their taxonomy using 16SrRNA sequencing (figure 3 A) To avoid confounding factorsfrom IMD therapies we sorted IgG-bound bacteria from un-treated patients We first examined microbial diversity by cal-culating the Shannon index and found a similar diversity inIgG-only and Ig-negative bacteria (206 [114ndash27] vs 212[18ndash232] n = 5 figure 3 B) In contrast dually coated bacteriatended to exhibit a decreased microbial diversity in comparisonwith the previous fractions (074 [053ndash135] n = 5 p = 0062figure 3 B) suggesting a state of restricted antimicrobiota se-cretory IgA diversity in MS We observed that individualIgA+IgG+ and IgAminusIgG+ bacterial repertoires were remarkablydistinct in all tested patients (figure 3 C for back-to-back

Figure 3 MS IgG Binds a Diverse Repertoire of Commensals

(A) Sorting strategy of IgG+IgA+ and IgG+IgAminus-coated bacteria in 5 patients with RR-MS Composition of sorted fractions was next analyzed by using 16S rRNAsequencing (B) Genera diversity in IgG+IgA+ and IgG+IgAminus-sorted fractions calculated by using the Shannon index (C) Median relative abundance of the 30most frequent genera in sorted fractions from 1 representative RR-MS patient MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 7

taxonomic analysis of fecal IgA+IgG+ IgAminusIgG+ and IgAminusIgG-

fractions in a representative patient) supporting an IgG re-sponse spread beyond IgA-surveyed taxa To quantitativelycompare IgAminusIgG+ and IgA+IgG+microbial compositions in thestudied population we thereafter narrowed the analysis down tothe 30 most frequent genera that were present in at least 80 ofthe Ig-sorted fractions Three genera derived from FirmicutesBlautia Clostridium and Eubacterium appeared preferentiallytargeted by MS serum IgG alone as deduced from their com-pared frequencies in IgAminusIgG+ bacteria and IgA+IgG+ sortedfractions (006 [002ndash02] vs 0004 [0002ndash0033] 007[0055ndash045] vs 0003 [0009ndash0041] and 0023 [0003ndash004] vs0002 [0001ndash0003] respectively p lt 005 figure 4 AndashC) Wesought to validate these observations by calculating a log-basedEI that is not influenced by genera representation in the studiedsamples We confirmed that both Clostridium and Eubacteriumwere predominant in IgAminusIgG+- compared with IgA+IgG+-

sorted fractions (EI minus06 [minus083 to 022] vs minus137 [minus166 to064] and minus018 [minus039 to 105] vs minus093 [minus174 to 019] forClostridium and Eubacterium respectively p lt 005 figure 4 B)

Taken together these results demonstrate thatMS is associatedwith a broad antimicrobiota systemic IgG response with avariety of bacterial genera being preferentially targeted by MSIgG compared with MS IgA Because systemic IgG could re-flect silent gut microbiota translocation episodes232829 theseobservations could support an enhanced bacterial translocationfrom the gut lumen to systemic compartments in patientswith MS

Increased MS IgG Antibacterial ResponsesConfirmed at the Strain LevelBacterial identification remains at the genus level using 16SrRNA sequencing Using bacterial flow cytometry we sought

Figure 4 IgG-Bound Commensal Shift in Patients With MS

(A) Relative abundance of the 30 most frequent genera in IgG+IgA+ and IgG+IgAminus-sorted fractions in blue and red respectively (B) EI of the 30 most frequentgenera in IgG+IgA+ and IgG+IgAminus-sorted fractions compared in blue and red respectively (C) Blautia Clostridium and Eubacterium frequencies in IgG+IgA+ andIgG+IgAminus fractions For all box plots each dot represents 1 donor errors bars represent maximum andminimum values andmedians are indicated p Valueswere calculated by using the nonparametric Mann-Whitney test (p lt 005 p lt 001) EI = enrichment index MS = multiple sclerosis

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

to determine whether increased antimicrobiota IgG reactiv-ities could also be measured at the species level in patientswith MS We therefore selected representative bacterialstrains that were previously described as secretory IgA targetsin humans (A muciniphila B longum and R obeum) and 3others that are not preferentially bound by IgA in humans (Bcaccae F nucleatum and P oris)30-33 A majority of strainstested (4 of 6) tended to be more brightly stained by MS (RRandor CIS) IgG that by control serum IgG (figure 5 AndashC)Preferred MS IgG targets are not systematically preferredcontrol IgA targets as neither B longum nor B caccae werepreferentially bound by MS IgG compared with control IgG(figure e-5 linkslwwcomNXIA472) Of interest we ob-served that F nucleatum is particularly well opsonized by CISserum compared with healthy control serum but also com-pared with RR serum (IgG median fluorescence intensity 518[146ndash5505] vs 126 [407ndash414] and 156 [421ndash3350] in CIShealthy control and RR serums respectively figure 5 AndashB)We conclude from this part thatMS serum contains high titersof antimicrobiota IgG that can be measured against specificbacterial strains

DiscussionWe report an imbalance between systemic and secretoryantimicrobiota antibody responses in patients with MS

Antimicrobiota secretory IgA responses are impaired whereas atthe same time systemic IgG responses directed against autolo-gous microbiota are increased compared with healthy controlsImportantly although we found MS-associated gut microbiotaalterations the systemic IgG responses observed were not di-rected to taxa overrepresented in MS microbiota Furthermorelow proportions of IgA-coated bacteria among fecal microbiotaare associated with disease severity Consistently with the latterobservation Rojas et al34 have recently reported that proportionsof IgA-coated bacteria among fecal microbiota are decreased inpatients with relapsing MS compared with inactive patients Re-duced proportions of fecal IgA+ bacteria might result from re-duced secretory IgA levels in relation with IgA-secreting cellsegress out of the gutmucosa Previous works indeed shed light onthe dynamic trafficking of intraepithelial CD4T cells between gutand an inflamed CNS35 and also of IgA-producing cells betweengut and bone marrow36 In the EAE model commensal-reactiveIgA-producing cells regulate inflammation via the production IL-10 after their migration in the CNS Probstel et al recentlydemonstrated that gut microbiota IgA-producing cells infiltratethe CNS in patients with MS leading to an increase in in situ IgAproduction during MS relapses However commensal-specificIgA does not cross-react with brain tissue37 Further studies arerequired to assess the role of gut-derived IgA-producing cells inMS pathophysiology Defective IgA binding to gut microbiota indisabled patients with MS might be also related to microbialcommunity alterations

Figure 5 MS Antimicrobiota IgG Signature Measured on Cultivable Bacterial Strains

(A) Representative flow cytometry analysis ofserum IgG binding to F nucleatum Gray histo-gram represents isotype control blue red andred dotted lines represent a healthy controlpatient with RR-MS and patient with CIS re-spectively (B) Flow cytometry analysis of serumIgG binding to IgA-unbound bacteria (from leftto right F nucleatum and P oris) in healthy do-nors (n = 30) patients with RR-MS (=32) andpatients with CIS (n = 12) (C) Flow cytometryanalysis of serum IgG binding to IgA-boundbacteria (from left to right A muciniphila and Robeum) in healthy donors (n = 30) patients withRR-MS (=32) and patients with CIS (n = 12) Darkbars represent medians p Values were calcu-lated by using the nonparametric Mann-Whit-ney test (p lt 005 p lt 001 p lt 0001)p lt 00001) MS = multiple sclerosis RR =relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 9

Impaired IgA-mediated microbiota survey might also favorbacterial translocation and induction of systemic IgG re-sponses as we previously described in patients with IgA de-ficiency23 Numerous studies underline that IgG typicallypresent in the CSF of the patients might play a role in MSpathogenesis38-40 IgG purified from patients withMS increasesmouse CNS demyelination and exacerbates EAE41 The mainclinical evidence for a potential pathogenic role for antibodiesin MS is the benefit of antibody removal in a subset of patientswith acute neurologic symptoms4243 However what triggersoligoclonal antibody responses observed in MS CSF remains amystery38-40 Antimicrobiota antibodies are frequently cross-reactive with self-antigens44-46 Cross-reactivity is probably anessential antimicrobial antibody feature enabling control of alarge diversity of gut commensals and invading pathogens4748

but is also associated with a theoretical risk of autoimmunity Ithas indeed been very recently proposed that some pathogenicimmune responses associated with lupus4950 or myocarditis51

can be triggered by commensal bacteria through a molecularmimicry mechanism Of interest more than 20 bacterial spe-cies well represented in human gut microbiota express pro-teins that contain potentially encephalitogenic peptides iehighly homologous to myelin basic protein (MBP) or myelinoligodendrocyte protein In particular 4 proteins expressed byF nucleatum have been identified as potentially encephalito-genic52 Moreover 3 proteins expressed by members of theClostridium genera were described to be homologous toMBP52 We report that RR-MS and even more so CIS IgGvery brightly stain F nucleatum We also report that the Clos-tridium genus is significantly more targeted by systemicMS IgGthan by IgA These observations open new avenues for ex-ploring the hypothesis that immune responses induced bycommensal antigens might play a role in MS pathogenesis

Enhanced induction of antimicrobiota IgG could also be aconsequence of intestinal barrier dysfunction in MS Indeednot only blood-brain barrier defects have been reported inMSbut also a state of increased intestinal permeability charac-terized by higher lactulosemannitol urinary ratios increasedlipopolysaccharide plasma levels and associated with in-creased plasma zonulin a regulator of epithelial and endo-thelial barrier functions53-55 In the EAE model suchalterations preceded the onset of neurologic symptoms56 It istherefore conceivable that bacterial translocation could occurat early stages of the disease and account for the increasedantimicrobiota IgG responses we observe as early as the CISstage Systemic antimicrobiota IgG responses are likely tomirror microbiota composition at the individual level Wetherefore tested some of the bacterial species differentiallyidentified between MS and healthy donor gut microbiota245

We observed that patients with RR-MS and CISharbor ele-vated serum antindashA muciniphila IgG reactivities comparedwith controls These results are in line with the higher levels ofantindashA muciniphila antibodies detected in CSF of patientswith MS57 Of interest a greater A muciniphila prevalence inMS gut microbiota has been shown in previous reports25 Apositive correlation between A muciniphila abundance and

gene expression in T cells and monocytes involved in keypathways previously implicated in MS pathogenesis was alsoreported5 Located in close vicinity with the intestinal cells Amuciniphila has also been reported to promoteTh1 lymphocyte differentiation a T-cell subset classicallyassociated with MS pathogenesis258 Altogether althoughonly correlative these observations suggest a role for Amuciniphila in driving exacerbated host immune responsein MS

As mentioned above signs of increased intestinal permeabilitypreceded the onset of neurologic symptoms in the EAEmodel56 It would now remain to determine to which extentantimicrobiota IgG monitoring could represent a tool topredict MS progression Not all patients with CIS progress tofull blown MS Baseline lesions age at onset and short timedelay between the first 2 relapses provide some predictivevalue for disease progression yet remain flawed5960 Whethermicrobialhost signatures including immune responses tocommensals could help to distinguish between stable casesand future progressive cases should be now tested in largerstudies

In summary we have found quantitative and qualitative al-terations of the antimicrobiota antibody response in patientswith MS involving peculiar bacterial taxa We propose thatsuch peculiar IgG responses associated with MS could rep-resent a lead in the quest for antigenic drivers possibly im-plicated in MS pathogenesis through a molecular mimicrymechanism We also propose that antimicrobiota fingerprintsshould be more widely used in further studies to seek forcorrelates with disease status Finally microbiota manipula-tion is now a recognized therapeutic option in the clinic61

How our results could translate into treatments shouldtherefore be tested in murine MS models

AcknowledgmentThe authors thank the patients who agreed to participate inthis study and doctors and nurses from the NeurologyDepartment (Hopital Pitie-Salpetriere 75013 Paris Franceand Hopital Fondation Adolphe de Rotschild 75019 ParisFrance) who participated in this study

Study FundingThe study was supported by a grant from Fondation pourlrsquoAide a la Recherche sur la Sclerose en Plaques (ARSEP) andby a proof of concept grant (CoPoc) from Inserm TransfertParis France

DisclosureG Gorochov is a scientific advisor for Luxia Scientific FranceThe other authors report no disclosures relevant to themanuscript Go to NeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 7 2020 Accepted in final form March 9 2021

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

This information is current as of May 11 2021

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References httpnnneurologyorgcontent84e997fullhtmlref-list-1

This article cites 60 articles 11 of which you can access for free at

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is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Page 3: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

treated DNA concentration was estimated by Nanodrop(Thermo Scientific)

Bacterial Strains and Culture ConditionsPrevotella oris and Fusobacterium nucleatum were isolated fromhuman samples and identified by matrix-assisted laser desorptionionizationndashtime of flight mass spectrometry (Microbiology De-partment Pitie-Salpetriere Hospital Paris) Both bacterial strainswere cultured on sheep red blood agar plates at 37degC underanaerobic conditions for 48 hours Ruminococcus obeum (VPI B3-21) Bacteroides caccae (VPI 3452A) Bifidobacterium longum(E194v variant A) and Akkermansia muciniphila (DSM-22959)were characterized at the Institut National de Recherche Agron-omique (Jouy-en-Josas France) A muciniphila was cultured inL-YHBHI4 medium (Liquid-Yeast extract Hemin Brain HeartInfusion) with mucin whereas R obeum was cultured in PYGmedium (peptonendashyeast extractndashdextrose) at 37degC under anaer-obic conditions Bacterial cultures were suspended in 1xPBSndash10glycerol (109 colony-forming units [CFUs]mL) and frozen atminus80degC Quantification of CFUs was performed by addingcounting beads (Beckman Coulter) to bacterial suspensions andthen analyzed by flow cytometry (FACSCanto II BD)

Bacterial Flow CytometrySystemic IgG and secretory IgA binding to microbiota wasassessed by bacterial flow cytometry as previously described21

Briefly thawed microbiota or bacterial strains (107 bacteriaconditions) were fixed in a solution of 4 paraformaldehyde andstained with Cell Proliferation Dye eFluor 450 (eBioscience)After washing with 1xPBS (10 minutes 4000g 4degC) cells weresuspended in 1xPBS 2 bovine serum albumin (Sigma) and002 sodium azide (Sigma) and incubated in a 96-V bottomwell plate with a 10 μgmL IgG solution (from either humanserum or pooled human IgG HizentrandashCSL Behring France orhuman anti-tumor necrosis factor (TNF) RemicadendashMSDFrance) per condition All buffers were passed through sterile022-μm filters before use After washing secondary conjugatedantibodies (goat anti-human IgA-FITC and goat anti-humanIgG-A647) or isotype controls (both from Jackson ImmunoR-esearch Laboratories West Grove) were added for 20 minutes at4degC Then bacteria were suspended in sterile PBS Thirtythousand bacterial events were acquired on a FACSCanto II flowcytometer (Becton Dickinson) Analysis was performed withFlowJo software (Treestar) Frequencies of Ig-bound microbiotawere expressed as percentages median minimum andmaximumvalues throughout the article Medians of fluorescence were usedto measure IgG-binding levels for pure bacterial strains

16S Ribosomal RNA Phylogenetic Analysis ofTotal Gut Microbiota and IgG-Coated FractionsPurified microbiota (108condition) was washed in 1xPBSand stained with secondary conjugated antibodies (goat anti-human IgA-FITC and goat anti-human IgG-A647) or isotypecontrols (both from Jackson ImmunoResearch LaboratoriesWest Grove) After washing sorting was performed using amicrobiota-dedicated single laser S3 cell sorter (Bio-RadLaboratories CA) 105 bacteria per fraction was collected in1xPBS centrifuged and immediately stored as dry pellets atminus80degC Purity for both fractions was systematically verifiedafter sorting DNA was extracted and the V3ndashV4 region ofthe 16S ribosomal ribonucleic acid (rRNA) gene was ampli-fied by seminested polymerase chain reaction (PCR) PrimersV3fwd (+357) 59 TACGGRAGGCAGCAG 39 and V4rev(+857) 59 ATCTTACCAGGGTATCTAATCCT 39 wereused during the first round of PCR (10 cycles) PrimersV3fwd and X926_Rev (+926) 59 CCGTCAATTCMTT-TRAGT 39 were used in the second PCR round (40 cycles)PCR amplicon libraries were sequenced using a MiSeq Illu-mina platform (Genotoul Toulouse France) The resultingsequences from whole microbiota were analyzed using theopen source software package Quantitative Insights IntoMicrobial Ecology22 as previously described23 Demultiplexedreads from IgG-coated bacteria were processed using MG-RAST analysis pipeline Sequencing artifacts host DNAcontamination and sequences less than 200 bp in length wereremoved Insufficient quality reads were discarded (lt5 oftotal reads) Sequences were then clustered into operationaltaxonomic units (OTUs) with a 97 homology usingGreengenes database OTUs containing only a single se-quence were discarded OTUs detected at gt01 relative

Table Demographic and Clinical Features of the Cohort

Healthydonors RR-MS CIS-MS

Sex (HF) 1220 1119 513

BMI 218(187ndash319)

226(164ndash327)

229(184ndash391)

Age 329(236ndash613)

384(189ndash606)

318(181ndash548)

Age at onset NA 298(123ndash532)

315(18ndash547)

Duration of thedisease

NA 7 (05ndash26) NA

EDSS score

lt3 NA 18 14

3ndash5 NA 10 4

gt5 NA 2 0

Treatment

IFN-β 0 8 NA

Glatirameracetate

0 6 NA

Mitoxantrone 0 1 NA

Solumedrol 0 1 NA

Natalizumab 0 1 NA

wo (or ND) 10 (3) NA

Abbreviations BMI = bodymass index IFN = interferon NA = not applicablewo = withoutNumbers for ages and duration of the disease refer to years

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 3

abundance in at least 2 samples were finally conserved Thisprocess reduced the total OTU count from 297 down to 102OTU table was rarefied to the minimum samplersquos depth(18727 reads) Shannon index was calculated according tothe following equation Shannon index = minusΣpiln(pi) where pi isthe relative abundance of the ith OTU in the data set Incalculating the enrichment index (EI) we scored a pseudo-relative abundance equal to 00001 which was the lower limitof detection if a taxon was not detected in a given fractionIgAminusIgG+ EI refers to

log10

IgA minus IgG+ taxon abundanceIgA minus IgGminus taxon abundance

and IgA+IgG+ EI to log10

IgA + IgG+ taxon abundanceIgA minus IgGminus taxon abundance

Statistical AnalysisStatistical analysis was performed using Graphpad Prism v6The Wilcoxon paired rank test was used when comparingpaired groups whereas the Mann-Whitney test was usedwhen comparing 2 independent groups For multiple com-parisons the Kruskall-Wallis test with post hoc Dunn test wasconducted Significant p values are indicated on plots (p lt005 p lt 001 lt0001)

Standard Protocol Approvals Registrationsand Patient ConsentsThe study had been approved by the local ethics committee ofPitie-Salpetriere Hospital (CPP Ile de France VI) A priorwritten consent was obtained from all the patients and con-trols before inclusion in the study

Data AvailabilityAll relevant data are available within the article Anonymizeddata are available and will be shared on request from anyqualified investigator

ResultsCollapsed IgA Interactions With FecalMicrobiota in Patients With Severe MSWe postulated that not only microbiota composition345 butalso the immunemicrobiota interface could be perturbed inMS We first compared IgA-bound fecal microbiota levels inhealthy donors and patients with MS In accordance withprevious studies19 we observed in the control group used forthe present study that IgA binds a median percentage of 76(08ndash188 n = 30) of the whole fecal microbiota (figure 1 Aand B) The proportion of IgA-bound fecal microbiota is notsignificantly decreased in patients with MS (median [minndashmax] 51 [02ndash272] n = 32 figure 1 A and B) comparedwith controls (p = 033) We then evaluated whether the IgAmicrobiota interface could be more perturbed in severely af-fected patients Disease severity at the time of serum andmicrobiota sampling was evaluated using the Extended Dis-ability Status Scale (EDSS)24 We observed that disabled

patients with RR-MS (EDSS score gt4) had significantly reducedproportions of IgA-bound fecal bacteria compared with patientswith RR-MS without disability (EDSS score lt2) (102[024ndash602] vs 123 [149ndash2723] respectively p lt 0001figure 1 C) Strikingly disease severity is inversely correlated withthe IgA-coated fecalmicrobiota fraction at the individual level (r=minus0647 p lt 00001 figure 1 D) Because the majority of severepatients were on immunosuppressive treatment we sought toexplore whether IMD therapies might alter IgA responses Wefound no difference in IgA-coated proportions of fecal microbiotabetween untreated and treated patients (figure e-1 A linkslwwcomNXIA468) Moreover interferon- and glatiramer-treatedpatients exhibited similar IgA-bound fecal microbiota levelscompared with untreated patients with MS (figure e-1 A linkslwwcomNXIA468) These results argue for a defective surveyof the gut microbiota by IgA in disabled patients with MS

MS-Associated Gut Microbiota DysbiosisWe reasoned that defective IgA responses in disabled patientswith MS might be related to differences in gut microbiotacomposition We therefore performed 16S rRNA sequencingand identified bacterial taxa at the genus level We first examinedthe microbial diversity by calculating the Shannon index Wefound that alpha-diversity ie the number of different specieswithin a sample was not different between healthy donors andpatients with MS (median [minndashmax] 051 [030ndash059] vs 053[039ndash061] p = 019 figure e-2 A linkslwwcomNXIA469)However neither disease severity nor treatments appeared toaffect bacterial diversity (figure e-2 A linkslwwcomNXIA469) We next investigated the frequency of prevalent genera(gt1 in any sample group) Consistent with previous findings8

the relative abundance of Clostridium cluster IV was increased inpatients withMS compared with healthy donors (51 times 10minus3 [54times 10minus4ndash003] vs 002 [15 times 10minus3 times 011] p = 002 figure e-2 BlinkslwwcomNXIA469) This difference was mainly drivenby a drastic increase ofClostridium cluster IV in disabled patients(0022 [31 times 10minus3ndash011] in patients with EDSS score gt2 p =00011 figure e-2 C linkslwwcomNXIA469) As previouslydescribed5 we found that Prevotella tended to be un-derrepresented in patients with MS (15 times 10minus3 [0ndash036] vs 004[0ndash06] p = 009 figure e-2 B linkslwwcomNXIA469) Wealso observed a decrease in the genus Coprococcus (001 [2 times10minus4ndash007] vs 0017 [0ndash008] p = 012 figure e-2 A linkslwwcomNXIA469) In both cases these alterations tended to bemore profound in severe cases (figure e-2 C linkslwwcomNXIA469) Previous reports described effects of IMD therapieson gut microbiota composition589 We therefore comparedmost prevalent genera in untreated interferon- and glatiramer-treated patients and healthy donors Although unclassified Clos-tridiales tended to be increased in interferon-treated patients(008 [17 times 10minus4ndash014] vs 0009 [93 times 10minus5ndash006] in healthydonors p = 006 figure e-3 linkslwwcomNXIA470) we didnot observe statistically significant differences between groupswhich is likely explained by the limited number of patients ineach subgroup Taken together these results indicate subtledifferences in the gutmicrobial communities in patients withMSthat might modulate gut IgA responses

4 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

Exacerbated Antimicrobiota IgG Response inPatients With MSWe then postulated that the perturbed gut IgAmicrobiota in-terface observed above could translate into abnormal systemicanticommensal immune responses in patients Indeed the hu-moral response to microbiota is not gut confined as high anti-commensal IgGs are detected in healthy donorrsquos serum232526

We used a previously described flow cytometric assay21 to con-comitantly detect secretory IgA and serum IgG binding to au-tologous fecal microbiota (figure 2 A) Of note IgG is about 5times more abundant in serum than IgA consequently thisprotocol allows us to detect serum IgG but not serum IgAbinding to fecal microbiota as shown in figure e-4 A linkslwwcomNXIA471 Moreover IgG is not transferred in the gutlumen leading to a negligible IgG-bound bacteria fraction inhuman feces (figure e-4 B linkslwwcomNXIA471)23 Alto-gether this assay measured specifically secretory IgA preboundlocally in the gut and serum IgG binding without interference ofserum IgA nor intestinal IgG We found both quantitative andqualitative antimicrobiota IgG alterations in patients with MSregardless of disease severity As shown MS serum IgG interactswith a broader proportion of fecal microbiota compared withcontrols (67 [05ndash171] n = 32 vs 11 [02ndash32] n = 30 p lt00001 figure 2 B)

To investigate whether IgG binding to fecal bacteria is ratherFc or Fab dependent we tested the reactivity of an irrelevant

human IgG (chimeric anti-human TNF) against MS fecalmicrobiota As shown irrelevant human IgG binding to MSmicrobiota is significantly reduced compared with autologousserum IgG (06 [0ndash22] vs 67 [05ndash171] p lt 00001figure 2 C) indicating that serum IgG targets fecal microbiotain a mostly Fab-mediated manner

We then askedwhether disease duration or treatments could skewantimicrobiota IgG response As shown recently diagnosed anduntreated patients with CIS also exhibit an enhanced anti-microbiota IgG response comparedwith controls (55 [34ndash84]n = 12 vs 11 [02ndash32] n = 30 p lt 00001 figure 2 B) FinallyIgG+ proportions of autologous microbiota did not significantlydiffer between treated and untreated patients with RR-MS (72[05ndash171] n = 16 vs untreated patients 54 [05ndash85] n = 10 p= 041 figure e-1 B linkslwwcomNXIA468) Taken togetherthese data reveal an enhanced triggering of systemic IgG immunityagainst gut microbiota occurring early in MS

Loss of Antimicrobiota IgAIgG Convergencein MSWe previously observed that serum antimicrobiota IgG con-verges with secretory IgA to target the same bacterial cells inhealthy individuals23 As expected serum IgG exclusivelycolabels secretory IgA-bound fecal bacteria in healthy donors23

but preferentially targets IgA-unbound bacteria in patients withMS (19 [02ndash69] in healthy donors (n = 30) vs 41

Figure 1 IgA-Coated Bacteria Are Reduced in Patients With Severe MS

(A) Representative Flow Cytometry Dot Plot Showing Endogenous Secretory IgA Coating on Indicated Fractions of Fecal Microbiota From aHealthy Donor or aPatientWith RR-MS (B) Flow cytometry analysis of the fraction of fecalmicrobiota bound by secretory IgA in healthy donors (n = 32) and patients with RR-MS (n= 32) Median values are indicated and groups were compared with a nonparametric Mann-Whitney test (ns not significant) (C) Flow cytometry analysis ofthe fraction of fecal microbiota bound by secretory IgA in healthy donors (n = 32) and patients with RR-MS classified according to disease severity measuredby the EDSS Median values are indicated Error bars represent minimum and maximum values p Values were calculated using the nonparametric Mann-Whitney test (p lt 005 p lt 0001) (D) Disease severity evaluated by the EDSS correlatedwith the percentage of secretory IgA-boundmicrobiota in patientswith MS-RR Spearman coefficient (r) and p values are indicated MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 5

Figure 2 Enhanced IgG Antimicrobiota Response in Patients With MS

(A) Procedure (top) and representative flow cytometry detection (bottom) of endogenous secretory IgA and autologous systemic IgG (10 μgmL) coating onhealthy or RR-MS fecal microbiota is presented (middle panels) (B) Proportions of IgG+IgAplusmn (left) and IgG+IgAminus (right) coated bacteria are compared in healthydonors (n = 30) patients with RR-MS (n = 32) or patients with CIS (n = 12) Median values are indicated p Values were calculated using the nonparametricMann-Whitney test (p lt 0001) (C) Representative flow cytometry dot plot (left) and analysis (right) of irrelevant IgG (anti-TNFα 10 μgmL) binding to RR-MSand CIS fecalmicrobiota (n = 44) (D) Procedure (top) representative flow cytometry dot plot (middle panel) and analysis (bottom) of autologous IgG or pooledhealthy IgG binding to RR-MS fecal microbiota (n = 32) Proportions of IgG+IgAplusmn coated bacteria (bottom left) IgG+IgA+ and IgG+IgAminus coated bacteria (bottomright) are shown p Value was calculated by using the Wilcoxon paired test (p lt 00001 ns = nonsignificant) CIS = clinically isolated syndrome MS =multiple sclerosis RR = relapsing-remitting

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

[01ndash164] and 25 [13ndash54] in patients with RR-MS (n =32) and CIS (n = 12) respectively p lt 0001 figure 2 B)

We then asked whether this observed loss of convergence wasrelated to IgG responses only found in MS or to an increasedMS microbiota reactivity to IgG in general To address thisissue we compared MS and pooled healthy IgG reactivity toMS microbiota As shown in figure 2 D although pooledhealthy IgG bound a smaller fraction of RR-MS microbiotacompared with autologous RR-MS IgG (19 [0ndash10] vs 67[05ndash171] n = 32 p lt 00001 figure 2 D) pooled healthyIgG targets not only in vivo IgA-bound but also IgA-free RR-MS microbiota (07 [0ndash64] vs 06 [0ndash62] p = 079figure 2 D) suggesting that MSmicrobiota are more prone tointeract with IgG regardless of their origin

From this part we conclude that the IgAIgG convergencerule does not apply to MS A previously IgA-ignored fractionof MS microbiota is able to interact with IgG

Antimicrobiota IgG Spreading in MSIt could be deduced from above that IgG binding to MSmicrobiota might extend beyond the usual bacterial repertoireof IgA targets that we and others previously defined2327 Todecipher antimicrobiota IgG specificities in patients with MSwith higher resolution we next isolated IgG-bound bacteria byflow cytometry sorting and identified their taxonomy using 16SrRNA sequencing (figure 3 A) To avoid confounding factorsfrom IMD therapies we sorted IgG-bound bacteria from un-treated patients We first examined microbial diversity by cal-culating the Shannon index and found a similar diversity inIgG-only and Ig-negative bacteria (206 [114ndash27] vs 212[18ndash232] n = 5 figure 3 B) In contrast dually coated bacteriatended to exhibit a decreased microbial diversity in comparisonwith the previous fractions (074 [053ndash135] n = 5 p = 0062figure 3 B) suggesting a state of restricted antimicrobiota se-cretory IgA diversity in MS We observed that individualIgA+IgG+ and IgAminusIgG+ bacterial repertoires were remarkablydistinct in all tested patients (figure 3 C for back-to-back

Figure 3 MS IgG Binds a Diverse Repertoire of Commensals

(A) Sorting strategy of IgG+IgA+ and IgG+IgAminus-coated bacteria in 5 patients with RR-MS Composition of sorted fractions was next analyzed by using 16S rRNAsequencing (B) Genera diversity in IgG+IgA+ and IgG+IgAminus-sorted fractions calculated by using the Shannon index (C) Median relative abundance of the 30most frequent genera in sorted fractions from 1 representative RR-MS patient MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 7

taxonomic analysis of fecal IgA+IgG+ IgAminusIgG+ and IgAminusIgG-

fractions in a representative patient) supporting an IgG re-sponse spread beyond IgA-surveyed taxa To quantitativelycompare IgAminusIgG+ and IgA+IgG+microbial compositions in thestudied population we thereafter narrowed the analysis down tothe 30 most frequent genera that were present in at least 80 ofthe Ig-sorted fractions Three genera derived from FirmicutesBlautia Clostridium and Eubacterium appeared preferentiallytargeted by MS serum IgG alone as deduced from their com-pared frequencies in IgAminusIgG+ bacteria and IgA+IgG+ sortedfractions (006 [002ndash02] vs 0004 [0002ndash0033] 007[0055ndash045] vs 0003 [0009ndash0041] and 0023 [0003ndash004] vs0002 [0001ndash0003] respectively p lt 005 figure 4 AndashC) Wesought to validate these observations by calculating a log-basedEI that is not influenced by genera representation in the studiedsamples We confirmed that both Clostridium and Eubacteriumwere predominant in IgAminusIgG+- compared with IgA+IgG+-

sorted fractions (EI minus06 [minus083 to 022] vs minus137 [minus166 to064] and minus018 [minus039 to 105] vs minus093 [minus174 to 019] forClostridium and Eubacterium respectively p lt 005 figure 4 B)

Taken together these results demonstrate thatMS is associatedwith a broad antimicrobiota systemic IgG response with avariety of bacterial genera being preferentially targeted by MSIgG compared with MS IgA Because systemic IgG could re-flect silent gut microbiota translocation episodes232829 theseobservations could support an enhanced bacterial translocationfrom the gut lumen to systemic compartments in patientswith MS

Increased MS IgG Antibacterial ResponsesConfirmed at the Strain LevelBacterial identification remains at the genus level using 16SrRNA sequencing Using bacterial flow cytometry we sought

Figure 4 IgG-Bound Commensal Shift in Patients With MS

(A) Relative abundance of the 30 most frequent genera in IgG+IgA+ and IgG+IgAminus-sorted fractions in blue and red respectively (B) EI of the 30 most frequentgenera in IgG+IgA+ and IgG+IgAminus-sorted fractions compared in blue and red respectively (C) Blautia Clostridium and Eubacterium frequencies in IgG+IgA+ andIgG+IgAminus fractions For all box plots each dot represents 1 donor errors bars represent maximum andminimum values andmedians are indicated p Valueswere calculated by using the nonparametric Mann-Whitney test (p lt 005 p lt 001) EI = enrichment index MS = multiple sclerosis

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

to determine whether increased antimicrobiota IgG reactiv-ities could also be measured at the species level in patientswith MS We therefore selected representative bacterialstrains that were previously described as secretory IgA targetsin humans (A muciniphila B longum and R obeum) and 3others that are not preferentially bound by IgA in humans (Bcaccae F nucleatum and P oris)30-33 A majority of strainstested (4 of 6) tended to be more brightly stained by MS (RRandor CIS) IgG that by control serum IgG (figure 5 AndashC)Preferred MS IgG targets are not systematically preferredcontrol IgA targets as neither B longum nor B caccae werepreferentially bound by MS IgG compared with control IgG(figure e-5 linkslwwcomNXIA472) Of interest we ob-served that F nucleatum is particularly well opsonized by CISserum compared with healthy control serum but also com-pared with RR serum (IgG median fluorescence intensity 518[146ndash5505] vs 126 [407ndash414] and 156 [421ndash3350] in CIShealthy control and RR serums respectively figure 5 AndashB)We conclude from this part thatMS serum contains high titersof antimicrobiota IgG that can be measured against specificbacterial strains

DiscussionWe report an imbalance between systemic and secretoryantimicrobiota antibody responses in patients with MS

Antimicrobiota secretory IgA responses are impaired whereas atthe same time systemic IgG responses directed against autolo-gous microbiota are increased compared with healthy controlsImportantly although we found MS-associated gut microbiotaalterations the systemic IgG responses observed were not di-rected to taxa overrepresented in MS microbiota Furthermorelow proportions of IgA-coated bacteria among fecal microbiotaare associated with disease severity Consistently with the latterobservation Rojas et al34 have recently reported that proportionsof IgA-coated bacteria among fecal microbiota are decreased inpatients with relapsing MS compared with inactive patients Re-duced proportions of fecal IgA+ bacteria might result from re-duced secretory IgA levels in relation with IgA-secreting cellsegress out of the gutmucosa Previous works indeed shed light onthe dynamic trafficking of intraepithelial CD4T cells between gutand an inflamed CNS35 and also of IgA-producing cells betweengut and bone marrow36 In the EAE model commensal-reactiveIgA-producing cells regulate inflammation via the production IL-10 after their migration in the CNS Probstel et al recentlydemonstrated that gut microbiota IgA-producing cells infiltratethe CNS in patients with MS leading to an increase in in situ IgAproduction during MS relapses However commensal-specificIgA does not cross-react with brain tissue37 Further studies arerequired to assess the role of gut-derived IgA-producing cells inMS pathophysiology Defective IgA binding to gut microbiota indisabled patients with MS might be also related to microbialcommunity alterations

Figure 5 MS Antimicrobiota IgG Signature Measured on Cultivable Bacterial Strains

(A) Representative flow cytometry analysis ofserum IgG binding to F nucleatum Gray histo-gram represents isotype control blue red andred dotted lines represent a healthy controlpatient with RR-MS and patient with CIS re-spectively (B) Flow cytometry analysis of serumIgG binding to IgA-unbound bacteria (from leftto right F nucleatum and P oris) in healthy do-nors (n = 30) patients with RR-MS (=32) andpatients with CIS (n = 12) (C) Flow cytometryanalysis of serum IgG binding to IgA-boundbacteria (from left to right A muciniphila and Robeum) in healthy donors (n = 30) patients withRR-MS (=32) and patients with CIS (n = 12) Darkbars represent medians p Values were calcu-lated by using the nonparametric Mann-Whit-ney test (p lt 005 p lt 001 p lt 0001)p lt 00001) MS = multiple sclerosis RR =relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 9

Impaired IgA-mediated microbiota survey might also favorbacterial translocation and induction of systemic IgG re-sponses as we previously described in patients with IgA de-ficiency23 Numerous studies underline that IgG typicallypresent in the CSF of the patients might play a role in MSpathogenesis38-40 IgG purified from patients withMS increasesmouse CNS demyelination and exacerbates EAE41 The mainclinical evidence for a potential pathogenic role for antibodiesin MS is the benefit of antibody removal in a subset of patientswith acute neurologic symptoms4243 However what triggersoligoclonal antibody responses observed in MS CSF remains amystery38-40 Antimicrobiota antibodies are frequently cross-reactive with self-antigens44-46 Cross-reactivity is probably anessential antimicrobial antibody feature enabling control of alarge diversity of gut commensals and invading pathogens4748

but is also associated with a theoretical risk of autoimmunity Ithas indeed been very recently proposed that some pathogenicimmune responses associated with lupus4950 or myocarditis51

can be triggered by commensal bacteria through a molecularmimicry mechanism Of interest more than 20 bacterial spe-cies well represented in human gut microbiota express pro-teins that contain potentially encephalitogenic peptides iehighly homologous to myelin basic protein (MBP) or myelinoligodendrocyte protein In particular 4 proteins expressed byF nucleatum have been identified as potentially encephalito-genic52 Moreover 3 proteins expressed by members of theClostridium genera were described to be homologous toMBP52 We report that RR-MS and even more so CIS IgGvery brightly stain F nucleatum We also report that the Clos-tridium genus is significantly more targeted by systemicMS IgGthan by IgA These observations open new avenues for ex-ploring the hypothesis that immune responses induced bycommensal antigens might play a role in MS pathogenesis

Enhanced induction of antimicrobiota IgG could also be aconsequence of intestinal barrier dysfunction in MS Indeednot only blood-brain barrier defects have been reported inMSbut also a state of increased intestinal permeability charac-terized by higher lactulosemannitol urinary ratios increasedlipopolysaccharide plasma levels and associated with in-creased plasma zonulin a regulator of epithelial and endo-thelial barrier functions53-55 In the EAE model suchalterations preceded the onset of neurologic symptoms56 It istherefore conceivable that bacterial translocation could occurat early stages of the disease and account for the increasedantimicrobiota IgG responses we observe as early as the CISstage Systemic antimicrobiota IgG responses are likely tomirror microbiota composition at the individual level Wetherefore tested some of the bacterial species differentiallyidentified between MS and healthy donor gut microbiota245

We observed that patients with RR-MS and CISharbor ele-vated serum antindashA muciniphila IgG reactivities comparedwith controls These results are in line with the higher levels ofantindashA muciniphila antibodies detected in CSF of patientswith MS57 Of interest a greater A muciniphila prevalence inMS gut microbiota has been shown in previous reports25 Apositive correlation between A muciniphila abundance and

gene expression in T cells and monocytes involved in keypathways previously implicated in MS pathogenesis was alsoreported5 Located in close vicinity with the intestinal cells Amuciniphila has also been reported to promoteTh1 lymphocyte differentiation a T-cell subset classicallyassociated with MS pathogenesis258 Altogether althoughonly correlative these observations suggest a role for Amuciniphila in driving exacerbated host immune responsein MS

As mentioned above signs of increased intestinal permeabilitypreceded the onset of neurologic symptoms in the EAEmodel56 It would now remain to determine to which extentantimicrobiota IgG monitoring could represent a tool topredict MS progression Not all patients with CIS progress tofull blown MS Baseline lesions age at onset and short timedelay between the first 2 relapses provide some predictivevalue for disease progression yet remain flawed5960 Whethermicrobialhost signatures including immune responses tocommensals could help to distinguish between stable casesand future progressive cases should be now tested in largerstudies

In summary we have found quantitative and qualitative al-terations of the antimicrobiota antibody response in patientswith MS involving peculiar bacterial taxa We propose thatsuch peculiar IgG responses associated with MS could rep-resent a lead in the quest for antigenic drivers possibly im-plicated in MS pathogenesis through a molecular mimicrymechanism We also propose that antimicrobiota fingerprintsshould be more widely used in further studies to seek forcorrelates with disease status Finally microbiota manipula-tion is now a recognized therapeutic option in the clinic61

How our results could translate into treatments shouldtherefore be tested in murine MS models

AcknowledgmentThe authors thank the patients who agreed to participate inthis study and doctors and nurses from the NeurologyDepartment (Hopital Pitie-Salpetriere 75013 Paris Franceand Hopital Fondation Adolphe de Rotschild 75019 ParisFrance) who participated in this study

Study FundingThe study was supported by a grant from Fondation pourlrsquoAide a la Recherche sur la Sclerose en Plaques (ARSEP) andby a proof of concept grant (CoPoc) from Inserm TransfertParis France

DisclosureG Gorochov is a scientific advisor for Luxia Scientific FranceThe other authors report no disclosures relevant to themanuscript Go to NeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 7 2020 Accepted in final form March 9 2021

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

This information is current as of May 11 2021

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httpnnneurologyorgcontent84e997fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent84e997fullhtmlref-list-1

This article cites 60 articles 11 of which you can access for free at

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Page 4: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

abundance in at least 2 samples were finally conserved Thisprocess reduced the total OTU count from 297 down to 102OTU table was rarefied to the minimum samplersquos depth(18727 reads) Shannon index was calculated according tothe following equation Shannon index = minusΣpiln(pi) where pi isthe relative abundance of the ith OTU in the data set Incalculating the enrichment index (EI) we scored a pseudo-relative abundance equal to 00001 which was the lower limitof detection if a taxon was not detected in a given fractionIgAminusIgG+ EI refers to

log10

IgA minus IgG+ taxon abundanceIgA minus IgGminus taxon abundance

and IgA+IgG+ EI to log10

IgA + IgG+ taxon abundanceIgA minus IgGminus taxon abundance

Statistical AnalysisStatistical analysis was performed using Graphpad Prism v6The Wilcoxon paired rank test was used when comparingpaired groups whereas the Mann-Whitney test was usedwhen comparing 2 independent groups For multiple com-parisons the Kruskall-Wallis test with post hoc Dunn test wasconducted Significant p values are indicated on plots (p lt005 p lt 001 lt0001)

Standard Protocol Approvals Registrationsand Patient ConsentsThe study had been approved by the local ethics committee ofPitie-Salpetriere Hospital (CPP Ile de France VI) A priorwritten consent was obtained from all the patients and con-trols before inclusion in the study

Data AvailabilityAll relevant data are available within the article Anonymizeddata are available and will be shared on request from anyqualified investigator

ResultsCollapsed IgA Interactions With FecalMicrobiota in Patients With Severe MSWe postulated that not only microbiota composition345 butalso the immunemicrobiota interface could be perturbed inMS We first compared IgA-bound fecal microbiota levels inhealthy donors and patients with MS In accordance withprevious studies19 we observed in the control group used forthe present study that IgA binds a median percentage of 76(08ndash188 n = 30) of the whole fecal microbiota (figure 1 Aand B) The proportion of IgA-bound fecal microbiota is notsignificantly decreased in patients with MS (median [minndashmax] 51 [02ndash272] n = 32 figure 1 A and B) comparedwith controls (p = 033) We then evaluated whether the IgAmicrobiota interface could be more perturbed in severely af-fected patients Disease severity at the time of serum andmicrobiota sampling was evaluated using the Extended Dis-ability Status Scale (EDSS)24 We observed that disabled

patients with RR-MS (EDSS score gt4) had significantly reducedproportions of IgA-bound fecal bacteria compared with patientswith RR-MS without disability (EDSS score lt2) (102[024ndash602] vs 123 [149ndash2723] respectively p lt 0001figure 1 C) Strikingly disease severity is inversely correlated withthe IgA-coated fecalmicrobiota fraction at the individual level (r=minus0647 p lt 00001 figure 1 D) Because the majority of severepatients were on immunosuppressive treatment we sought toexplore whether IMD therapies might alter IgA responses Wefound no difference in IgA-coated proportions of fecal microbiotabetween untreated and treated patients (figure e-1 A linkslwwcomNXIA468) Moreover interferon- and glatiramer-treatedpatients exhibited similar IgA-bound fecal microbiota levelscompared with untreated patients with MS (figure e-1 A linkslwwcomNXIA468) These results argue for a defective surveyof the gut microbiota by IgA in disabled patients with MS

MS-Associated Gut Microbiota DysbiosisWe reasoned that defective IgA responses in disabled patientswith MS might be related to differences in gut microbiotacomposition We therefore performed 16S rRNA sequencingand identified bacterial taxa at the genus level We first examinedthe microbial diversity by calculating the Shannon index Wefound that alpha-diversity ie the number of different specieswithin a sample was not different between healthy donors andpatients with MS (median [minndashmax] 051 [030ndash059] vs 053[039ndash061] p = 019 figure e-2 A linkslwwcomNXIA469)However neither disease severity nor treatments appeared toaffect bacterial diversity (figure e-2 A linkslwwcomNXIA469) We next investigated the frequency of prevalent genera(gt1 in any sample group) Consistent with previous findings8

the relative abundance of Clostridium cluster IV was increased inpatients withMS compared with healthy donors (51 times 10minus3 [54times 10minus4ndash003] vs 002 [15 times 10minus3 times 011] p = 002 figure e-2 BlinkslwwcomNXIA469) This difference was mainly drivenby a drastic increase ofClostridium cluster IV in disabled patients(0022 [31 times 10minus3ndash011] in patients with EDSS score gt2 p =00011 figure e-2 C linkslwwcomNXIA469) As previouslydescribed5 we found that Prevotella tended to be un-derrepresented in patients with MS (15 times 10minus3 [0ndash036] vs 004[0ndash06] p = 009 figure e-2 B linkslwwcomNXIA469) Wealso observed a decrease in the genus Coprococcus (001 [2 times10minus4ndash007] vs 0017 [0ndash008] p = 012 figure e-2 A linkslwwcomNXIA469) In both cases these alterations tended to bemore profound in severe cases (figure e-2 C linkslwwcomNXIA469) Previous reports described effects of IMD therapieson gut microbiota composition589 We therefore comparedmost prevalent genera in untreated interferon- and glatiramer-treated patients and healthy donors Although unclassified Clos-tridiales tended to be increased in interferon-treated patients(008 [17 times 10minus4ndash014] vs 0009 [93 times 10minus5ndash006] in healthydonors p = 006 figure e-3 linkslwwcomNXIA470) we didnot observe statistically significant differences between groupswhich is likely explained by the limited number of patients ineach subgroup Taken together these results indicate subtledifferences in the gutmicrobial communities in patients withMSthat might modulate gut IgA responses

4 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

Exacerbated Antimicrobiota IgG Response inPatients With MSWe then postulated that the perturbed gut IgAmicrobiota in-terface observed above could translate into abnormal systemicanticommensal immune responses in patients Indeed the hu-moral response to microbiota is not gut confined as high anti-commensal IgGs are detected in healthy donorrsquos serum232526

We used a previously described flow cytometric assay21 to con-comitantly detect secretory IgA and serum IgG binding to au-tologous fecal microbiota (figure 2 A) Of note IgG is about 5times more abundant in serum than IgA consequently thisprotocol allows us to detect serum IgG but not serum IgAbinding to fecal microbiota as shown in figure e-4 A linkslwwcomNXIA471 Moreover IgG is not transferred in the gutlumen leading to a negligible IgG-bound bacteria fraction inhuman feces (figure e-4 B linkslwwcomNXIA471)23 Alto-gether this assay measured specifically secretory IgA preboundlocally in the gut and serum IgG binding without interference ofserum IgA nor intestinal IgG We found both quantitative andqualitative antimicrobiota IgG alterations in patients with MSregardless of disease severity As shown MS serum IgG interactswith a broader proportion of fecal microbiota compared withcontrols (67 [05ndash171] n = 32 vs 11 [02ndash32] n = 30 p lt00001 figure 2 B)

To investigate whether IgG binding to fecal bacteria is ratherFc or Fab dependent we tested the reactivity of an irrelevant

human IgG (chimeric anti-human TNF) against MS fecalmicrobiota As shown irrelevant human IgG binding to MSmicrobiota is significantly reduced compared with autologousserum IgG (06 [0ndash22] vs 67 [05ndash171] p lt 00001figure 2 C) indicating that serum IgG targets fecal microbiotain a mostly Fab-mediated manner

We then askedwhether disease duration or treatments could skewantimicrobiota IgG response As shown recently diagnosed anduntreated patients with CIS also exhibit an enhanced anti-microbiota IgG response comparedwith controls (55 [34ndash84]n = 12 vs 11 [02ndash32] n = 30 p lt 00001 figure 2 B) FinallyIgG+ proportions of autologous microbiota did not significantlydiffer between treated and untreated patients with RR-MS (72[05ndash171] n = 16 vs untreated patients 54 [05ndash85] n = 10 p= 041 figure e-1 B linkslwwcomNXIA468) Taken togetherthese data reveal an enhanced triggering of systemic IgG immunityagainst gut microbiota occurring early in MS

Loss of Antimicrobiota IgAIgG Convergencein MSWe previously observed that serum antimicrobiota IgG con-verges with secretory IgA to target the same bacterial cells inhealthy individuals23 As expected serum IgG exclusivelycolabels secretory IgA-bound fecal bacteria in healthy donors23

but preferentially targets IgA-unbound bacteria in patients withMS (19 [02ndash69] in healthy donors (n = 30) vs 41

Figure 1 IgA-Coated Bacteria Are Reduced in Patients With Severe MS

(A) Representative Flow Cytometry Dot Plot Showing Endogenous Secretory IgA Coating on Indicated Fractions of Fecal Microbiota From aHealthy Donor or aPatientWith RR-MS (B) Flow cytometry analysis of the fraction of fecalmicrobiota bound by secretory IgA in healthy donors (n = 32) and patients with RR-MS (n= 32) Median values are indicated and groups were compared with a nonparametric Mann-Whitney test (ns not significant) (C) Flow cytometry analysis ofthe fraction of fecal microbiota bound by secretory IgA in healthy donors (n = 32) and patients with RR-MS classified according to disease severity measuredby the EDSS Median values are indicated Error bars represent minimum and maximum values p Values were calculated using the nonparametric Mann-Whitney test (p lt 005 p lt 0001) (D) Disease severity evaluated by the EDSS correlatedwith the percentage of secretory IgA-boundmicrobiota in patientswith MS-RR Spearman coefficient (r) and p values are indicated MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 5

Figure 2 Enhanced IgG Antimicrobiota Response in Patients With MS

(A) Procedure (top) and representative flow cytometry detection (bottom) of endogenous secretory IgA and autologous systemic IgG (10 μgmL) coating onhealthy or RR-MS fecal microbiota is presented (middle panels) (B) Proportions of IgG+IgAplusmn (left) and IgG+IgAminus (right) coated bacteria are compared in healthydonors (n = 30) patients with RR-MS (n = 32) or patients with CIS (n = 12) Median values are indicated p Values were calculated using the nonparametricMann-Whitney test (p lt 0001) (C) Representative flow cytometry dot plot (left) and analysis (right) of irrelevant IgG (anti-TNFα 10 μgmL) binding to RR-MSand CIS fecalmicrobiota (n = 44) (D) Procedure (top) representative flow cytometry dot plot (middle panel) and analysis (bottom) of autologous IgG or pooledhealthy IgG binding to RR-MS fecal microbiota (n = 32) Proportions of IgG+IgAplusmn coated bacteria (bottom left) IgG+IgA+ and IgG+IgAminus coated bacteria (bottomright) are shown p Value was calculated by using the Wilcoxon paired test (p lt 00001 ns = nonsignificant) CIS = clinically isolated syndrome MS =multiple sclerosis RR = relapsing-remitting

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

[01ndash164] and 25 [13ndash54] in patients with RR-MS (n =32) and CIS (n = 12) respectively p lt 0001 figure 2 B)

We then asked whether this observed loss of convergence wasrelated to IgG responses only found in MS or to an increasedMS microbiota reactivity to IgG in general To address thisissue we compared MS and pooled healthy IgG reactivity toMS microbiota As shown in figure 2 D although pooledhealthy IgG bound a smaller fraction of RR-MS microbiotacompared with autologous RR-MS IgG (19 [0ndash10] vs 67[05ndash171] n = 32 p lt 00001 figure 2 D) pooled healthyIgG targets not only in vivo IgA-bound but also IgA-free RR-MS microbiota (07 [0ndash64] vs 06 [0ndash62] p = 079figure 2 D) suggesting that MSmicrobiota are more prone tointeract with IgG regardless of their origin

From this part we conclude that the IgAIgG convergencerule does not apply to MS A previously IgA-ignored fractionof MS microbiota is able to interact with IgG

Antimicrobiota IgG Spreading in MSIt could be deduced from above that IgG binding to MSmicrobiota might extend beyond the usual bacterial repertoireof IgA targets that we and others previously defined2327 Todecipher antimicrobiota IgG specificities in patients with MSwith higher resolution we next isolated IgG-bound bacteria byflow cytometry sorting and identified their taxonomy using 16SrRNA sequencing (figure 3 A) To avoid confounding factorsfrom IMD therapies we sorted IgG-bound bacteria from un-treated patients We first examined microbial diversity by cal-culating the Shannon index and found a similar diversity inIgG-only and Ig-negative bacteria (206 [114ndash27] vs 212[18ndash232] n = 5 figure 3 B) In contrast dually coated bacteriatended to exhibit a decreased microbial diversity in comparisonwith the previous fractions (074 [053ndash135] n = 5 p = 0062figure 3 B) suggesting a state of restricted antimicrobiota se-cretory IgA diversity in MS We observed that individualIgA+IgG+ and IgAminusIgG+ bacterial repertoires were remarkablydistinct in all tested patients (figure 3 C for back-to-back

Figure 3 MS IgG Binds a Diverse Repertoire of Commensals

(A) Sorting strategy of IgG+IgA+ and IgG+IgAminus-coated bacteria in 5 patients with RR-MS Composition of sorted fractions was next analyzed by using 16S rRNAsequencing (B) Genera diversity in IgG+IgA+ and IgG+IgAminus-sorted fractions calculated by using the Shannon index (C) Median relative abundance of the 30most frequent genera in sorted fractions from 1 representative RR-MS patient MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 7

taxonomic analysis of fecal IgA+IgG+ IgAminusIgG+ and IgAminusIgG-

fractions in a representative patient) supporting an IgG re-sponse spread beyond IgA-surveyed taxa To quantitativelycompare IgAminusIgG+ and IgA+IgG+microbial compositions in thestudied population we thereafter narrowed the analysis down tothe 30 most frequent genera that were present in at least 80 ofthe Ig-sorted fractions Three genera derived from FirmicutesBlautia Clostridium and Eubacterium appeared preferentiallytargeted by MS serum IgG alone as deduced from their com-pared frequencies in IgAminusIgG+ bacteria and IgA+IgG+ sortedfractions (006 [002ndash02] vs 0004 [0002ndash0033] 007[0055ndash045] vs 0003 [0009ndash0041] and 0023 [0003ndash004] vs0002 [0001ndash0003] respectively p lt 005 figure 4 AndashC) Wesought to validate these observations by calculating a log-basedEI that is not influenced by genera representation in the studiedsamples We confirmed that both Clostridium and Eubacteriumwere predominant in IgAminusIgG+- compared with IgA+IgG+-

sorted fractions (EI minus06 [minus083 to 022] vs minus137 [minus166 to064] and minus018 [minus039 to 105] vs minus093 [minus174 to 019] forClostridium and Eubacterium respectively p lt 005 figure 4 B)

Taken together these results demonstrate thatMS is associatedwith a broad antimicrobiota systemic IgG response with avariety of bacterial genera being preferentially targeted by MSIgG compared with MS IgA Because systemic IgG could re-flect silent gut microbiota translocation episodes232829 theseobservations could support an enhanced bacterial translocationfrom the gut lumen to systemic compartments in patientswith MS

Increased MS IgG Antibacterial ResponsesConfirmed at the Strain LevelBacterial identification remains at the genus level using 16SrRNA sequencing Using bacterial flow cytometry we sought

Figure 4 IgG-Bound Commensal Shift in Patients With MS

(A) Relative abundance of the 30 most frequent genera in IgG+IgA+ and IgG+IgAminus-sorted fractions in blue and red respectively (B) EI of the 30 most frequentgenera in IgG+IgA+ and IgG+IgAminus-sorted fractions compared in blue and red respectively (C) Blautia Clostridium and Eubacterium frequencies in IgG+IgA+ andIgG+IgAminus fractions For all box plots each dot represents 1 donor errors bars represent maximum andminimum values andmedians are indicated p Valueswere calculated by using the nonparametric Mann-Whitney test (p lt 005 p lt 001) EI = enrichment index MS = multiple sclerosis

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

to determine whether increased antimicrobiota IgG reactiv-ities could also be measured at the species level in patientswith MS We therefore selected representative bacterialstrains that were previously described as secretory IgA targetsin humans (A muciniphila B longum and R obeum) and 3others that are not preferentially bound by IgA in humans (Bcaccae F nucleatum and P oris)30-33 A majority of strainstested (4 of 6) tended to be more brightly stained by MS (RRandor CIS) IgG that by control serum IgG (figure 5 AndashC)Preferred MS IgG targets are not systematically preferredcontrol IgA targets as neither B longum nor B caccae werepreferentially bound by MS IgG compared with control IgG(figure e-5 linkslwwcomNXIA472) Of interest we ob-served that F nucleatum is particularly well opsonized by CISserum compared with healthy control serum but also com-pared with RR serum (IgG median fluorescence intensity 518[146ndash5505] vs 126 [407ndash414] and 156 [421ndash3350] in CIShealthy control and RR serums respectively figure 5 AndashB)We conclude from this part thatMS serum contains high titersof antimicrobiota IgG that can be measured against specificbacterial strains

DiscussionWe report an imbalance between systemic and secretoryantimicrobiota antibody responses in patients with MS

Antimicrobiota secretory IgA responses are impaired whereas atthe same time systemic IgG responses directed against autolo-gous microbiota are increased compared with healthy controlsImportantly although we found MS-associated gut microbiotaalterations the systemic IgG responses observed were not di-rected to taxa overrepresented in MS microbiota Furthermorelow proportions of IgA-coated bacteria among fecal microbiotaare associated with disease severity Consistently with the latterobservation Rojas et al34 have recently reported that proportionsof IgA-coated bacteria among fecal microbiota are decreased inpatients with relapsing MS compared with inactive patients Re-duced proportions of fecal IgA+ bacteria might result from re-duced secretory IgA levels in relation with IgA-secreting cellsegress out of the gutmucosa Previous works indeed shed light onthe dynamic trafficking of intraepithelial CD4T cells between gutand an inflamed CNS35 and also of IgA-producing cells betweengut and bone marrow36 In the EAE model commensal-reactiveIgA-producing cells regulate inflammation via the production IL-10 after their migration in the CNS Probstel et al recentlydemonstrated that gut microbiota IgA-producing cells infiltratethe CNS in patients with MS leading to an increase in in situ IgAproduction during MS relapses However commensal-specificIgA does not cross-react with brain tissue37 Further studies arerequired to assess the role of gut-derived IgA-producing cells inMS pathophysiology Defective IgA binding to gut microbiota indisabled patients with MS might be also related to microbialcommunity alterations

Figure 5 MS Antimicrobiota IgG Signature Measured on Cultivable Bacterial Strains

(A) Representative flow cytometry analysis ofserum IgG binding to F nucleatum Gray histo-gram represents isotype control blue red andred dotted lines represent a healthy controlpatient with RR-MS and patient with CIS re-spectively (B) Flow cytometry analysis of serumIgG binding to IgA-unbound bacteria (from leftto right F nucleatum and P oris) in healthy do-nors (n = 30) patients with RR-MS (=32) andpatients with CIS (n = 12) (C) Flow cytometryanalysis of serum IgG binding to IgA-boundbacteria (from left to right A muciniphila and Robeum) in healthy donors (n = 30) patients withRR-MS (=32) and patients with CIS (n = 12) Darkbars represent medians p Values were calcu-lated by using the nonparametric Mann-Whit-ney test (p lt 005 p lt 001 p lt 0001)p lt 00001) MS = multiple sclerosis RR =relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 9

Impaired IgA-mediated microbiota survey might also favorbacterial translocation and induction of systemic IgG re-sponses as we previously described in patients with IgA de-ficiency23 Numerous studies underline that IgG typicallypresent in the CSF of the patients might play a role in MSpathogenesis38-40 IgG purified from patients withMS increasesmouse CNS demyelination and exacerbates EAE41 The mainclinical evidence for a potential pathogenic role for antibodiesin MS is the benefit of antibody removal in a subset of patientswith acute neurologic symptoms4243 However what triggersoligoclonal antibody responses observed in MS CSF remains amystery38-40 Antimicrobiota antibodies are frequently cross-reactive with self-antigens44-46 Cross-reactivity is probably anessential antimicrobial antibody feature enabling control of alarge diversity of gut commensals and invading pathogens4748

but is also associated with a theoretical risk of autoimmunity Ithas indeed been very recently proposed that some pathogenicimmune responses associated with lupus4950 or myocarditis51

can be triggered by commensal bacteria through a molecularmimicry mechanism Of interest more than 20 bacterial spe-cies well represented in human gut microbiota express pro-teins that contain potentially encephalitogenic peptides iehighly homologous to myelin basic protein (MBP) or myelinoligodendrocyte protein In particular 4 proteins expressed byF nucleatum have been identified as potentially encephalito-genic52 Moreover 3 proteins expressed by members of theClostridium genera were described to be homologous toMBP52 We report that RR-MS and even more so CIS IgGvery brightly stain F nucleatum We also report that the Clos-tridium genus is significantly more targeted by systemicMS IgGthan by IgA These observations open new avenues for ex-ploring the hypothesis that immune responses induced bycommensal antigens might play a role in MS pathogenesis

Enhanced induction of antimicrobiota IgG could also be aconsequence of intestinal barrier dysfunction in MS Indeednot only blood-brain barrier defects have been reported inMSbut also a state of increased intestinal permeability charac-terized by higher lactulosemannitol urinary ratios increasedlipopolysaccharide plasma levels and associated with in-creased plasma zonulin a regulator of epithelial and endo-thelial barrier functions53-55 In the EAE model suchalterations preceded the onset of neurologic symptoms56 It istherefore conceivable that bacterial translocation could occurat early stages of the disease and account for the increasedantimicrobiota IgG responses we observe as early as the CISstage Systemic antimicrobiota IgG responses are likely tomirror microbiota composition at the individual level Wetherefore tested some of the bacterial species differentiallyidentified between MS and healthy donor gut microbiota245

We observed that patients with RR-MS and CISharbor ele-vated serum antindashA muciniphila IgG reactivities comparedwith controls These results are in line with the higher levels ofantindashA muciniphila antibodies detected in CSF of patientswith MS57 Of interest a greater A muciniphila prevalence inMS gut microbiota has been shown in previous reports25 Apositive correlation between A muciniphila abundance and

gene expression in T cells and monocytes involved in keypathways previously implicated in MS pathogenesis was alsoreported5 Located in close vicinity with the intestinal cells Amuciniphila has also been reported to promoteTh1 lymphocyte differentiation a T-cell subset classicallyassociated with MS pathogenesis258 Altogether althoughonly correlative these observations suggest a role for Amuciniphila in driving exacerbated host immune responsein MS

As mentioned above signs of increased intestinal permeabilitypreceded the onset of neurologic symptoms in the EAEmodel56 It would now remain to determine to which extentantimicrobiota IgG monitoring could represent a tool topredict MS progression Not all patients with CIS progress tofull blown MS Baseline lesions age at onset and short timedelay between the first 2 relapses provide some predictivevalue for disease progression yet remain flawed5960 Whethermicrobialhost signatures including immune responses tocommensals could help to distinguish between stable casesand future progressive cases should be now tested in largerstudies

In summary we have found quantitative and qualitative al-terations of the antimicrobiota antibody response in patientswith MS involving peculiar bacterial taxa We propose thatsuch peculiar IgG responses associated with MS could rep-resent a lead in the quest for antigenic drivers possibly im-plicated in MS pathogenesis through a molecular mimicrymechanism We also propose that antimicrobiota fingerprintsshould be more widely used in further studies to seek forcorrelates with disease status Finally microbiota manipula-tion is now a recognized therapeutic option in the clinic61

How our results could translate into treatments shouldtherefore be tested in murine MS models

AcknowledgmentThe authors thank the patients who agreed to participate inthis study and doctors and nurses from the NeurologyDepartment (Hopital Pitie-Salpetriere 75013 Paris Franceand Hopital Fondation Adolphe de Rotschild 75019 ParisFrance) who participated in this study

Study FundingThe study was supported by a grant from Fondation pourlrsquoAide a la Recherche sur la Sclerose en Plaques (ARSEP) andby a proof of concept grant (CoPoc) from Inserm TransfertParis France

DisclosureG Gorochov is a scientific advisor for Luxia Scientific FranceThe other authors report no disclosures relevant to themanuscript Go to NeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 7 2020 Accepted in final form March 9 2021

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2021 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Page 5: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

Exacerbated Antimicrobiota IgG Response inPatients With MSWe then postulated that the perturbed gut IgAmicrobiota in-terface observed above could translate into abnormal systemicanticommensal immune responses in patients Indeed the hu-moral response to microbiota is not gut confined as high anti-commensal IgGs are detected in healthy donorrsquos serum232526

We used a previously described flow cytometric assay21 to con-comitantly detect secretory IgA and serum IgG binding to au-tologous fecal microbiota (figure 2 A) Of note IgG is about 5times more abundant in serum than IgA consequently thisprotocol allows us to detect serum IgG but not serum IgAbinding to fecal microbiota as shown in figure e-4 A linkslwwcomNXIA471 Moreover IgG is not transferred in the gutlumen leading to a negligible IgG-bound bacteria fraction inhuman feces (figure e-4 B linkslwwcomNXIA471)23 Alto-gether this assay measured specifically secretory IgA preboundlocally in the gut and serum IgG binding without interference ofserum IgA nor intestinal IgG We found both quantitative andqualitative antimicrobiota IgG alterations in patients with MSregardless of disease severity As shown MS serum IgG interactswith a broader proportion of fecal microbiota compared withcontrols (67 [05ndash171] n = 32 vs 11 [02ndash32] n = 30 p lt00001 figure 2 B)

To investigate whether IgG binding to fecal bacteria is ratherFc or Fab dependent we tested the reactivity of an irrelevant

human IgG (chimeric anti-human TNF) against MS fecalmicrobiota As shown irrelevant human IgG binding to MSmicrobiota is significantly reduced compared with autologousserum IgG (06 [0ndash22] vs 67 [05ndash171] p lt 00001figure 2 C) indicating that serum IgG targets fecal microbiotain a mostly Fab-mediated manner

We then askedwhether disease duration or treatments could skewantimicrobiota IgG response As shown recently diagnosed anduntreated patients with CIS also exhibit an enhanced anti-microbiota IgG response comparedwith controls (55 [34ndash84]n = 12 vs 11 [02ndash32] n = 30 p lt 00001 figure 2 B) FinallyIgG+ proportions of autologous microbiota did not significantlydiffer between treated and untreated patients with RR-MS (72[05ndash171] n = 16 vs untreated patients 54 [05ndash85] n = 10 p= 041 figure e-1 B linkslwwcomNXIA468) Taken togetherthese data reveal an enhanced triggering of systemic IgG immunityagainst gut microbiota occurring early in MS

Loss of Antimicrobiota IgAIgG Convergencein MSWe previously observed that serum antimicrobiota IgG con-verges with secretory IgA to target the same bacterial cells inhealthy individuals23 As expected serum IgG exclusivelycolabels secretory IgA-bound fecal bacteria in healthy donors23

but preferentially targets IgA-unbound bacteria in patients withMS (19 [02ndash69] in healthy donors (n = 30) vs 41

Figure 1 IgA-Coated Bacteria Are Reduced in Patients With Severe MS

(A) Representative Flow Cytometry Dot Plot Showing Endogenous Secretory IgA Coating on Indicated Fractions of Fecal Microbiota From aHealthy Donor or aPatientWith RR-MS (B) Flow cytometry analysis of the fraction of fecalmicrobiota bound by secretory IgA in healthy donors (n = 32) and patients with RR-MS (n= 32) Median values are indicated and groups were compared with a nonparametric Mann-Whitney test (ns not significant) (C) Flow cytometry analysis ofthe fraction of fecal microbiota bound by secretory IgA in healthy donors (n = 32) and patients with RR-MS classified according to disease severity measuredby the EDSS Median values are indicated Error bars represent minimum and maximum values p Values were calculated using the nonparametric Mann-Whitney test (p lt 005 p lt 0001) (D) Disease severity evaluated by the EDSS correlatedwith the percentage of secretory IgA-boundmicrobiota in patientswith MS-RR Spearman coefficient (r) and p values are indicated MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 5

Figure 2 Enhanced IgG Antimicrobiota Response in Patients With MS

(A) Procedure (top) and representative flow cytometry detection (bottom) of endogenous secretory IgA and autologous systemic IgG (10 μgmL) coating onhealthy or RR-MS fecal microbiota is presented (middle panels) (B) Proportions of IgG+IgAplusmn (left) and IgG+IgAminus (right) coated bacteria are compared in healthydonors (n = 30) patients with RR-MS (n = 32) or patients with CIS (n = 12) Median values are indicated p Values were calculated using the nonparametricMann-Whitney test (p lt 0001) (C) Representative flow cytometry dot plot (left) and analysis (right) of irrelevant IgG (anti-TNFα 10 μgmL) binding to RR-MSand CIS fecalmicrobiota (n = 44) (D) Procedure (top) representative flow cytometry dot plot (middle panel) and analysis (bottom) of autologous IgG or pooledhealthy IgG binding to RR-MS fecal microbiota (n = 32) Proportions of IgG+IgAplusmn coated bacteria (bottom left) IgG+IgA+ and IgG+IgAminus coated bacteria (bottomright) are shown p Value was calculated by using the Wilcoxon paired test (p lt 00001 ns = nonsignificant) CIS = clinically isolated syndrome MS =multiple sclerosis RR = relapsing-remitting

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

[01ndash164] and 25 [13ndash54] in patients with RR-MS (n =32) and CIS (n = 12) respectively p lt 0001 figure 2 B)

We then asked whether this observed loss of convergence wasrelated to IgG responses only found in MS or to an increasedMS microbiota reactivity to IgG in general To address thisissue we compared MS and pooled healthy IgG reactivity toMS microbiota As shown in figure 2 D although pooledhealthy IgG bound a smaller fraction of RR-MS microbiotacompared with autologous RR-MS IgG (19 [0ndash10] vs 67[05ndash171] n = 32 p lt 00001 figure 2 D) pooled healthyIgG targets not only in vivo IgA-bound but also IgA-free RR-MS microbiota (07 [0ndash64] vs 06 [0ndash62] p = 079figure 2 D) suggesting that MSmicrobiota are more prone tointeract with IgG regardless of their origin

From this part we conclude that the IgAIgG convergencerule does not apply to MS A previously IgA-ignored fractionof MS microbiota is able to interact with IgG

Antimicrobiota IgG Spreading in MSIt could be deduced from above that IgG binding to MSmicrobiota might extend beyond the usual bacterial repertoireof IgA targets that we and others previously defined2327 Todecipher antimicrobiota IgG specificities in patients with MSwith higher resolution we next isolated IgG-bound bacteria byflow cytometry sorting and identified their taxonomy using 16SrRNA sequencing (figure 3 A) To avoid confounding factorsfrom IMD therapies we sorted IgG-bound bacteria from un-treated patients We first examined microbial diversity by cal-culating the Shannon index and found a similar diversity inIgG-only and Ig-negative bacteria (206 [114ndash27] vs 212[18ndash232] n = 5 figure 3 B) In contrast dually coated bacteriatended to exhibit a decreased microbial diversity in comparisonwith the previous fractions (074 [053ndash135] n = 5 p = 0062figure 3 B) suggesting a state of restricted antimicrobiota se-cretory IgA diversity in MS We observed that individualIgA+IgG+ and IgAminusIgG+ bacterial repertoires were remarkablydistinct in all tested patients (figure 3 C for back-to-back

Figure 3 MS IgG Binds a Diverse Repertoire of Commensals

(A) Sorting strategy of IgG+IgA+ and IgG+IgAminus-coated bacteria in 5 patients with RR-MS Composition of sorted fractions was next analyzed by using 16S rRNAsequencing (B) Genera diversity in IgG+IgA+ and IgG+IgAminus-sorted fractions calculated by using the Shannon index (C) Median relative abundance of the 30most frequent genera in sorted fractions from 1 representative RR-MS patient MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 7

taxonomic analysis of fecal IgA+IgG+ IgAminusIgG+ and IgAminusIgG-

fractions in a representative patient) supporting an IgG re-sponse spread beyond IgA-surveyed taxa To quantitativelycompare IgAminusIgG+ and IgA+IgG+microbial compositions in thestudied population we thereafter narrowed the analysis down tothe 30 most frequent genera that were present in at least 80 ofthe Ig-sorted fractions Three genera derived from FirmicutesBlautia Clostridium and Eubacterium appeared preferentiallytargeted by MS serum IgG alone as deduced from their com-pared frequencies in IgAminusIgG+ bacteria and IgA+IgG+ sortedfractions (006 [002ndash02] vs 0004 [0002ndash0033] 007[0055ndash045] vs 0003 [0009ndash0041] and 0023 [0003ndash004] vs0002 [0001ndash0003] respectively p lt 005 figure 4 AndashC) Wesought to validate these observations by calculating a log-basedEI that is not influenced by genera representation in the studiedsamples We confirmed that both Clostridium and Eubacteriumwere predominant in IgAminusIgG+- compared with IgA+IgG+-

sorted fractions (EI minus06 [minus083 to 022] vs minus137 [minus166 to064] and minus018 [minus039 to 105] vs minus093 [minus174 to 019] forClostridium and Eubacterium respectively p lt 005 figure 4 B)

Taken together these results demonstrate thatMS is associatedwith a broad antimicrobiota systemic IgG response with avariety of bacterial genera being preferentially targeted by MSIgG compared with MS IgA Because systemic IgG could re-flect silent gut microbiota translocation episodes232829 theseobservations could support an enhanced bacterial translocationfrom the gut lumen to systemic compartments in patientswith MS

Increased MS IgG Antibacterial ResponsesConfirmed at the Strain LevelBacterial identification remains at the genus level using 16SrRNA sequencing Using bacterial flow cytometry we sought

Figure 4 IgG-Bound Commensal Shift in Patients With MS

(A) Relative abundance of the 30 most frequent genera in IgG+IgA+ and IgG+IgAminus-sorted fractions in blue and red respectively (B) EI of the 30 most frequentgenera in IgG+IgA+ and IgG+IgAminus-sorted fractions compared in blue and red respectively (C) Blautia Clostridium and Eubacterium frequencies in IgG+IgA+ andIgG+IgAminus fractions For all box plots each dot represents 1 donor errors bars represent maximum andminimum values andmedians are indicated p Valueswere calculated by using the nonparametric Mann-Whitney test (p lt 005 p lt 001) EI = enrichment index MS = multiple sclerosis

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

to determine whether increased antimicrobiota IgG reactiv-ities could also be measured at the species level in patientswith MS We therefore selected representative bacterialstrains that were previously described as secretory IgA targetsin humans (A muciniphila B longum and R obeum) and 3others that are not preferentially bound by IgA in humans (Bcaccae F nucleatum and P oris)30-33 A majority of strainstested (4 of 6) tended to be more brightly stained by MS (RRandor CIS) IgG that by control serum IgG (figure 5 AndashC)Preferred MS IgG targets are not systematically preferredcontrol IgA targets as neither B longum nor B caccae werepreferentially bound by MS IgG compared with control IgG(figure e-5 linkslwwcomNXIA472) Of interest we ob-served that F nucleatum is particularly well opsonized by CISserum compared with healthy control serum but also com-pared with RR serum (IgG median fluorescence intensity 518[146ndash5505] vs 126 [407ndash414] and 156 [421ndash3350] in CIShealthy control and RR serums respectively figure 5 AndashB)We conclude from this part thatMS serum contains high titersof antimicrobiota IgG that can be measured against specificbacterial strains

DiscussionWe report an imbalance between systemic and secretoryantimicrobiota antibody responses in patients with MS

Antimicrobiota secretory IgA responses are impaired whereas atthe same time systemic IgG responses directed against autolo-gous microbiota are increased compared with healthy controlsImportantly although we found MS-associated gut microbiotaalterations the systemic IgG responses observed were not di-rected to taxa overrepresented in MS microbiota Furthermorelow proportions of IgA-coated bacteria among fecal microbiotaare associated with disease severity Consistently with the latterobservation Rojas et al34 have recently reported that proportionsof IgA-coated bacteria among fecal microbiota are decreased inpatients with relapsing MS compared with inactive patients Re-duced proportions of fecal IgA+ bacteria might result from re-duced secretory IgA levels in relation with IgA-secreting cellsegress out of the gutmucosa Previous works indeed shed light onthe dynamic trafficking of intraepithelial CD4T cells between gutand an inflamed CNS35 and also of IgA-producing cells betweengut and bone marrow36 In the EAE model commensal-reactiveIgA-producing cells regulate inflammation via the production IL-10 after their migration in the CNS Probstel et al recentlydemonstrated that gut microbiota IgA-producing cells infiltratethe CNS in patients with MS leading to an increase in in situ IgAproduction during MS relapses However commensal-specificIgA does not cross-react with brain tissue37 Further studies arerequired to assess the role of gut-derived IgA-producing cells inMS pathophysiology Defective IgA binding to gut microbiota indisabled patients with MS might be also related to microbialcommunity alterations

Figure 5 MS Antimicrobiota IgG Signature Measured on Cultivable Bacterial Strains

(A) Representative flow cytometry analysis ofserum IgG binding to F nucleatum Gray histo-gram represents isotype control blue red andred dotted lines represent a healthy controlpatient with RR-MS and patient with CIS re-spectively (B) Flow cytometry analysis of serumIgG binding to IgA-unbound bacteria (from leftto right F nucleatum and P oris) in healthy do-nors (n = 30) patients with RR-MS (=32) andpatients with CIS (n = 12) (C) Flow cytometryanalysis of serum IgG binding to IgA-boundbacteria (from left to right A muciniphila and Robeum) in healthy donors (n = 30) patients withRR-MS (=32) and patients with CIS (n = 12) Darkbars represent medians p Values were calcu-lated by using the nonparametric Mann-Whit-ney test (p lt 005 p lt 001 p lt 0001)p lt 00001) MS = multiple sclerosis RR =relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 9

Impaired IgA-mediated microbiota survey might also favorbacterial translocation and induction of systemic IgG re-sponses as we previously described in patients with IgA de-ficiency23 Numerous studies underline that IgG typicallypresent in the CSF of the patients might play a role in MSpathogenesis38-40 IgG purified from patients withMS increasesmouse CNS demyelination and exacerbates EAE41 The mainclinical evidence for a potential pathogenic role for antibodiesin MS is the benefit of antibody removal in a subset of patientswith acute neurologic symptoms4243 However what triggersoligoclonal antibody responses observed in MS CSF remains amystery38-40 Antimicrobiota antibodies are frequently cross-reactive with self-antigens44-46 Cross-reactivity is probably anessential antimicrobial antibody feature enabling control of alarge diversity of gut commensals and invading pathogens4748

but is also associated with a theoretical risk of autoimmunity Ithas indeed been very recently proposed that some pathogenicimmune responses associated with lupus4950 or myocarditis51

can be triggered by commensal bacteria through a molecularmimicry mechanism Of interest more than 20 bacterial spe-cies well represented in human gut microbiota express pro-teins that contain potentially encephalitogenic peptides iehighly homologous to myelin basic protein (MBP) or myelinoligodendrocyte protein In particular 4 proteins expressed byF nucleatum have been identified as potentially encephalito-genic52 Moreover 3 proteins expressed by members of theClostridium genera were described to be homologous toMBP52 We report that RR-MS and even more so CIS IgGvery brightly stain F nucleatum We also report that the Clos-tridium genus is significantly more targeted by systemicMS IgGthan by IgA These observations open new avenues for ex-ploring the hypothesis that immune responses induced bycommensal antigens might play a role in MS pathogenesis

Enhanced induction of antimicrobiota IgG could also be aconsequence of intestinal barrier dysfunction in MS Indeednot only blood-brain barrier defects have been reported inMSbut also a state of increased intestinal permeability charac-terized by higher lactulosemannitol urinary ratios increasedlipopolysaccharide plasma levels and associated with in-creased plasma zonulin a regulator of epithelial and endo-thelial barrier functions53-55 In the EAE model suchalterations preceded the onset of neurologic symptoms56 It istherefore conceivable that bacterial translocation could occurat early stages of the disease and account for the increasedantimicrobiota IgG responses we observe as early as the CISstage Systemic antimicrobiota IgG responses are likely tomirror microbiota composition at the individual level Wetherefore tested some of the bacterial species differentiallyidentified between MS and healthy donor gut microbiota245

We observed that patients with RR-MS and CISharbor ele-vated serum antindashA muciniphila IgG reactivities comparedwith controls These results are in line with the higher levels ofantindashA muciniphila antibodies detected in CSF of patientswith MS57 Of interest a greater A muciniphila prevalence inMS gut microbiota has been shown in previous reports25 Apositive correlation between A muciniphila abundance and

gene expression in T cells and monocytes involved in keypathways previously implicated in MS pathogenesis was alsoreported5 Located in close vicinity with the intestinal cells Amuciniphila has also been reported to promoteTh1 lymphocyte differentiation a T-cell subset classicallyassociated with MS pathogenesis258 Altogether althoughonly correlative these observations suggest a role for Amuciniphila in driving exacerbated host immune responsein MS

As mentioned above signs of increased intestinal permeabilitypreceded the onset of neurologic symptoms in the EAEmodel56 It would now remain to determine to which extentantimicrobiota IgG monitoring could represent a tool topredict MS progression Not all patients with CIS progress tofull blown MS Baseline lesions age at onset and short timedelay between the first 2 relapses provide some predictivevalue for disease progression yet remain flawed5960 Whethermicrobialhost signatures including immune responses tocommensals could help to distinguish between stable casesand future progressive cases should be now tested in largerstudies

In summary we have found quantitative and qualitative al-terations of the antimicrobiota antibody response in patientswith MS involving peculiar bacterial taxa We propose thatsuch peculiar IgG responses associated with MS could rep-resent a lead in the quest for antigenic drivers possibly im-plicated in MS pathogenesis through a molecular mimicrymechanism We also propose that antimicrobiota fingerprintsshould be more widely used in further studies to seek forcorrelates with disease status Finally microbiota manipula-tion is now a recognized therapeutic option in the clinic61

How our results could translate into treatments shouldtherefore be tested in murine MS models

AcknowledgmentThe authors thank the patients who agreed to participate inthis study and doctors and nurses from the NeurologyDepartment (Hopital Pitie-Salpetriere 75013 Paris Franceand Hopital Fondation Adolphe de Rotschild 75019 ParisFrance) who participated in this study

Study FundingThe study was supported by a grant from Fondation pourlrsquoAide a la Recherche sur la Sclerose en Plaques (ARSEP) andby a proof of concept grant (CoPoc) from Inserm TransfertParis France

DisclosureG Gorochov is a scientific advisor for Luxia Scientific FranceThe other authors report no disclosures relevant to themanuscript Go to NeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 7 2020 Accepted in final form March 9 2021

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

This information is current as of May 11 2021

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is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Page 6: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

Figure 2 Enhanced IgG Antimicrobiota Response in Patients With MS

(A) Procedure (top) and representative flow cytometry detection (bottom) of endogenous secretory IgA and autologous systemic IgG (10 μgmL) coating onhealthy or RR-MS fecal microbiota is presented (middle panels) (B) Proportions of IgG+IgAplusmn (left) and IgG+IgAminus (right) coated bacteria are compared in healthydonors (n = 30) patients with RR-MS (n = 32) or patients with CIS (n = 12) Median values are indicated p Values were calculated using the nonparametricMann-Whitney test (p lt 0001) (C) Representative flow cytometry dot plot (left) and analysis (right) of irrelevant IgG (anti-TNFα 10 μgmL) binding to RR-MSand CIS fecalmicrobiota (n = 44) (D) Procedure (top) representative flow cytometry dot plot (middle panel) and analysis (bottom) of autologous IgG or pooledhealthy IgG binding to RR-MS fecal microbiota (n = 32) Proportions of IgG+IgAplusmn coated bacteria (bottom left) IgG+IgA+ and IgG+IgAminus coated bacteria (bottomright) are shown p Value was calculated by using the Wilcoxon paired test (p lt 00001 ns = nonsignificant) CIS = clinically isolated syndrome MS =multiple sclerosis RR = relapsing-remitting

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

[01ndash164] and 25 [13ndash54] in patients with RR-MS (n =32) and CIS (n = 12) respectively p lt 0001 figure 2 B)

We then asked whether this observed loss of convergence wasrelated to IgG responses only found in MS or to an increasedMS microbiota reactivity to IgG in general To address thisissue we compared MS and pooled healthy IgG reactivity toMS microbiota As shown in figure 2 D although pooledhealthy IgG bound a smaller fraction of RR-MS microbiotacompared with autologous RR-MS IgG (19 [0ndash10] vs 67[05ndash171] n = 32 p lt 00001 figure 2 D) pooled healthyIgG targets not only in vivo IgA-bound but also IgA-free RR-MS microbiota (07 [0ndash64] vs 06 [0ndash62] p = 079figure 2 D) suggesting that MSmicrobiota are more prone tointeract with IgG regardless of their origin

From this part we conclude that the IgAIgG convergencerule does not apply to MS A previously IgA-ignored fractionof MS microbiota is able to interact with IgG

Antimicrobiota IgG Spreading in MSIt could be deduced from above that IgG binding to MSmicrobiota might extend beyond the usual bacterial repertoireof IgA targets that we and others previously defined2327 Todecipher antimicrobiota IgG specificities in patients with MSwith higher resolution we next isolated IgG-bound bacteria byflow cytometry sorting and identified their taxonomy using 16SrRNA sequencing (figure 3 A) To avoid confounding factorsfrom IMD therapies we sorted IgG-bound bacteria from un-treated patients We first examined microbial diversity by cal-culating the Shannon index and found a similar diversity inIgG-only and Ig-negative bacteria (206 [114ndash27] vs 212[18ndash232] n = 5 figure 3 B) In contrast dually coated bacteriatended to exhibit a decreased microbial diversity in comparisonwith the previous fractions (074 [053ndash135] n = 5 p = 0062figure 3 B) suggesting a state of restricted antimicrobiota se-cretory IgA diversity in MS We observed that individualIgA+IgG+ and IgAminusIgG+ bacterial repertoires were remarkablydistinct in all tested patients (figure 3 C for back-to-back

Figure 3 MS IgG Binds a Diverse Repertoire of Commensals

(A) Sorting strategy of IgG+IgA+ and IgG+IgAminus-coated bacteria in 5 patients with RR-MS Composition of sorted fractions was next analyzed by using 16S rRNAsequencing (B) Genera diversity in IgG+IgA+ and IgG+IgAminus-sorted fractions calculated by using the Shannon index (C) Median relative abundance of the 30most frequent genera in sorted fractions from 1 representative RR-MS patient MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 7

taxonomic analysis of fecal IgA+IgG+ IgAminusIgG+ and IgAminusIgG-

fractions in a representative patient) supporting an IgG re-sponse spread beyond IgA-surveyed taxa To quantitativelycompare IgAminusIgG+ and IgA+IgG+microbial compositions in thestudied population we thereafter narrowed the analysis down tothe 30 most frequent genera that were present in at least 80 ofthe Ig-sorted fractions Three genera derived from FirmicutesBlautia Clostridium and Eubacterium appeared preferentiallytargeted by MS serum IgG alone as deduced from their com-pared frequencies in IgAminusIgG+ bacteria and IgA+IgG+ sortedfractions (006 [002ndash02] vs 0004 [0002ndash0033] 007[0055ndash045] vs 0003 [0009ndash0041] and 0023 [0003ndash004] vs0002 [0001ndash0003] respectively p lt 005 figure 4 AndashC) Wesought to validate these observations by calculating a log-basedEI that is not influenced by genera representation in the studiedsamples We confirmed that both Clostridium and Eubacteriumwere predominant in IgAminusIgG+- compared with IgA+IgG+-

sorted fractions (EI minus06 [minus083 to 022] vs minus137 [minus166 to064] and minus018 [minus039 to 105] vs minus093 [minus174 to 019] forClostridium and Eubacterium respectively p lt 005 figure 4 B)

Taken together these results demonstrate thatMS is associatedwith a broad antimicrobiota systemic IgG response with avariety of bacterial genera being preferentially targeted by MSIgG compared with MS IgA Because systemic IgG could re-flect silent gut microbiota translocation episodes232829 theseobservations could support an enhanced bacterial translocationfrom the gut lumen to systemic compartments in patientswith MS

Increased MS IgG Antibacterial ResponsesConfirmed at the Strain LevelBacterial identification remains at the genus level using 16SrRNA sequencing Using bacterial flow cytometry we sought

Figure 4 IgG-Bound Commensal Shift in Patients With MS

(A) Relative abundance of the 30 most frequent genera in IgG+IgA+ and IgG+IgAminus-sorted fractions in blue and red respectively (B) EI of the 30 most frequentgenera in IgG+IgA+ and IgG+IgAminus-sorted fractions compared in blue and red respectively (C) Blautia Clostridium and Eubacterium frequencies in IgG+IgA+ andIgG+IgAminus fractions For all box plots each dot represents 1 donor errors bars represent maximum andminimum values andmedians are indicated p Valueswere calculated by using the nonparametric Mann-Whitney test (p lt 005 p lt 001) EI = enrichment index MS = multiple sclerosis

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

to determine whether increased antimicrobiota IgG reactiv-ities could also be measured at the species level in patientswith MS We therefore selected representative bacterialstrains that were previously described as secretory IgA targetsin humans (A muciniphila B longum and R obeum) and 3others that are not preferentially bound by IgA in humans (Bcaccae F nucleatum and P oris)30-33 A majority of strainstested (4 of 6) tended to be more brightly stained by MS (RRandor CIS) IgG that by control serum IgG (figure 5 AndashC)Preferred MS IgG targets are not systematically preferredcontrol IgA targets as neither B longum nor B caccae werepreferentially bound by MS IgG compared with control IgG(figure e-5 linkslwwcomNXIA472) Of interest we ob-served that F nucleatum is particularly well opsonized by CISserum compared with healthy control serum but also com-pared with RR serum (IgG median fluorescence intensity 518[146ndash5505] vs 126 [407ndash414] and 156 [421ndash3350] in CIShealthy control and RR serums respectively figure 5 AndashB)We conclude from this part thatMS serum contains high titersof antimicrobiota IgG that can be measured against specificbacterial strains

DiscussionWe report an imbalance between systemic and secretoryantimicrobiota antibody responses in patients with MS

Antimicrobiota secretory IgA responses are impaired whereas atthe same time systemic IgG responses directed against autolo-gous microbiota are increased compared with healthy controlsImportantly although we found MS-associated gut microbiotaalterations the systemic IgG responses observed were not di-rected to taxa overrepresented in MS microbiota Furthermorelow proportions of IgA-coated bacteria among fecal microbiotaare associated with disease severity Consistently with the latterobservation Rojas et al34 have recently reported that proportionsof IgA-coated bacteria among fecal microbiota are decreased inpatients with relapsing MS compared with inactive patients Re-duced proportions of fecal IgA+ bacteria might result from re-duced secretory IgA levels in relation with IgA-secreting cellsegress out of the gutmucosa Previous works indeed shed light onthe dynamic trafficking of intraepithelial CD4T cells between gutand an inflamed CNS35 and also of IgA-producing cells betweengut and bone marrow36 In the EAE model commensal-reactiveIgA-producing cells regulate inflammation via the production IL-10 after their migration in the CNS Probstel et al recentlydemonstrated that gut microbiota IgA-producing cells infiltratethe CNS in patients with MS leading to an increase in in situ IgAproduction during MS relapses However commensal-specificIgA does not cross-react with brain tissue37 Further studies arerequired to assess the role of gut-derived IgA-producing cells inMS pathophysiology Defective IgA binding to gut microbiota indisabled patients with MS might be also related to microbialcommunity alterations

Figure 5 MS Antimicrobiota IgG Signature Measured on Cultivable Bacterial Strains

(A) Representative flow cytometry analysis ofserum IgG binding to F nucleatum Gray histo-gram represents isotype control blue red andred dotted lines represent a healthy controlpatient with RR-MS and patient with CIS re-spectively (B) Flow cytometry analysis of serumIgG binding to IgA-unbound bacteria (from leftto right F nucleatum and P oris) in healthy do-nors (n = 30) patients with RR-MS (=32) andpatients with CIS (n = 12) (C) Flow cytometryanalysis of serum IgG binding to IgA-boundbacteria (from left to right A muciniphila and Robeum) in healthy donors (n = 30) patients withRR-MS (=32) and patients with CIS (n = 12) Darkbars represent medians p Values were calcu-lated by using the nonparametric Mann-Whit-ney test (p lt 005 p lt 001 p lt 0001)p lt 00001) MS = multiple sclerosis RR =relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 9

Impaired IgA-mediated microbiota survey might also favorbacterial translocation and induction of systemic IgG re-sponses as we previously described in patients with IgA de-ficiency23 Numerous studies underline that IgG typicallypresent in the CSF of the patients might play a role in MSpathogenesis38-40 IgG purified from patients withMS increasesmouse CNS demyelination and exacerbates EAE41 The mainclinical evidence for a potential pathogenic role for antibodiesin MS is the benefit of antibody removal in a subset of patientswith acute neurologic symptoms4243 However what triggersoligoclonal antibody responses observed in MS CSF remains amystery38-40 Antimicrobiota antibodies are frequently cross-reactive with self-antigens44-46 Cross-reactivity is probably anessential antimicrobial antibody feature enabling control of alarge diversity of gut commensals and invading pathogens4748

but is also associated with a theoretical risk of autoimmunity Ithas indeed been very recently proposed that some pathogenicimmune responses associated with lupus4950 or myocarditis51

can be triggered by commensal bacteria through a molecularmimicry mechanism Of interest more than 20 bacterial spe-cies well represented in human gut microbiota express pro-teins that contain potentially encephalitogenic peptides iehighly homologous to myelin basic protein (MBP) or myelinoligodendrocyte protein In particular 4 proteins expressed byF nucleatum have been identified as potentially encephalito-genic52 Moreover 3 proteins expressed by members of theClostridium genera were described to be homologous toMBP52 We report that RR-MS and even more so CIS IgGvery brightly stain F nucleatum We also report that the Clos-tridium genus is significantly more targeted by systemicMS IgGthan by IgA These observations open new avenues for ex-ploring the hypothesis that immune responses induced bycommensal antigens might play a role in MS pathogenesis

Enhanced induction of antimicrobiota IgG could also be aconsequence of intestinal barrier dysfunction in MS Indeednot only blood-brain barrier defects have been reported inMSbut also a state of increased intestinal permeability charac-terized by higher lactulosemannitol urinary ratios increasedlipopolysaccharide plasma levels and associated with in-creased plasma zonulin a regulator of epithelial and endo-thelial barrier functions53-55 In the EAE model suchalterations preceded the onset of neurologic symptoms56 It istherefore conceivable that bacterial translocation could occurat early stages of the disease and account for the increasedantimicrobiota IgG responses we observe as early as the CISstage Systemic antimicrobiota IgG responses are likely tomirror microbiota composition at the individual level Wetherefore tested some of the bacterial species differentiallyidentified between MS and healthy donor gut microbiota245

We observed that patients with RR-MS and CISharbor ele-vated serum antindashA muciniphila IgG reactivities comparedwith controls These results are in line with the higher levels ofantindashA muciniphila antibodies detected in CSF of patientswith MS57 Of interest a greater A muciniphila prevalence inMS gut microbiota has been shown in previous reports25 Apositive correlation between A muciniphila abundance and

gene expression in T cells and monocytes involved in keypathways previously implicated in MS pathogenesis was alsoreported5 Located in close vicinity with the intestinal cells Amuciniphila has also been reported to promoteTh1 lymphocyte differentiation a T-cell subset classicallyassociated with MS pathogenesis258 Altogether althoughonly correlative these observations suggest a role for Amuciniphila in driving exacerbated host immune responsein MS

As mentioned above signs of increased intestinal permeabilitypreceded the onset of neurologic symptoms in the EAEmodel56 It would now remain to determine to which extentantimicrobiota IgG monitoring could represent a tool topredict MS progression Not all patients with CIS progress tofull blown MS Baseline lesions age at onset and short timedelay between the first 2 relapses provide some predictivevalue for disease progression yet remain flawed5960 Whethermicrobialhost signatures including immune responses tocommensals could help to distinguish between stable casesand future progressive cases should be now tested in largerstudies

In summary we have found quantitative and qualitative al-terations of the antimicrobiota antibody response in patientswith MS involving peculiar bacterial taxa We propose thatsuch peculiar IgG responses associated with MS could rep-resent a lead in the quest for antigenic drivers possibly im-plicated in MS pathogenesis through a molecular mimicrymechanism We also propose that antimicrobiota fingerprintsshould be more widely used in further studies to seek forcorrelates with disease status Finally microbiota manipula-tion is now a recognized therapeutic option in the clinic61

How our results could translate into treatments shouldtherefore be tested in murine MS models

AcknowledgmentThe authors thank the patients who agreed to participate inthis study and doctors and nurses from the NeurologyDepartment (Hopital Pitie-Salpetriere 75013 Paris Franceand Hopital Fondation Adolphe de Rotschild 75019 ParisFrance) who participated in this study

Study FundingThe study was supported by a grant from Fondation pourlrsquoAide a la Recherche sur la Sclerose en Plaques (ARSEP) andby a proof of concept grant (CoPoc) from Inserm TransfertParis France

DisclosureG Gorochov is a scientific advisor for Luxia Scientific FranceThe other authors report no disclosures relevant to themanuscript Go to NeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 7 2020 Accepted in final form March 9 2021

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

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is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Page 7: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

[01ndash164] and 25 [13ndash54] in patients with RR-MS (n =32) and CIS (n = 12) respectively p lt 0001 figure 2 B)

We then asked whether this observed loss of convergence wasrelated to IgG responses only found in MS or to an increasedMS microbiota reactivity to IgG in general To address thisissue we compared MS and pooled healthy IgG reactivity toMS microbiota As shown in figure 2 D although pooledhealthy IgG bound a smaller fraction of RR-MS microbiotacompared with autologous RR-MS IgG (19 [0ndash10] vs 67[05ndash171] n = 32 p lt 00001 figure 2 D) pooled healthyIgG targets not only in vivo IgA-bound but also IgA-free RR-MS microbiota (07 [0ndash64] vs 06 [0ndash62] p = 079figure 2 D) suggesting that MSmicrobiota are more prone tointeract with IgG regardless of their origin

From this part we conclude that the IgAIgG convergencerule does not apply to MS A previously IgA-ignored fractionof MS microbiota is able to interact with IgG

Antimicrobiota IgG Spreading in MSIt could be deduced from above that IgG binding to MSmicrobiota might extend beyond the usual bacterial repertoireof IgA targets that we and others previously defined2327 Todecipher antimicrobiota IgG specificities in patients with MSwith higher resolution we next isolated IgG-bound bacteria byflow cytometry sorting and identified their taxonomy using 16SrRNA sequencing (figure 3 A) To avoid confounding factorsfrom IMD therapies we sorted IgG-bound bacteria from un-treated patients We first examined microbial diversity by cal-culating the Shannon index and found a similar diversity inIgG-only and Ig-negative bacteria (206 [114ndash27] vs 212[18ndash232] n = 5 figure 3 B) In contrast dually coated bacteriatended to exhibit a decreased microbial diversity in comparisonwith the previous fractions (074 [053ndash135] n = 5 p = 0062figure 3 B) suggesting a state of restricted antimicrobiota se-cretory IgA diversity in MS We observed that individualIgA+IgG+ and IgAminusIgG+ bacterial repertoires were remarkablydistinct in all tested patients (figure 3 C for back-to-back

Figure 3 MS IgG Binds a Diverse Repertoire of Commensals

(A) Sorting strategy of IgG+IgA+ and IgG+IgAminus-coated bacteria in 5 patients with RR-MS Composition of sorted fractions was next analyzed by using 16S rRNAsequencing (B) Genera diversity in IgG+IgA+ and IgG+IgAminus-sorted fractions calculated by using the Shannon index (C) Median relative abundance of the 30most frequent genera in sorted fractions from 1 representative RR-MS patient MS = multiple sclerosis RR = relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 7

taxonomic analysis of fecal IgA+IgG+ IgAminusIgG+ and IgAminusIgG-

fractions in a representative patient) supporting an IgG re-sponse spread beyond IgA-surveyed taxa To quantitativelycompare IgAminusIgG+ and IgA+IgG+microbial compositions in thestudied population we thereafter narrowed the analysis down tothe 30 most frequent genera that were present in at least 80 ofthe Ig-sorted fractions Three genera derived from FirmicutesBlautia Clostridium and Eubacterium appeared preferentiallytargeted by MS serum IgG alone as deduced from their com-pared frequencies in IgAminusIgG+ bacteria and IgA+IgG+ sortedfractions (006 [002ndash02] vs 0004 [0002ndash0033] 007[0055ndash045] vs 0003 [0009ndash0041] and 0023 [0003ndash004] vs0002 [0001ndash0003] respectively p lt 005 figure 4 AndashC) Wesought to validate these observations by calculating a log-basedEI that is not influenced by genera representation in the studiedsamples We confirmed that both Clostridium and Eubacteriumwere predominant in IgAminusIgG+- compared with IgA+IgG+-

sorted fractions (EI minus06 [minus083 to 022] vs minus137 [minus166 to064] and minus018 [minus039 to 105] vs minus093 [minus174 to 019] forClostridium and Eubacterium respectively p lt 005 figure 4 B)

Taken together these results demonstrate thatMS is associatedwith a broad antimicrobiota systemic IgG response with avariety of bacterial genera being preferentially targeted by MSIgG compared with MS IgA Because systemic IgG could re-flect silent gut microbiota translocation episodes232829 theseobservations could support an enhanced bacterial translocationfrom the gut lumen to systemic compartments in patientswith MS

Increased MS IgG Antibacterial ResponsesConfirmed at the Strain LevelBacterial identification remains at the genus level using 16SrRNA sequencing Using bacterial flow cytometry we sought

Figure 4 IgG-Bound Commensal Shift in Patients With MS

(A) Relative abundance of the 30 most frequent genera in IgG+IgA+ and IgG+IgAminus-sorted fractions in blue and red respectively (B) EI of the 30 most frequentgenera in IgG+IgA+ and IgG+IgAminus-sorted fractions compared in blue and red respectively (C) Blautia Clostridium and Eubacterium frequencies in IgG+IgA+ andIgG+IgAminus fractions For all box plots each dot represents 1 donor errors bars represent maximum andminimum values andmedians are indicated p Valueswere calculated by using the nonparametric Mann-Whitney test (p lt 005 p lt 001) EI = enrichment index MS = multiple sclerosis

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

to determine whether increased antimicrobiota IgG reactiv-ities could also be measured at the species level in patientswith MS We therefore selected representative bacterialstrains that were previously described as secretory IgA targetsin humans (A muciniphila B longum and R obeum) and 3others that are not preferentially bound by IgA in humans (Bcaccae F nucleatum and P oris)30-33 A majority of strainstested (4 of 6) tended to be more brightly stained by MS (RRandor CIS) IgG that by control serum IgG (figure 5 AndashC)Preferred MS IgG targets are not systematically preferredcontrol IgA targets as neither B longum nor B caccae werepreferentially bound by MS IgG compared with control IgG(figure e-5 linkslwwcomNXIA472) Of interest we ob-served that F nucleatum is particularly well opsonized by CISserum compared with healthy control serum but also com-pared with RR serum (IgG median fluorescence intensity 518[146ndash5505] vs 126 [407ndash414] and 156 [421ndash3350] in CIShealthy control and RR serums respectively figure 5 AndashB)We conclude from this part thatMS serum contains high titersof antimicrobiota IgG that can be measured against specificbacterial strains

DiscussionWe report an imbalance between systemic and secretoryantimicrobiota antibody responses in patients with MS

Antimicrobiota secretory IgA responses are impaired whereas atthe same time systemic IgG responses directed against autolo-gous microbiota are increased compared with healthy controlsImportantly although we found MS-associated gut microbiotaalterations the systemic IgG responses observed were not di-rected to taxa overrepresented in MS microbiota Furthermorelow proportions of IgA-coated bacteria among fecal microbiotaare associated with disease severity Consistently with the latterobservation Rojas et al34 have recently reported that proportionsof IgA-coated bacteria among fecal microbiota are decreased inpatients with relapsing MS compared with inactive patients Re-duced proportions of fecal IgA+ bacteria might result from re-duced secretory IgA levels in relation with IgA-secreting cellsegress out of the gutmucosa Previous works indeed shed light onthe dynamic trafficking of intraepithelial CD4T cells between gutand an inflamed CNS35 and also of IgA-producing cells betweengut and bone marrow36 In the EAE model commensal-reactiveIgA-producing cells regulate inflammation via the production IL-10 after their migration in the CNS Probstel et al recentlydemonstrated that gut microbiota IgA-producing cells infiltratethe CNS in patients with MS leading to an increase in in situ IgAproduction during MS relapses However commensal-specificIgA does not cross-react with brain tissue37 Further studies arerequired to assess the role of gut-derived IgA-producing cells inMS pathophysiology Defective IgA binding to gut microbiota indisabled patients with MS might be also related to microbialcommunity alterations

Figure 5 MS Antimicrobiota IgG Signature Measured on Cultivable Bacterial Strains

(A) Representative flow cytometry analysis ofserum IgG binding to F nucleatum Gray histo-gram represents isotype control blue red andred dotted lines represent a healthy controlpatient with RR-MS and patient with CIS re-spectively (B) Flow cytometry analysis of serumIgG binding to IgA-unbound bacteria (from leftto right F nucleatum and P oris) in healthy do-nors (n = 30) patients with RR-MS (=32) andpatients with CIS (n = 12) (C) Flow cytometryanalysis of serum IgG binding to IgA-boundbacteria (from left to right A muciniphila and Robeum) in healthy donors (n = 30) patients withRR-MS (=32) and patients with CIS (n = 12) Darkbars represent medians p Values were calcu-lated by using the nonparametric Mann-Whit-ney test (p lt 005 p lt 001 p lt 0001)p lt 00001) MS = multiple sclerosis RR =relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 9

Impaired IgA-mediated microbiota survey might also favorbacterial translocation and induction of systemic IgG re-sponses as we previously described in patients with IgA de-ficiency23 Numerous studies underline that IgG typicallypresent in the CSF of the patients might play a role in MSpathogenesis38-40 IgG purified from patients withMS increasesmouse CNS demyelination and exacerbates EAE41 The mainclinical evidence for a potential pathogenic role for antibodiesin MS is the benefit of antibody removal in a subset of patientswith acute neurologic symptoms4243 However what triggersoligoclonal antibody responses observed in MS CSF remains amystery38-40 Antimicrobiota antibodies are frequently cross-reactive with self-antigens44-46 Cross-reactivity is probably anessential antimicrobial antibody feature enabling control of alarge diversity of gut commensals and invading pathogens4748

but is also associated with a theoretical risk of autoimmunity Ithas indeed been very recently proposed that some pathogenicimmune responses associated with lupus4950 or myocarditis51

can be triggered by commensal bacteria through a molecularmimicry mechanism Of interest more than 20 bacterial spe-cies well represented in human gut microbiota express pro-teins that contain potentially encephalitogenic peptides iehighly homologous to myelin basic protein (MBP) or myelinoligodendrocyte protein In particular 4 proteins expressed byF nucleatum have been identified as potentially encephalito-genic52 Moreover 3 proteins expressed by members of theClostridium genera were described to be homologous toMBP52 We report that RR-MS and even more so CIS IgGvery brightly stain F nucleatum We also report that the Clos-tridium genus is significantly more targeted by systemicMS IgGthan by IgA These observations open new avenues for ex-ploring the hypothesis that immune responses induced bycommensal antigens might play a role in MS pathogenesis

Enhanced induction of antimicrobiota IgG could also be aconsequence of intestinal barrier dysfunction in MS Indeednot only blood-brain barrier defects have been reported inMSbut also a state of increased intestinal permeability charac-terized by higher lactulosemannitol urinary ratios increasedlipopolysaccharide plasma levels and associated with in-creased plasma zonulin a regulator of epithelial and endo-thelial barrier functions53-55 In the EAE model suchalterations preceded the onset of neurologic symptoms56 It istherefore conceivable that bacterial translocation could occurat early stages of the disease and account for the increasedantimicrobiota IgG responses we observe as early as the CISstage Systemic antimicrobiota IgG responses are likely tomirror microbiota composition at the individual level Wetherefore tested some of the bacterial species differentiallyidentified between MS and healthy donor gut microbiota245

We observed that patients with RR-MS and CISharbor ele-vated serum antindashA muciniphila IgG reactivities comparedwith controls These results are in line with the higher levels ofantindashA muciniphila antibodies detected in CSF of patientswith MS57 Of interest a greater A muciniphila prevalence inMS gut microbiota has been shown in previous reports25 Apositive correlation between A muciniphila abundance and

gene expression in T cells and monocytes involved in keypathways previously implicated in MS pathogenesis was alsoreported5 Located in close vicinity with the intestinal cells Amuciniphila has also been reported to promoteTh1 lymphocyte differentiation a T-cell subset classicallyassociated with MS pathogenesis258 Altogether althoughonly correlative these observations suggest a role for Amuciniphila in driving exacerbated host immune responsein MS

As mentioned above signs of increased intestinal permeabilitypreceded the onset of neurologic symptoms in the EAEmodel56 It would now remain to determine to which extentantimicrobiota IgG monitoring could represent a tool topredict MS progression Not all patients with CIS progress tofull blown MS Baseline lesions age at onset and short timedelay between the first 2 relapses provide some predictivevalue for disease progression yet remain flawed5960 Whethermicrobialhost signatures including immune responses tocommensals could help to distinguish between stable casesand future progressive cases should be now tested in largerstudies

In summary we have found quantitative and qualitative al-terations of the antimicrobiota antibody response in patientswith MS involving peculiar bacterial taxa We propose thatsuch peculiar IgG responses associated with MS could rep-resent a lead in the quest for antigenic drivers possibly im-plicated in MS pathogenesis through a molecular mimicrymechanism We also propose that antimicrobiota fingerprintsshould be more widely used in further studies to seek forcorrelates with disease status Finally microbiota manipula-tion is now a recognized therapeutic option in the clinic61

How our results could translate into treatments shouldtherefore be tested in murine MS models

AcknowledgmentThe authors thank the patients who agreed to participate inthis study and doctors and nurses from the NeurologyDepartment (Hopital Pitie-Salpetriere 75013 Paris Franceand Hopital Fondation Adolphe de Rotschild 75019 ParisFrance) who participated in this study

Study FundingThe study was supported by a grant from Fondation pourlrsquoAide a la Recherche sur la Sclerose en Plaques (ARSEP) andby a proof of concept grant (CoPoc) from Inserm TransfertParis France

DisclosureG Gorochov is a scientific advisor for Luxia Scientific FranceThe other authors report no disclosures relevant to themanuscript Go to NeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 7 2020 Accepted in final form March 9 2021

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

This information is current as of May 11 2021

ServicesUpdated Information amp

httpnnneurologyorgcontent84e997fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent84e997fullhtmlref-list-1

This article cites 60 articles 11 of which you can access for free at

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunologyfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2021 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Page 8: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

taxonomic analysis of fecal IgA+IgG+ IgAminusIgG+ and IgAminusIgG-

fractions in a representative patient) supporting an IgG re-sponse spread beyond IgA-surveyed taxa To quantitativelycompare IgAminusIgG+ and IgA+IgG+microbial compositions in thestudied population we thereafter narrowed the analysis down tothe 30 most frequent genera that were present in at least 80 ofthe Ig-sorted fractions Three genera derived from FirmicutesBlautia Clostridium and Eubacterium appeared preferentiallytargeted by MS serum IgG alone as deduced from their com-pared frequencies in IgAminusIgG+ bacteria and IgA+IgG+ sortedfractions (006 [002ndash02] vs 0004 [0002ndash0033] 007[0055ndash045] vs 0003 [0009ndash0041] and 0023 [0003ndash004] vs0002 [0001ndash0003] respectively p lt 005 figure 4 AndashC) Wesought to validate these observations by calculating a log-basedEI that is not influenced by genera representation in the studiedsamples We confirmed that both Clostridium and Eubacteriumwere predominant in IgAminusIgG+- compared with IgA+IgG+-

sorted fractions (EI minus06 [minus083 to 022] vs minus137 [minus166 to064] and minus018 [minus039 to 105] vs minus093 [minus174 to 019] forClostridium and Eubacterium respectively p lt 005 figure 4 B)

Taken together these results demonstrate thatMS is associatedwith a broad antimicrobiota systemic IgG response with avariety of bacterial genera being preferentially targeted by MSIgG compared with MS IgA Because systemic IgG could re-flect silent gut microbiota translocation episodes232829 theseobservations could support an enhanced bacterial translocationfrom the gut lumen to systemic compartments in patientswith MS

Increased MS IgG Antibacterial ResponsesConfirmed at the Strain LevelBacterial identification remains at the genus level using 16SrRNA sequencing Using bacterial flow cytometry we sought

Figure 4 IgG-Bound Commensal Shift in Patients With MS

(A) Relative abundance of the 30 most frequent genera in IgG+IgA+ and IgG+IgAminus-sorted fractions in blue and red respectively (B) EI of the 30 most frequentgenera in IgG+IgA+ and IgG+IgAminus-sorted fractions compared in blue and red respectively (C) Blautia Clostridium and Eubacterium frequencies in IgG+IgA+ andIgG+IgAminus fractions For all box plots each dot represents 1 donor errors bars represent maximum andminimum values andmedians are indicated p Valueswere calculated by using the nonparametric Mann-Whitney test (p lt 005 p lt 001) EI = enrichment index MS = multiple sclerosis

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

to determine whether increased antimicrobiota IgG reactiv-ities could also be measured at the species level in patientswith MS We therefore selected representative bacterialstrains that were previously described as secretory IgA targetsin humans (A muciniphila B longum and R obeum) and 3others that are not preferentially bound by IgA in humans (Bcaccae F nucleatum and P oris)30-33 A majority of strainstested (4 of 6) tended to be more brightly stained by MS (RRandor CIS) IgG that by control serum IgG (figure 5 AndashC)Preferred MS IgG targets are not systematically preferredcontrol IgA targets as neither B longum nor B caccae werepreferentially bound by MS IgG compared with control IgG(figure e-5 linkslwwcomNXIA472) Of interest we ob-served that F nucleatum is particularly well opsonized by CISserum compared with healthy control serum but also com-pared with RR serum (IgG median fluorescence intensity 518[146ndash5505] vs 126 [407ndash414] and 156 [421ndash3350] in CIShealthy control and RR serums respectively figure 5 AndashB)We conclude from this part thatMS serum contains high titersof antimicrobiota IgG that can be measured against specificbacterial strains

DiscussionWe report an imbalance between systemic and secretoryantimicrobiota antibody responses in patients with MS

Antimicrobiota secretory IgA responses are impaired whereas atthe same time systemic IgG responses directed against autolo-gous microbiota are increased compared with healthy controlsImportantly although we found MS-associated gut microbiotaalterations the systemic IgG responses observed were not di-rected to taxa overrepresented in MS microbiota Furthermorelow proportions of IgA-coated bacteria among fecal microbiotaare associated with disease severity Consistently with the latterobservation Rojas et al34 have recently reported that proportionsof IgA-coated bacteria among fecal microbiota are decreased inpatients with relapsing MS compared with inactive patients Re-duced proportions of fecal IgA+ bacteria might result from re-duced secretory IgA levels in relation with IgA-secreting cellsegress out of the gutmucosa Previous works indeed shed light onthe dynamic trafficking of intraepithelial CD4T cells between gutand an inflamed CNS35 and also of IgA-producing cells betweengut and bone marrow36 In the EAE model commensal-reactiveIgA-producing cells regulate inflammation via the production IL-10 after their migration in the CNS Probstel et al recentlydemonstrated that gut microbiota IgA-producing cells infiltratethe CNS in patients with MS leading to an increase in in situ IgAproduction during MS relapses However commensal-specificIgA does not cross-react with brain tissue37 Further studies arerequired to assess the role of gut-derived IgA-producing cells inMS pathophysiology Defective IgA binding to gut microbiota indisabled patients with MS might be also related to microbialcommunity alterations

Figure 5 MS Antimicrobiota IgG Signature Measured on Cultivable Bacterial Strains

(A) Representative flow cytometry analysis ofserum IgG binding to F nucleatum Gray histo-gram represents isotype control blue red andred dotted lines represent a healthy controlpatient with RR-MS and patient with CIS re-spectively (B) Flow cytometry analysis of serumIgG binding to IgA-unbound bacteria (from leftto right F nucleatum and P oris) in healthy do-nors (n = 30) patients with RR-MS (=32) andpatients with CIS (n = 12) (C) Flow cytometryanalysis of serum IgG binding to IgA-boundbacteria (from left to right A muciniphila and Robeum) in healthy donors (n = 30) patients withRR-MS (=32) and patients with CIS (n = 12) Darkbars represent medians p Values were calcu-lated by using the nonparametric Mann-Whit-ney test (p lt 005 p lt 001 p lt 0001)p lt 00001) MS = multiple sclerosis RR =relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 9

Impaired IgA-mediated microbiota survey might also favorbacterial translocation and induction of systemic IgG re-sponses as we previously described in patients with IgA de-ficiency23 Numerous studies underline that IgG typicallypresent in the CSF of the patients might play a role in MSpathogenesis38-40 IgG purified from patients withMS increasesmouse CNS demyelination and exacerbates EAE41 The mainclinical evidence for a potential pathogenic role for antibodiesin MS is the benefit of antibody removal in a subset of patientswith acute neurologic symptoms4243 However what triggersoligoclonal antibody responses observed in MS CSF remains amystery38-40 Antimicrobiota antibodies are frequently cross-reactive with self-antigens44-46 Cross-reactivity is probably anessential antimicrobial antibody feature enabling control of alarge diversity of gut commensals and invading pathogens4748

but is also associated with a theoretical risk of autoimmunity Ithas indeed been very recently proposed that some pathogenicimmune responses associated with lupus4950 or myocarditis51

can be triggered by commensal bacteria through a molecularmimicry mechanism Of interest more than 20 bacterial spe-cies well represented in human gut microbiota express pro-teins that contain potentially encephalitogenic peptides iehighly homologous to myelin basic protein (MBP) or myelinoligodendrocyte protein In particular 4 proteins expressed byF nucleatum have been identified as potentially encephalito-genic52 Moreover 3 proteins expressed by members of theClostridium genera were described to be homologous toMBP52 We report that RR-MS and even more so CIS IgGvery brightly stain F nucleatum We also report that the Clos-tridium genus is significantly more targeted by systemicMS IgGthan by IgA These observations open new avenues for ex-ploring the hypothesis that immune responses induced bycommensal antigens might play a role in MS pathogenesis

Enhanced induction of antimicrobiota IgG could also be aconsequence of intestinal barrier dysfunction in MS Indeednot only blood-brain barrier defects have been reported inMSbut also a state of increased intestinal permeability charac-terized by higher lactulosemannitol urinary ratios increasedlipopolysaccharide plasma levels and associated with in-creased plasma zonulin a regulator of epithelial and endo-thelial barrier functions53-55 In the EAE model suchalterations preceded the onset of neurologic symptoms56 It istherefore conceivable that bacterial translocation could occurat early stages of the disease and account for the increasedantimicrobiota IgG responses we observe as early as the CISstage Systemic antimicrobiota IgG responses are likely tomirror microbiota composition at the individual level Wetherefore tested some of the bacterial species differentiallyidentified between MS and healthy donor gut microbiota245

We observed that patients with RR-MS and CISharbor ele-vated serum antindashA muciniphila IgG reactivities comparedwith controls These results are in line with the higher levels ofantindashA muciniphila antibodies detected in CSF of patientswith MS57 Of interest a greater A muciniphila prevalence inMS gut microbiota has been shown in previous reports25 Apositive correlation between A muciniphila abundance and

gene expression in T cells and monocytes involved in keypathways previously implicated in MS pathogenesis was alsoreported5 Located in close vicinity with the intestinal cells Amuciniphila has also been reported to promoteTh1 lymphocyte differentiation a T-cell subset classicallyassociated with MS pathogenesis258 Altogether althoughonly correlative these observations suggest a role for Amuciniphila in driving exacerbated host immune responsein MS

As mentioned above signs of increased intestinal permeabilitypreceded the onset of neurologic symptoms in the EAEmodel56 It would now remain to determine to which extentantimicrobiota IgG monitoring could represent a tool topredict MS progression Not all patients with CIS progress tofull blown MS Baseline lesions age at onset and short timedelay between the first 2 relapses provide some predictivevalue for disease progression yet remain flawed5960 Whethermicrobialhost signatures including immune responses tocommensals could help to distinguish between stable casesand future progressive cases should be now tested in largerstudies

In summary we have found quantitative and qualitative al-terations of the antimicrobiota antibody response in patientswith MS involving peculiar bacterial taxa We propose thatsuch peculiar IgG responses associated with MS could rep-resent a lead in the quest for antigenic drivers possibly im-plicated in MS pathogenesis through a molecular mimicrymechanism We also propose that antimicrobiota fingerprintsshould be more widely used in further studies to seek forcorrelates with disease status Finally microbiota manipula-tion is now a recognized therapeutic option in the clinic61

How our results could translate into treatments shouldtherefore be tested in murine MS models

AcknowledgmentThe authors thank the patients who agreed to participate inthis study and doctors and nurses from the NeurologyDepartment (Hopital Pitie-Salpetriere 75013 Paris Franceand Hopital Fondation Adolphe de Rotschild 75019 ParisFrance) who participated in this study

Study FundingThe study was supported by a grant from Fondation pourlrsquoAide a la Recherche sur la Sclerose en Plaques (ARSEP) andby a proof of concept grant (CoPoc) from Inserm TransfertParis France

DisclosureG Gorochov is a scientific advisor for Luxia Scientific FranceThe other authors report no disclosures relevant to themanuscript Go to NeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 7 2020 Accepted in final form March 9 2021

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

This information is current as of May 11 2021

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Page 9: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

to determine whether increased antimicrobiota IgG reactiv-ities could also be measured at the species level in patientswith MS We therefore selected representative bacterialstrains that were previously described as secretory IgA targetsin humans (A muciniphila B longum and R obeum) and 3others that are not preferentially bound by IgA in humans (Bcaccae F nucleatum and P oris)30-33 A majority of strainstested (4 of 6) tended to be more brightly stained by MS (RRandor CIS) IgG that by control serum IgG (figure 5 AndashC)Preferred MS IgG targets are not systematically preferredcontrol IgA targets as neither B longum nor B caccae werepreferentially bound by MS IgG compared with control IgG(figure e-5 linkslwwcomNXIA472) Of interest we ob-served that F nucleatum is particularly well opsonized by CISserum compared with healthy control serum but also com-pared with RR serum (IgG median fluorescence intensity 518[146ndash5505] vs 126 [407ndash414] and 156 [421ndash3350] in CIShealthy control and RR serums respectively figure 5 AndashB)We conclude from this part thatMS serum contains high titersof antimicrobiota IgG that can be measured against specificbacterial strains

DiscussionWe report an imbalance between systemic and secretoryantimicrobiota antibody responses in patients with MS

Antimicrobiota secretory IgA responses are impaired whereas atthe same time systemic IgG responses directed against autolo-gous microbiota are increased compared with healthy controlsImportantly although we found MS-associated gut microbiotaalterations the systemic IgG responses observed were not di-rected to taxa overrepresented in MS microbiota Furthermorelow proportions of IgA-coated bacteria among fecal microbiotaare associated with disease severity Consistently with the latterobservation Rojas et al34 have recently reported that proportionsof IgA-coated bacteria among fecal microbiota are decreased inpatients with relapsing MS compared with inactive patients Re-duced proportions of fecal IgA+ bacteria might result from re-duced secretory IgA levels in relation with IgA-secreting cellsegress out of the gutmucosa Previous works indeed shed light onthe dynamic trafficking of intraepithelial CD4T cells between gutand an inflamed CNS35 and also of IgA-producing cells betweengut and bone marrow36 In the EAE model commensal-reactiveIgA-producing cells regulate inflammation via the production IL-10 after their migration in the CNS Probstel et al recentlydemonstrated that gut microbiota IgA-producing cells infiltratethe CNS in patients with MS leading to an increase in in situ IgAproduction during MS relapses However commensal-specificIgA does not cross-react with brain tissue37 Further studies arerequired to assess the role of gut-derived IgA-producing cells inMS pathophysiology Defective IgA binding to gut microbiota indisabled patients with MS might be also related to microbialcommunity alterations

Figure 5 MS Antimicrobiota IgG Signature Measured on Cultivable Bacterial Strains

(A) Representative flow cytometry analysis ofserum IgG binding to F nucleatum Gray histo-gram represents isotype control blue red andred dotted lines represent a healthy controlpatient with RR-MS and patient with CIS re-spectively (B) Flow cytometry analysis of serumIgG binding to IgA-unbound bacteria (from leftto right F nucleatum and P oris) in healthy do-nors (n = 30) patients with RR-MS (=32) andpatients with CIS (n = 12) (C) Flow cytometryanalysis of serum IgG binding to IgA-boundbacteria (from left to right A muciniphila and Robeum) in healthy donors (n = 30) patients withRR-MS (=32) and patients with CIS (n = 12) Darkbars represent medians p Values were calcu-lated by using the nonparametric Mann-Whit-ney test (p lt 005 p lt 001 p lt 0001)p lt 00001) MS = multiple sclerosis RR =relapsing-remitting

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 9

Impaired IgA-mediated microbiota survey might also favorbacterial translocation and induction of systemic IgG re-sponses as we previously described in patients with IgA de-ficiency23 Numerous studies underline that IgG typicallypresent in the CSF of the patients might play a role in MSpathogenesis38-40 IgG purified from patients withMS increasesmouse CNS demyelination and exacerbates EAE41 The mainclinical evidence for a potential pathogenic role for antibodiesin MS is the benefit of antibody removal in a subset of patientswith acute neurologic symptoms4243 However what triggersoligoclonal antibody responses observed in MS CSF remains amystery38-40 Antimicrobiota antibodies are frequently cross-reactive with self-antigens44-46 Cross-reactivity is probably anessential antimicrobial antibody feature enabling control of alarge diversity of gut commensals and invading pathogens4748

but is also associated with a theoretical risk of autoimmunity Ithas indeed been very recently proposed that some pathogenicimmune responses associated with lupus4950 or myocarditis51

can be triggered by commensal bacteria through a molecularmimicry mechanism Of interest more than 20 bacterial spe-cies well represented in human gut microbiota express pro-teins that contain potentially encephalitogenic peptides iehighly homologous to myelin basic protein (MBP) or myelinoligodendrocyte protein In particular 4 proteins expressed byF nucleatum have been identified as potentially encephalito-genic52 Moreover 3 proteins expressed by members of theClostridium genera were described to be homologous toMBP52 We report that RR-MS and even more so CIS IgGvery brightly stain F nucleatum We also report that the Clos-tridium genus is significantly more targeted by systemicMS IgGthan by IgA These observations open new avenues for ex-ploring the hypothesis that immune responses induced bycommensal antigens might play a role in MS pathogenesis

Enhanced induction of antimicrobiota IgG could also be aconsequence of intestinal barrier dysfunction in MS Indeednot only blood-brain barrier defects have been reported inMSbut also a state of increased intestinal permeability charac-terized by higher lactulosemannitol urinary ratios increasedlipopolysaccharide plasma levels and associated with in-creased plasma zonulin a regulator of epithelial and endo-thelial barrier functions53-55 In the EAE model suchalterations preceded the onset of neurologic symptoms56 It istherefore conceivable that bacterial translocation could occurat early stages of the disease and account for the increasedantimicrobiota IgG responses we observe as early as the CISstage Systemic antimicrobiota IgG responses are likely tomirror microbiota composition at the individual level Wetherefore tested some of the bacterial species differentiallyidentified between MS and healthy donor gut microbiota245

We observed that patients with RR-MS and CISharbor ele-vated serum antindashA muciniphila IgG reactivities comparedwith controls These results are in line with the higher levels ofantindashA muciniphila antibodies detected in CSF of patientswith MS57 Of interest a greater A muciniphila prevalence inMS gut microbiota has been shown in previous reports25 Apositive correlation between A muciniphila abundance and

gene expression in T cells and monocytes involved in keypathways previously implicated in MS pathogenesis was alsoreported5 Located in close vicinity with the intestinal cells Amuciniphila has also been reported to promoteTh1 lymphocyte differentiation a T-cell subset classicallyassociated with MS pathogenesis258 Altogether althoughonly correlative these observations suggest a role for Amuciniphila in driving exacerbated host immune responsein MS

As mentioned above signs of increased intestinal permeabilitypreceded the onset of neurologic symptoms in the EAEmodel56 It would now remain to determine to which extentantimicrobiota IgG monitoring could represent a tool topredict MS progression Not all patients with CIS progress tofull blown MS Baseline lesions age at onset and short timedelay between the first 2 relapses provide some predictivevalue for disease progression yet remain flawed5960 Whethermicrobialhost signatures including immune responses tocommensals could help to distinguish between stable casesand future progressive cases should be now tested in largerstudies

In summary we have found quantitative and qualitative al-terations of the antimicrobiota antibody response in patientswith MS involving peculiar bacterial taxa We propose thatsuch peculiar IgG responses associated with MS could rep-resent a lead in the quest for antigenic drivers possibly im-plicated in MS pathogenesis through a molecular mimicrymechanism We also propose that antimicrobiota fingerprintsshould be more widely used in further studies to seek forcorrelates with disease status Finally microbiota manipula-tion is now a recognized therapeutic option in the clinic61

How our results could translate into treatments shouldtherefore be tested in murine MS models

AcknowledgmentThe authors thank the patients who agreed to participate inthis study and doctors and nurses from the NeurologyDepartment (Hopital Pitie-Salpetriere 75013 Paris Franceand Hopital Fondation Adolphe de Rotschild 75019 ParisFrance) who participated in this study

Study FundingThe study was supported by a grant from Fondation pourlrsquoAide a la Recherche sur la Sclerose en Plaques (ARSEP) andby a proof of concept grant (CoPoc) from Inserm TransfertParis France

DisclosureG Gorochov is a scientific advisor for Luxia Scientific FranceThe other authors report no disclosures relevant to themanuscript Go to NeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 7 2020 Accepted in final form March 9 2021

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

This information is current as of May 11 2021

ServicesUpdated Information amp

httpnnneurologyorgcontent84e997fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent84e997fullhtmlref-list-1

This article cites 60 articles 11 of which you can access for free at

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httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

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httpnnneurologyorgcgicollectionall_immunologyAll Immunologyfollowing collection(s) This article along with others on similar topics appears in the

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is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Page 10: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

Impaired IgA-mediated microbiota survey might also favorbacterial translocation and induction of systemic IgG re-sponses as we previously described in patients with IgA de-ficiency23 Numerous studies underline that IgG typicallypresent in the CSF of the patients might play a role in MSpathogenesis38-40 IgG purified from patients withMS increasesmouse CNS demyelination and exacerbates EAE41 The mainclinical evidence for a potential pathogenic role for antibodiesin MS is the benefit of antibody removal in a subset of patientswith acute neurologic symptoms4243 However what triggersoligoclonal antibody responses observed in MS CSF remains amystery38-40 Antimicrobiota antibodies are frequently cross-reactive with self-antigens44-46 Cross-reactivity is probably anessential antimicrobial antibody feature enabling control of alarge diversity of gut commensals and invading pathogens4748

but is also associated with a theoretical risk of autoimmunity Ithas indeed been very recently proposed that some pathogenicimmune responses associated with lupus4950 or myocarditis51

can be triggered by commensal bacteria through a molecularmimicry mechanism Of interest more than 20 bacterial spe-cies well represented in human gut microbiota express pro-teins that contain potentially encephalitogenic peptides iehighly homologous to myelin basic protein (MBP) or myelinoligodendrocyte protein In particular 4 proteins expressed byF nucleatum have been identified as potentially encephalito-genic52 Moreover 3 proteins expressed by members of theClostridium genera were described to be homologous toMBP52 We report that RR-MS and even more so CIS IgGvery brightly stain F nucleatum We also report that the Clos-tridium genus is significantly more targeted by systemicMS IgGthan by IgA These observations open new avenues for ex-ploring the hypothesis that immune responses induced bycommensal antigens might play a role in MS pathogenesis

Enhanced induction of antimicrobiota IgG could also be aconsequence of intestinal barrier dysfunction in MS Indeednot only blood-brain barrier defects have been reported inMSbut also a state of increased intestinal permeability charac-terized by higher lactulosemannitol urinary ratios increasedlipopolysaccharide plasma levels and associated with in-creased plasma zonulin a regulator of epithelial and endo-thelial barrier functions53-55 In the EAE model suchalterations preceded the onset of neurologic symptoms56 It istherefore conceivable that bacterial translocation could occurat early stages of the disease and account for the increasedantimicrobiota IgG responses we observe as early as the CISstage Systemic antimicrobiota IgG responses are likely tomirror microbiota composition at the individual level Wetherefore tested some of the bacterial species differentiallyidentified between MS and healthy donor gut microbiota245

We observed that patients with RR-MS and CISharbor ele-vated serum antindashA muciniphila IgG reactivities comparedwith controls These results are in line with the higher levels ofantindashA muciniphila antibodies detected in CSF of patientswith MS57 Of interest a greater A muciniphila prevalence inMS gut microbiota has been shown in previous reports25 Apositive correlation between A muciniphila abundance and

gene expression in T cells and monocytes involved in keypathways previously implicated in MS pathogenesis was alsoreported5 Located in close vicinity with the intestinal cells Amuciniphila has also been reported to promoteTh1 lymphocyte differentiation a T-cell subset classicallyassociated with MS pathogenesis258 Altogether althoughonly correlative these observations suggest a role for Amuciniphila in driving exacerbated host immune responsein MS

As mentioned above signs of increased intestinal permeabilitypreceded the onset of neurologic symptoms in the EAEmodel56 It would now remain to determine to which extentantimicrobiota IgG monitoring could represent a tool topredict MS progression Not all patients with CIS progress tofull blown MS Baseline lesions age at onset and short timedelay between the first 2 relapses provide some predictivevalue for disease progression yet remain flawed5960 Whethermicrobialhost signatures including immune responses tocommensals could help to distinguish between stable casesand future progressive cases should be now tested in largerstudies

In summary we have found quantitative and qualitative al-terations of the antimicrobiota antibody response in patientswith MS involving peculiar bacterial taxa We propose thatsuch peculiar IgG responses associated with MS could rep-resent a lead in the quest for antigenic drivers possibly im-plicated in MS pathogenesis through a molecular mimicrymechanism We also propose that antimicrobiota fingerprintsshould be more widely used in further studies to seek forcorrelates with disease status Finally microbiota manipula-tion is now a recognized therapeutic option in the clinic61

How our results could translate into treatments shouldtherefore be tested in murine MS models

AcknowledgmentThe authors thank the patients who agreed to participate inthis study and doctors and nurses from the NeurologyDepartment (Hopital Pitie-Salpetriere 75013 Paris Franceand Hopital Fondation Adolphe de Rotschild 75019 ParisFrance) who participated in this study

Study FundingThe study was supported by a grant from Fondation pourlrsquoAide a la Recherche sur la Sclerose en Plaques (ARSEP) andby a proof of concept grant (CoPoc) from Inserm TransfertParis France

DisclosureG Gorochov is a scientific advisor for Luxia Scientific FranceThe other authors report no disclosures relevant to themanuscript Go to NeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 7 2020 Accepted in final form March 9 2021

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

This information is current as of May 11 2021

ServicesUpdated Information amp

httpnnneurologyorgcontent84e997fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent84e997fullhtmlref-list-1

This article cites 60 articles 11 of which you can access for free at

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunologyfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2021 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Page 11: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

References1 Berer K Mues M Koutrolos M et al Commensal microbiota and myelin autoantigen

cooperate to trigger autoimmune demyelination Nature 2011479538-5412 Cekanaviciute E Yoo BB Runia TF et al Gut bacteria frommultiple sclerosis patients

modulate human T cells and exacerbate symptoms in mouse models Proc Natl AcadSci USA 201111410713-10718

3 Miyake S Kim S Suda W et al Dysbiosis in the gut microbiota of patients withmultiple sclerosis with a striking depletion of species belonging to clostridia XIVa andIV clusters PLoS One 201510e0137429

4 Chen J Chia N Kalari KR et al Multiple sclerosis patients have a distinct gutmicrobiota compared to healthy controls Sci Rep 2016628484

5 Jangi S Gandhi R Cox LM et al Alterations of the human gut microbiome inmultiplesclerosis Nat Commun 2016712015

6 Berer K Gerdes LA Cekanaviciute E et al Gut microbiota from multiple sclerosispatients enables spontaneous autoimmune encephalomyelitis in mice Proc Natl AcadSci USA 201711410719-10724

7 Tremlett H Fadrosh DW Faruqi AA et al Gut microbiota in early pediatric multiplesclerosis a case-control study Eur J Neurol 2016231308-1321

8 Reynders T Devolder L Valles-Colomer M et al Gut microbiome variation isassociated to Multiple Sclerosis phenotypic subtypes Ann Clin Transl Neurol 20207406-419

9 Katz Sand I Zhu Y Ntranos A et al Disease-modifying therapies alter gut microbialcomposition in MS Neurol Neuroimmunol Neuroinflamm 20196e517

10 Varrin-Doyer M Spencer CM Schulze-TopphoffU et al Aquaporin 4-specific T cellsin neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC trans-porter Ann Neurol 20127253-64

11 Cree BA Spencer CM Varrin-Doyer M Baranzini SE Zamvil SS Gut microbiomeanalysis in neuromyelitis optica reveals overabundance of Clostridium perfringensAnn Neurol 201680443-447

12 Jacobs BC van Doorn PA Schmitz PI et al Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-Barre syndrome Ann Neurol 199640181-187

13 Shu XM Cai FC Zhang XP Carbohydrate mimicry of Campylobacter jejuni lip-ooligosaccharide is critical for the induction of anti-GM1 antibody and neuropathyMuscle Nerve 200633225-231

14 Polman CH Reingold SC Banwell B et al Diagnostic criteria for multiple sclerosis2010 Revisions to the McDonald criteria Ann Neurol 201169292-302

15 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

Appendix Authors

Name Location Contribution

DelphineSterlinPharmDPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data anddrafted the manuscript

MartinLarsen PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the study

JehaneFadlallahMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

ChristopheParizot MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in theacquisition analysis andinterpretation of data

MarinaVignesPharmD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

GaelleAutaa MS

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

KarimDorghamPhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data

CatherineJuste PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

PatriciaLepage PhD

Universite Paris-SaclayINRAE AgroParisTechMicalis Institute Jouy-en-Josas France

Major role in the acquisitionof data

JenniferAboab MD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Major role in the acquisitionof data

SavineVicart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Appendix (continued)

Name Location Contribution

ElisabethMaillart MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Major role in the acquisitionof data and revised themanuscript for intellectualcontent

Olivier GoutMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CatherineLubetzkiMD PhD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

RomainDeschampsMD

Hopital OphtalmologiqueAdolphe de RothschildDepartement deNeurologie Paris France

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

CarolinePapeix MD

Sorbonne UniversiteDepartement deNeurologie AP-HP HopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand revised the manuscriptfor intellectual content

GuyGorochovMD PhD

Sorbonne UniversiteInserm CentredrsquoImmunologie et desMaladies Infectieuses(CIMI-Paris) AP-HPHopitalPitie-Salpetriere ParisFrance

Designed andconceptualized the studyand drafted and revised themanuscript for intellectualcontent

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 11

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

60 Tintore M Arrambide G Otero-Romero S et al The long-term outcomes of CISpatients in the Barcelona inception cohort looking back to recognize aggressive MSMult Scler 2020261658-1669

61 van Nood E Vrieze A Nieuwdorp M et al Duodenal infusion of donor feces forrecurrent Clostridium difficile N Engl J Med 2013368407-415

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

This information is current as of May 11 2021

ServicesUpdated Information amp

httpnnneurologyorgcontent84e997fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent84e997fullhtmlref-list-1

This article cites 60 articles 11 of which you can access for free at

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunologyfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2021 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Page 12: Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis

16 DaltonCMBrexPAMiszkiel KA et al Application of the newMcDonald criteria to patientswith clinically isolated syndromes suggestive of multiple sclerosisAnnNeurol 20025247-53

17 Miller DH Chard DT Ciccarelli O Clinically isolated syndromes Lancet Neurol201211157-169

18 Juste C Kreil DP Beauvallet C et al Bacterial protein signals are associated withCrohnrsquos disease Gut 2014631566-1577

19 Fadlallah J El Kafsi H Sterlin D et al Microbial ecology perturbation in human IgAdeficiency Sci Transl Med 201810eaan1217

20 Godon JJ Zumstein E Dabert P Habouzit F Moletta RMolecular microbial diversityof an anaerobic digestor as determined by small-subunit rDNA sequence analysisAppl Environ Microbiol 1997632802-2813

21 Moor K Fadlallah J Toska A et al Analysis of bacterial-surface-specific antibodies inbody fluids using bacterial flow cytometry Nat Protoc 2016111531-1553

22 Caporaso JG Kuczynski J Stombaugh J et al QIIME allows analysis of high-throughput community sequencing data Nat Methods 20107335-336

23 Fadlallah J Sterlin D Fieschi C et al Synergistic convergence of microbiota-specificsystemic IgG and secretory IgA J Allergy Clin Immunol 20191431575-1585e4

24 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444-1452

25 Haas A Zimmermann K Graw F et al Systemic antibody responses to gut com-mensal bacteria during chronic HIV-1 infection Gut 2011601506-1519

26 HarmsenHJM Pouwels SD Funke A BosNADijkstraGCrohnrsquos disease patients havemore IgG-binding fecal bacteria than controls Clin Vaccin Immunol 201219515-521

27 Koch MA Reiner GL Lugo KA et al Maternal IgG and IgA antibodies dampenmucosal T helper cell responses in early life Cell 2016165827-841

28 Christmann BS Abrahamsson TR Bernstein CN et al Human seroreactivity to gutmicrobiota antigens J Allergy Clin Immunol 20151361378-1385e1-5

29 Mannon PJ Systemic antibody responses to gut commensal bacteria how and why doI know you J Allergy Clin Immunol 20191431353-1354

30 DrsquoAuria G Peris-Bondia F Dzunkova M et al Active and secreted IgA-coated bac-terial fractions from the human gut reveal an under-represented microbiota core SciRep 201333515

31 Palm NW de Zoete MR Cullen TW et al Immunoglobulin A coating identifiescolitogenic bacteria in inflammatory bowel disease Cell 20141581000-1010

32 Kau AL Planer JD Liu J et al Functional characterization of IgA-targeted bacterialtaxa from undernourished Malawian children that produce diet-dependent enterop-athy Sci Transl Med 20157276ra24

33 Magri G Comerma L Pybus M et al Human secretory IgM emerges from plasmacells clonally related to gut memory B cells and targets highly diverse commensalsImmunity 201747118-134e8

34 Rojas OL Probstel AK Porfilio EA et al Recirculating intestinal IgA-producing cellsregulate neuroinflammation via IL-10 Cell 2019176610-624e18

35 Kadowaki A Miyake S Saga R Chiba A Mochizuki H Yamamura T Gutenvironment-induced intraepithelial autoreactive CD4(+) T cells suppress centralnervous system autoimmunity via LAG-3 Nat Commun 2016711639

36 Wilmore JR Gaudette BT Gomez Atria D et al Commensal microbes induce serum IgAresponses that protect against polymicrobial sepsis Cell Host Microbe 201823302-311e3

37 Probstel A-K Zhou X Baumann R et al Gut microbiota-specific IgA(+) B cells trafficto the CNS in active multiple sclerosis Sci Immunol 20205eabc7191

38 Thompson AJ Banwell BL Barkhof F et al Diagnosis of multiple sclerosis 2017revisions of the McDonald criteria Lancet Neurol 201817162-173

39 Tomescu-Baciu A Johansen JN Holmoslashy T et al Persistence of intrathecal oligo-clonal B cells and IgG in multiple sclerosis J Neuroimmunol 2019333576966

40 Graner M Pointon TManton S et al Oligoclonal IgG antibodies in multiple sclerosistarget patient-specific peptides PLoS One 202015e0228883

41 Pedotti R Musio S Scabeni S et al Exacerbation of experimental autoimmuneencephalomyelitis by passive transfer of IgG antibodies from a multiple sclerosispatient responsive to immunoadsorption J Neuroimmunology 201326219-26

42 Keegan M Konig F McClelland R et al Relation between humoral pathologicalchanges in multiple sclerosis and response to therapeutic plasma exchange Lancet2005366579-582

43 Magantildea SM Keegan BM Weinshenker BG et al Beneficial plasma exchange re-sponse in central nervous system inflammatory demyelination Arch Neurol 201168870-878

44 Benckert J Schmolka N Kreschel C et al The majority of intestinal IgA+ and IgG+plasmablasts in the human gut are antigen-specific J Clin Invest 20111211946-1955

45 Rollenske T Szijarto V Lukasiewicz J et al Cross-specificity of protective humanantibodies against Klebsiella pneumoniae LPS O-antigen Nat Immunol 201819617-624

46 Sterlin D Fadlallah J Adams O et al Human IgA binds a diverse array of commensalbacteria J Exp Med 2020217217

47 Pabst O Slack E IgA and the intestinal microbiota the importance of being specificMucosal Immunol 20201312-21

48 Sterlin D Fadlallah J Slack E Gorochov G The antibodymicrobiota interface inhealth and disease Mucosal Immunol 2020133-11

49 Greiling TM Dehner C Chen X et al Commensal orthologs of the human auto-antigen Ro60 as triggers of autoimmunity in lupus Sci Transl Med 201810eaan2306

50 Manfredo Vieira S Hiltensperger M Kumar V et al Translocation of a gut pathobiontdrives autoimmunity in mice and humans Science 20183591156-1161

51 Gil-Cruz C Perez-Shibayama C De Martin A et al Microbiota-derived peptidemimics drive lethal inflammatory cardiomyopathy Science 2019366881-886

52 Westall FC Molecular mimicry revisited gut bacteria and multiple sclerosis J ClinMicrobiol 2006442099-2104

53 Fasano A Zonulin and its regulation of intestinal barrier function the biological doorto inflammation autoimmunity and cancer Physiol Rev 201191151-175

54 Teixeira B Bittencourt VC Ferreira TB et al Low sensitivity to glucocorticoidinhibition of in vitro Th17-related cytokine production in multiple sclerosis patients isrelated to elevated plasma lipopolysaccharide levels Clin Immunol 2013148209-218

55 Buscarinu MC Cerasoli B Annibali V et al Altered intestinal permeability in patientswith relapsing-remitting multiple sclerosis a pilot studyMult Scler 201723442-446

56 Nouri M Bredberg A Westrom B Lavasani S Intestinal barrier dysfunction developsat the onset of experimental autoimmune encephalomyelitis and can be induced byadoptive transfer of auto-reactive T cells PLoS One 20149e106335

57 Vallino A Dos Santos A Mathe CV et al Gut bacteria Akkermansia elicit a specific IgGresponse in CSF of patients with MS Neurol Neuroimmunol Neuroinflamm 20207

58 Derrien M Van Baarlen P Hooiveld G Norin E Muller M de Vos WM Modulationof mucosal immune response tolerance and proliferation in mice colonized by themucin-degrader Akkermansia muciniphila Front Microbiol 20112166

59 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013-1024

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12 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 4 | July 2021 NeurologyorgNN

DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

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DOI 101212NXI000000000000099720218 Neurol Neuroimmunol Neuroinflamm

Delphine Sterlin Martin Larsen Jehane Fadlallah et al Perturbed MicrobiotaImmune Homeostasis in Multiple Sclerosis

This information is current as of May 11 2021

ServicesUpdated Information amp

httpnnneurologyorgcontent84e997fullhtmlincluding high resolution figures can be found at

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This article cites 60 articles 11 of which you can access for free at

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunologyfollowing collection(s) This article along with others on similar topics appears in the

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httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

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httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2021 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm