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of September 29, 2014.This information is current as
jejuni CampylobacterInflammasome Activation by
PuttenBleumink-Pluym, Richard A. Flavell and Jos P. M. van
Lieneke I. Bouwman, Marcel R. de Zoete, Nancy M. C.
ol.1400648http://www.jimmunol.org/content/early/2014/09/28/jimmun
published online 29 September 2014J Immunol
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The Journal of Immunology
Inflammasome Activation by Campylobacter jejuni
Lieneke I. Bouwman,* Marcel R. de Zoete,, Nancy M. C.
Bleumink-Pluym,*
Richard A. Flavell,, and Jos P. M. van Putten*
The Gram-negative pathogen Campylobacter jejuni is the most
common cause of bacterial foodborne disease worldwide. The
mechanisms that lead to bacterial invasion of eukaryotic cells
and massive intestinal inflammation are still unknown. In this
study,
we report that C. jejuni infection of mouse macrophages induces
upregulation of proIL-1b transcript and secretion of IL-1b
without eliciting cell death. Immunoblotting indicated cleavage
of caspase-1 and IL-1b in infected cells. In bone marrowderived
macrophages from different knockout mice, IL-1b secretion was
found to require NLRP3, ASC, and caspase-1/11 but not NLRC4.
In contrast to NLRP3 activation by ATP, C. jejuni activation did
not require priming of these macrophages. C. jejuni also
activated
the NLRP3 inflammasome in human macrophages as indicated by the
presence of ASC foci and caspase-1positive cells. Analysis
of a vast array of C. jejuni mutants with defects in capsule
formation, LPS biosynthesis, chemotaxis, flagella synthesis and
flagellin
(-like) secretion, type 6 secretion system needle protein, or
cytolethal distending toxin revealed a direct correlation between
the
number of intracellular bacteria and NLRP3 inflammasome
activation. The C. jejuni invasionrelated activation of the
NLRP3
inflammasome without cytotoxicity and even in nonprimed cells
extends the known repertoire of bacterial inflammasome acti-
vation and likely contributes to C. jejuniinduced intestinal
inflammation. The Journal of Immunology, 2014, 193: 000000.
Inflammasomes are multiprotein complexes that form in thecytosol
following sensing of intracellular threats like in-truding bacteria
and viruses or cell damage. Inflammasome
complexes generally consist of sensor proteins (members of
theNod-like receptor [NLR] or Pyrin and HIN200 domain
[PYHIN]protein family) and effector procaspases (mainly
caspase-1),which are bridged by the adaptor protein
apoptosis-associatedspeck-like protein (ASC). After assembly,
inflammasomes in-duce the activation of caspase-1 through
autocleavage, whichsubsequently activates cytokines IL-1b and
IL-18, and inducea form of cell death referred to as pyroptosis
(15).The best studied inflammasomes in relation to bacterial
infection
are the NLR family, CARD domaincontaining 4 (NLRC4) and theNLR
family, pyrin domaincontaining 3 (NLRP3) inflammasomes(6, 7). The
NLRC4 inflammasome is formed after sensing cyto-solic bacterial
flagellin or components of the bacterial type IIIsecretion system
(T3SS) by distinct neuronal apoptosis inhibitoryprotein (NAIP)
receptors in the cell (810). NLRP3 inflamma-some activation
requires a two signal process. The first (priming)
signal leads to the expression of NLRP3 and proIL-1b
throughactivation of NF-kB via stimulation of TLRs, other pattern
rec-
ognition receptors, or endogenous cytokines. Then, a second
stimulus (e.g., pore-forming toxins, bacterial invasion, or
uptake
of large particulates) induces the formation of the NLRP3
inflammasome (4). Although NLRP3 ligands are highly diverse,
they all seem to converse in the efflux of K+ from the cell,
which is
proposed to be the common trigger (11, 12).The bacterial
pathogen Campylobacter jejuni is the most
common cause of bacterial foodborne disease worldwide. Symp-
tomatic infection typically involves intestinal inflammation
with
abdominal pain, fever, and (bloody) diarrhea. In 1% of the
cases,serious complications may develop such as the acute
autoimmune
paralyzing neuropathy GuillainBarre syndrome (13, 14). In
contrast to most enteropathogens including Salmonella, C.
jejuni
lacks traditional virulence factors like T3SSs. The
molecular
cause of C. jejuni intestinal inflammation is still largely
unknown.
After ingestion, the bacteria travel deep down into the
intestinal
crypts of the colon where they colonize and replicate. At
some
point the epithelial barrier is breached, resulting in acute
inflam-
mation accompanied by strong neutrophil recruitment and
acti-
vation of T- and B-cell responses (15, 16). Bacterial motility
and
chemotaxis are crucial for causing disease (1720). Other
pro-
posed virulence traits include the polysaccharide capsule,
secreted
proteins (Cia proteins, HtrA protease), type 6 secretion
(T6SS)
effector molecules, apoptosis-inducing proteins (cytolethal
dis-
tending toxin, FspA2), and bacterial adhesion and invasion
pro-
moting factors (FlaC, PEB1, JlpA, CapA, and CadF) (for
review,
see Refs. 2123). The role of these factors in the development
of
human infection, however, remains to be demonstrated.The
induction of acute intestinal inflammation in response to
C. jejuni infection suggests the activation of innate pattern
rec-
ognition receptors (24, 25). Although C. jejuni flagellin and
DNA
escape TLR recognition, the bacterial LPS and lipoproteins
po-
tently activate the TLR4 and TLR2 pathways (25). In
addition,
C. jejuni is internalized by monocytes and macrophages and
activates NOD1 (24, 2629). Cellular infection is accompanied
by
the secretion of several proinflammatory cytokines such as
IL-6,
*Department of Infectious Diseases and Immunology, Utrecht
University, 3584 CLUtrecht, the Netherlands; Department of
Immunobiology, Yale University School ofMedicine, New Haven, CT
06520; and Howard Hughes Medical Institute, YaleUniversity, New
Haven, CT 06520
Received for publication March 11, 2014. Accepted for
publication August 27, 2014.
This work was supported by a Rubicon fellowship from the
Netherlands and theOrganization of Scientific Research (to
M.R.d.Z.).
Address correspondence and reprint requests to Dr. Jos P.M. van
Putten, Departmentof Infectious Diseases and Immunology, Utrecht
University, Yalelaan 1, 3584 CLUtrecht, the Netherlands. E-mail
address: [email protected]
The online version of this article contains supplemental
material.
Abbreviations used in this article: ASC, apoptosis-associated
speck-like protein;BMM, bone marrow macrophage; cat,
chloramphenicol; CDT, cytolethal distendingtoxin; F.I.,
fluorescence intensity; FLICA, fluorescent labeled inhibitor of
caspases;HI, heart infusion; kana, kanamycin; LB, LuriaBertani;
LDH, lactate dehydroge-nase; LOS, lipooligosaccharide; m.o.i.,
multiplicity of infection; NAIP, neuronalapoptosis inhibitory
protein; NLR, Nod-like receptor; NLRC4, NLR family,
CARDdomaincontaining 4; NLRP3, NLR family, pyrin domaincontaining
3; PI, propi-dium iodide; T3SS, type 3 secretion system; T4SS, type
4 secretion system; T6SS,type 6 secretion system.
Copyright 2014 by The American Association of Immunologists,
Inc. 0022-1767/14/$16.00
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IL-8, TNF-a, and IL-1b (3032). Considering the potential
im-portant role of IL-1b in the clinical manifestation of C.
jejuniinfection (33), we investigated in the current study the
ability ofC. jejuni to activate the inflammasome. Our results
reveal thatC. jejuni can induce inflammasome activation without
cytotoxic-ity, which has thus far not been observed for other
pathogens.
Materials and MethodsCell culture and reagents
J774.A1 cells (ATCC TIB-67) were routinely cultured in DMEM plus
10%FCS at 37C and 10% CO2. THP-1 null and THP-1 defNLRP3
(InvivoGen;thp-null, thp-dnlp) were grown according to the
manufacturers protocol inRPMI 1640 medium plus 10% FCS in the
presence of 200 mg/mlHygrogold (every other passage) at 37C and 5%
CO2. L929 cells werecultured in RPMI 1640 medium plus 10% FCS at
37C and 5% CO2.
The following reagents were used: gentamicin, kanamycin
(kana),chloramphenicol (cat), Triton X-100, Tween 20, Tris,
paraformaldehyde,PMA, TCA, and goat anti-rabbit IgG-HRP (A4914)
(Sigma-Aldrich);FCS and Dulbeccos PBS (PAA); DMEM, RPMI 1640
medium, Opti-MEM, penicillin, and streptomycin (Life Technologies);
BCA ProteinAssay Kit, Concentrators, 9K MWCO, and SuperSignal West
FemtoChemiluminescent Substrate (Pierce); cOmplete ULTRA Tablets
EDTA-Free, lactate dehydrogenase (LDH), and Cytotoxicity Detection
KitPLUS
(LDH) (Roche); WGA-Alexa Fluor 633, goat anti-rabbit-Alexa Fluor
488,primers, Pfx DNA polymerase (Life Technologies); ATP,
DNase,29-deoxynucleoside 59-triphosphates, BamHI, KpnI, SacI,
SacII, PhusionDNA Polymerase, GeneJET Gel Extraction Kit, CloneJET
PCR CloningKit, and Rapid DNA ligation kit (Thermo); Mouse IL-1b
ELISA Ready-SET-Go and Human IL-1b ELISA Ready-SET-Go
(eBioscience); BrilliantIII Ultra-Fast Sybr Green qRT-PCR kit
(Agilent); reporter lysis buffer andLuciferase Assay Agent, pGEM-T
easy (Promega); Saponin agar plates,Mueller Hinton plates, heart
infusion (HI) plates, LuriaBertani (LB)plates, LB broth, and HI
broth (Biotrading); Campylobacter selectivesupplement and charcoal
cefoperazone desoxycholate agar (SR0155)(Oxoid); RNA Bee
(Bio-connect); Fluorsave (Calbiochem); Qiaex II gelextraction kit
(Qiagen); FAM fluorescent labeled inhibitor of caspases(FLICA)
Caspase-1 Assay Kit (Immunochemistry); and rabbit anticaspase-1
(ab17820), rabbit anti-TMS1 (ASC) (ab64808), and rabbit antiIL-1b
(ab9722) (Abcam).
Cultivation of primary mouse macrophages
Bone marrow cells were isolated as described previously (34).
Uponthawing, cells were collected (5 min, 4853 g, 20C), resuspended
in 10 mlbone marrowderived macrophage (BMM) medium (RPMI 1640
mediumplus 10% heat-inactivated FCS and 30% L929 conditioned
medium) withpenicillin (100 IU) and streptomycin (100 mg/ml) and
allowed to differ-entiate for 6 d into BMM at 37C and 5% CO2. After
3 d, an additional10 ml BMM medium was added to the cells. After
differentiation cellswere collected, counted, and seeded into a
96-well (1 3 105 cells/well) or24-well (2.5 3 105 cells/well) plate
in BMM medium, and used the nextday. L929 conditioned medium was
collected from L929 cells grown in40 ml medium for 10 d in a T75
flask, filter sterilized (0.22-mm pore size),and stored at 220C
until use.
Bacterial culture
All C. jejuni strains (Supplemental Table I) were routinely
grown undermicroaerophilic conditions at 37C on saponin agar plates
containing 4%lysed horse blood or in 5 ml HI broth at 160 rpm for
16 h. Kana (50 mg/ml)or cat (20 mg/ml) was added to the medium when
appropriate. All C. jejunistrains had similar growth rates.
Escherichia coli DH5a (Netherlands Cul-ture Collection of Bacteria)
was grown on LB agar plates or in 5 ml LBbroth at 37C in air.
Salmonella was grown on LB agar with ampicillin(100 mg/ml), or in
HI medium for 2 h at 37C in air. C. jejuni lysate wasprepared as
described previously (25). Shortly, bacteria were grown for 16
hunder standard conditions, collected by centrifugation (10 min,
3000 3 g),and resuspended in DPBS to a final OD550 of 1. The
suspended bacteria wereheat killed at 65C for 30 min and
subsequently sonicated on ice (15-s pulse,30-s pause, six times),
aliquoted, and stored at 220C until further use.
Generation of C. jejuni mutants
To generate C. jejuni mutants, the target genes and their
flanking regionswere PCR amplified from chromosomal DNA from
strains 108 or 81116using the primers gene name fwd and gene name
rev (Table I) andPhusion polymerase or PFX polymerase using the
manufacturers protocol
in a Bio-Rad iCycler. The PCR products were gel purified using
the QiaexII gel extraction kit or the GeneJET gel extraction kit,
ligated into pGEM-T easy (cheY, cetA, flaC) or pJet1.2 (cdt) using
the Rapid DNA ligation kitor CloneJET PCR cloning kit, and
transformed into E. coli DH5a. Plas-mids were isolated from the
transformants using the GeneJET plasmidminiprep kit and used to
inactivate the cdt, cheY, and flaC genes via anoutward PCR with
primers gene name BamHI/KpnI fwd and genename BamHI/KpnI rev. BamHI
or KpnI sites were introduced to enableinsertion of the cat
cassette from pAV35. This yielded the vectors pcdt::cat,pcheY::cat,
and pflaC::cat. The cetA gene already contains a BamHI siteenabling
direct insertion of the cat cassette after BamHI digestion,
yieldingpcetA::cat. Gene inactivation constructs were verified by
sequencing. Pri-mers T7 and Sp6 were used for the pGEM-Teasy
inserts. The pJet1.2forward and reverse primers were used to
sequence the pJet1.2 inserts(Baseclear). The vectors were
introduced individually via electroporation(pcdt::cat, pcheY::cat,
and pcetA::cat) or natural transformation (pflaC::cat)into C.
jejuni using cat (20 mg/ml) selection. The
81116flaAB::kana/flaC::cat mutant was constructed via natural
transformation of 81116flaC::catwith 81116flaAB::kana chromosomal
DNA. All gene disruptions wereconfirmed via PCR. For cheY
complementation, the cheY gene was am-plified from the chromosomal
DNA from strain 108 using the primerscheY-sacI-fwd and
cheY-SacII-rev and introduced into pWM1007 via SacIand SacII
resulting in pcheY. pcheY was introduced into 108cheY::cat
viaelectroporation resulting in 108cheY::cat+pcheY. Sequences were
analyzedand aligned using Clone Manager 9 software (Sci-Ed).
Construction of C. jejuni luciferase reporter strains
andfluorescent strains
The pMA5-metK-luc plasmid was introduced into several C. jejuni
strains(81116, 108cheY::cat, 108kpsM::cat, and 108cetA::cat) via
conjugation (35).Strain 108cheY::cat became either GFP or mCherry
positive by introducingplasmid pMA1 containing GFP or mCherry via
conjugation. In short, a 16-hculture of E. coli S17.1 containing
the pMA5-metK-luc, pMA1-GFP, orpMA1-mCherry plasmid was diluted to
an OD550 of 0.05 in 5 ml LBmedium. Analogous 16-h cultures of the
C. jejuni strains were diluted to anOD550 of 0.5 in 5 ml HI broth.
When the E. coli culture reached an OD550 of0.4, 1 ml C. jejuni
culture was collected by centrifugation (10 min, 50003 g)and
suspended in 1 ml E. coli culture. The C. jejuni and E. coli mix
wasreleased on a Mueller Hinton plate and incubated for 5 h (37C)
undermicroaerophilic conditions. Then, bacteria were collected in 1
ml HI, pel-leted (10 min, 5000 3 g), suspended in 100 ml HI, and
plated on saponinagar plates containing 4% lysed horse blood,
charcoal cefoperazone des-oxycholate agar, Campylobacter selective
supplement, 50 mg/ml kana, andwhen required, 20 mg/ml cat. Single
antibiotic resistant colonies werecollected after 48 h of
incubation. The pMA1-mCherry plasmid was in-troduced in E. coli via
chemical transformation.
Infection assay
J774.A1macrophages were seeded into 24- or 96-well plates in
DMEMplus10% FCS. BMMs were seeded as described above. The next day,
cells wereprimed by the addition of C. jejuni 108 lysate
(equivalent of multiplicity ofinfection [m.o.i.] 20) for 16 h when
appropriate. THP-1 monocytes (2 3105 cells/well in a 96-well plate)
were differentiated with 100 nM PMA for48 h in RPMI 1640 medium
plus 10% FCS. Prior to inoculation, the THP-1cells were rinsed, and
RPMI 1640 medium plus 10% FCS without PMAwas added. The macrophages
were inoculated with the indicated amountsof C. jejuni, Salmonella,
or E. coli or stimulated with 2.5 or 5 mM ATP.After 20 min, the ATP
was removed, and fresh medium was given. Ex-tracellular Salmonella
and E. coli were removed after 2 h by replacing themedium with
fresh medium containing 50 mg/ml gentamicin. The exper-iment was
stopped, and samples were collected after 12 h of incubationunless
indicated otherwise.
Real time RT-PCR
J774.A1 cells were seeded into a 24-well plate and stimulated
the next dayby the addition of 50 ng/ml lipooligosaccharides (LOS)
of N. meningitidis,lysate of C. jejuni strain 108 (equivalent of
m.o.i 20) or inoculated withviable C. jejuni 108 (m.o.i of 20).
After 4 h of stimulation, RNA wasisolated using RNA Bee, according
to the manufacturers protocol. RNAwas treated with 1 mg DNAse/mg
RNA for 30 min at 37C. The DNase wasinactivated by heating for 10
min at 65C in the presence of 2.5 mMEDTA. mRNA levels were
determined in the LightCycler 480 Real-TimePCR System using the
Brilliant III Ultra-Fast Sybr-Green qRT-PCR kit,according to the
manufacturers protocol with the primers listed in Table I.Per
reaction 50 ng DNase-treated RNA was used as template.
Real-timecycle conditions: 3 min at 95C, 40 cycles 5 s 95C and 10 s
60C, 3 min
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95C. mRNA levels were calculated by subtracting the
corresponding Ctvalues obtained for samples before (1) and after
(2) treatment using thefollowing formula: 1) DCt control = Ct
target gene control Ct Actin control and 2)DCt target gene treat Ct
Actin treated. The fold change in mRNA was deter-mined by fold
change = 2(DCt[treated]-(DCt[control]) (36). Presented results
arefrom three individual assays performed in duplicate.
ELISA
Cell culture supernatants were collected in a fresh plate,
centrifuged(10 min, 485 3 g) to remove dead cells, bacteria, and
other debris, andstored at 280C until further analysis. IL-b levels
were determined byELISA using the manufacturers protocol. Samples
were diluted in assaydiluent to stay within the range of the assay
(8 - 1000 pg/ml for mice and4 - 500 pg/ml for human). Plates were
measured at 450 and 570 nm forwavelength correction on the FLUOstar
Omega (BMG Labtech). Presentedresults are from three individual
assays performed in duplicate.
Detection of caspase-1 and IL-1b cleavage
For caspase-1 cleavage J774.A1 macrophages were seeded into a
6-wellin DMEM+10% FCS. The next day, the medium was replaced with
Opti-MEM and primed by the addition of C. jejuni 108 lysate
(equivalent ofm.o.i. 20) for 16 h when appropriate. The macrophages
were inoculatedwith C. jejuni or E. coli. After 2 h of infection,
250 mg/ml gentamicin wasadded to the E. coliinoculated wells. After
6 h of infection, the super-natant was collected and frozen at 280C
for a minimum of 1 h witha protease inhibitor. For the detection of
cleaved IL-1b in the supernatant,J774.A1 macrophages or THP-1
monocytes were seeded in a T25 flask andinfected as described for
the infection assay with one minor change. Beforeinfection, the
medium was replaced with Opti-MEM. Postinfection (12 h)the
supernatant was collected and frozen at 280C for a minimum of 1
hwith a protease inhibitor. The thawed supernatant was concentrated
viaa concentrator (9K MWCO) (IL-1b detection) or TCA
precipitation(caspase-1). TCA (40%) was added to the supernatant in
a 1:1 volume andincubated for 30 min at 4C. The precipitate was
collected by centri-fugation (10 min, 21,100 3 g), washed twice
with ice cold acetone(10 min, 21,100 3 g), dried (10 min at 50C),
and taken up in radio-immunoprecipitation assay buffer. Protein
concentration was determinedvia BCA protein concentration kit,
according to the manufacturers pro-tocol. Protein (10 mg for
caspase-1 detection and 50 mg for IL-1b detec-tion) was loaded and
run on a 12% SDS-Page gel. After transfer of theproteins via
blotting, the polyvinylidene difluoride membrane was blockedin TBST
plus 5% milk (1 h) and incubated (16 h) with rabbit anticaspase-1
(ab17820) (1:1000) or rabbit antiIL-1b (ab9722) (1:2500) in TBST
plus5% milk at 4C. The next day, the membrane was washed three
times withTBST plus 5% milk (10 min per wash), incubated (1 h) with
goat anti-rabbit IgG-HRP (A4914) (1:10,000), and washed (10 min per
wash) withTBST plus 5% milk, TBST, and TBS. HRP signal was detected
usingSuperSignal West Femto Chemiluminescent Substrate on the
ChemiDocMP System (Bio-Rad). Data were analyzed using Image Lab
software(Bio-Rad). The images have been cropped.
Luciferase reporter assay
Bacteria were cultured for 16 h under standard conditions with a
start OD550of 0.01 from an 8-h preculture. Infection assays were
performed withC. jejuni strains containing pMA5-metK-luc as
described above. After 6 h,the cells were washed twice, lysed with
13 reporter lysis buffer supple-mented with 1% Triton-X100, and
placed at 280C for at least 30 min.The cell lysate was analyzed for
luciferase activity in a luminometer(TD20/20; Turner Designs)
immediately after adding 50 ml Promega Lu-ciferase Assay Agent to
the sample as described previously (20). Tomeasure intracellular
survival, the medium was replaced after 6 h withmedium containing
gentamicin (250 mg/ml). After an additional incuba-tion (2 h), the
gentamicin concentration (50 mg/ml) was reduced andremained present
throughout the assay. Presented results are from threeindividual
assays performed in duplicate.
Cytotoxicity assay
Primed J774.A1 macrophages were infected in 96-well plate as
describedabove with some minor changes. The assay was performed in
DMEMwithout FCS. After 12 h of infection, the total and secreted
amount of LDHwas determined using the Cytotoxicity Detection
KitPLUS (LDH), accordingto the manufacturers protocol. Plates were
measured at 492 and 690 nmfor wavelength correction on the FLUOstar
Omega (BMG Labtech).C. jejuni by itself had no effect on LDH and
did not influence the assay.The percentage of cytotoxicity was
calculated as the percentage of LDHrelease compared with the total
LDH concentration (percentage of cyto-
toxicity = 100 3 [LDH released/total LDH]). Presented results
are fromthree individual assays performed in triplicate.
Propidium iodide uptake
Primed J774.A1 macrophages were infected in a 96-blackwell plate
witha transparent bottom (see infection assay) with some minor
changes. Theassay was performed in Opti-MEM without phenol red.
After 11 h of in-fection, propidium iodide (PI) (3 mM) was added to
the wells, and incu-bated for 1 h after which the plate was
measured. Plates were excited at492 nm and measured at 610 nm using
a fixed gain, with bottom optics(orbital averaging, 35 flashes) on
the FLUOstar Omega (BMG Labtech).Fluorescence intensity (F.I.) was
corrected for background fluorescencefrom an unstained non
stimulated well. Presented results are from threeindividual assays
performed in triplicate.
Confocal microscopy
J774.A1 cells or BMMs were grown on 12 mm circular glass slides,
primedfor 16 h with the indicated stimulus, and inoculated with
fluorescentC. jejuni. THP-1 monocytes were PMA differentiated, as
mentioned pre-viously, on 12 mm circular glass slides and
inoculated with fluorescentC. jejuni. For active caspase-1
detection cells were incubated (1 h, 37C)with FAM-FLICA (0.53) and
washed twice (10 min, 37C) before fixa-tion. After incubation,
cells were washed twice with DPBS and fixed with4% of
paraformaldehyde in 100 mM phosphate buffer (pH 7.4) (1 h,
roomtemperature). The fixed cells were washed with DPBS and stained
withWGA Alexa Fluor 633 (1:500 for 1 h in DPBS). When needed cells
werepermeabilized with 1% Triton X-100 plus 1% BSA in DPBS (30
min,20C). ASC was stained by incubation (16 h, 4C) of the cells
with anti-TMS1 plus 0.01% Triton X-100 plus 2% BSA in DPBS followed
with thegoat anti-rabbit-Alexa Fluor 488 (1:100) secondary Ab in
DPBS plus 2%BSA (1 h, 20C). After staining the slides were washed
three times withDPBS, once with MilliQ, embedded in Fluorsave, and
viewed in the Bio-Rad radiance2000 system or Leica SPE-II system.
The slides used forC. jejuni uptake were viewed using fixed
settings for section thickness(2 mm) and magnification. Per slide 4
random images were captured. Datawere analyzed with ImageJ
software.
Statistical analysis
Results were analyzed using GraphPad Prism 5 software.Where
appropriatesignificance was calculated using a paired Student t
test.
ResultsC. jejuni infection primes the macrophages for
inflammasomeactivation
As activation of the inflammasome may involve a two-step
process(priming and activation), we first determined whether C.
jejuni wascapable of priming the cells for inflammasome activation
(Fig. 1).The transcription levels of proIL-1b in J774.A1
macrophageswere determined using real-time RT-PCR with the primers
listedin Table I. Incubation of macrophages with C. jejuni strain
108 ledto a 125-fold induction of proIL-1b mRNA at 4 h of
infection(Fig. 1A). Lysed C. jejuni yielded an even stronger effect
probablydue to increased availability of TLR ligands after
bacterial dis-integration (25). The transcriptional levels of ASC,
NLRP3,caspase-1, or caspase-11 were similar upon stimulation withC.
jejuni lysate or LPS (4 h) (Fig. 1BE).To functionally verify the
effect of priming, J774.A1 macro-
phages were incubated (for 16 h) with C. jejuni lysate and
sub-sequently infected (12 h) with E. coli or stimulated with
ATP(known inflammasome activators). The amount of secreted IL-1bin
the culture supernatant was determined using ELISA. IL-1bsecretion
was only observed in primed cells with additional E. colior ATP
stimulation (Fig. 1F). Taken together, these result showthat C.
jejuni lysate primes the cells for inflammasome activation.
C. jejuni infection activates the inflammasome
ProIL-1b is processed by activated inflammasomes and
subse-quently secreted from the cell. To assess the activation of
theinflammasome by C. jejuni, the amount of IL-1b secreted
fromprimed macrophages was determined using ELISA. Infection of
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primed macrophages with live C. jejuni resulted in a
dose-dependent secretion of IL-1b (Fig. 2A) and was observed
forseveral C. jejuni strains (Fig. 2B). Western blot analysis of
the
supernatant confirmed the presence of the active IL-1b (17
kDa)form upon infection with C. jejuni strain 108 (Fig. 2C). TheC.
jejuni lysate alone did not cause IL-1b secretion, suggesting
therequirement for intact bacteria to activate inflammasomes.To
ascertain that the release of IL-1b from the macrophages
was caused by activation of the inflammasome, we
determinedwhether caspase-1 was cleaved and secreted into the
supernatantupon infection (6 h) with E. coli or C. jejuni. The
presence of thep20 cleavage fragment (containing the active domain)
of caspase-1in the supernatant was determined using Western
blotting. BothE. coli and C. jejuni strain 108 induced caspase-1
cleavage, asevident from the increased appearance of the p20
protein band(Fig. 2D). For unknown reasons, the cleavage pattern
forE. coli and C. jejuniinfected macrophages were different.
Asexpected, cleavage was most evident for primed
macrophages.Inflammasome activation was also confirmed by the
formation ofthe ASC speck (a large multiprotein complex is being
formedcontaining ASC) upon infection with C. jejuni or E. coli (2
h) asdetermined by confocal microscopy (Fig. 2E). Clearly,
theseresults demonstrate that C. jejuni is capable to activate
theinflammasome.
Cellular infectiondependent inflammasome activation byC.
jejuni
To learn more about the mechanism(s) of C.
jejuniinducedinflammasome activation, a series of C. jejuni mutants
with defectsin putative virulence determinants was tested for their
ability toinduce IL-1b secretion (Fig. 3AC). In these experiments
an in-termediate dose of C. jejuni (m.o.i. of 20) was used to
infect thecells to avoid IL-1b secretion as result of bacterial
depletion ofmedia components or possible C. jejuni induced cell
toxicity. Ge-netic manipulation resulted in successful inactivation
of a vastnumber of putative virulence genes, albeit in different C.
jejunistrains. Infection assays demonstrated that genetically
definedmutants with defects in bacterial capsule assembly
(kpsM::cat),LOS assembly (waaF::cat), bacterial motility
(motAB::cat), pro-duction of flagellin and flagellin-like proteins
(flaAB::cat; flaAB::cat + flaC::kana), cytolethal distending toxin
(CDT) production(cdt::cat), or the type 6 secretion apparatus
(hcp::kana + kpsM::cat)induced similar levels of IL-1b secretion as
their correspondingparental strain (Fig. 3AC). In contrast, mutants
lacking thechemotaxis protein CheY or the energy taxis protein CetA
eliciteda strongly increased (cheY::cat) or reduced (cetA::cat)
IL-1b secretioncompared with the parent strain (Fig. 3A). Western
blotting con-firmed the presence of more cleaved IL-1b in the
supernatant of cellsinfected with strain 108cheY::cat compared with
the parent strain(108) (Fig, 2C). This suggests that bacterial
taxis strongly influencesthe level of inflammasome activation.The
CheY- and CetA-defective bacterial phenotypes show, re-
spectively, hyperinvasive and hypoinvasive behavior toward
epi-thelial cells (18, 19, 37, 38). Therefore, we examined a
possiblecorrelation between the number of bacteria that infected
themacrophages and IL-1b secretion for both taxis mutants and
theparental strain 108 using a luciferase reporter assay (20).
Mac-rophages were infected with different strains of C.
jejunipro-ducing luciferase. The amount of luciferase produced
correlates tothe number of viable intracellular bacteria. After 6 h
of incubation,the supernatant of the cells was removed, and the
cells were lysedto determine bacterial luciferase activity. In
addition, after 12 h ofinfection, the IL-1b secretion in the
supernatant was determined.This demonstrated that the cheY::cat
mutant yielded highest lu-ciferase activity, whereas the cetA::cat
mutant yielded lower levelsthan the parent strain C. jejuni 108
(Fig. 3D), thus followinga similar pattern as observed for the
IL-1b secretion. Although
FIGURE 1. C. jejuni lysate primes macrophages for inflammasome
ac-
tivation. (AE) mRNA expression levels of IL-1b, NLRP3, ASC,
caspase-1,
and caspase-11 in J774.A1 macrophages postinfection (4 h) with
viable
C. jejuni 108 (m.o.i. 20) or stimulation with LPS (50 ng/ml) or
C. jejuni
lysate (m.o.i. 20). (A) LPS, viable, and lysate C. jejuni showed
significantupregulation of IL-1b mRNA (p , 0.01). (B and C) No
differences inmRNA levels were observed for NLRP3 or ASC after
stimulation with
C. jejuni (viable or lysate) compared with LPS. (D) Caspase-1
mRNA
expression levels were upregulated after stimulation with LPS (p
, 0.001),C. jejuni (p , 0.01), and lysate (p , 0.001). (E)
Caspase-11 mRNAtranscript showed a minimal increase (p , 0.05)
after stimulation with LPSor C. jejuni (viable or lysate). (F)
Macrophage IL-1b secretion after stim-
ulation (12 h) of nonprimed (N) or lysate primed (n) cells with
ATP (5 mM)
or E. coli (m.o.i. 20). In primed macrophages, ATP and E. coli
significantly
increased IL-1b secretion (p , 0.01). Values are presented as
the mean 6SEM of three independent experiments performed in
duplicate.
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several strain of C. jejuni induced IL-1b secretion (Fig. 2B),
therewas some variation among the strains. This variation again
cor-responded with the amount of intracellular bacteria as
determinedwith the luciferase reporter assay (Fig. 3E). Taken
together, theseresults highlight a correlation between the amount
of intracellularbacteria and inflammasome activation.
C. jejuni does not cause cell death
Besides IL-1b secretion, a common downstream effect of
in-flammasome activation is cell death via pyroptosis. Indeed,
in-cubation of primed J774.A1 macrophages with ATP, Salmonella,or
E. coli (12 h) induced cytotoxicity as estimated from the releaseof
LDH in the culture supernatants (Fig. 4A). In contrast, incu-bation
of the cells with C. jejuni did not result in the release
ofdetectable amounts of LDH despite activation of the inflamma-some
(Fig. 3AC). Even the hyperinvasive cheY::cat mutant didnot cause
any LDH release. Measurement of PI uptake in J774.A1macrophages
infected with C. jejuni (strain 108 and cheY::cat,12 h) revealed a
minor yet not significant increase (Figure 4B). Incontrast,
infection with Salmonella did increase PI uptake in thesecells,
whereas E. coli had a minimal effect under the conditionsused.
Taken together, these results suggest that C. jejuni activatesthe
inflammasome without causing cell death.To assess whether the lack
of cytotoxicity may be related to rapid
killing of the intracellular C. jejuni, we measured the
intracellularsurvival in primed J774.A1 macrophages using the
bacterial lu-ciferase reporter assay (Fig. 4C). After 12 h of
infection, very fewviable intracellular bacteria were detected and
none after 24 h.There was also no major difference in survival
between strain 108and the hyperinvasive strain 108cheY::cat.
C. jejuni activates the NLRP3 inflammasome in primary
mousemacrophages
To determine which type of inflammasome is activated byC.
jejuni, we used BMMs derived from either C57BL/6 wild-typeor
knockout mice deficient in distinct inflammasome components.The
wild-type BMMs showed the expected secretion of IL-1bafter
stimulation with ATP and the enhanced IL-1b secretionpostinfection
with Salmonella (Fig. 5A). Both ATP and Salmo-nella required
priming of the primary macrophages to be effective.Infection of the
wild-type macrophages with C. jejuni strain108cheY::cat also
elicited the release of IL-1b but without theneed to previous prime
the macrophages. In fact, in primed cells,C. jejuni did not induce
IL-1b secretion (Fig. 5A).The type of inflammasome that was
activated by the various
stimuli was determined using BMMs from
caspase-12/2/112/2,ASC2/2, NLRP32/2, and NLRC42/2mice (Fig. 5B,
5C). As expected,ATP induced IL-1b secretion in wild-type and
NLRC42/2 BMMbut not in BMMs deficient in NLRP3, ASC, and
caspase1/11, whichall are components of the NLRP3 inflammasome
(Fig. 5B). Infectionwith Salmonella induced IL-1b secretion in both
wild-type andNLRP32/2 BMMs, whereas secretion was severely reduced
inNLRC42/2 and ASC2/2 macrophages, consistent with previ-ous
reports (Fig. 5B) (39). C. jejuni strain 108 and the
cheY::catmutant induced equal levels of IL-1b secretion in
wild-typeBMM and BMMs isolated from NLRC42/2 mice (Fig. 5C).C.
jejuniinduced IL-b secretion was not detected postinfectionof
caspase-12/2/112/2 and ASC2/2 BMMs and severely reducedin NLRP32/2
BMMs. This suggests that C. jejuni activates theNLRP3 inflammasome
and that caspase-1/11 and ASC are criticalfor the activation.
Table I. Primers used in this study
Primer Name Sequence
Real-time RT-PCRmActin RT fwd 59-TCCTGTGGCATCCACGAAACT-39mActin
RT rev 59-GGAGCAATGATCCTGATCTTC-39mIL-1b RT fwd
59-CCCAAGCAATACCCAAAGAAGAAG-39mIL-1b RT rev
59-TGTCCTGACCACTGTTGTTTCC-39mNLRP3 RT fwd
59-CGAGACCTCTGGGAAAAAGCT-39mNLRP3 RT rev
59-GCATACCATAGAGGAATGTGATGTACA-39mASC RT fwd
59-AAAAGTTCAAGATGAAGCTGCTG-39mASC RT rev
59-CTCCTGTAAGCCCATGTCTCTAA-39mCaspase-1 RT fwd
59-TTTCAGTAGCTCTGCGGTGT-39mCaspase-1 RT rev
59-TTTCTTCCTGATTCAGCACTCTC-39mCaspase-11 RT fwd
59-GCCACTTGCCAGGTCTACGAG-39mCaspase-11 RT rev
59-AGGCCTGCACAATGATGACTTT-39
PCRcdt fwd 59-CTACACCCAAGGCCAAAG-39cdt rev
59-GCCTCGATAATATGGCGTCC-39cdt BamHI fwd
59-CCGGATCCAATTCGCCAAATGAACG-39cdt BamHI rev
59-CCGGATCCCTTTAACAGCTGCTACCC-39cheY fwd
59-AACTACACCACTCATTGATTT-39cheY rev 59-GCTGAGGCAGTGCAACTTGT-39cheY
BamHI fwd 59-CGGGATCCTTCTGGCATATTCCAATCTG-39cheY BamHI rev
59-CGGGATCCGCCTATCATCATGGTTACAA-39cetA fwd
59-TCCCGCCATAAAGCCTTGTG-39cetA rev 59-TAGAGCCGCAAGCGTACTTC-39cheY
sacI fwd 59-TCCGAGCTCTAAAAAACTTTGAAAGGACGAAAT-39cheY sacII rev
59-TCCCCGCGGTTAAAAATCAGCCTTTACTCAG-39FlaC fwd
59-AATCATTTTACCGCAGAACC-39FlaC rev 59-ATCAATCCCAAAGCCTTAGA-39FlaC
KpnI fwd 59-GGGGTACCATAGTTGCATCAGAGATCAT-39FlaC KpnI rev
59-GGGGTACCAAATAGGCTCAGGTATCAAT-39T7 59-TATTTAGGTGACACTATAG-39SP6
59-TAATACGACTCACTATAGGG-39pJet1.2 forward
59-CGACTCACTATAGGGAGAGCGGC-39pJet1.2 reverse
59-AAGAACATCGATTTTCCATGGCAG-39
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To ensure that the lack of inflammasome activation in C.
jejuniinfected NLRP32/2 BMMs was not caused by poor infection
ofthese cells, we determined the number of intracellular
bacteria.Wild-type and NLRP32/2 cells were incubated (1 h)
withmCherry-producing C. jejuni strains 108 and 108cheY::cat,
andbacteria were visualized by confocal microscopy (Fig. 5E,
5F).Both cell types contained equal numbers of C. jejuni strains
108and 108cheY::cat. The cheY::cat mutant was more
infective(4-fold) than the parent strain, as was already noted for
the J774.A1 macrophages (Fig. 3D). The increased infectivity of the
cheY::cat mutant compared with the parent strain likely explains
thehigher production of IL-1b (Fig. 5C). Complementation of
thecheY::cat mutant with the plasmid pcheY restored IL-1b
secretionto parent levels (Fig. 5D).
Activation of the human NLRP3 inflammasome by C. jejuni
Although C. jejuni efficiently infects both mouse and
humanmacrophages, mice normally do not establish infection afterC.
jejuni exposure in contrast to humans. In addition,
speciesdifferences between the human and mouse inflammasomes
have
been reported (40). To ascertain that C. jejuni also activates
thehuman inflammasome, PMA-differentiated human THP-1 mono-cytes
and THP-1 cells deficient in NLRP3 (THP-1 defNLRP3)were infected
with Salmonella, E. coli, or C. jejuni. This experi-mental setup
abolished the need for additional priming of the cellsbecause the
PMA treatment (100 nM PMA for 48 h) alreadyactivates NF-kB.
Infection of the cells with Salmonella and E. coliinduced IL-1b
secretion in the THP-1 cells but not in THP-1defNLRP3 cells (Fig.
6A), indicating that these bacteria activatethe human NLRP3
inflammasome. All C. jejuni strains tested alsoinduced IL-1b
secretion in a NLRP3-dependent fashion (Fig. 6B).Furthermore, the
C. jejuni 108cheY::cat mutant caused more andthe C. jejuni
108cetA::cat less secretion than the parent strain, aswas found for
the mouse macrophages (Figs. 3A, 5C). Inflam-masome activation by
C. jejuni in PMA-differentiated THP-1 cells(2 h infection) was
confirmed by the visualization of an ASCspeck (Fig. 6C) and the
presence of active caspase-1 (FLICA-positive cells) (Fig. 6D) as
determined by confocal microscopyand the secretion of cleaved IL-1b
(17 kDa) into the culture su-pernatant as shown via Western
blotting (12 h infection) (Fig. 6E).
FIGURE 2. C. jejuni activates the inflammasome. (A and
B) IL-1b secretion by primed J774.A1 macrophages infected
(12 h) with different numbers of C. jejuni, C. jejuni lysate
(m.o.i. 20) (A) or with different C. jejuni strains (m.o.i.200)
(B). Infection with an m.o.i. higher than 20 (p , 0.05)or with
different strains (p , 0.01) increased IL-1b secre-tion. Values are
presented as the mean 6 SEM of three in-dependent experiments
performed in duplicate. (C) Western
blot probed for active IL-1b (17 kDa) (arrowhead) in the
supernatant of primed J774.A1 macrophages postinfection
(12 h) with C. jejuni strain 108 (m.o.i. 20 or 200),
108cheY::
cat (m.o.i. 20), E. coli (m.o.i 20), or without bacteria
(non-
stimulated). Lanes were loaded with 50 mg protein. (D)
Western blot probed for the cleaved caspase-1 p20 fragment
in the supernatant of nonprimed or primed J774.A1 macro-
phages postinfection (6 h) with C. jejuni strain 108 (m.o.i.
200), E. coli (m.o.i. 20), or without bacteria
(nonstimulated).
Lanes were loaded with 10 mg protein. (E) Confocal mi-
croscopy showing ASC speck formation (,) in primed J774.A1
macrophages postinfection (2 h) with mCherry fluores-
cent C. jejuni strain 108 (m.o.i. 40) or E. coli (m.o.i. 20)
(red). ASC foci were stained with anti-ASC Ab in combi-
nation with goat-anti-rabbit-Alexa Fluor 488 (green). Cell
surface was stained with WGA-Alexa Fluor 633 (blue). Scale
bar, 10 mm.
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Luciferase assays on the infected THP-1 cells demonstrated
higherbacterial values for the 108cheY::cat than for the parent
strain(Fig. 6F), suggesting that also in human cells inflammasome
ac-tivation varies with cellular infection levels. THP-1
defNLRP3macrophages showed similar levels of luciferase activity
asmeasured for the infected THP-1 cells excluding that the
differ-ence in IL-1b secretion in the deficient cells was caused by
lowinfection rates. Finally, intracellular survival of C. jejuni
strain108 (Fig. 6G) and 108cheY::cat (Fig. 6H) was followed by
theluciferase reporter assay in differentiated THP-1 and
THP-1defNLRP3 cells. Intracellular levels of both strains severely
re-duced overtime and were almost absent after 24 h of infection.
Nodifference in bacterial survival was observed between the
THP-1
and the THP-1 defNLRP3 cells, indicating that the poor
intra-cellular survival of C. jejuni occurs independent of
inflammasomeactivation. Overall, these results show that C. jejuni
also activatesthe human NLRP3 inflammasome and that activation
varies withthe amount of infection.
DiscussionIn the current study, we provide evidence that the
principal bacterialfood-borne pathogen C. jejuni induces the
secretion of IL-1b viaactivation of the NLRP3 but not the NLRC4
inflammasome. Theeffect required the inflammasome components NLRP3,
ASC andcaspase-1/11 and was observed upon infection of both mouse
andhuman macrophages. Inflammasome activation required viable
FIGURE 3. Cellular infection-induced acti-
vation of the inflammasome. (AC) Induction of
IL-1b secretion in primed J774.A1 macro-
phages (12 h) exposed to different C. jejuni
mutants and their respective parent strains 108,
81176, and 81116 (m.o.i. 20). (D) Effect of
C. jejuni infection of primed J774.A1 macro-
phages (m.o.i. 20) on IL-1b secretion (N) (after
12 h) and bacterial viability (n) (after 6 h) as
measured via the luciferase reporter assay (rel-
ative light unit [RLU]). The p values (AD) for
all mutant strains compared with the parent
strain were not significantly different, except for
the cheY::cat and cetA::cat (p , 0.05). (E) In-tracellular
bacterial viability of several C. jejuni
strains (m.o.i. 20) in primed J774.A1 macro-
phages (6 h) as measured via the luciferase re-
porter assay. Strain 81176 was significantly
(p, 0.01) more present intracellular than strain108. Values are
the mean 6 SEM of three in-dependent experiments performed in
duplicate.
FIGURE 4. Cell viability and intracellular survival after
C. jejuni induced inflammasome activation. (AC) Cyto-
toxicity and bacterial survival in infected primed J774.A1
macrophages. (A) LDH release from primed cells at 12 h of
incubation with ATP (5 mM), Salmonella (m.o.i. 20),
E. coli (m.o.i. 20), or several C. jejuni (m.o.i. 20) or
mutants of strain 108. ATP (p , 0.001), Salmonella (p ,0.01),
and E. coli (p , 0.01) caused significant cytotox-icity. None of
the C. jejuni strains induced cytotoxicity. (B)
PI uptake by primed macrophages incubated (12 h) with
the indicated strains. Salmonella (p , 0.001) causeda
significant increase in F.I. The increase F.I. induced by
C. jejuni cheY mutant was not statistically significant. (C)
Intracellular survival of C. jejuni strain 108 and its cheY
derivative (m.o.i. 20) in primed J774.A1 macrophages as
measured via the luciferase reporter assay. Luciferase ac-
tivity (relative light unit [RLU]) after 6 h infection was
set
as 100% value. Values are the mean 6 SEM of three in-dependent
experiments performed in triplicate.
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bacteria and varied with the severity of the cellular
infection.Strikingly, inflammasome activation by C. jejuni did not
lead tocell death and occurred in primary mouse macrophages
withoutthe need of a priming signal.The secretion of IL-1b and the
structurally related IL-18 are
important in the innate immunity and systemic response
againstbacterial infections (41). Many flagellated bacterial
pathogens(Legionella pneumophila, Pseudomonas aeruginosa,
SalmonellaTyphimurium, Shigella flexneri, and enteropathogenic E.
coli) acti-vate the NLRC4 inflammasome (42, 43). Crucial for this
activation isthe translocation of flagellin or components of the
T3SS into thecytosol, which are sensed by members of the NAIP
family (810, 4446). Our results with mouse NLRP32/2 and NLRC42/2
knockoutcells and human NLRP32/2 macrophages demonstrate that C.
jejunifails to activate the NLRC4 inflammasome. This is consistent
withthe absence of a T3SS or T4SS in this pathogen. The apparent
ab-sence of translocation of flagellin or other NAIP ligands into
thecytosol makes C. jejuni invisible for the NLRC4 inflammasome.In
this study, a large body of evidence indicates that C. jejuni
activates the NLRP3 inflammasome. The C. jejuniinduced
se-cretion of mature IL-1b in primed J774.A1 macrophages,
theformation of an ASC speck, the generation of caspase-1
cleavagefragments, and the absence and severe reduction of IL-1b
secre-tion in human and mouse NLRP3-negative cells respectively,
all
indicate NLRP3-dependent IL-1b secretion. Interestingly, some
lowresidual IL-1b secretion remained in the NLRP3-deficient
mousemacrophages, whereas this was not observed for the
caspase-1/11 andASC-deficient macrophages. This suggests a possible
role for anadditional inflammasome contributing to the response.The
NLRP3 inflammasome can be formed in response to a di-
verse array of agents (4, 7, 43) but, to our knowledge, for none
ofthem binding of a specific ligand to NLRP3 has been
demon-strated. The mechanism driving the activation of the NLRP3
byC. jejuni was investigated using different C. jejuni strains
andseries of genetically defined mutants. All of the tested strains
in-duced IL-1b secretion, suggesting that inflammasome activation
isa stable trait of the pathogen. This trait was preserved in
mutantswith defects in capsule formation, LOS biosynthesis,
flagellasynthesis and flagellin(-like) secretion, T6SS needle
protein, CDT,and several assumed bacterial adhesion/invasion
promoting fac-tors. Inflammasome activation was affected after
disruption of thecheY gene and, to a lesser extent, the cetA gene.
The strong in-crease in IL-1b production observed for the CheY
mutant wasaccompanied by increased cellular infection. The
hyperinvasiveCheY phenotype was evident from microscopy and
luciferasebacterial gene reporter assays and was observed for both
mouseand human macrophages. The exact signal(s) that drive(s)C.
jejuniinduced NLRP3 formation remain to be defined but
FIGURE 5. C. jejuni activation of the NLRP3 inflam-
masome in primary mouse macrophages. (A) Secretion of
IL-1b by non-primed (N) and primed (n) BMMs incubated
(12 h) with Salmonella (m.o.i. 2), C. jejuni (m.o.i. 20), or
ATP (2.5 mM). ATP (p , 0.05) and Salmonella (p ,0.001)
significantly increased IL-1b secretion; C. jejuni
significantly increased IL-1b in nonprimed cells only (p
,0.001). (B) Similar assay but with primed BMMs isolated
from the indicated knockout mice and incubated with
Salmonella (N) (m.o.i. 2) or ATP (2.5 mM) (n). IL-1b se-
cretion was significantly lower in caspase-12/2/112/2 and
NLRC42/2 BMMs upon infection with Salmonella (p ,0.05). Upon ATP
stimulation, IL-1b secretion was signifi-
cantly reduced in caspase-12/2/112/2, ASC2/2, or
NLRP32/2 BMMs (p , 0.05). (C) IL-1b secretion bynonprimed BMMs
from several knockout mice infected
(12 h) with C. jejuni strain 108 (N) or 108cheY::cat (n)
(m.o.i. 20). Significant lower secretion was observed for
caspase-12/2/112/2 (p , 0.001), ASC2/2 (p , 0.001),and NLRP32/2
(p , 0.01) BMMs upon stimulation withboth C. jejuni strains. Strain
cheY::cat induced more IL-1b
secretion (p, 0.05) than the parent strain. (D) Secretion
ofIL-1b in nonprimed BMMs infected (12 h) with C. jejuni
strain 108, 108cheY::cat, or 108cheY::cat+pcheY (m.o.i.
20). Complementation of the defective cheY restored the
high IL-1b secretion induced by strain 108cheY::cat (p ,0.01) to
parental levels (p . 0.0.05). (E) Confocal mi-croscopy on BMMs (wt
and NLRP32/2) infected (1 h)
with mCherry fluorescent (red) C. jejuni 108 and 108cheY::
cat (m.o.i 200); cells were counterstained with WGA-
Alexa Fluor 633 (green). (F) Quantification of the number
of intracellular bacteria in the wild-type (wt) (N) and
NLRP32/2 (n) BMMs showed no significant difference in
the number of intracellular bacteria between the wt and
NLRP32/2 BMMs. Strain 108cheY::cat was more invasive
(p , 0.001) than the parent strain in the BMMs. Values arethe
mean 6 SEM of three independent experiments per-formed in
duplicate.
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likely cause a transmembrane ion flux (e.g., K+-efflux),
whichseems the common denominator of NLRP3 activation (12). At
thistime, it is tempting to speculate that C. jejuniinduced cell
dam-age caused by the cellular infection contributes to the
NLRP3activation, although this was not evident from increased
release ofLDH from the cells (Fig. 4).Our results indicate that C.
jejuni activates the NLRP3 in-
flammasome in murine J774.A1 macrophage cells, primary
mouseBMMs, and human-differentiated THP-1 cells. A striking
differ-ence among these cell types however, was the apparent lack
ofneed to prime the primary cells to establish a strong IL-1b
pro-duction upon C. jejuni infection. ATP only activated the
inflamma-some in primed BMMs, indicating that the cells were not
alreadyprimed. Viable C. jejuni were most effective in inflammasome
ac-tivation in nonprimed primary cells. In primed cells, IL-1b
pro-duction was minimal probably because of the more efficient
bacterialuptake and killing in activated macrophages.Another
unexpected finding was that C. jejuni activates the
inflammasome without apparent cytotoxicity as evidenced by
theabsence of LDH release or an increased PI uptake by the
infectedcells. LDH release was noted postinfection with E. coli or
Sal-monella, indicating that the pyroptosis pathway is intact in
thesecells. Inflammasome activation by other bacterial species
alwaysseems to be followed by pyroptosis (2, 4, 6, 7, 42, 47). This
hasbeen linked with the presence of LPS in the cytosol (48, 49). It
canbe imagined that different bacterial metabolic demands, a
lowlevel of bacterial LPS in the cytosol, and/or the relative poor
in-tracellular survival of C. jejuni prevent C. jejuniinduced
cyto-toxicity. Activation of the inflammasome was not required to
killthe intracellular C. jejuni. Alternatively, the pathogen may
haveevolved a strategy to prevent bacteria-induced pyroptosis.Our
results that C. jejuni induces inflammasome activation in
both murine and human cells without apparent cytotoxicity and
inprimary cells without a need of priming extends the known
rep-ertoire of inflammasome activation by bacterial pathogens.
Thedata provide a molecular basis for the observed IL-1b
secretionduring C. jejuni infection and for key features in C.
jejuni path-ogenesis (22, 23, 31, 33, 5052).
FIGURE 6. C. jejuni activation of the NLRP3 inflammasome in
human
macrophages. (A and B) Secretion of IL-1b by PMA differentiated
THP-1
(N) and THP-1 defNLRP3 (n) cells infected with Salmonella
(m.o.i. 2),
E. coli (m.o.i. 20), or different C. jejuni strains (m.o.i. 20)
(12 h). Sal-
monella (p , 0.01), E. coli (p , 0.01), and all C. jejuni
strains (p , 0.05)induced IL-1b secretion upon infection. Strain
cheY::cat induced more
IL-1b secretion (p , 0.01) than the parent strain. (C and D)
Confocalmicroscopy on PMA-differentiated THP-1 cells infected (2 h)
with
mCherry positive C. jejuni strain 108 (m.o.i. 40) and E. coli
(m.o.i. 20)
(red). Cell surface was stained with WGA-Alexa Fluor 633 (blue).
ASC
foci [, and . in (C)] were stained with an anti-ASC Ab in
combinationwith goat-anti-rabbit-Alexa Fluor 488 (green). Active
caspase-1 (D) was
detected with FLICA (green) at 1 h of infection. Scale bars, 10
mm. (E)
Western blot showing the presence of active (cleaved) IL-1b (17
kDa) in
the supernatant of noninfected and C. jejuni strain 108 (m.o.i.
20)infected
(12 h) PMA-differentiated THP-1 cells. Lanes were loaded with 50
mg
protein. (F) Intracellular viability of C. jejuni mutants and
parent strain in
6 h infected PMA-differentiated THP-1 (N) and THP-1 defNLRP3 (n)
as
measured with the bacterial luciferase reporter assay. No
significant dif-
ferences in RLU were measured between the THP-1 and THP-1
defNLRP3. Strain 108cheY::cat was more invasive (p , 0.001) than
theparent strain. (G and H) Intracellular survival (24 h) of C.
jejuni strains 108
(G) or 108cheY::cat (H) in THP-1 (N) or THP-1 defNLRP3 (n)
cells. Lu-
ciferase activity at 6 h of infection was set as 100%. The
decrease in in-
tracellular C. jejuni (108 and 108cheY::cat) at 12 h of
infection was
statistically significant (p , 0.05). Values are the mean 6 SEM
of threeindependent experiments performed in duplicate.
The Journal of Immunology 9
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AcknowledgmentsWe thank Dr. Dietmar Zaiss (Utrecht University)
for providing the L929
cells.
DisclosuresThe authors have no financial conflicts of
interest.
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