HAL Id: hal-01603303 https://hal.archives-ouvertes.fr/hal-01603303 Submitted on 26 May 2020 HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Paneth cell defects induce microbiota dysbiosis In mice and promote visceral hypersensitivity Ambre Riba, Maïwenn Olier, Sonia Lacroix-Lamandé, Corinne Lencina, Valérie Alquier-Bacquié, Cherryl Harkat, Marion Gillet, Marine Baron, Caroline Sommer, Virginie Mallet, et al. To cite this version: Ambre Riba, Maïwenn Olier, Sonia Lacroix-Lamandé, Corinne Lencina, Valérie Alquier-Bacquié, et al.. Paneth cell defects induce microbiota dysbiosis In mice and promote visceral hypersensitivity. Gas- troenterology, WB Saunders, 2017, 153, pp.1594-1606. 10.1053/j.gastro.2017.08.044. hal-01603303
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HAL Id: hal-01603303https://hal.archives-ouvertes.fr/hal-01603303
Submitted on 26 May 2020
HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.
To appear in: GastroenterologyAccepted Date: 24 August 2017
Please cite this article as: Riba A, Olier M, Lacroix-Lamandé S, Lencina C, Bacquié V, Harkat C, GilletM, Baron M, Sommer C, Mallet V, Salvador-Cartier C, Laurent F, Théodorou V, Ménard S, Paneth CellDefects Induce Microbiota Dysbiosis In Mice And Promote Visceral Hypersensitivity, Gastroenterology(2017), doi: 10.1053/j.gastro.2017.08.044.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.
of EMG in response to colorectal distension with increasing volumes (0.02 to 0.1ml) in
Sox9flox/flox-vil-cre (grey square plain line) compared to littermate (white circle dashed line) (n
= 14 to 15 mice per group). ** P<0,01 compared to identical volumes for controls. (Two-way
ANOVA followed by Holm-Sidak’s post-test) (B) Responses to colorectal distension are
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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depicted as AUC (n = 14 to 15 mice per group). * P<0,05 compared to littermate (C) Fecal
antimicrobial activity against commensal E. coli (fecal protein concentration: 20 mg/ml) (n =
9 to 10 mice per group) * P<0,05 compared to littermate. (D) TNFα concentration in the
ileum fragment measured by ELISA (n = 14 to 15 mice per group). (Mann-Whitney test).
Figure 4 - Fecal microbiota analysis on maternally separated (MS) and Sox9flox/flox-vil-
cre adult mice. PLS-DA score plot of the relative quantitative abundances (Log10 No) of 21
microbial taxa in feces of MS and control mice (n= 10 mice per group, confidence ellipse
level =0.5) (A) and of Sox9flox/flox-vil-cre mice and littermate (n= 10 mice per group,
confidence ellipse level =0.5) (B). VIP plot representing important features (microbial taxa)
identified by PLS-DA in a descending order of importance (Increase of relative abundances
appeared in red and decrease in green) in MS and control mice (C) and in Sox9flox/flox-vil-cre
mice and littermate (D).
Figure 5 – Fecal E. coli analysis on maternally separated (MS) and Sox9flox/flox-vil-cre
adult mice correlated with AUC of EMG. Relative abundances per g feces of E. coli by
real-time qPCR in MS and control mice (A) and Sox9flox/flox-vil-cre mice and littermate (B)
(n= 9 – 10 mice per group) *: P<0.05 compared to littermate. Counting of E. coli colonies on
ChromID coli selective plates reported per g feces in MS and control mice (n = 8 mice per
group) ***: P<0.001 compared to control (C) and Sox9flox/flox vil-cre mice and littermate (n=
11 to 13 mice per group) *: P<0.05 compared to littermate (D). (Mann-Whitney test).
Correlation between E. coli CFU and AUC of EMG of control and MS mice (Pearson
r=0.6949, P=0.0028) (E) and littermate and Sox9flox/flox-vil-cre (Pearson r=0.5513, P=0.0331)
(F).
Figure 6 - Fecal microbiota, inflammation and visceral sensitivity analysis on adult mice
receiving commensal E. coli by oral gavage. (A) PLS-DA score plot of the relative
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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quantitative abundances (Log10 No) of 21 microbial taxa in feces of mice receiving
commensal E. coli or vehicle by oral gavage (n= 10 mice per group, confidence ellipse level
=0.5). (B) VIP plot representing important features (microbial taxa) identified by PLS-DA in
a descending order of importance (Increase of relative abundances appeared in red and
decrease in green). (C) Counting of fecal E. coli colonies on ChromID coli selective plates
after 15 days of oral gavage with E. coli. (n = 11 - 12 mice per group) ***: P<0.001 compared
to control. (Mann-Whitney test) (D) Relative abundances per g feces of E. coli by real-time
PCR (n = 10 - 11 mice per group) *: P<0.05 compared to control. (Mann-Whitney test) (E)
Representative curves of EMG in response to colorectal distension with increasing volumes
(0.02 to 0.1ml) in mice receiving E. coli by oral gavage (grey squares plain line) compared to
control/vehicle (white circles dashed line) (n = 8 mice per group). **: P<0,01 and ***:
P<0.001 compared to identical volumes for controls. (Two-way ANOVA followed by Holm-
Sidak’s post-test). (F) Responses to colorectal distension are depicted as Area Under the
Curve (AUC) (n = 8 mice per group). ***: P<0,001 compared to vehicle. (G) TNFα
concentration in the ileum fragment measured by ELISA (n = 10 to 11 mice per group).
(Mann-Whitney test).
Figure 7 - Fecal microbiota, inflammation and visceral sensitivity analysis on MS adult
mice orally treated or not with lysozyme for 15 days. (A) Counting of E. coli colonies on
ChromID selective plates after 15 days of oral lysozyme treatment (D50/G15) (n = 14 to 15
mice per group). *: P<0,05 compared to controls; µµµ: P<0.001 compared to MS+LZM; #:
P<0.05 compared to control+LZM; ##: P<0.01 compared to control+LZM. controls. (Two-
way ANOVA followed by Newman-keuls post-test). (B) TNFα concentration in the ileum
fragment measured by ELISA (n = 14 to 22 mice per group). *P<0.05 compared to control.
(Two-way ANOVA followed by Newman-keuls post-test). (C) Representative curves of
EMG in response to colorectal distension with increasing volumes (0.02 to 0.1ml) in MS
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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(grey square), MS treated with LZM (pale grey squares), control (white circles) and control
treated with LZM (grey circles) (n = 8-9 mice per group). *: P<0,05 compared to identical
volumes for controls; ** P<0,01 compared to identical volumes for controls; *** P<0,001
compared to identical volumes for controls; µµ: P<0.01 compared to MS+LZM; µµµ:
P<0.001 compared to MS+LZM; ## P<0.01 compared to control+LZM; ### P<0.001
compared to control+LZM. (Two-way ANOVA followed by Holm-Sidak’s post-test) (D)
Responses to colorectal distension are depicted as AUC (n = 8 - 9 mice per group). ***:
P<0,001 compared to control; µµµ: p<0.001 compared to MS+LZM and # p<0.05 compared
to control+LZM. (Two-way ANOVA followed by Holm-Sidak’s post-test).
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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Supplementary Materials and Methods
Mouse models
In all experiments, mice were kept at a constant temperature (22+/-1°C) and maintained on a 12:12h light/dark cycle (light on at 7h30 am). Food (Harlan, Gannat, France) and water were available ad libitum. After delivery (D1), litters were homogenized to 6±1 pups. Weaning was performed on D21, siblings were sex matched.
Maternal Separation protocol
Nulliparous female C3H/HeN mice (Janvier, Roubaix, France) were mated with male for 4 days. Maternal separation (MS) was performed daily for three consecutive hours (from 9 am to 12 pm). Pups were kept in controlled temperature (27+/-1°C). MS was repeated for 10 working days, weekend excluded, between D2 and D15. Control pups were left with their dam. From D15 to D21, all pups were maintained with their dam.
Oral gavage of commensal E. coli streptomycin resistant
Live E. coli were isolated from feces of naïve healthy C3H/HeN mice by culture on selective ChromID coli plates (Biomérieux, Marcy L’étoile, France). Grown colonies belonged to B1 phylogenetic group. Further characterization of one representative isolate revealed to belong to the O18 serogroup and none of checked virulence genes was harbored (lt, sTa, sTb, stx1, stx2, eae, hly, cnf, Afa, cdt, pks). In order to facilitate monitoring of this commensal E. coli isolate in feces of mice after gavage, a spontaneous streptomycin resistant mutant of the commensal isolate was generated by plating a PBS-washed overnight culture on LB 1% agar plates supplemented with 50µg/ml streptomycin. Growing colonies were then subcultured either on LB 1% agar plate alone, LB 1% agar supplemented with 50µg/ml or 500µg/ml streptomycin in parallel. Colony able to growth on all three conditions was selected and frozen at -80°C until use for gavage.
IL17, IL22 and IL10 measurement in the ileum
IL17, IL22 and IL10 were measured in supernatant of ileal fragments previously treated as follow. Frozen ileal fragments were suspended in RIPA buffer (0.5% deoxycholate, 0.1% SDS and 1% Igepal in TBS) containing complete anti protease cocktail (Roche), protein concentrations were measured using BCA uptima kit (Interchim).
IL17, IL22 and IL10 present in ileal tissues were assayed using commercial enzyme linked immunosorbent assays (ELISA kits; Duoset R&D Systems, Lille, France). Cytokines concentrations were normalized per mg of proteins in the supernatant and results expressed in pg of cytokine per mg of proteins.
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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Lysozyme expression in Paneth cells
Ileal samples were fixed in 4% formol, dehydrated through graded ethanol and embedded in paraffin. Sections (5µm) were rehydrated and submerged in antigen retrieval solution (citrate buffer, 10mM, pH6, 99°C) for 30 minutes. After incubation in blocking solution (PBS 0,01% Tween 20, 1% bovine serum albumin and 2% donkey normal serum) for 15 min, sections were incubated with rabbit anti-mouse lysozyme antibody (1/100, overnight, +4°C ;Abcam, Paris, France) followed by a Alexa fluor 488-conjugated donkey anti-rabbit IgG (0.75µg/ml, 1H, Room temperature, Jackson, Sullfolk). Antibody used would stained the mouse M and P lysozyme isoforms. Sections were incubated with Alexa fluor 594-conjugated Wheat Germ Agglutinin (WGA, 10µg/ml, 45 min; Invitrogen, Life Technology, Cergy Pontoise, France), mounted in Prolong gold antifade mounting medium with DAPI (Invitrogen) and examined under a Nikon 90i fluorescence microscope. Lysozyme fluorescence intensity in Paneth cells were quantified employing the software Nis-Elements Ar (Nikon, Champigny sur Marne, France) and results were expressed in fluorescence intensity per cell. Analyses were done on five well preserved crypts per animal and on five animals from each group.
Lysozyme activity in fecal content
Fecal proteins were mechanically extracted after rehydration of frozen feces in 500µl of PBS (10mM; pH7.2). After 10 minutes of centrifugation at 1600g, supernatants were sterilized with a 0.22 µm filter and frozen (-80°C). Fecal protein concentration was measured using BCA protein Assay kit, Uptima (Interchim, Montluçon, France). Activity of lysozyme against the peptidoglycan was determined using the EnzChek® Lysozyme Assay Kit (Molecular probes, life technology, St Aubin, France).
Fecal microbiota composition
Changes in the relative abundance of 21 relevant microbial 16S rRNA gene targets (Supplementary table 1) was obtained using the GULDA platform approach (Bergström et al, 2012b, 2012a) with minor adaptations (Yvon et al, 2016). According to this method, the universal bacterial primer set (All phyla) was included as the reference gene (four technical replicas of each amplification). Each 384-well PCR plate (MicroAmp optical reaction plates, Applied Biosystems, Naerum, Denmark) accommodated simultaneous analysis of 8 DNA samples per group. Quantitative real-time PCR was performed in duplicate on an ViiA7 from Applied Biosystems in a total volume of 5 µl containing 2,5 µl 2x Power SYBR Green PCR Master Mix (Applied biosystems), 0,18 µl of each primer (10 µM), 1 µl template DNA, and 1,14 µl nuclease-free water. Liquid handling was performed with a Bravo platform (Agilent Technologies, Santa Clara, USA). Following the previously described thermocycling program, the raw fluorescence data recorded by the ViiA7 RUO Software were exported to the LinRegPCR program to perform baseline correction, calculate the mean PCR efficiency per amplicon group and calculate the initial quantities No (arbitrary fluorescence units) for each amplicon. The relative abundance of the 21 specific amplicon groups were obtained by normalization to the No value obtained for the universal bacterial amplicon group determined
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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in the same array (No, specific/No, universal). A limit of detection of 10-6 (No, specific/No, universal) was set and samples below this limit were set to 5.10-6.
REFERENCES
Bergström A, Kristensen MB, Bahl MI, Metzdorff SB, Fink LN, Frøkiær H & Licht TR Nature of bacterial colonization influences transcription of mucin genes in mice during the first week of life. BMC Res. Notes (2012a) 5: 402
Bergström A, Licht TR, Wilcks A, Andersen JB, Schmidt LR, Grønlund HA, Vigsnæs LK, Michaelsen KF & Bahl MI Introducing GUt Low-Density Array (GULDA) - a validated approach for qPCR-based intestinal microbial community analysis. FEMS Microbiol. Lett. (2012b) 337: 38–47
Yvon S, Olier M, Leveque M, Jard G, Tormo H, Haimoud-Lekhal DA, Peter M & Eutamène H Donkey milk consumption exerts anti-inflammatory properties by normalizing antimicrobial peptides levels in Paneth’s cells in a model of ileitis in mice. Eur. J. Nutr. (2016) Available at: http://link.springer.com/10.1007/s00394-016-1304-z
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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Supplementary Table 1
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
Supplementary Figure 1‐ Experimental protocols. Experimental protocol of Maternal Separation (MS) (A). Experimental protocol of E. coli gavage (B). Experimental protocol of lysozyme gavage (C)
Supplementary Figure 1
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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Supplementary Figure 2‐ Representative, illustrative EMG recording of one mice per group: control, MS mice of MS mice receiving oral gavage of LZM (A) littermate and Sox9flox/flox‐vil‐cre (B) and vehicle or mice receiving E. coli by oral gavage (C).
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Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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Supplementary Figure 3‐ Number of crypts stained with lysozyme in small intestine on MS mice (n=6 per group)
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Supplementary Figure 4
Supplementary Figure 4‐ Immunostaining of ileum paraffin section with anti‐lysozyme‐(FITC) (Green) Wheat germ agglutinin‐(Texas red) (red) and DAPI (blue) (10 fold magnification) in control mice (A) and maternally separated mice (B) (n=5‐6 per group)
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[IL17
] (pg
/mg
of il
eal p
rote
in)
[IL22
] (pg
/mg
of il
eal p
rote
in)
[IL10
] (pg
/mg
of il
eal p
rote
in)
Supplementary Figure 5
Supplementary Figure 5‐ Ileal concentration of IL17, IL22 and IL10 measured by ELISA (n=12‐21 per group)
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Comment citer ce document :Riba, A., Olier, M., Lacroix Lamandé, S., Lencina, C., Bacquié, V., Harkat, C., Gillet, M.,
Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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Supplementary Figure 6‐ Relative abundances of fecal microbial communities identified as important contributors in addition to these of E. coli to differences between MS mice and control (n=9 to 10 mice per group). *: p<0.05 with control.
Supplementary Figure 6
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Comment citer ce document :Riba, A., Olier, M., Lacroix Lamandé, S., Lencina, C., Bacquié, V., Harkat, C., Gillet, M.,
Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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ACCEPTED
ACCEPTED MANUSCRIPT
Supplementary Figure 7 ‐ Relative abundances of fecal microbial communities identified as important contributors in addition to these of E. coli to differences between Sox9 flox/flox‐vil‐cre mice and littermate (n=9 to 10 mice per group). *: p<0.05 with littermate.
Supplementary Figure 7
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Comment citer ce document :Riba, A., Olier, M., Lacroix Lamandé, S., Lencina, C., Bacquié, V., Harkat, C., Gillet, M.,
Baron, M., Sommer, C., Mallet, V., Salvador Cartier, C., Laurent, F., Théodorou, V., Ménard, S.(2017). Paneth cell defects induce microbiota dysbiosis In mice and promote visceral
hypersensitivity. Gastroenterology, 153 (6), 1594-1606. DOI : 10.1053/j.gastro.2017.08.044
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ACCEPTED
ACCEPTED MANUSCRIPT
Supplementary Figure 8
Supplementary Figure 8 – Microbial genomic DNA extracted per gram feces (n=9 to 10 mice per group).