DOI: 10.6084/m9.figshare.26579468.v1
title: Additional file 1 of Diet prevents the expansion of segmented filamentous bacteria and ileo-colonic inflammation in a model of Crohn’s disease

description: Additional file 1: Supplementary Figure S1. SFB abundance correlates with ileitis severity in SPF-house Tnf ΔARE (A) SPF-housed 8-week-old ARE and WT mice were cohoused with SFB-monoassociated NOD-SCID mice for 10 weeks (B) Litter and cage effect on ileitis development in SPF-housed Tnf ΔARE mice. Squares represent males; circles indicate females. Green, orange, and red symbols indicate Tnf ΔARE mice at 18-week endpoint with no (score 0), low (score &lt;4) and high (score &gt;4) ileitis histopathological score, respectively; grey symbols indicate WT littermates that do not develop ileitis; white symbols indicate male mice of unknown ileitis status. Each cage is delineated with the brackets below and a cage number. (C) Quantitative Analysis of SFB in recolonized Tnf ΔARE mice (F0, F1, F2). Color-code represents the severity of inflammation as described above. CT value &gt;30 is regarded as non-specificity threshold. (D) Ileitis scores of recolonized 18-weeks-old Tnf ΔARE mice including 2 breeding generations (F0, F1, F2). Mice are color-coded based on inflammation severity with green (score 0); orange (score &lt;4) (orange); and red (score &gt;4). (E) Cladogram obtained from Linear discriminant analysis effect size (LEfSe) analysis of taxonomic profiling using 16S rRNA gene sequencing of intestinal microbiota in WT and Tnf ΔARE mice. (F) Comparison of relative abundance of bacterial genera between WT and Tnf ΔARE mice using LEfSe analysis. Taxa meeting an LDA significant threshold 2 are shown, taxa enriched in Tnf ΔARE mice (red) and taxa enriched in WT mice (blue). (G) Representative H&E-stained tissue sections from distal ileum, caecum, and proximal colon of Tnf ΔARE mice colonized with single bacterial strains (Alistipes, Lactobacillus murinus and E. coli LF82) or with MIBAC, a minimal consortium of 7 bacterial strains showing no signs of inflammation. Supplementary Figure S2. Enhanced numbers of IL-17- and IFNg-expressing CD4-positive cells as well as neutrophilic granulocytes in inflamed mucosal tissue of Tnf ΔARE mice. Immune cells were isolated from spleen, MLN, and intestinal mucosa of jejunum, ileum, cecum, and colon of 12 weeks old Tnf ΔARE mice (n = 3) and WT controls (n = 4). (A) Quantitative analysis of Ifng transcript levels in mucosal tissue of distal ileum, cecum, and proximal colon from 4 and 12 weeks-old Tnf ΔARE mice and 12 weeks-old WT controls. Statistical significance was assessed by xy. *p&lt;0.05; **p&lt;0.01; ***p&lt;0.001 was considered statistically significant. (B) Gating strategy used for the assessment of IL-17- and IFNg-expressing CD3+CD4+ immune cells. Discrimination of live and dead cell population was performed by applying specific fluorescent dye. Percentage of IL-17- (C) and IFNg- (D) expressing cells within the CD3+CD4+ population. (E) Gating strategy used for the assessment of neutrophilic granulocytes. Discrimination of live and dead cell population was performed by applying specific fluorescent dye. Population of neutrophils was identified by applying antibodies against CD45, CD11b, MHCII, Ly6C, and Ly6G. (F) Percentage of neutrophilic granulocytes was assessed within the CD45+CD11b+ population by gating for MHCII-Ly6CintLy6Ghigh cells. (G) Immunofluorescence staining of tissue sections from distal ileum of WT and Tnf ΔARE mice applying Ly6G and E-Cadherin. DAPI was used for nuclear visualization. (H) PAS-AB goblet cell staining in ileal tissue sections from 4 weeks and 12 weeks mono-colonized WT and Tnf ΔARE mice (400x). Lower panel represents higher magnification (1200x) of the indicated sections. Graph represents the quantification of goblet cells (x1000) in 1μm2 selected crypt area. Statistical analyses were performed by One-way analysis of variance (ANOVA) followed by Tukey test. *p&lt;0.05; **p&lt;0.01; ***p&lt;0.001 was considered statistically significant. Extended Supplementary Figure S2. B. adolescentis monoassociation does not trigger inflammation in Tnf ΔARE mice. (A) Quantitative Analysis of B.adolescentis in recolonized Tnf ΔARE mice and WT control mice monoassociated with B.adolescentis for 4 weeks (B) Representative H&E-stained tissue sections from distal ileum, caecum, and colon of 12-week old ARE and WT control mice monoassociated with B.adolescentis for 4 weeks. (C) Tissue pathology scores of distal ileum (dI), caecum (CC), proximal colon (pC) and distal colon (dC) of Tnf ΔARE mice monoassociated with B. adolescentis for 4 weeks. (D) Quantitative analysis of Tnf, Il-17 and Ifng transcript levels in mucosal tissue of distal ileum from 12 weeks-old Tnf ΔARE mice mice monoassociated with B. adolescentis for 4 weeks. (E) Immunofluorescence (IF) co-staining of Lysozyme (red) and E-cadherin (IEC borders, blue) counterstained with Dapi (nuclei, cyan) in ileal tissue sections from 12 weeks-old Tnf ΔARE mice mice and their WT littermate controls monoassociated with B. adolescentis for 4 weeks (600x) lower panel: higher magnification of the indicated sections (3600x) and quantification of the total number of Lysozyme positive Paneth cells per crypt based on IF staining. Statistical analyses were performed by One-way analysis of variance (ANOVA) followed by Tukey test. Supplementary Figure S3. SFB initiate inflammation in humanized Tnf ΔARE mice. (A) Tnf ΔARE mice and WT controls were colonized either with fecal microbiota from CD patient only or in combination with SFB. Both groups were colonized at 8 weeks of age and assessed for tissue pathology at 12 weeks of age. (B) Severity of inflammation was assessed by evaluating the tissue pathology score in tissue sections of distal ileum, cecum and proximal colon of humanized or SFB + humanized Tnf ΔARE mice. (C) Quantitative analysis of SFB abundance in intestinal contents collected from ileal, colonic and cecal compartments of humanized or SFB + humanized Tnf ΔARE mice. (D) H&E-stained tissue sections of Tnf ΔARE mice revealed enhanced intestinal inflammation in the distal ileum and to a lower degree in cecum tissue of humanized Tnf ΔARE mice co-colonized with SFB. (E, F) Immunofluorescence (IF) co-staining of Lysozyme (red) and E-cadherin (IEC borders, blue) counterstained with Dapi (nuclei, cyan) in ileal tissue sections from Tnf ΔARE and WT mice colonized with human CD microbiota alone (hARE, hWT) or in co-colonization with SFB (hARE+SFB, hWT+SFB) (600x) lower panel: higher magnification of the indicated sections (3600x). Graph represents quantification of the total number of Lysozyme positive Paneth cells per crypt based on IF staining. Statistical analyses were performed by One-way analysis of variance (ANOVA) followed by Tukey test. (G) Beta-diversity analysis of bacterial community profiles in Tnf ΔARE and WT mice colonized with human CD microbiota alone (hARE, hWT) or in co-colonization with SFB (hARE+SFB, hWT+SFB) (H) Significantly increased percentage relative abundance of Alistipes and Blautia in humanized Tnf ΔARE and WT mice co-colonized with SFB (hARE+SFB, hWT+SFB). Extended Supplementary Figure S3. (A, B) Melting curves and CT values (C, D) Alignment to control human SFB sequence yielding 99% similarity. Supplementary Figure S4. (A) Correlation analysis between ileitis score and SFB abundance in cecal contents of chow-fed or PD-fed Specific pathogen-free (SPF) Tnf ΔARE mice and. (B) MDS analysis show separation of caecal bacterial communities according to diet among chow-fed WT, chow-fed or PD-fed Tnf ΔARE mice (C) Comparison of relative abundance of bacterial genera between SPF-housed Tnf ΔARE mice fed with chow or PD under SPF conditions, using LEfSe analysis. Taxa meeting an LDA significant threshold 2 are shown (D) Histopathology section of ileal and colonic tissue in ileal and colonic tissue of ex-GF Tnf ΔARE mice monocolonized with SFB and fed with chow or PD (E) Quantitative analysis of Tnf and Il-17A transcript levels in mucosal tissue of distal ileum from ex-GF Tnf ΔARE mice monocolonized with SFB and fed with chow or PD. PD: purified diet.