Fig 2: MHC-II deficiency in intestinal epithelial cells mitigates CDI outcomes(A) Body weight and clinical score during CDI in WT or H2-Ab1−/− mice on different diets (n = 5).(B) C. difficile burden in feces 4 d.p.i. (n= 4).(C) Intestinal permeability assessed by FITC-dextran 4 d.p.i. (n= 4).(D) Number of CD11b+ Ly6G+ neutrophils in the colonic LP 4 d.p.i. (n = 4).(E) Experimental design of bone marrow (BM) chimera: CD45.2 WT or H2-Ab1−/− mice were reconstituted with CD45.1 WT BM (n = 5). Mice were maintained on SD.(F) Body weight and clinical score of WT or H2-Ab1−/− BM chimeric mice in CDI (n= 5).(G) C. difficile burden 4 d.p.i. (n = 5).(H) Intestinal permeability measured by FITC-dextran assay 4 d.p.i. (n = 5).(I) Body weight and clinical score of H2-Ab1fl/fl and H2-Ab1ΔIEC mice during CDI (n= 5). Mice were fed SD.(J) Fecal C. difficile burden 5 d.p.i. (n= 5).(K) Colon length in H2-Ab1fl/fl and H2-Ab1ΔIEC mice uninfected 5 d.p.i. (n = 4–5).(L) Bacterial translocation in liver, spleen, and mLN (n= 5).(M) Representative H&E colon sections and cumulative histopathological score 5 d.p.i. (n = 5). Scale bars, 100 μm.(N) Fecal lipocalin-2 levels 5 d.p.i. (n= 3).(O) Number of CD11b+ Ly6G+ neutrophils and CD11b+ Ly6C+ monocytes in colonic LP 5 d.p.i. (n= 4–9).(P) S100a8 mRNA levels in proximal colon 5 d.p.i. (n = 4–5).(Q and R) Frequency of CD3+ CD4+ T cells (Q) and CD4+ Foxp3+ Treg or CD4+ T-bet+ Th1 (R) cells in the epithelium of H2-Ab1fl/fl and H2-Ab1ΔIEC mice either uninfected or 5 d.p.i. (n= 4–9).(S) Quantification of CD3+ CD4+, CD4+ Foxp3+, and CD4+ T-bet+ T cells in colonic LP 5 d.p.i. (n = 4–9).(T) mRNA expression of cytokines in the proximal colon 5 d.p.i. (n = 4–5).Error bars, mean ± SEM. Statistical analysis: one-way ANOVA with Tukey’s post hoc test. *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001; ns, not significant.

Fig 3: FFAR2-deficiency exacerbates gut inflammation during CDI(A) Body weight and clinical score of Ffar2−/− mice on different fiber diets during CDI (n = 5).(B–E) Colon length (B), fecal lipocalin-2 levels (C), bacterial translocation to the liver, spleen, and mLN (D), and representative H&E colon sections with histopathological scores (E) 4 d.p.i. of Ffar2−/− mice on SD, LFiD, and HFiD. Scale bars, 100 μm.(F) H2-Ab1 and Ciita mRNA levels in colonic epithelial cells from Ffar2−/− mice 4 d.p.i., normalized to SD group (n= 4).(G) mRNA levels of Ifng and Il10 in the proximal colon of Ffar2−/− mice 4 d.p.i. (n = 4).(H) Body weight, clinical score, and colon length of Ffar2fl/fl and Ffar2ΔIEC mice maintained on SD during CDI (n = 5).(I–J) Representative H&E colon sections with histopathological scores (I), and bacterial translocation (J) 5 d.p.i. Scale bars, 100 μm.(K) MHC-II expression in CD45− EpCAM+ epithelial cells 5 d.p.i. (n = 4).(L) Frequency of colonic CD3+ CD4+ IELs 5 d.p.i. (n= 4).(M) Ifng and Il10 mRNA expression in the proximal colon 5 d.p.i. (n = 4).(N) Schematic of experimental design and representative immunofluorescence images of small intestine organoids for MHC-II and EpCAM expression, with or without IFN-γ and acetate (n= 3). Scale bars, 50 μm. DAPI stains nuclei.(O) FACS plot and MHC-II gMFI of CD45− EpCAM+ cells from organoid cultures stimulated or not with IFN-γ and acetate (n = 4).Error bars, mean ± SEM. Statistical analysis: t test or one-way ANOVA with Tukey’s post hoc test. *p< 0.05, **p < 0.01; ns, not significant.