Extended Data Fig. 8: IEC-specific IL6ST ablation exacerbates ethanol-induced liver disease, inhibits SI GAP formation, and alters the LP-immune landscape.

(a–u) WT and gp130^ΔIEC littermates were fed either control (n = 4–13) or ethanol-containing (n = 4–49) Lieber DeCarli diets for 10 weeks. A group of gp130^ΔIEC mice were treated with the mAChR4 PAM VU0467154 (5 mg kg⁻¹) dissolved in the diet during the last 29 days (n = 4–19); 65 independent experiments. (a) Plasma ALT. Note that WT samples were also used as controls in Fig. 5c, all littermates. (b) Representative H&E-stained liver sections. Scale bar=200 μm. (c) Quantification of ORO staining. WT samples were also used as controls in Fig. 5e. (d) Representative ORO-stained liver sections. Scale bar = 100 μm. (e–g) Liver Ccl2, Cxcl5, and Col1a1 mRNA amounts. (h) Survival rate curves. Statistical analysis was performed using the Gehan-Breslow-Wilcoxon test (Chi-square=4.33, P = 0.0374). (i) Plasma ethanol. WT samples were also used in Extended Data Fig. 9p. (j) Liquid diet intake. WT samples were also used as controls in Extended Data Fig. 9o. (k) Duodenal GAPs per villus. WT samples were also used as controls in Fig. 5a. (l) Representative images of TMR-dextran (red) showing GAPs (white arrowheads), Muc2 (green), and (DAPI) (blue) stained SI sections. Scale bar=25 μm. (m–n) Chrm4 mRNA amounts in isolated GCs (m) and duodenal samples (n). (o) Percentage of Muc2-stained area in the PSI. (p) Representative sections stained with Muc2 (red) showing GCs, and DAPI (blue) stained nuclei. 5 μm sections. Scale bar=20 μm. (q) Number of CFUs of anaerobically cultured bacteria from sterile collected MLN (100 mg) and liver (200 mg). WT samples were also used as WT controls in Fig. 5m (middle and right panels). (r–u) Isolated LP cells were stimulated with PMA (10 ng ml⁻¹) plus ionomycin (500 ng ml⁻¹) for 4 h. (r) Frequencies of CD4⁺/CD25⁺/FOXP3⁺ Tregs within total lymphocytes and total numbers. Gating strategy (Supplementary Fig. 8a). (s) Duodenal Il10 mRNA. (t) Frequencies of CD8⁺ Tregs (CD45⁺/CD8⁺/CD25⁺/FOXP3⁺) within total lymphocytes and total numbers. Gating strategy (Supplementary Fig. 8b). (u) Total IL-10 expressing CD8⁺ Treg (CD45⁺/CD8⁺/CD25⁺/FOXP3⁺/IL-10⁺) numbers. Diagonal ticks indicate a break in the y-axis scale (a, e–g, q–s). P values were determined using two-way ANOVA with Tukey’s test (j, q–left). or the BKY FDR (c, q–right), one-way ANOVA with Tukey’s test (i) or the BKY FDR (a, k, o), Kruskal-Wallis with the BKY FDR test (r), or either the two-sided unpaired Student’s t-test (s, t–left panel, u), unpaired Welch’s test (n), or the Mann-Whitney U test (e–g, m, t–right panel). Survival curves were compared using the Gehan–Breslow–Wilcoxon test (h). Results are presented as mean ± s.e.m. P (or q) *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001. Q when applying the BKY FDR method. The illustration in p was created using BioRender.