Initiation of colitis in mice lead to hepatic Rela and Stat3 activation

(A) GO pathway enrichment analysis was done for DEGs with adjusted p-value < 0.05 on days 6 post DSS treatment. Bubble plot depicts the enrichment of pathways on day 6 for different genotypes, where the coordinate on x-axis represents gene ratio, size of bubble represents the gene count and colour represents the p-value. (B) Heatmap represents normalized transcript count of the Rela and Stat3 pathway obtained from the RNA-seq experiment of three biological replicates. Scale is normalized across each row and colour from blue to red represents minimum and the maximum values respectively. (C) Representative confocal microscopy images show Rela and Stat3 activation in untreated and 6 days DSS-treated liver tissue of C57BL/6 mice. Blue, orange, red and green color represents represents the nuclei, p-Rela (Ser536), p-Stat3 (Ser727) and p-Stat3 (Tyr705). Images were taken at 40X. Scale is 20 μm. (D) Western blot revealing the abundance of total Rela and total Stat3, and their phosphorylated functionally active forms, in the liver extracts prepared from wildtype C57BL/6 mice either left untreated or subjected to DSS treatment for two, four and six days. (E) The signal intensity of bands corresponding to the indicated phosphorylated proteins was quantified from western blots, normalized against beta-actin, and presented as a bar plot. The data represent the means of three biological replicates ± SEM.

rela and stat3 deficiency in hepatocytes ameliorates DSS-induced acute colitis in mice

(A) Line plot charting disease activity index of wild type, relaΔhep, stat3Δhepand relaΔhepstat3Δhep littermate mice subjected to treatment with 2% DSS for six days. (B) Bar plot revealing colon length measured on day six post-onset of DSS treatment of mice of the indicated genotypes. Untreated mice were used as controls. (C) Bar plot quantifying gut permeability. Briefly, untreated and DSS-treated wildtype and relaΔhepstat3Δhepmice were orally gavaged with FITC-dextran and serum concentration of FITC was measured in another six hours. (D) Colon sections from untreated and DSS-treated mice of the indicated genotypes were examined for histological features involving H&E staining [upper panel] and alcian blue staining [lower panel]. Data were obtained in 10X magnification and represent three experimental replicates; two fields per section and a total of three sections from each set were examined. (E) RT-qPCR revealing the relative abundance of the indicated mRNAs encoding broadly IEC-specific or goblet cell-specific markers in untreated or DSS-treated mice of the indicated genotypes.

Charting hepatic gene expressions in colitogenic wildype and relaΔhep stat3 Δhep mice

(A) PCA plot illustrating the hepatic transcriptome, identified through global RNA-seq analyses, of untreated or DSS treated wildtype and relaΔhepstat3Δhep mice (n=3). DSS treatment was carried out for six days. (B) Bubble plot depicting the relative enrichment of GO biological terms among genes differentially expressed in wildtype or relaΔhepstat3Δhep mice. The gene ratio for a given term and the adjusted p value associated with the enrichment score has been presented for the individual genetic backgrounds. (C) Dot plot of dinor-cholic acid and dinor-chenodeoxycholic acid detected in an untargeted LC-MS based quantification of bile acid in the mucosal biopsy samples from IBD and Non-IBD patients. (D) Schematic presentation of classical and alternate pathways of bile synthesis in mice. CA, CDCA, MCA and UDCA represent cholic, chenodeoxycholic, muricholic and ursodeoxycholic acids, respectively. (E) RT-qPCR analyses comparing the hepatic abundance of indicated mRNAs encoding enzymes involved in bile metabolism in DSS-treated wildtype and relaΔhepstat3Δhepmice (n=3).

Altered accumulation of primary bile acids in relaΔhep Stat3 Δhep mice accompanies a less severe inflammatory signature in the colitogenic gut

Targeted LC-MS based quantification of primary bile acid in the liver (A) and the colon (B) of DSS-treated wildtype and relaΔhepstat3Δhep mice (n=5). (C) RT-qPCR analyses comparing DSS-treated wildtype and relaΔhepstat3Δhepmice for the colonic abundance of indicated mRNAs encoding pro-inflammatory cytokines (n=3). (D) Dot-plot representing the frequency of F4/80+, Ly-6G+ and CD11c+ cells among total DAPI-stained cells in the colon sections derived from DSS treated wildtype and relaΔhepstat3Δhep mice. 23

Supplementing Chenodeoxycholic acid restores the colitogenic sensitivity in relaΔhep stat3Δhep mice

(A) Line plot charting the disease activity in a time course of wildtype and relaΔhepstat3Δhep mice subjected to DSS treatment while being daily supplemented with 10 mg/kg CDCA. Mice devoid of CDCA supplementation were treated with DMSO as controls. (B) Bar plot comparing the colon length of relaΔhepstat3Δhep mice subjected to DSS treatment for six days in the absence or presence of CDCA supplementation. (C) Similarly, colon sections from DSS-treated relaΔhepstat3Δhep mice either not supplemented or supplemented with CDCA were examined by H&E staining. Data were obtained in 10X magnification and represent four experimental replicates. The data represent n = 3; a total of four sections from each set were examined. RT-qPCR analyses comparing the colonic abundance of indicated mRNAs encoding entrocytic markers (D) and pro-inflammatory cytokines (E) in mice subjected to DSS treatment for six days in the absence or presence of CDCA supplementation (n=4). Untread mice were used as controls.

A model depicting the immuno-metabolic network linking the inflammation induced hepatic signaling pathway to intestinal pathologies in mice

(a) Violin plot comparing the clinical parameter in serum of untreated and DSS treated wildtype mice (n=3) (b) Liver sections from untreated and DSS-treated wildtype mice were examined for histological features involving H&E staining [upper panel] and Sirius red staining [lower panel]. Data were obtained in 10X magnification and represent three experimental replicates; two fields per section and a total of three sections from each set were examined. (c) PCA plot illustrating the hepatic transcriptome, identified through global RNA-seq analyses, of untreated or DSS treated wildtype mice (n=3).

Characterization of mice strain by (a) genotyping and (b) western blotting. (c) Representative images of colon from untreated and DSS-treated wildtype and relaΔhep stat3Δhep mice

Heatmap represents relative transcript abundance of (a) biosynthesis pathway (b) regulators of bile acid metabolic pathways and (c) modification enzymes of bile acid metabolic pathway of indicated genotypes obtained from the RNA-seq experiment of three biological replicates.

Representative images of colon tissue stained with indicated immune cell marker, DAPI and merged section in the DSS-treated wildtype and relaΔhep stat3Δhep mice

(a) Line plot charting the disease activity in a time course of relaΔhepstat3Δhep mice subjected to daily supplementation of 10 mg/kg CDCA. Mice devoid of CDCA supplementation were treated with DMSO as controls. (b) Bar plot comparing the colon length of relaΔhepstat3Δhep mice subjected to CDCA supplementation. (c) Colon sections from relaΔhepstat3Δhep mice supplemented with CDCA were examined by H&E staining.

List of antibodies

List of mice strain

List of genotyping primers

List of primers

List of reagents

Control patient demography

UC patient demography