Figures and data
Initiation of colitis in mice leads 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. 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 wild type C57BL/6 mice either left untreated or subjected to DSS treatment for two, four and six days respectively. (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 depicting colon length measured on day six post-onset of DSS treatment of mice of the indicated genotypes. Untreated wild type littermates of corresponding genotypes were used as controls. (C) Colon sections from untreated and DSS-treated mice of the indicated genotypes were examined for histological features involving H&E staining [left panel] and alcian blue staining [right 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. (D) Bar plot quantifying gut permeability of untreated and DSS-treated wild type and relaΔhepstat3Δhepmice. Briefly, the serum concentration of FITC was measured six hours after oral gavaging the mice with FITC-dextran. (E) RT-qPCR revealing the relative abundance of the indicated mRNAs encoding broadly enterocyte-specific (above panel) or goblet cell-specific (below panel) markers in untreated or DSS-treated mice of the indicated genotypes.
Charting hepatic gene expressions in colitogenic wild type and relaΔhepstat3Δhepmice.
(A) PCA plot illustrating the hepatic transcriptome, identified through global RNA-seq analyses, of untreated or DSS-treated wild type and relaΔhepstat3Δhepmice (n=3). DSS treatment was carried out for six days. (B) Bubble plot depicting the relative enrichment of GO biological terms for differentially expressed genes in wild type 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 as detected in an untargeted LC-MS based quantification of bile acids in the mucosal biopsy samples from IBD and Non-IBD patients. (D) Schematic presentation of classical and alternate pathways of bile synthesis in mice liver tissue. 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 wild type and relaΔhepstat3Δhep mice (n=3). Fold change is relative to their corresponding wild type littermates.
Altered accumulation of primary bile acids in relaΔhepstat3Δhepmice 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 wild type and relaΔhepstat3Δhep mice (n=5). (C) RT-qPCR analyses comparing the colonic abundance of indicated mRNAs encoding pro-inflammatory cytokines (n=3) for DSS-treated wild type and relaΔhepstat3Δhepmice. Fold change is relative to their corresponding wild type littermates. (D) Dot-plot representing the frequency of F4/80+, CD11c+ and Ly6G+ cells among total DAPI-stained cells in the colon sections derived from DSS-treated wild type and relaΔhepstat3Δhep mice. 26
Supplementing chenodeoxycholic acid (CDCA) restores the colitogenic sensitivity in relaΔhepstat3Δhepmice.
(A) Line plot charting the disease activity in a time course of wild type and relaΔhepstat3Δhep mice subjected to DSS treatment while being supplemented with 10 mg/kg CDCA daily. 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) H&E stained colon sections from DSS-treated relaΔhepstat3Δhepmice with and without CDCA supplementation. Data were obtained in 10X magnification, this is a representative of three experimental replicates and a total of four sections from each set were examined. RT-qPCR analyses comparing the colonic abundance of indicated mRNAs encoding (D) IEC-specific markers and (E) pro-inflammatory cytokines in mice subjected to DSS treatment for six days in the absence or presence of CDCA supplementation (n=4). Untreated relaΔhepstat3Δhep mice were used as controls.
