A Pilot Integrative Analysis of Colonic Gene Expression, Gut Microbiota, and Immune Infiltration in Primary Sclerosing Cholangitis-Inflammatory Bowel Disease: Association of Disease With Bile Acid Pathways

Mohammed Nabil Quraishi, Animesh Acharjee, Andrew D Beggs, Richard Horniblow, Chris Tselepis, Georgios Gkoutos, Subrata Ghosh, A E Rossiter, Nicholas Loman, Willem van Schaik, David Withers, Julian R F Walters, Gideon M Hirschfield, Tariq H Iqbal, Mohammed Nabil Quraishi, Animesh Acharjee, Andrew D Beggs, Richard Horniblow, Chris Tselepis, Georgios Gkoutos, Subrata Ghosh, A E Rossiter, Nicholas Loman, Willem van Schaik, David Withers, Julian R F Walters, Gideon M Hirschfield, Tariq H Iqbal

Abstract

Background: Although a majority of patients with PSC have colitis [PSC-IBD; primary sclerosing cholangitis-inflammatory bowel disease], this is phenotypically different from ulcerative colitis [UC]. We sought to define further the pathophysiological differences between PSC-IBD and UC, by applying a comparative and integrative approach to colonic gene expression, gut microbiota and immune infiltration data.

Methods: Colonic biopsies were collected from patients with PSC-IBD [n = 10], UC [n = 10], and healthy controls [HC; n = 10]. Shotgun RNA-sequencing for differentially expressed colonic mucosal genes [DEGs], 16S rRNA analysis for microbial profiling, and immunophenotyping were performed followed by multi-omic integration.

Results: The colonic transcriptome differed significantly between groups [p = 0.01]. Colonic transcriptomes from HC were different from both UC [1343 DEGs] and PSC-IBD [4312 DEGs]. Of these genes, only 939 had shared differential gene expression in both UC and PSC-IBD compared with HC. Imputed pathways were predominantly associated with upregulation of immune response and microbial defense in both disease cohorts compared with HC. There were 1692 DEGs between PSC-IBD and UC. Bile acid signalling pathways were upregulated in PSC-IBD compared with UC [p = 0.02]. Microbiota profiles were different between the three groups [p = 0.01]; with inferred function in PSC-IBD also being consistent with dysregulation of bile acid metabolism. Th17 cells and IL17-producing CD4 cells were increased in both PSC-IBD and UC when compared with HC [p < 0.05]. Multi-omic integration revealed networks involved in bile acid homeostasis and cancer regulation in PSC-IBD.

Conclusions: Colonic transcriptomic and microbiota analysis in PSC-IBD point toward dysregulation of colonic bile acid homeostasis compared with UC. This highlights important mechanisms and suggests the possibility of novel approaches in treating PSC-IBD.

Keywords: Autoimmune liver disease; bioinformatics; colitis; dysbiosis.

© European Crohn’s and Colitis Organisation 2020.

Figures

Figure 1.
Figure 1.
Principal component analysis [PCA] score plot performed on the mucosal transcriptome datasets demonstrating clustering of subjects within, and variation between, cohorts. Dots represents samples and are coloured according to the subject cohort. Ellipse represents 95% confidence. Results are plotted according to the PC1 and PC2 scores, with the percent variation explained by the respective axis. [a] PSC-IBD versus HC; [b] UC versus HC; [c] PSC-UC versus UC; [d] PCA plots demonstrating variation between HC, UC, and PSC-UC samples. The three groups were significantly different from each other [p = 0.01]. PSC, primary sclerosing cholangitis; IBD, inflammatory bowel disease; UC, ulcerative colitis; HC, healthy controls.
Figure 2.
Figure 2.
Differential gene expression profiles and volcano plots highlighting differentially expressed genes. [a] PSC-IBD versus HC; [b] UC versus HC; [c] PSC-IBD versus UC. Differentially expressed genes with logFC >1 are shown; 939 genes had shared differential expression in PSC-IBD and UC when compared with healthy controls [588 upregulated; 351 downregulated]. PSC, primary sclerosing cholangitis; IBD, inflammatory bowel disease; UC, ulcerative colitis; HC, healthy controls.
Figure 3.
Figure 3.
Bioinformatic analysis identifies pathways including bile acid signalling as relevant to disease distinctions. [a] Detailed enrichment analysis of gene clusters in functionally grouped network. Gene networks and pathways involving key nuclear receptors involved in bile acid homeostasis such as FXR, PPAR, conjugation, binding, and transport are significantly upregulated in PSC-IBD compared with controls; [b] top 20 gene ontology biological processes PSC-IBD vs UC demonstrate metabolic pathways, many of which are involved in bile acid homeostasis are upregulated in PSC-IBD compared with UC, whereas immune activation and defense pathways are upregulated in UC compared with PSC-IBD. PSC, primary sclerosing cholangitis; IBD, inflammatory bowel disease; UC, ulcerative colitis; HC.
Figure 4.
Figure 4.
Distinct microbiota taxa in patients with PSC-IBD. [a] Phylum level differences in the three cohorts—UC and PSC-IBD are characterised by a relative expansion of Proteobacteria and Fusobacteria and reduction of Bacteroidetes compared with HC; [b] microbial taxa comparing PSC-IBD and UC. Association of specific microbiota taxa with PSC-UC and UC by linear discriminant analysis [LDA] effect size [LEfSe]. Red indicates taxa enriched in PSC-IBD and green indicates taxa enriched in UC; [c] Cladogram representation of the gut microbial taxa associated with PSC-IBD and UC. PSC-IBD is associated with an increased abundance of bacteria expressing bile salt hydrolase and hydroxysteroid dehydrogenases [such as Bacteroides fragilis, Roseburia spp, Shewanella spp.,and Clostridium ramosum] in comparison with UC. PSC, primary sclerosing cholangitis; IBD, inflammatory bowel disease; UC, ulcerative colitis.
Figure 5.
Figure 5.
Functional classification of the predicted metagenome content of the microbiota of PSC-IBD compared with UC. [a] MetaCyc pathways; and [b] KEGG pathways. There is significant enrichment of primary bile acid biosynthesis [associated with significantly higher expression of bile salt hydrolase and hydroxysteroid dehydrogenases], pentose and glucoronate interconversions, and galactose metabolism in PSC-IBD compared with UC. PSC, primary sclerosing cholangitis; IBD, inflammatory bowel disease; UC, ulcerative colitis.
Figure 6.
Figure 6.
Correlation networks between the mucosal transcriptome, 16S microbial profiles, and immunophenotype in patients with PSC-IBD. The mucosal transcriptome is in red, 16S microbial profiles purple. Genes were selected based on supervised and unsupervised dimension reduction as described in Methods. Microbial taxa were selected based on the loading values more than log2[3] values. Th17- and IL17-expressing cells were selected as they were significantly upregulated in PSC-IBD and UC compared with HC. Red line indicates negative correlation and green positive correlation. Thickness of the line shows strength of the correlation between each features. Clusters are defined based on a high inter-feature correlation [r = 0.8]. Two main clusters were identified—cluster A consists of genes involved in bile acid homeostasis; cluster B demonstrates genes associated with cancer regulatory pathways. PSC, primary sclerosing cholangitis; IBD, inflammatory bowel disease; UC, ulcerative colitis; HC, healthy controls.

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