The Microbial Metabolite Sensor Pregnane X Receptor (PXR) Restrains Fibroblasts from Promoting Gastrointestinal Inflammation and Fibrosis in Mice

INTRODUCTION Fibrosis contributes to intestinal strictures and obstruction in 30–50% of Crohn's disease patients. The pathogenesis of intestinal fibrosis is incompletely understood and has virtually no effective treatments. The pregnane X receptor (PXR), a xenobiotic receptor involved in detoxification responses, has recently been identified as a modulator of fibrosis in hepatic stellate cells. Of the number of foreign ligands for the PXR, the tryptophan metabolite indole‐3‐propionic acid (IPA) produced by the intestinal commensal Clostridium sporogenes can bind to the PXR to mediate signalling events that protect intestinal barrier function. The role of the PXR in intestinal fibrosis and if microbial metabolite sensing can affect intestinal fibrotic responses is unknown. AIMS To examine the role of the PXR and its ability to sense microbial metabolites in the modulation of intestinal fibrosis. METHODS Intestinal inflammation was induced using DSS (3.5%) for 5 days followed by healing for 25 days. Fibrosis was assessed using Masson‐trichrome and Serius Red staining of histological sections. Mouse primary fibroblasts were grown from wild type (WT) and PXR −/minus; mice. Fibroblasts were stimulated with cytomix (TNF‐α, IL‐1β, and IFN‐γ) for 24 hours after which cell supernatants were assessed for cytokine concentrations using Luminex. Fibroblasts were also stimulated with cytomix in the presence of the PXR agonists pregnenolone 16α‐carbonitrile (PCN) or IPA and assessed for gene expression using qPCR. NFκB activity was assessed via Western blot for the p65 subunit. To examine the microbiota's role in fibrosis, the microbiota was depleted after the 5‐day course of DSS for 25 days using an antibiotic cocktail of vancomycin, neomycin, metronidazole and ampicillin. FINDINGS Following a 25‐day recovery after DSS, WT mice showed clear evidence of fibrosis as indicated by Masson‐trichrome and Serius Red staining. When compared to WT mice, PXR −/− mice demonstrated significantly greater levels of fibrosis. Mouse primary fibroblasts were found to express the PXR, and PXR −/− fibroblasts displayed morphological differences compared to WT fibroblasts. Following stimulation with cytomix, PXR −/− fibroblasts produced dramatically higher levels of proinflammatory cytokines including eotaxin, CXCL2, GM‐CSF, G‐CSF and IL‐9. Importantly, PCN and the microbial metabolite IPA were able to block the expression of these cytokines. These effects in PXR − / − fibroblasts may be linked to the increased activity of NFκB that was observed both basally and after stimulation with cytomix compared to WT fibroblasts. Depletion of the microbiota led to exacerbated intestinal fibrosis. CONCLUSIONS PXR signaling in intestinal fibroblasts appears to be required to restrain inflammation and fibrosis. Luminal sensing of bacterial derived indoles (ie. IPA) via PXR may be involved in this process, highlighting a xenobiotic receptor‐microbiota axis that could be targeted to prevent the intestinal fibrosis observed in Crohn's disease. Support or Funding Information Crohn's & Colitis Canada, Dr. Keith Sharkey's CCFC Chair in IBD Research, The Dr. Lloyd Sutherland Investigator in IBD/GI Research, Canadian Foundation for Innovation, Canada Research Chairs Program, US Department of Defense, Beverley Philips Rising Star Fellowship.

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