Abstract
BACKGROUND & AIMS: Anti-tumor necrosis factor (anti-TNF) is a mainstay of inflammatory bowel disease (IBD) therapy but fails in many patients. Although TNF has pro-inflammatory effects, depletion of TNF receptor 1 (TNFR1) paradoxically exacerbates chronic colitis. Because colitis induces remodeling of mesenchymal cell populations, which provide a niche for epithelial stem cells involved in mucosal healing, we hypothesized that TNFR1 promotes colonic mesenchymal cell diversity and stem cell niche function. METHODS: Mesenchymal TNFR1 function was studied using TNFR1 (-/-) , platelet derived growth factor receptor alpha (PDGFRα)-Cre;TNFR1 (fl/fl) mice, and mixed-genotype mesenchymal-epithelial co-cultures. Mesenchymal cell diversity and gene function were assessed using single-cell RNA-Seq of primary colonic myofibroblasts (CMFs) and via anti-integrin A6 (ITGA6) antibody treatment and exogenous R-spondin 3 (RSPO3) supplementation. RESULTS: TNFR1 (-/-) mesenchyme exhibits reduced cell diversity, with specific depletion of specialized TNF- and interferon-signaling pericryptal cell-type. Deletion of TNFR1 in the pericryptal mesenchyme diminished the (PDGFRα)+ CMF population and reduced RSPO3 expression, but increased ITGA6 expression relative to controls (TNFR1 (+/-) ). Moreover, inhibition of ITGA6 reversed the proliferative and migratory phenotype of TNFR1 (-/-) CMFs and restored expression of PDGFRα and RSPO3. Co-cultures of colonoids with TNFR1 (-/-) CMFs resulted in downregulation of stem cell marker expression; this was rescued by supplementation with RSPO3. Supporting the role for mesenchymal TNFR1 in regulating colonic epithelial stem cells, mice deficient for TNFR1 in PDGFRα+ cells showed a 40% loss of Lgr5+ stem cells, consistent with the global TNFR1-deficient mouse. CONCLUSION: TNFR1-mediated signaling regulates specification and function of colonic mesenchyme, performing an integral role in the maintenance of the crypt stem cell population.