Microbiota regulate the induction and proliferation of revival intestinal stem cells through inflammatory cytokines

Abstract Gut microbiota are critical mediators of inflammation and regeneration of the intestinal epithelium. Intestinal restitution is a coordinated response that involves the dedifferentiation of mature epithelial cell lineages, the proliferation of Lgr5+ intestinal stem cells, and the induction of Clu+ revival stem cells (RSCs). How the gut microbiota directly impacts this regenerative process remains unclear. Using irradiation as a model for small intestinal epithelium restitution, we demonstrate that microbiota regulate the induction and subsequent proliferation of Clu+ RSCs. Our results report that specific pathogen-free (SPF) mice induce greater RSCs 3 days post-IR in comparison to germ-free (GF) mice. This microbiota-dependent increase in RSCs was matched by an increase in BrdU+ proliferating cells and an increase in TUNEL+ apoptotic cells in SPF mice. Using intestinal organoids as an in vitro model of intestinal restitution, SPF and GF intestinal crypts demonstrated equal propensity to generate mature organoids. Transcriptional analysis of GF and SPF RSCs by single-cell RNA sequencing highlighted unique regenerative and inflammatory gene signatures. The altered regeneration kinetics observed in SPF mice was accompanied by an increase in Tnfa and Cxcl1 at the peak of RSC induction and an increase in Ifnγ post RSC induction. Altogether, these findings suggest that microbiota-dependent expression of inflammatory cytokines may be key regulators in facilitating the expansion and proliferation of RSCs to efficiently repair the intestinal epithelium following damage.