The small intestinal epithelial barrier inputs signals from the gut microbiota in order to balance physiological inflammation and tolerance, and to promote homeostasis. Understanding the dynamic relationship between microbes and intestinal epithelial cells has been a challenge given the cellular heterogeneity associated with the epithelium and the inherent difficulty of isolating and identifying individual cell types. Here, we used single-cell RNA sequencing of small intestinal epithelial cells from germ-free and specific pathogen-free mice to study microbe-epithelium crosstalk at the single-cell resolution. The presence of microbiota did not impact overall cellular composition of the epithelium, except for an increase in Paneth cell numbers. Contrary to expectations, pattern recognition receptors and their adaptors were not induced by the microbiota but showed concentrated expression in a small proportion of epithelial cell subsets. The presence of the microbiota induced the expression of host defense- and glycosylation-associated genes in distinct epithelial cell compartments. Moreover, the microbiota altered the metabolic gene expression profile of epithelial cells, consequently inducing mTOR signaling thereby suggesting microbe-derived metabolites directly activate and regulate mTOR signaling. Altogether, these findings present a resource of the homeostatic transcriptional and cellular impact of the microbiota on the small intestinal epithelium.

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.