Deciphering a Toll-like receptor-mediated intestinal stem cells microbiota cross-talk

The gut is composed of a single layer of intestinal epithelial cells (IECs) lines between the external environment and the immune compartment. The epithelial monolayer physically impedes the penetration of microorganisms, but it can also interact with them to maintain homeostasis. Under homeostasis, the IEC secrete anti-microbial peptides (AMP) to limit the bacterial load mainly by Paneth cells. The mechanism for orchestrating AMP secretion from epithelial cells remains unclear. In our study, we aim to illuminate the AMP secretion mechanism by IEC and how it is regulated by Toll like receptor (TLR)-dependent sensing of the gut lumen. We employed TLR-deficient in-vivo models to follow AMP expression and secretion. Single-cell RNA-sequencing (scRNA seq) was used to analyze epithelial populations gene expression profiles. Our methodology also involved microbiome manipulations to study the interactions of the epithelium with the environment. Additionally, we used a reductional approach of two-dimensional and three-dimensional in-vitro systems to control the cellular environment and dissect the contribution of the different compartments. We observed that specific intestinal stem cells (ISC) TLR depletion led to changes in Paneth cell differentiation and a marked reduction in AMP profiles in the two AMP-enriched epithelial cells, enterocytes and Paneth cells. This reduction was also accompanied by decreased gene expression related to innate immunity and cellular response to microbial stimuli in the epithelium. In-vitro experiments revealed a polarized nature of TLR response in ISC. Gram-specific microbiome manipulation further highlighted its impact on epithelial cells innate responses and AMP expression. Our results demonstrate how the epithelial cells of the gut can sense the microbes via TLR-mediated responses to elevate AMP secretion in IEC. These findings suggest a symbiotic relationship of the ISC with microbiota mediated by TLRs, with potential implications for gut pathologies such as bacterial infections and Inflammatory bowel diseases (IBD).