Exploring Connections between Commensal Bacteria, Metabolites and Small Intestine Stromal Cells using ‘Omics Approaches

Abstract

The intestine is colonized by a complex community of bacteria, termed the intestinal microbiota, that have considerable effects on intestinal immunity. One mechanism by which the microbiota influences the host is by altering the complement of metabolites that are available to the host. Stromal cells, inclusive of lymphatic endothelial cells (LEC) and mesenchymal stromal cells (MSC), support immune responses by providing structural support and direct signals to immune cells. Despite established microbiota-immune and stromal-immune interactions, how the microbiota influences intestinal MSC (iMSC) and LEC is not well defined. This thesis was driven by the hypothesis that bacteria-induced metabolites influence the development and regulation of intestinal stromal cells. To assess this, we profiled metabolites in the intestinal tract, serum, peritoneal fluid, liver, spleen and urine of germ-free (GF), gnotobiotic and specific-pathogen-free (SPF) mice. We analyzed metabolites in weanling and adult mice and male and female mice. This revealed the relative impact of microbiota, age and sex on metabolism and highlighted cases where microbial-induced metabolites were dependent on age and sex. We found that colonization impacts the structure and function of intestinal lymphatics by performing in vivo lipid transport assays and whole mount imaging of small intestine tissues. By performing single cell RNA and ATAC sequencing, we uncover 4 main subclusters of iMSC: FB1, FB2, telocytes and myocytes. We found that all subclusters were present in GF and SPF mice, however there was an expansion of telocytes and contraction of FB2 in female SPF mice compared to GF. We uncover subset-specific regulation of genes relating to epithelial, immune and extracellular matrix deposition and found that myocytes were particularly responsive to the microbiota. In response to microbial colonization, myocytes upregulate interleukin-33 (IL33), potentially through epigenetic modification. Finally, ablation of IL33 in iMSC decreases tissue Il33 levels in the jejunum and increase inflammatory tone, suggesting that iMSC-derived IL33 is a regulator of tissue homeostasis. Collectively, this thesis contributes to our understanding of how microbes regulate metabolism and how the microbiota affects the structure and function of stromal cells in the intestine. This information could be leveraged to develop stromal-based therapies to maintain intestinal health.