Mechanism of action of dietary fibre in the regulation of intestinal epithelial barrier function

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The intestinal epithelium provides an essential, multi-functional barrier that: (1) establishes the osmotic, chemical and electrical potential gradients that facilitate absorption of nutrients, water and electrolytes; (2) secretes mucus, antimicrobial peptides and immunoglobulins that are important for host defence; and (3) prevents excessive mucosal penetration of bacterial and luminal antigens and, consequently, prevents over-activation of the mucosal immune system. This Discovery Grant proposal describes a new research program that will determine the mechanisms underlying how plant- and fungus-based polysaccharides (PS) act as important physiological regulators of the intestinal epithelial barrier. Our preliminary data show that the plant-derived PS, rhamnogalacturonan (RGal), directly alters epithelial behaviour (cell migration, barrier function) in the Caco-2 epithelial cell line, although the exact mechanism of action has not been elucidated. ******Our hypothesis is that PS enhance intestinal epithelial barrier function either directly or through the gut microbiota, to elicit specific signaling responses that regulate tight junction structure and function to maintain epithelial homeostasis. Three aims will test this hypothesis.***Aim 1. Determine the structural and functional changes in intestinal epithelium promoted by PS. Effects of PS on epithelial function (migration, proliferation, death) will be determined with a high-throughput screening system we developed on the IncuCyte platform. Dynamic effects on tight junction structure will be assessed using fluorescence recovery after photobleaching (FRAP) to track PS-induced changes in tight junction protein mobility. ***Aim 2. Analyze the genes and signalling pathways activated by PS. Using RNAseq, we will identify PS-induced changes in the transcription of genes with known roles in regulating epithelial barrier function. We will then target identified pathways with siRNA, CRISPR-Cas9 or pharmacological inhibitors to determine their functional role in epithelial barrier function. ***Aim 3. Determine the role of the gut microbiota in PS-induced effects on the epithelial barrier. Through the new Western Canadian Microbiome Centre at University of Calgary, we will have access to germ-free mice that will help us to understand the epithelial effects of PS in the absence of gut microbiota. If a dependence on bacterial metabolism is observed in the barrier-specific effects of PS, we will subsequently investigate the PS metabolites that have relevant biological activity and determine their cellular mechanism of action. ******Our program will characterize the largely unexplored mechanisms of interactions among PS, the gut microbiome and the epithelium. This information will contribute to food science by mechanistically highlighting the benefits of dietary fibre on epithelial function and overall intestinal homeostasis.