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

肠上皮提供了一种基本的多功能屏障：(1)建立渗透、化学和电势梯度，促进营养物质、水和电解质的吸收；(2)分泌对宿主防御重要的粘液、抗菌肽和免疫球蛋白；以及(3)防止细菌和管腔抗原过度渗透粘膜，从而防止粘膜免疫系统过度激活。这项发现拨款提案描述了一项新的研究计划，该计划将确定植物和真菌多糖(PS)如何作为肠道上皮屏障的重要生理调节因子的潜在机制。我们的初步数据显示，植物来源的PS，鼠李半乳糖(RGal)，直接改变Caco-2上皮细胞系的上皮行为(细胞迁移，屏障功能)，尽管确切的作用机制尚未阐明。我们的假设是，PS直接或通过肠道微生物区系增强肠上皮屏障功能，以诱导特定的信号反应，调节紧密连接的结构和功能，以维持上皮的动态平衡。三个目标将验证这一假说。目的1.确定PS促进的肠上皮结构和功能的变化。PS对上皮细胞功能(迁移、增殖、死亡)的影响将通过我们在IncuCyte平台上开发的高通量筛选系统来确定。将使用光漂白后荧光恢复(FRAP)来跟踪PS诱导的紧密连接蛋白质迁移率的变化，以评估对紧密连接结构的动态影响。目的2.分析PS激活的基因和信号通路。利用RNAseq，我们将识别PS诱导的基因转录的变化，这些基因在调节上皮屏障功能方面具有已知的作用。然后，我们将以siRNA、CRISPR-Cas9或药物抑制剂为靶点，以确定它们在上皮屏障功能中的功能作用。目的3.确定肠道微生物区系在PS诱导的上皮屏障效应中的作用。通过卡尔加里大学新的加拿大西部微生物中心，我们将获得无菌小鼠，这将帮助我们了解在没有肠道微生物区系的情况下PS对上皮细胞的影响。如果在PS的屏障特异性效应中观察到对细菌代谢的依赖，我们将随后研究具有相关生物活性的PS代谢物，并确定它们的细胞作用机制。我们的计划将描述PS、肠道微生物群和上皮之间大部分未被探索的相互作用机制。这些信息将通过机械地强调膳食纤维对上皮功能和整体肠道稳态的好处，为食品科学做出贡献。

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.