Summary Dysfunction of the intestinal barrier established by tight junctions between intestinal epithelial cells (IECs) is a critical issue for many diseases, including inflammatory bowel diseases and mucositis as a severe complication of chemotherapy. There is an unmet need in efficiently preventing dysfunction of the intestinal barrier. Observational evidence suggests that oligosaccharides in human milk are associated with beneficial effects in infancy and in adulthood. However, neither basic research nor clinical studies have revealed the exact biological functions or mechanisms of action of individual oligosaccharides during development or in adulthood. Thus, it remains largely unknown whether these oligosaccharides could become effective therapeutics. 2’- fucosyllactose (2’-FL) is the most abundant oligosaccharide in human milk but are not present in dairy milk. We have found a previously unrecognized role of 2’-FL in protecting intestinal barrier and ameliorating intestinal injury during colitis and mucositis in adult mice. Our preliminary studies have demonstrated that dysregulated microbial metabolic pathways in ulcerative colitis patients are regulated by 2-FL in adult mice. We have further found that 2’-FL promotes Bifidobacterium infantis to produce metabolites, such as pantothenate that protects the intestinal barrier against oxidative stress. Another important finding is that 2’-FL directly stimulates transactivation of a cell protective signaling, epidermal growth factor receptor (EGFR) in IECs. We will test the hypothesis that 2’-FL protects the intestinal barrier against injury during intestinal inflammation in adulthood through two mechanisms: by promoting the growth of beneficial bacteria capable of metabolizing 2’-FL, and by stimulating transactivation of EGFR in IECs in a microbially-independent manner. The mechanisms underlying protecting the mucosal barrier in intestinal inflammation by human gut microbiota consuming 2’-FL will be defined in Aim 1. We will define the impact of 2’-FL on the adult human gut microbial compositional balance, particularly growth of 2’-FL consuming bacteria, and metabolite production and determine the exact roles of 2’-FL-regulated microbial profile and metabolites, such as pantothenate, in protecting the intestinal barrier against injury in colitis and mucositis in adult mice. We will further identify 2’-FL-directed pathways that mediate growth and metabolism in B. infantis and B. breve. In Aim 2, we will determine the mechanisms by which 2’-FL directly stimulates cellular responses in IECs that ameliorate inflammation-induced disruption of the intestinal barrier through identifying novel targets of 2’-FL in IECs, including targets for EGFR activation, revealing protective cellular response through 2’-FL-stimulated EGFR signaling in IECs and further define these cellular programs at single cell resolution in the human intestinal epithelium, and defining the contribution of 2’-FL-stimulated EGFR...