The intestinal epithelium plays an essential role in homeostasis and health by maintaining a barrier and responding appropriately to luminal antigens and bacterial signals. The epithelium accomplishes these functions via thousands of actin-rich protrusions known as microvilli. Microvilli harbor proteins which relay information to the epithelial cell. One important protein at the tips of the microvilli is intestinal alkaline phosphatase -an enzyme that detoxify pro-inflammatory bacterial and host derived compounds and minimizes epithelial insults. Microvilli are maintained at the proper height, distance and density by linker proteins known as the intermicrovillar adhesion complex (IMAC). Our preliminary data indicates that IMAC compounds and intestinal alkaline phosphatase are delivered to the apical member of epithelial cells by the molecular motor Myosin 5b. Loss of functional Myosin 5b and subsequent mislocalization of IMACs and intestinal alkaline phosphatase alters the structure and function of microvilli in our animal model, and we postulate that this sets the stage for epithelial dysfunction. Recently it has been demonstrated that patients with Inflammatory Bowel Disease (IBD) have decreased gene expression of Myosin 5b and have shortened, disordered microvilli on their epithelial cells. Our preliminary data indicates that Myosin 5b protein is decreased in colonic biopsies of IBD patients and in a TNBS mouse model of colitis. We hypothesize that inflammation decreases Myosin 5b gene expression and protein levels, which leads to the mislocalization of IMAC and intestinal alkaline phosphatase protein and perpetuates a cycle of inflammation. To address this question, we propose to define the role of Myosin 5b in intestinal inflammation using acute (DSS) and chronic (T-cell transfer) animal colitis models (Aim 1) and define the mechanisms by which pro-inflammatory stimuli downregulate Myosin 5b and how decreased Myosin 5b sets the stage for further insults using mouse and human intestinal organoids (Aim 2). This work has the potential to identify key pathways involved in intestinal inflammation; targets which could be used to develop next generation therapeutics. This research may lead to treatments which enhance patient health outcomes and quality of life.