Immune dysregulation and intestinal barrier defects are key pathogenic factors driving inflammatory bowel disease (IBD). These pathogenic factors are influenced by both genetic and environmental factors and have been the target of numerous therapeutic agents which have had limited clinical success. Given the current therapeutic options and recent failures, it’s critical that we enhance our understanding of the basic science behind immune dysregulation and intestinal barrier defects to identify new therapeutic targets. The studies proposed in this grant application seek to bridge this gap in IBD knowledge and provide insight into possible novel therapeutic targets. The goals of this application are to investigate how inflammatory macrophages contribute to barrier dysfunction through Notch signaling and to determine the efficacy of targeting Notch ligands to prevent and treat IBD. Our preliminary data show macrophages in the inflamed gut upregulate Notch ligands, Notch is activated in colonocytes, and inflammatory macrophages alter organoid differentiation and barrier integrity. Based on our preliminary data, we advance a novel hypothesis that inflammatory macrophages through Notch signaling i) alters intestinal stem cell (ISC) differentiation; ii) disrupts intestinal tight junction permeability and cellular metabolism; and iii) that autophagy plays a role in regulating cell surface Notch ligand expression in macrophages. Additionally, we hypothesize the in vivo targeting of Notch ligands in animal models of IBD will ameliorate intestinal inflammation. The following are the specific aims of the project: In aim 1, we will delineate the pathogenic role of Notch ligand-positive macrophages in intestinal barrier differentiation and function and the therapeutic efficacy of targeting Notch ligands in limiting intestinal inflammation. In aim 2, we will determine the mechanism behind increased Notch ligand expression. Specifically, we will determine the role of autophagy in regulating Notch ligand expression. The information generated from this project will provide a ground-breaking step with important long-term implications in understanding how inflammatory macrophages affect intestinal barrier integrity through modulation of both ISC and differentiated epithelium functions. Project Summary Page