PROJECT SUMMARY The bacterial pathogen enterotoxigenic E. coli (ETEC) contributes to the global burden of diarrheal disease because vaccine protection and therapy remain inadequate. A long-term goal is to improve knowledge of innate immune responses to ETEC infection, as they are likely important for initiating durable adaptive immunity. The objective of this project is to understand the innate immune defenses employed by human intestine to sense and eliminate ETEC, thus informing strategies for vaccine and/or drug design. We will (Aim 1) characterize the recognition and response mechanisms employed by tissue resident macrophages and neutrophils against ETEC infection. In a complementary inquiry, we will (Aim 2) assess antimicrobial peptides (AMPs) released by epithelial and immune cells in response to ETEC that could serve as potential treatment. These aims will be explored in primary co-culture models of human small intestinal epithelium (tissue-derived enteroids) and immune cell types from peripheral blood that are assembled on scaffolds to facilitate physiologically relevant crosstalk between bacteria, epithelia, and immune cell populations. Model development, application of anaerobic environment to mimic conditions in the intestinal lumen, and characterization will combine the expertise of the Enteroid and Immunology Cores that support this P01. The proposed studies are significant in that they will indicate the molecular signal transduction involved in reacting to and resolving ETEC infection without clinically overt inflammation, an understudied aspect of acute pathogenic diarrheal disease. Results from these studies will be compared to findings from enteroaggregative E. coli and Shigella pathogenesis proposals in this P01 to identify common effectors for therapeutic targeting. The projects of this P01 will cumulatively innovate human primary epithelial and immune cell co-culture strategies for host-pathogen research.