PROJECT SUMMARY Microphysiological systems have great potential for modeling human disease but advanced in vitro models of parasitic infection and immunity are severely underrepresented. Parasites are major causes of morbidity and mortality globally, infecting millions of people every year, yet, there are no effective vaccines available for any enteric parasitic infection. Oral transmission via contaminated food or water is the most common route of parasitic infection for humans, but our knowledge of parasite/host interactions, including how parasites interact with immune cells to either cause disease or elicit a protective immune response, within the intestinal tract is very limited. There is a critical need to create improved in vitro models of human immune-parasite interactions to capture key features present during parasitic infection establishment and disease progression. To address this need we will develop a microphysiological gut vasculature lumen system based on the LumeNEXT microfluidic device system. This 3-dimensional cell culture device recapitulates the gut architecture and includes a human intestinal epithelial lumen flanked by blood and lymphatic vasculature. With these advanced in vitro models, we will introduce parasites into the intestinal epithelium and human immune cells into the vasculature to monitor parasitic disease and immune response. Our goal is to create a microphysiological system that can be used for the study of any protozoan parasite. For this proposal, we will use the protozoan parasites Toxoplasma gondii (T. gondii) and Entamoeba histolytica (E. histolytica) because 1) they are human pathogens of global importance, 2) they are on distinct branches of the protist evolutionarily tree, 3) they have defined lab growth conditions and genetically tagged marker strains, 4) our lab has recently developed the unique ability to produce large numbers of the highly infectious oocysts and cysts forms of both T. gondii. Our data shows that T. gondii infection of the intestinal epithelial lumen in our in vitro model system elicits an active immune response and migration of human immune cells from the vasculature. In this project, we will use these 3D biomimetic gut-vasculature lumen models to address critical knowledge gaps of the human immune responses to T. gondii and E. histolytica. We will incorporate an anaerobic environment so that the immune responses can be defined under hypoxic conditions. These experiments will provide the foundational understanding of the human innate immune responses to intestinal T. gondii infection that are essential for vaccine development. We will also model E. histolytica invasive disease using nutrient limitation and co-culture with Clostridiodes difficile. The advances we will achieve in this proposal will allow the microbiology and immunology fields to determine the immune responses to the biologically relevant stages of intestinal parasites in human models.