Project Summary Diet is critical modulator of both the composition and function of the microbiota. The Western diet, which is high in both fat and simple host-accessible sugars is used to model diet-induced obesity. Many studies have shown that the Western diet alters the composition of the microbiota and the immune system. Clostridioides (Clostridium) difficile is the leading cause of infectious nosocomial diarrhea. Primary risk factors for C. difficile infection (CDI) include antibiotic exposure, age, and proton pump inhibitor use, with more recent data also implicating obesity as a risk factor. Host factors determining the outcome of acute CDI include the gut microbiota and immune response. Recent work from our lab demonstrated that antibiotic-induced changes to the gut microbiota results in decreased type 2 immunity due to decreased epithelial ‘alarmins’ IL-25 and IL-33. In this proposal will explore how the Western diet alters the composition of the microbiota and the immune system to increase susceptibility to CDI. I have discovered that a Western diet increases severity of CDI. Additionally, I found that IL-33, which is an orchestrator of type 2 immunity, is suppressed in the gut of wild- type mice fed a Western diet while levels of Ly6G+ neutrophils are increased. Additionally, I found that transfer of microbiota from mice fed a control diet protects mice on the Western diet from severe disease. Together these data suggest that i) a diet high in dietary fats and simple carbohydrates alters the microbiota leading to (ii) alterations in immune homeostasis and gut metabolism (iii) resulting in increased severity of CDI. This hypothesis will be tested through a series of integrated specific aims. Specific aim 1: determine if the Western diet increases severity of CDI via alteration of colonic immunity. Specific aim 2: identify the metabolites and microbes which protect from severe CDI in WD-fed mice exposed to CD-microbiota. Successful completion of this fellowship will determine the impact of high-fat dietary components on the gut microbiome and mucosal immune response, specifically in their downstream ramifications for CDI susceptibility.