PROJECT SUMMARY Food allergy is associated with a hypersensitive type 2 immune response that develops following sensitization to food proteins. Allergic sensitization elicits the development of adaptive immune memory, characterized by antigen specific B-cells that produce antibodies of the IgE and IgG1 isotypes. IgE antibodies bind to tissue- resident mast cells, and these IgE-mast cell units enable a rapid and exuberant responses to low quantities of food antigen. Pre-clinical and clinical studies underscore the importance of IgE antibodies in the gastrointestinal manifestation of food allergy, such as abdominal pain, nausea, and vomiting; however, how this hypersensitive state is established and what mast-cells induce to initiate these symptoms are poorly understood. The overarching hypothesis of this proposal is that close symbiotic interactions between mast cells and epithelial cells enable intraepithelial mast cell expansion within the allergic intestines, and that mast cells in turn alter both the transcriptional state and differentiation of intestinal epithelium to mediate their effects on food allergy symptoms. Preliminary data in in vivo food allergy models demonstrates marked intraepithelial mast cell expansion in food allergic mice, with the greatest frequencies found in the proximal small intestine in the crypt/transit amplifying zone. We further identified a leukotriene transcriptional signature in the intestines of food allergic mice, which was largely confined to the intraepithelial layer and was dependent on IgE and FceR1 expressing cells. Consistently, pharmacological and genetic disruption of cysteinyl leukotriene synthesis was found to dramatically blunt intestinal symptoms of food allergen ingestion, with separate cysteinyl leukotriene receptors mediating the acute behavioral (CysLTR2) and physiological effects (CysLTR1) to allergen. We hypothesized that mast-cell epithelial cross talk through cysteinyl leukotrienes may contribute to symptoms such as allergen induced malaise. In support of this, we find mast cells induce transcription of the stress- related TGFb cytokine, GDF15, from colonic crypt epithelial cells via a CysLTR1 dependent mechanism, and that GDF15 blockade ameliorates late phase avoidance behavior to allergen. This proposal aims to build upon these findings through the following aims: Aim 1 will examine IgE-dependent transcriptional and cell composition changes in the intestinal epithelium and will identify candidate epithelium-derived mast cell growth factors in vitro and in vivo, Aim 2 will clarify the role of cysteinyl leukotrienes and their receptors in food allergy pathogenesis using pharmacological inhibitors, genetic knockouts, and CysLTR1-3 floxed mice, and Aim 3 will examine the transcriptional state of GDF15+ epithelial cells induced by food allergy and will investigate the behavioral and immunological effect of GDF15 and GFRAL deficiency using newly generated KO mice on the BALB/cJ background. Togeth...