PROJECT SUMMARY The goal of this proposal is to identify new ways of preventing anaphylaxis in those with food allergy by defining cellular and molecular mechanisms that transport intact food allergens across the gut epithelium. In its most severe form, food allergy can trigger life-threatening anaphylaxis. One factor that can determine risk of anaphylaxis is the integrity of the gut barrier, but there are no treatments to reduce intestinal permeability to food allergens. A major mode of allergen transport across the gut epithelium occurs transcellularly through secretory and goblet cell-associated antigen passages (GAPs). Using mouse models of peanut and egg allergy, we have made the exciting discovery that susceptibility to oral anaphylaxis was genetically determined and associated with an increased number and function of GAPs. Using a forward genetic screen we identified a single chromosomal region that tracks with this phenotype. Moreover, drug treatments targeting goblet cell biology reduced anaphylaxis in vivo. We hypothesize that the genetic regulation of gut goblet cell quantity and transport capability determines susceptibility to anaphylaxis by controlling intestinal permeability to intact food allergens. In Aim 1, using complementary mouse strains described above, we will perform quantitative trait locus (QTL) mapping with SNP genotyping to identify genetic resistance loci for oral anaphylaxis associated with reduced gut permeability. These data will be integrated with scRNA-Seq analyses of intestinal epithelium from anaphylaxis susceptible vs. resistant littermates to identify cell type-specific regulators of allergen transport. Using CRISPR/Cas9 gene editing in mice, we will test the contribution to gut permeability and oral anaphylaxis in vivo of known and novel candidate genes. In Aim 2 we will determine goblet cell-intrinsic vs. goblet cell extrinsic pathways that inhibit allergen transport. Our preliminary data in mice suggests that the number and function of GAPs prominently contribute to susceptibility to oral anaphylaxis. Using bone marrow chimeras and in vitro human or mouse enteroid cultures we will directly test whether transcellular transport of allergens is increased in anaphylaxis susceptible humans or mice and will determine whether these phenotypes are epithelial cell-intrinsic or -extrinsic. In Aim 3 we will perform a targeted screen of FDA approved drugs that could inhibit goblet cell differentiation and/or function for their ability to block fluorescent allergen uptake in vitro and oral anaphylaxis in vivo. Effective compounds will be validated using human gut enteroids from donors with or without food allergy. We have already identified multiple drugs that reduce allergen transport in oral anaphylaxis susceptible mice, highlighting that pharmaceutical blockade of these pathways could potentially prevent anaphylaxis in patients with food allergy. Impact: Identifying genes and cellular pathways regulating food allerg...