Project Summary Eosinophilic gastrointestinal disorders (EGIDs) are a group of allergic inflammatory disorders characterized by excessive accumulation and persistence of eosinophils and a Th2-dominant immune response. EGID symptoms and treatments carry heavy social and psychological tolls, particularly in affected children. Despite a worldwide increase in prevalence, the natural disease course of EGIDs is unknown, and treatment options limited to food allergen avoidance and oral corticosteroids. Eosinophils are innate immune leukocytes that constitutively home to the small and large intestine in the steady state where they contribute to immune, tissue, and microbiome homeostasis. Why and how eosinophils shift to pathologic roles, and what those pathologic roles are within the context of EGIDs, is unknown and represents a fundamental knowledge gap hindering the development of needed therapeutic approaches. Our published studies during the previous funding cycle contributed to an evolving paradigm in eosinophil biology that now recognizes the existence of tissue-specific adaptations and distinct functional phenotypes within mucosal organs. We demonstrated morphologic, spatial and phenotypic heterogeneity of intestinal eosinophils, proposed a framework to stratify tissue eosinophils with “homeostatic” versus “inflammatory” potentials, and traced sub-phenotype versatility in situ within the contexts of local allergic, and systemic non-specific, inflammation. These studies provide a scaffolding for functional outcomes and spatial mapping proposed in this renewal application. Our preliminary data also show that notch 2 signaling is active in human blood eosinophils, drives a cytokine-activated transcriptome, and is required for cytokine-mediated survival. To explore notch 2 signaling in vivo we generated a new mouse line with eosinophil-targeted deletion (EoN2). EoN2 mice exhibit impaired intestinal tissue-specific adaptations of eosinophils at baseline, and the selective loss of a villus-migrated, putatively “inflammatory” eosinophil subset in an allergen-driven EGID model. Building on and extending these findings, this renewal application applies novel tools created in the first cycle to explore the overarching hypothesis that notch 2 signaling underlies intestinal tissue specific adaptations of eosinophils in the steady state and regulates their functional phenotypes in inflammation. Aim 1 uses complementary high dimensional flow cytometry and multispectral imaging to define and integrate eosinophil functional subsets with spatial mapping and translate advances in profiling and spatial mapping to active EGID patient biopsies. Aim 2 exploits notch 2-dependent regulation of eosinophils to investigate functional phenotypes within the context of allergic inflammation. Aim 3 uses genetic and pharmacologic approaches to trial therapeutic targeting of notch 2 in mouse models of EGIDs. Completion of these aims will provide fundamental insights into the regu...