Project Summary/Abstract The long-term goal of this study is to determine the regulation and function of gastrointestinal (GI) eosinophils, a cell associated with multiple inflammatory diseases, especially a new set of emerging chronic allergic diseases referred to as eosinophilic GI diseases (EGID). Whether GI eosinophils exist as heterogeneous cells and subpopulations is currently not known. This is an important subject as distinct cell populations may have different roles in disease and during homeostasis. Understanding this subject has significant clinical implications in view of the emerging class of new drugs that target eosinophils. Perhaps only a specific population of eosinophils should be ablated and only in certain patients. The central hypothesis is that GI eosinophils are dynamic, transcriptionally active cells regulated epigenetically by local microenvironmental cues and that these processes lead to heterogeneous cell populations, under homeostatic and disease conditions. The rationale for this hypothesis is based on the findings that eosinophils express dynamic transcriptomes characterized by distal regulatory elements associated with histone 3 lysine 27 acetylation (H3K27ac) and binding sites for the transcription factor PU.1. We will test this hypothesis using recently developed innovations (e.g., ChIP-seq, ATAC-seq, transcriptomics, ex vivo co-culture) that we have applied to study murine and human eosinophils. In Aim 1, we will examine the epigenetic landscape and function of eosinophils. We will test the hypothesis that eosinophils express dynamic transcriptomes and epigenomes under basal and IL-33–activated conditions. We will mechanistically uncover the involvement of PU.1 as a prototypic transcription factor that regulates eosinophil enhancer function. We will elucidate the epigenetic and transcriptional mechanisms underlying eosinophil responses to a microenvironmental stimulus, IL-33. In Aim 2, we will examine GI eosinophil heterogeneity, aiming to test the hypothesis that murine GI eosinophils exist as heterogeneous populations defined by their epigenomic and transcriptomic landscapes and their proteomic content and functional capacity. In Aim 3, we will examine eosinophil heterogeneity in EGID. We will compare the epigenome, transcriptome, and proteome of human blood and GI tissue eosinophils in healthy and diseased individuals. We will model the interplay of eosinophils with key microenvironmental factors that are likely to influence these processes (e.g., co-culture with epithelial cells and relevant activating cytokines [IL-13]). We are hopeful that the renewal of this grant will allow the elucidation of the fundamental properties of GI eosinophils to continue. We anticipate that the findings from this grant will lay the foundation for defining eosinophil subpopulations in mice and humans. The importance of this undertaking (identifying subtypes and functions of eosinophils unique to different tissues) was highlighte...