Summary The long-term goal of this study is to elucidate the immunologic features of Eosinophilic Esophagitis (EoE). This is an important subject as EoE is an emerging, chronic disease that often starts in childhood and continues into adulthood and is associated with substantial morbidity, especially the other atopic diseases being studied in this U19 proposal, yet has no FDA-approved therapies. Understanding this subject has significant implications as elucidating its fundamental immunologic features has potential to lay the foundation for improved diagnostics and therapies. Our central hypothesis is that the aryl hydrocarbon receptor (AHR) serves as an esophageal sensor and mediates its action via the transcription factor OVOL1, which induces transcription of the antiprotease SPINK7, and that IL-4 and IL-13 repress this pathway. The rationale for this hypothesis is based on findings from our current U19 grant that have provided evidence for a central role of serine peptidase inhibitor Kazal type 7 (SPINK7) in EoE pathogenesis. Acquired loss of SPINK7 is sufficient to unleash uncontrolled esophageal protease activity, which in turn induces loss of epithelial cell differentiation, loss of desmosomal protein expression, and impaired barrier function, as well as overproduction of proinflammatory mediators, including TSLP. Yet, there is virtually nothing known about the molecular regulation of SPINK7 expression, which is the subject of this grant renewal. We have uncovered that the esophageal epithelial enriched transcription factor, Ovo-like transcriptional repressor 1 (OVOL1) regulates SPINK7 promoter activity and expression. Furthermore, OVOL1 overexpression increases SPINK7 expression, whereas OVOL1 depletion decreases SPINK7, impairs the epithelial barrier, and importantly increases TSLP production. Mechanistically, ligands of the AHR induce OVOL1 nuclear translocation, which in turn promotes SPINK7 expression; conversely, AHR antagonists inhibit SPINK7 expression. A link with type 2 immunity is revealed by the finding that IL-4 and IL-13 reduce AHRinduced OVOL1 nuclear translocation and OVOL1-induced SPINK7 expression. Furthermore, overexpression of CAPN14, which is encoded for by a chief EoE genetic susceptibility locus, decreases OVOL1 expression. Translational studies demonstrate a decrease in esophageal expression of OVOL1 protein in patients with EoE and blockade of serine protease activity attenuates murine experimental EoE. The clinical significance of these data are underscored by the proton pump inhibitor omeprazole, which is used to treat EoE, being an AHR ligand that induces SPINK7. We will test the central hypothesis via 3 complementary aims using innovative approaches that combine molecular, genomic, and biological studies. In Aim 1, we will test the role of OVOL1 in esophageal epithelium by regulating the expression of differentiation genes, including SPINK7, testing the hypothesis that OVOL1 is critical ...