Understanding Interferon-γ-Induced Esophageal Epithelial Barrier Dysfunction and Apoptosis using Int

Megha Lal, PhD1, Ravi Gautam, PhD1, Zoe Mrozek, BS1, Yusen Zhou, PhD3, Jarad Beers, MS1, Margaret C. Carroll, MS1, Melanie A. Ruffner, MD, PhD1,2. 1Division of Allergy and Immunology, Children's Hospital of Philadelphia 2Department of Pediatrics, Perelman School of Medicine at University of Pennsylvania 3Department of Biomedical and Health Informatics, Children's Hospital of Philadelphia

Eosinophilic esophagitis (EoE) is a chronic, allergic inflammatory disease triggered by specific food antigens. EoE has elevated type 2 cytokines (i.e., interleukin (IL)-13) yet treating type 2 inflammation alone in EoE is not successful for all patients. Recent whole tissue RNA-sequencing highlight the upregulation of type I and II interferon (IFN) response in adult and pediatric EoE patients. However, the role of interferon signaling EoE immunopathology remains unresolved, and our goal was to determine the impact of IFN signaling on esophageal epithelium to elucidate its role in EoE. Biopsy tissue from pediatric patients with active EoE and control patients was dissociated and CD45+ cells were removed using negative isolation beads. RNA sequencing of epithelial cell enriched population identified IFN-γ response as the most significant upregulated pathways in EoE patients. Next, we compared the transcriptional response of esophageal epithelial cells cultured as 3D-organoids to IL-13, IFN-α and IFN-γ. We observed that the in-vitro response to IL-13 was comparable to previous published literature whereas IFN-α stimulation had marginal effect. Interestingly, IFN-γ treatment triggered upregulation of several hundred genes, including tissue differentiation, epithelial barrier and apoptotic genes replicating the active EoE mucosa. Further, single-cell RNA sequencing identified four sub-populations of epithelial cells in varying stages of differentiation featuring barrier dysfunction and apoptosis. The protein expression of relevant markers for tissue differentiation (IVL), epithelial barrier (CLDN1) and apoptosis (Caspase-3) were confirmed in 3D organoids through immunohistochemistry, validating the bioinformatic findings. In conclusion, IFN response gene signature is enriched in the epithelium of active EoE. IFN-γ stimulation triggers disruption of tissue differentiation, epithelial barrier integrity and cytotoxicity in human epithelium, features relevant to EoE immunopathology. Our findings offer new insights into EoE pathogenesis and might help in improving therapy options for EoE patients.