Loss of Peptidyl‐Arginine Deiminase 1‐mediated Citrullination Drives Esophageal Epithelial Barrier Impairment in Allergic Inflammation

Abstract

BackgroundCitrullination or deimination is a post‐translational modification in which neutral arginine residues are converted to positive citrulline by members of the peptidyl‐arginine deiminase (PADI) family. PADI1 is an esophageal enriched gene that is significantly downregulated in Eosinophilic Esophagitis (EoE), an eosinophilic‐driven chronic allergic inflammatory disorder characterized by reduced tissue differentiation and impaired esophageal epithelial barrier formation. While the role of PADI1 in deiminating structural proteins has been implicated in epithelial cell populations, including the skin, the functional significance and extent of PADI1‐mediated citrullination in allergic inflammation remains understudied.HypothesisHerein we hypothesize that the loss of PADI1‐mediated citrullination drives esophageal epithelial barrier impairment in allergic inflammation.MethodsPADI1 WT and KO EPC2 immortalized human esophageal epithelial cells generated by CRISPR‐Cas9 were grown in either monolayer or at Air‐Liquid Interface (ALI) to mimic esophageal differentiation. Transepithelial Electrical Resistance (TEER) measurements of EPC2 cells grown at ALI were measured as a function of barrier integrity. Transcriptional, protein and epigenetic analyses were conducted on EPC2 cells grown at ALI treated with or without IL‐13, a T‐cell derived cytokine known to induce responses involved in EoE. Immunofluorescence (IF) was used to visualize structural proteins and assess overall level of citrullination.ResultsPADI1 protein was found to be most highly expressed in the Esophageal Mucosa as compared to all other tissue types, specifically in the suprabasal layer of differentiated esophageal cells. PADI1 RNA expression was decreased eight‐fold (P < 0.0001) in esophageal biopsies of patients with EoE as compared to control patients. Treatment of EPC2 cells with IL‐13 (100 ng/ml) at confluence reduced PADI1 mRNA by three‐fold (P < 0.01). PADI1 KO cells demonstrate altered differentiation as assessed by decreased levels of citrullinated proteins and structural epithelial proteins, including filaggrin, involucrin, desmoglein‐1, E‐cadherin, and cornulin compared with wild‐type control cells.ConclusionOur data suggest that loss of PADI1‐mediated citrullination may drive esophageal epithelial barrier impairment in allergic inflammation.Support or Funding InformationThis work is supported by PHS Grant R01‐AI124355 and the Ohio Physiological Society.