LAUSR

BACKGROUND & AIMS In upper airway cells, Th2 cytokines that signal through IL-4 receptor alpha (IL4Rα) have been shown to stimulate eotaxin-3 secretion via a non-gastric proton pump (ngH+,K+ATPase). To seek novel targets for eosinophilic esophagitis (EoE) treatments, we evaluated ngH+,K+ATPase expression in EoE squamous cells, and explored molecular pathways involved in eotaxin-3 secretion by IL4Rα signaling. METHODS ngH+,K+ATPase expression in EoE cells was evaluated by qPCR and western blotting. IL-4-stimulated eotaxin-3 secretion was measured by ELISA after treatment with omeprazole, SCH 28080 (potassium-competitive acid blocker), EGTA-AM (calcium chelator), 2-APB (inhibitor of endoplasmic reticulum calcium release), verapamil and diltiazem (L-type calcium channel inhibitors). Intracellular calcium transients were measured by Fluo-4 fluorescence. Key experiments were confirmed in EoE primary cells and in RNA sequencing datasets from mucosal biopsies of EoE patients and controls. RESULTS EoE cells expressed ngH+,K+ATPase mRNA and protein. Omeprazole and SCH 28080 decreased IL-4-stimulated eotaxin-3 secretion. IL-4 increased intracellular calcium transients, and IL-4-stimulated eotaxin-3 secretion was blocked by EGTA-AM, 2-APB, verapamil, and diltiazem. The combination of omeprazole and verapamil suppressed IL-4-stimulated eotaxin-3 secretion more than either agent alone. EoE biopsies expressed higher ngH+,K+ATPase and exhibited more calcium signaling than controls. CONCLUSIONS EoE cells express a non-gastric proton pump that mediates Th2 cytokine-stimulated eotaxin-3 secretion. IL-4 induces calcium release from the endoplasmic reticulum and calcium entry via L-type calcium channels, increasing intracellular calcium that contributes to eotaxin-3 secretion by EoE cells. L-type calcium channel inhibitors block Th2 cytokine-stimulated eotaxin-3 secretion, suggesting a potential role for these agents in EoE treatment.