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,KATPase). To seek novel targets for eosinophilic esophagitis (EoE) treatments, we evaluated ngH,KATPase expression in EoE squamous cells, and explored molecular pathways involved in eotaxin-3 secretion by IL4Rα signaling.
ngH,KATPase 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.
EoE cells expressed ngH,KATPase 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.
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.