Allergen-induced activation of epithelial P2Y(2) receptors promotes adenosine triphosphate exocytosis and type 2 immunity in airways.

BACKGROUND: Environmental allergens induce the release of danger signals from the airway epithelium that trigger type 2 immune responses and promote airway inflammation. OBJECTIVE: We investigated the role of allergen-stimulated P2Y(2) receptor activation in regulating adenosine triphosphate (ATP), IL-33, and DNA release by human bronchial epithelial (hBE) cells and mouse airways. METHODS: The hBE cells were exposed to Alternaria alternata extract and secretion of ATP, IL-33, and DNA were studied in vitro. Molecular and cellular mechanisms were examined by biochemical and genetic approaches. Mice were treated intranasally with pharmacologic agents and exposed to Alternaria extract. RESULTS: Exposure of hBE cells to Alternaria extract stimulated P2Y(2) receptors coupled to phospholipase C β(3), leading to activation of multiple protein kinase C (PKC) isoforms and an increase in intracellular Ca(2+) concentration. Small interfering RNAs targeting PKC δ or inhibiting PKC δ activity with delcasertib blocked exocytosis of ATP and reduced IL-33 and DNA secretion. Moreover, a peptide antagonist for myristoylated alanine-rich C-kinase substrate (MARCKS) reduced vesicular ATP release. A proximity ligand assay showed that Alternaria extract stimulated MARCKS desorption from the plasma membrane and delcasertib prevented the response. Finally, the P2Y(2) receptor antagonist AR-C118925XX and delcasertib blocked IL-33, DNA, and type 2 cytokine secretion in vivo in mice exposed to Alternaria. CONCLUSION: P2Y(2) receptor stimulation after allergen exposure promoted activation of PLC β(3), PKC δ, and MARCKS protein desorption from the apical membrane, which facilitated ATP exocytosis and subsequent secretion of IL-33 and DNA. Epithelial P2Y(2) receptors serve as primary sensors for aeroallergen-induced alarmin release by airway epithelial cells.