Allergen-induced activation of epithelial P2Y₂ receptors promotes adenosine triphosphate exocytosis and type 2 immunity in airways.

Abstract

Environmental allergens induce the release of danger signals from the airway epithelium that trigger type 2 immune responses and promote airway inflammation. We investigated the role of allergen-stimulated P2Y₂ receptor activation in regulating adenosine triphosphate (ATP), IL-33, and DNA release by human bronchial epithelial (hBE) cells and mouse airways. 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. Exposure of hBE cells to Alternaria extract stimulated P2Y₂ receptors coupled to phospholipase C β₃, leading to activation of multiple protein kinase C (PKC) isoforms and an increase in intracellular Ca²⁺ 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 P2Y₂ receptor antagonist AR-C118925XX and delcasertib blocked IL-33, DNA, and type 2 cytokine secretion in vivo in mice exposed to Alternaria. P2Y₂ receptor stimulation after allergen exposure promoted activation of PLC β₃, PKC δ, and MARCKS protein desorption from the apical membrane, which facilitated ATP exocytosis and subsequent secretion of IL-33 and DNA. Epithelial P2Y₂ receptors serve as primary sensors for aeroallergen-induced alarmin release by airway epithelial cells.