Allergen-induced activation of epithelial P2Y2 receptors promotes ATP 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: To investigate the role of allergen-stimulated P2Y₂ receptor activation in regulating ATP, IL-33 and DNA release by human bronchial epithelial (hBE) cells and mouse airways.

Methods: 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 (i.n.) with pharmacologic agents and exposed to Alternaria extract.

Results: Exposure of hBE cells to Alternaria extract stimulated P2Y₂ receptors coupled to Phospholipase Cβ₃, leading to activation of multiple PKC isoforms and an increase in intracellular CA²⁺ concentration. Small interfering RNAs (siRNAs) targeting PKCδ or inhibiting PKCδ activity with delcasertib blocked exocytosis of ATP and reduced IL-33 and DNA secretion. Moreover, a peptide antagonist for 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.

Conclusion: P2Y₂ receptor stimulation following allergen exposure promoted activation of PLCβ₃, PKCδ 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.

DNA release; IL-33; P2Y(2) receptor; delcasertib; myristoylated alanine rich protein kinase C substrate (MARCKS); phosphatidylinositol 4,5-bisphosphate (PIP2); phospholipase C (PLCβ(3)); protein kinase C (PKCδ).