Protease-activated receptor-2 signaling through β-arrestin-2 mediates alternaria alkaline serine protease-induced airway inflammation

Al-ternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin pro-tease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR ₂ ) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR ₂ activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR ₂ signaling contributed to asthma symptoms via a PAR ₂ /β-arrestin signaling axis, identify the protease activity responsible for PAR ₂ signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR ₂ /β-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR ₂ ^/ and β-arrestin-2 ^/ mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR ₂ ^/ or β-arrestin-2 ^/ mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR ₂ signaling and induce β-arrestin-2 ^/ -dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyper-plasia largely via AASP using the PAR ₂ /β-arrestin signaling axis. Thus, β-arrestin-biased PAR ₂ antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.