Protease-activated Receptor-2 Signaling through β-Arrestin-2 Mediates Alternaria Alkaline Serine Protease-induced Airway Inflammation.

Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similar to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor, protease-activated receptor-2 (PAR2 ), to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR2 signaling contributed to asthma symptoms via a PAR2 /β-arrestin signaling axis, to identify the protease activity responsible for PAR2 signaling and determine if protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR2 /β-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR2 -/- 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 mice, but not PAR2 -/- 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 PAR2 signaling and induce β-arrestin-2-dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR2 /β-arrestin signaling axis. Thus, β-arrestin-biased PAR2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.