Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly 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, identify the protease activity responsible for PAR₂ 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 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 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.

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