Alternaria alternata serine proteases induce lung inflammation and airway epithelial cell activation via PAR₂

Allergens are diverse proteins from mammals, birds, arthropods, plants, and fungi. Allergens associated with asthma (asthmagens) share a common protease activity that may directly impact respiratory epithelial biology and lead to symptoms of asthma. Alternaria alternata is a strong asthmagen in semiarid regions. We examined the impact of proteases from A. alternata on lung inflammation in vivo and on cleaving protease-activated receptor-2 (PAR₂) in vitro. A. alternata filtrate applied to the airway in nonsensitized Balb/c mice induced a protease-dependent lung inflammation. Moreover, A. alternata filtrate applied to human bronchial epithelial cells (16HBE14o-) induced changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), consistent with PAR₂ activation. These effects were blocked by heat inactivation or by serine protease inhibition of A. alternata filtrates, and mimicked by PAR₂ specific ligands SLIGRL-NH₂ or 2-furoyl-LIGRLO-NH₂, but not the PAR₁-specific ligand TFLLR-NH₂. De-sensitization of PAR₂ in 16HBE14o- cells with 2-furoyl-LIGRLO-NH₂ or trypsin prevented A. aternata-induced [Ca²⁺]_i changes while desensitization of PAR₁, PAR₃, and PAR₄ with thrombin had no effect on A. aternata-induced Ca²⁺ responses. Furthermore, the Ca²⁺ response to A. alternata filtrates was dependent on PAR2 expression in stably transfected HeLa cell models. These data demonstrate that A. alternata proteases act through PAR2 to induce rapid increases in human airway epithelial [Ca²⁺]_i in vitro and cell recruitment in vivo. These responses are likely critical early steps in the development of allergic asthma.