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

Abstract

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 PAR2activation. These effects were blocked by heat inactivation or by serine protease inhibition of A. alternata filtrates, and mimicked by PAR2specific ligands SLIGRL-NH2or 2-furoyl-LIGRLO-NH2, but not the PAR1-specific ligand TFLLR-NH2. Desensitization of PAR2in 16HBE14o- cells with 2-furoyl-LIGRLO-NH2or trypsin prevented A. alternata-induced [Ca2+]ichanges while desensitization of PAR1, PAR3, and PAR4with thrombin had no effect on A. alternata-induced Ca2+responses. Furthermore, the Ca2+response to A. alternata filtrates was dependent on PAR2expression in stably transfected HeLa cell models. These data demonstrate that A. alternata proteases act through PAR2to induce rapid increases in human airway epithelial [Ca2+]iin vitro and cell recruitment in vivo . These responses are likely critical early steps in the development of allergic asthma.