Purification and characterization of a serine protease from organic dust and elucidation of its inductive effects on lung inflammatory mediators.

Organic dust inhalation is associated with the development of respiratory diseases. Serine protease activities in organic dusts were previously reported to contribute to the induction of lung inflammatory mediators however, the identities of the proteases and the mechanisms by which they induce inflammatory mediators are unknown. The goal of this study was to purify and characterize serine protease(s) from organic dust and elucidate mechanisms by which they induce lung inflammatory mediators. A serine protease was purified from poultry organic dust by benzamidine-agarose affinity chromatography. Mass spectrometry and amino-terminal sequence analysis identified the purified protease as chicken trypsin II-P29. Purified protease induced proinflammatory cytokine levels in Beas2B and NHBE epithelial and THP-1 macrophage cells. Treatment with the purified protease increased cellular and mitochondrial reactive oxygen species (ROS) generation. Induction of inflammatory mediators and ROS were suppressed by serine protease inhibitors and antioxidants. Purified protease activated protein kinase C (PKC), mitogen-activated protein kinase (MAPK)1/3 and MAPK14 signaling, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription 3 (Stat-3), and chemical inhibitors targeting these pathways suppressed induction of inflammatory mediators. Calcium mobilization studies showed that the purified protease activated protease-activated receptors (PAR) F2R, F2RL1, F2RL2, F2RL3, and F2R and F2RL1 knockdown suppressed the induction of inflammatory mediators. Intranasal instillation of purified protease increased lung chemokine (C-X-C motif) ligand (CXCL)1, interleukin (IL)-6, and tumor necrosis factor (TNF) levels in mice. Our studies have shown that chicken trypsin is a proinflammatory constituent of poultry organic dust, and induces lung inflammatory mediators via increased ROS and PAR activation in a cell signaling pathway involving PKC, MAPK1/3 and MAPK14, and NF-κB and Stat-3. Inhalation of dust in industrial agricultural operations is linked to the development of lung diseases. Our studies have isolated for the first time a trypsin protease from poultry farm dust and have shown that it stimulates lung inflammation. The protease stimulates the production of oxidants and cell signaling pathways to increase inflammatory mediator production. Targeting trypsin protease in poultry farm environment may be a useful strategy to counter the harmful effects of dust.