Excessive release of neutrophil extracellular traps (NETs) has been implicated in several organ fibrosis, including pulmonary fibrosis. NETs constitute a phenomenon in which decorated nuclear chromatin with cytosolic proteins is released into the extracellular space. PAD4 (peptidylarginine deiminase 4) plays an important role in the formation of NETs. However, the role of NETs in the pathogenesis of pulmonary fibrosis remains undefined. Here, we identified NETs in the alveolar and interstitial lung space of mice undergoing bleomycin (BLM)-induced lung fibrosis, which was suppressed by a pan-PAD inhibitor, Cl-amidine. In vitro, BLM directly induced NETs in blood neutrophils, which was also inhibited by Cl-amidine. Furthermore, Padi4 gene knockout (PAD4-KO) in mice led to the alleviation of BLM-induced NETs and pulmonary fibrosis and to the expression of inflammatory and fibrotic genes. PAD4 deficiency prevented decreases in alveolar epithelial and pulmonary vascular endothelial cell numbers and increases in ACTA2-positive mesenchymal cells and S100A4-positive fibroblasts in the lung. Hematopoietic cell grafts from PAD4-KO mice, not wild-type mice, resolved BLM-induced lung fibrosis and fibrotic gene expression in wild-type and PAD4-KO mice, suggesting that expression of PAD4 in hematopoietic cells may be involved in the development of lung fibrosis. These data suggest that PAD4 deficiency could ameliorate BLM-induced formation of NETs and lung fibrosis, suggesting that this pathway could serve as a therapeutic target for pulmonary fibrosis treatment.
Keywords: bleomycin; citrullinated histone H3; neutrophil extracellular trap; peptidylarginine deiminase 4; pulmonary fibrosis.