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.

嗜中性粒细胞细胞外捕获物（NETs）的过度释放与多种器官纤维化有关，包括肺纤维化。NETs构成一种现象，其中带有胞浆蛋白的修饰核染色质被释放到细胞外空间。PAD4（肽基精氨酸脱亚氨酶4）在NETs的形成中起重要作用。但是，NETs在肺纤维化发病机制中的作用仍不确定。在这里，我们确定了博莱霉素（BLM）诱导的小鼠肺纤维化小鼠的肺泡和间质性肺间隙中的NETs，该蛋白被pan-PAD抑制剂Cl-am抑制。在体外，BLM直接诱导血液中性粒细胞中的NET，这也被Cl-am抑制。此外，Padi4小鼠基因敲除（PAD4-KO）导致BLM诱导的NET和肺纤维化的减轻，并导致炎症和纤维化基因的表达。PAD4缺乏可防止肺泡中肺泡上皮和肺血管内皮细胞数量减少，以及ACTA2阳性间充质细胞和S100A4阳性成纤维细胞增加。PAD4-KO小鼠而非野生型小鼠的造血细胞移植物解决了BLM诱导的肺纤维化和野生型和PAD4-KO小鼠中纤维化基因的表达，提示造血细胞中PAD4的表达可能参与了PAD4-KO的发育。肺纤维化。这些数据表明，PAD4缺乏症可以改善BLM诱导的NETs和肺纤维化的形成，表明该途径可以作为肺纤维化治疗的治疗靶标。