Neutrophil extracellular traps contribute to pulmonary fibrosis induced by bleomycin

Abstract

Background: Increased neutrophil percentage in bronchoalveolar lavage fluids (BALF) in idiopathic pulmonary fibrosis is associated with poor prognosis. Neutrophil extracellular traps (NETs) exhibit a neutrophil antimicrobial function and induce systemic tissue damage and chronic inflammation under normal and diseased conditions such as acute respiratory distress syndrome and organ fibrosis. The enzyme peptidyl arginine deiminase, type Ⅳ (PAD4) mainly regulates NET release. However, the role of NETs in pulmonary fibrosis is unclear. Objectives: To verify the role of NET in murine pulmonary fibrosis induced by bleomycin (BLM) instillation. Methods: Wild type (C57BL/6) and PAD4-knockout (Pad4-/-, C57BL/6 background) mice were used to evaluate NET levels in BALF and lungs 2, 7, and 21 days after single bronchial instillation of saline and BLM (5U/kg body weight). Fibrosis was evaluated by Masson’s trichrome staining and modified Ashcroft Scale. Lung collagen load was evaluated by Sircol Collagen Assay 21 days after instillation. Results: In the BLM group, immunohistochemistry (IHC) showed that NETs increased in lavage 2 days after instillation (p = 0.013) and decreased in BALF and lungs 7 and 21 days after instillation. Two days after instillation, NETs in BALF and lungs of PAD4-KO mice decreased compared to wild type mice. After 21 days, pulmonary fibrosis score of PAD4-KO mice was lower than that of wild type mice (p = 0.003). In the BLM group, collagen load of PAD4-KO mice tended to be lower than that of wild type mice (408.8 ± 0.0 vs 472.1 ± 20.4 µg/whole lung). Conclusion: NETs may contribute to developing pulmonary fibrosis induced by BLM instillation.