Pirfenidone alleviates interstitial lung disease in mice by inhibiting neutrophil extracellular trap formation and NLRP3 inflammasome activation.

BACKGROUND: Idiopathic inflammatory myopathy (IIM) is a progressive autoimmune disease characterized by interstitial lung disease (ILD) with limited therapeutics available. Pirfenidone (PFD), a medication utilized for the treatment of idiopathic pulmonary fibrosis, exhibits notable antioxidant, anti-inflammatory, and inhibition of collagen synthesis. This study aims to clarify its efficacy and mechanism in treating IIM-ILD. METHODS: A murine myositis-associated interstitial lung disease (MAILD) model was used to assess the therapeutic effect of PFD. The serum levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). Pirfenidone was utilized to disrupt neutrophil extracellular traps (NETs) formation in vitro, and its inhibitory effect on NETs was assessed through immunohistochemistry of citrullinated histone H3 and myeloperoxidase in the lung tissue and the serum cfDNA level in mice. Immunohistochemical and western blot were utilized to examine alterations in epithelial-mesenchymal transition (EMT) and NOD-like receptor protein 3 (NLRP3) inflammasome markers. RESULTS: Pirfenidone treatment inhibited pulmonary inflammation and fibrosis in the MAILD model. Pirfenidone intervention reduced NETs formation in vitro. Pirfenidone treatment significantly reduces NETs infiltration in the lung tissue and the level of cfDNA in the serum of mice. Additionally, PFD downregulated EMT and NLRP3-related proteins in vivo. Pirfenidone treatment also notably reduced serum levels of IL-1β, IL-6, and TNF-α. After NETs stimulation, A549 cells exhibited EMT and activation of NLRP3 inflammasome. Pirfenidone attenuated EMT in A549 cells and suppressed the activation of NLRP3 inflammasome. CONCLUSION: Pirfenidone alleviates ILD in a murine MAILD model by inhibiting NETs formation and NLRP3 inflammasome activation, suggesting that PFD might be a potential therapeutic agent for IIM-ILD.