A109-16 Reprogramming of Alveolar Neutrophils by Mitochondria Drives Pulmonary Fibrosis

Abstract Rationale Neutrophils are increasingly recognized as functionally heterogeneous immune cells that play disease-specific roles beyond the defense against infection. Neutrophils mainly rely on glycolysis; however, recent studies have indicated that their mitochondria can be active under specific conditions. Although the involvement of neutrophils in pulmonary fibrosis has been suggested, the underlying mechanisms remain poorly understood. We hypothesized that fibrotic lung environment reprograms neutrophil function to promote tissue remodeling and fibrosis. Methods We analyzed publicly available transcriptomic datasets from patients with idiopathic pulmonary fibrosis (IPF) and performed single-cell RNA sequencing (scRNA-seq) of lung and bronchoalveolar lavage (BAL) cells in the bleomycin (BLM)-induced lung fibrosis mouse model. ATAC-seq and bulk RNA-seq were performed on neutrophils isolated from BALF from BLM-induced lung fibrosis mouse model to identify transcriptional regulatory mechanisms in the fibrotic alveolar environment. Results Neutrophils from IPF lungs exhibited elevated expression of mitochondrial genes. In the BLM model, circulating, lung-tissue-resident, and bronchoalveolar lavage (BAL) fluid neutrophils formed distinct clusters on scRNA-seq, and pseudotime trajectory analysis suggested that BAL neutrophils were elicited from lung-resident neutrophils. BAL neutrophils showed marked upregulation of mitochondrial ATP synthesis-related genes. Exposure to BAL fluid from BLM-treated mice as well as IPF patients enhanced mitochondrial ATP production in neutrophils. To directly assess the contribution of mitochondrial function, we generated neutrophil-specific mitochondrial DNA transcription factor knockout mice (TfamΔneu). TfamΔneu mice, in which mitochondrial function was selectively impaired in neutrophils, displayed significantly attenuated lung fibrosis histological scores, hydroxyproline deposition in the lung, and improved survival after BLM challenge. In contrast, acute inflammatory process such as intratracheal lipopolysaccharide instillation or Pseudomonas aeruginosa infection did not show significant difference in the lung disease severity, suggesting mitochondria in neutrophils specifically affect the fibrotic pathogenesis. ATAC-seq and bulk RNA-seq analyses of TfamΔneu neutrophils in the BAL fluid of BLM-treated mice revealed reduced chromatin accessibility at fibrosis-associated gene loci, accompanied by downregulation of profibrotic gene mRNA expression. Integrative analysis of public ChIP-seq databases identified IRF8 and NRF2 as key transcription factors linked to these profibrotic genes. IRF8 expression was induced in a mitochondria-dependent manner, whereas NRF2 protein levels increased in response to mitochondria-dependent reactive oxygen species. Conclusion Our findings demonstrate that alveolar neutrophils in fibrotic lungs acquire a mitochondria-dependent profibrotic phenotype mediated through chromatin remodeling and transcriptional reprogramming involving IRF8 and NRF2. Targeting mitochondrial metabolism in neutrophils may represent a potential therapeutic strategy for pulmonary fibrosis. This abstract is funded by: Japan Society for the Promotion of Science