Integrated multi-omics analyses reveal the pro-inflammatory and pro-fibrotic pulmonary macrophage subcluster in silicosis

Background: Silicosis is a lethal occupational disease caused by long-term exposure to respirable silica dust. Pulmonary macrophages play a crucial role in mediating the initiation of silicosis. However, the phenotypic and functional heterogeneities of pulmonary macrophages in silicosis have not been well-studied. Methods: The silicosis mouse model was established by intratracheal administration of silica suspension. Bronchoalveolar lavage fluids (BALFs) of mice were collected for the multiplex cytokine analysis. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics were performed to reveal the heterogeneity and spatial localization of macrophages in the lung tissues. The formation of the fibrotic nodules was characterized by histology, hydroxyproline assay, and immunohistochemical staining, respectively. The expression of the proinflammatory or pro-fibrotic genes was investigated by quantitative polymerase chain reaction (qPCR). Results: We found that the level of pro-inflammatory cytokines and chemokines is significantly increased in the BALFs of silicosis mice. Apparent collagen deposition can also be observed in the silicotic lung tissues. By scRNAseq, we have identified a subpopulation of Mmp12himacrophages hi macrophages significantly expanding in the lung tissues of mice with silicosis. Spatial transcriptomics analysis further confirmed that the Mmp12hi hi macrophages are mainly enriched in silicosis nodules. Pseudotime trajectory showed that these Mmp12hi hi macrophages, highly expressing both pro-inflammatory and pro-fibrotic genes, are derived from Ly6c+ + monocytes. Additionally, 4-octyl itaconate (4-OI) treatment, which can alleviate pulmonary fibrosis in silicosis mice, also reduces the enrichment of the Mmp12hi hi macrophages. Moreover, we found a subset of macrophages in BALFs derived from patients with silicosis exhibited similar characteristics of Mmp12hi hi macrophages in silicosis mice models. Conclusions: Our study suggested that a group of Mmp12hi hi macrophages highly express both pro-inflammatory and pro-fibrotic factors in silicosis mice, and thus may contribute to the progression of fibrosis. The findings have proposed new insights for understanding the heterogeneity of lung macrophages in silicosis, suggesting that the subset of Mmp12hi hi macrophages may be a potential therapy target to further halt the progression of silicosis.