Single-Cell RNA Sequencing Identifies Spp1+ Macrophage Subpopulation as a Target for Alleviating Silica-Induced Pulmonary Fibrosis

<bold>Background:</bold> Prolonged inhalation of crystalline silica results in the formation of silicotic nodules and progressive massive fibrosis, which are associated with the upregulation of secreted phosphoprotein 1 (Spp1). However, the underlying mechanism is still unclear. <bold>Objective:</bold> To investigate Spp1⁺ macrophage subpopulation involved in silica-induced pulmonary fibrosis and to elucidate its upstream regulation. <bold>Methods:</bold> We conducted an unbiased single cell RNA-seq (scRNA-seq) analysis using C57BL/6N male mice, with intratracheal instillation of silica (0.4 g/kg) and PBS as the control. Adeno-associated virus-mediated or lentivirus-mediated knockdown of the expression of Spp1 was administered in vivo or in vitro, respectively. <bold>Results:</bold> Analysis of scRNA-seq revealed twelve major cell types in lung tissue, notably showing an increase in macrophages in silicosis mice. The Spp1/Cd44 interaction was found to play a critical role in macrophage-mediated activation of fibroblast by exploring cell-cell communications among all cellular components. Additionally, Spp1⁺ macrophages were located at the end of the macrophage developmental trajectory, and they were regulated by transcription factors Mafb, Ets1, and Irf7. In vitro and in vivo experiments demonstrated that inhibition of macrophage Spp1 expression can effectively inhibit the proliferation and differentiation of fibroblasts in silicosis. <bold>Conclusions:</bold> Spp1⁺ macrophage subpopulation significantly contributed to the formation of pulmonary fibrosis induced by silica. Therapeutic strategies targeting transcription factors Mafb, Ets1, and Irf7 may reduce silica-induced lung fibrosis.