Coal Dust Exposure Triggers Heterogeneity of Transcriptional Profiles in Mouse Pneumoconiosis and Vitamin D Remedies

Abstract

Background: Coal dust particles (CDP), an inevitable by-product of coal mining for the environment, mainly causes coal workers’ pneumoconiosis (CWP). Long-term exposure to coal dust leads to a complex alternation of biological processes during regeneration and repair in the healing lung. However, the cellular and molecular mechanism of abnormal homeostasis in lung tissue are not explicit. Methods: This study mainly investigated the pulmonary toxicity of respirable-sized CDP in mice using unbiased single-cell RNA sequencing. CDP (less than 5μm) collected from the coal mine was analyzed by Scanning Electron Microscope (SEM) and Mass Spectrometer. In addition, western blotting, Elisa, QPCR was used to detect gene expression at mRNA or protein levels. Pathological analysis including HE staining, Masson staining, immunohistochemistry, and immunofluorescence staining were performed to characterize the structure and functional alternation in the pneumoconiosis mouse and verify the reliability of single-cell sequencing results.Results: SEM image and Mass Spectrometer analysis showed that coal dust particles generated during coal mine production have been crushed and screened with a diameter of less than 5 µm and contained less than 10% silica. Dust can intranasally enter the alveoli in mice. Compared with the control group, alveolar epithelial cells and macrophages showed apparent heterogeneity after long-term exposure to CDP. Alveolar structure and pulmonary microenvironment were destroyed, leading to pneumoconiosis after coal dust stimulation. A distinct abnormally increased 39 population of alveolar type 2 epithelial cells (AT2) was classified with a highly active state but reducing the antimicrobial-related protein expression of LYZ and Chia1 after CDP exposure. Unique 40 inflammatory factors and signaling pathways further characterized critical signal exchanges between epithelial cells and macrophages. Apoptosis, autophagy and necrosis pathways were activated. A new subset of macrophages with M2-type polarization double expressed MLPH+/CD206+ was found in mice having pneumoconiosis but markedly decreased after the vitamin D treatment. Activated MLPH+/CD206+ M2 macrophages secreted TGF-β1 and are sensitive to vitamin D treatment. Beclin1, LC3B, LAMP2, TGF-ß, and MLPH were up-regulated, promoting autophagy and pulmonary fibrosis. Vitamin D supplements can reduce the cytotoxicity induced by CDP. This study first reconstructed the pathologic progression and transcriptome landscape of coal-pneumoconiosis and the therapeutic opportunity of vitamin D in mice.