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 complete molecular changes associated with pulmonary homeostasis caused by respiratory coal dust particles remain unclear.
Methods
This study mainly investigated the pulmonary toxicity of respirable-sized CDP in mice using unbiased single-cell RNA sequencing. CDP (< 5 μm) 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. Alveolar structure and pulmonary microenvironment were destroyed, inflammatory and death (apoptosis, autophagy, and necrosis) pathways were activated, leading to pneumoconiosis in post 9 months coal dust stimulation. A distinct abnormally increased alveolar type 2 epithelial cell (AT2) were classified with a highly active state but reduced the antimicrobial-related protein expression of LYZ and Chia1 after CDP exposure. Beclin1, LC3B, LAMP2, TGF-ß, and MLPH were up-regulated induced by CDP, promoting autophagy and pulmonary fibrosis. 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.
Conclusions
This is the first study to reconstruct the pathologic progression and transcriptome pattern of coal pneumoconiosis in mice. Coal dust had obvious toxic effects on lung epithelial cells and macrophages and eventually induced pulmonary fibrosis. CDP-induced M2-type macrophages could be inhibited by VD, which may be related to the alleviation of the pulmonary fibrosis process.
Background
Silicosis is a severe pulmonary disease caused by inhaling dust containing crystalline silica. The progression of silicosis to pulmonary fibrosis is usually unavoidable. Recent studies have revealed positivity for the overexpression of C-X-C chemokine receptor type 4 (CXCR4) in pulmonary fibrosis and shown that the CXCR4 inhibitor AMD3100 attenuated pulmonary fibrosis after bleomycin challenge and paraquat exposure. However, it is unclear whether AMD3100 reduces crystalline silica-induced pulmonary fibrosis.
Methods
C57BL/6 male mice were instilled intranasally with a single dose of crystalline silica (12 mg/60 μL) to establish an acute silicosis mouse model. Twelve hours later, the mice were injected intraperitoneally with 5 mg/kg AMD3100 or control solution. Then, the mice were weighed daily and sacrificed on day 7, 14, or 28 to collect lung tissue and peripheral blood. Western blotting was also applied to determine the level of CXCR4, while different histological techniques were used to assess pulmonary inflammation and fibrosis. In addition, the level of B cells in peripheral blood was measured by flow cytometry.
Results
CXCR4 and its ligand CXCL12 were upregulated in the lung tissues of crystalline silica-exposed mice. Blocking CXCR4 with AMD3100 suppressed the upregulation of CXCR4/CXCL12, reduced the severity of lung injury, and prevented weight loss. It also inhibited neutrophil infiltration at inflammatory sites and neutrophil extracellular trap formation, as well as reduced B-lymphocyte aggregates in the lung. Additionally, it decreased the recruitment of circulating fibrocytes (CD45
+
collagen I
+
CXCR4
+
) to the lung and the deposition of collagen I and α-smooth muscle actin in lung tissue. AMD3100 also increased the level of B cells in peripheral blood, preventing circulating B cells from migrating to the injured lungs.
Conclusion
Blocking CXCR4 with AMD3100 delays pulmonary inflammation and fibrosis in a silicosis mouse model, suggesting the potential of AMD3100 as a drug for treating silicosis.
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