BHLHE40 driven CCL3+neutrophils promote emphysema after particulate matter exposure

<bold>Introduction:</bold> Exposure to particulate matter (PM) can lead to the occurrence and development of chronic obstructive pulmonary disease (COPD). Neutrophils are important immune cells in the formation of COPD. This study aims to explore the heterogeneity and mechanism of neutrophils in the formation of emphysema caused by PM exposure. Method: C57BL/6 mice were exposed to PM once a week for three consecutive months. Single-cell RNA sequencing and spatial transcriptomics were performed. Flow cytometry, immunofluorescence staining, in vitro experiments, and tracheal instillation of isolated cells were used to validate the results of bioinformatics analysis. <bold>Results:</bold> Our study found that there is a specific CCL3+neutrophil subtype in the lungs of mice after PM exposure. Compared with the CCL3-neutrophil subtype, it has the function of releasing ROS and extracellular vesicles, and is in an aging state. Meanwhile, CCL3+neutrophils express more elastase compared to CCL3-neutrophils. Intratracheal instillation of CCL3+neutrophils are more likely to lead to the formation of emphysema than CCL3- neutrophils. CCL3+neutrophils highly express the transcription factor BHLHE40, and overexpression of BHLHE40 in dhl-60 cells can lead to high expression of elastase. In addition, CCL3+neutrophils can induce MMP12+macrophage aggregation through CCL3-CCR1 pathway, and jointly participate in the formation of emphysema. <bold>Conclusion:</bold> Our study confirms that BHLHE40 is a key regulatory factor for the development of CCL3+neutrophils. CCL3+neutrophils can release more elastase and induce MMP12+macrophage aggregation to participate in emphysema formation through the CCL3-CCR1 pathway.