Integrating Single‐Cell and Experimental Analyses Uncover Macrophage Ferroptosis Is a Key Event in the Development of Chronic Obstructive Pulmonary Disease

Although ferroptosis, induced by cigarette smoke extracts (CSE), is crucial in chronic obstructive pulmonary disease (COPD) progression, its underlying mechanism remains unclear. The mRNA and protein expressions of relevant genes were investigated using immunofluorescence, immunohistochemistry, quantitative reverse-transcriptase PCR and western blotting. The proportion of macrophages in the lungs of COPD patients and the expression level of their ferroptosis-related genes were then investigated based on single-cell sequencing data retrieved from public databases (GSE171541 and GSE173896). Finally, cigarette smoke (CS)-induced ferroptosis in macrophages was investigated using in vitro (RAW264.7) and in vivo cell lines (BMDM), along with a mouse model of COPD. Single-cell sequencing revealed a significant decrease in the proportion of macrophages and a downregulation of their ferroptosis-related factors (Nrf2, SLC7A11, and GPX4) in COPD patients compared with the controls. Furthermore, gene and protein expressions of SLC7A11, Nrf2, and GPX4 decreased significantly, while those of NCOA4, ferritin, and TfR1 were significantly upregulated in CS-induced RAW264.7 cells and BMDM. Additionally, the macrophage proportions were markedly reduced in BALF samples from COPD mice. Finally, there was a significant increase in the total iron, ferrous ions and MDA content of lung tissues, while the GSH/GSSG content decreased drastically. CS-induced ferroptosis of macrophages is critical in COPD's pathogenesis, and targeting this process in macrophages could provide new strategies for treating the condition.