Single-cell analysis reveals smoke-induced neutrophil-macrophage crosstalk as a targetable mechanism in COPD pathogenesis

Background Chronic obstructive pulmonary disease (COPD) is projected to become the third leading cause of death globally by 2030, with cigarette smoke (CS) exposure accounting for 80-90% of cases. This study aimed to elucidate the intercellular communication networks amplified by CS exposure and identify targetable pathways driving smoking-related COPD pathogenesis. Methods We performed single-cell RNA sequencing (scRNA-seq) analysis of lung tissues from nine COPD patients stratified by smoking status (GSE173896), integrating ligand-receptor interaction inference (CellChat), pseudo-temporal trajectory analysis (Monocle), phenotype-cell linkage (Scissor), and weighted gene co-expression network analysis (WGCNA) with bulk RNA-seq data (GSE57148). Smoking-associated immune cell subpopulations were identified, and pathogenic gene modules were prioritized through functional enrichment analysis. Key computational predictions were validated in C57BL/6 mice exposed to cigarette smoke for 90 days, with assessment of pulmonary function, histopathology, serum cytokines (ELISA), immunofluorescence, Western blot, and lung myeloperoxidase (MPO) activity. Results After quality control, 40,500 cells were analyzed (28,630 from COPD and 11,870 from never-smoker control tissues). At the sample level, COPD tissues showed a lower proportion of NK cells than controls (FDR = 0.011) and a trend toward more macrophages. Intercellular communication analysis revealed neutrophils and macrophages as dominant signaling hubs, and ANXA1-FPR1/FPR2 was among the most prominent neutrophil-to-macrophage ligand-receptor pairs. Trajectory analysis followed by sub-clustering and Scissor analysis prioritized macrophage and neutrophil subpopulations associated with the COPD phenotype in bulk lung tissue. Enrichment analysis of these modules pointed to neutrophil extracellular trap (NET) formation and HIF-1 signaling. In vivo validation confirmed CS-induced pulmonary dysfunction, elevated serum cytokines (IL-1β, TNF-α, IL-6; all p < 0.05), increased macrophage infiltration with FPR2 upregulation (p < 0.01), neutrophil accumulation (p < 0.01) and NET formation, increased lung MPO activity (p < 0.001), and HIF-1α protein induction (p < 0.001). Conclusion CS exposure drives chronic pulmonary inflammation through a self-amplifying neutrophil-macrophage circuit mediated by HIF-1α-induced NET formation and dysregulated ANXA1-FPR2 signaling. This study establishes intercellular crosstalk networks as central mechanisms linking environmental tobacco exposure to COPD pathogenesis. Single-cell profiling stratified by environmental exposure may inform early biomarker development and guide pathway-specific interventions for smoke-related lung diseases.