Integrative Single-Cell RNA and ATAC Sequencing Reveals the Impact of Chronic Cigarette Smoking on Lung Epithelial Responses to Influenza and Hyperoxia

Cigarette smoke (CS) is a significant risk factor for developing acute respiratory distress syndrome (ARDS), but the cellular and molecular mechanisms linking smoking to ARDS susceptibility remain unclear. Our goal was to improve our understanding of these mechanisms. To address this, we established a mouse model comparing long-term CS exposure to non-smoking controls, examining responses to influenza infection and hyperoxia-induced lung injury. The mice were divided into six groups (n=1 per group): control (CON), cigarette smoke (CS), hyperoxia (HYP), influenza infection (FLU), cigarette smoke plus hyperoxia (CS+HYP), and cigarette smoke plus influenza infection (CS+FLU). Single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) were performed on lung tissues. Quality control analysis using Seurat (v5.1.0) and Signac (v1.13.1) retained 78,402 cells from scRNA-seq and 84,144 cells from scATAC-seq, with 32,305 matched cells identified across both datasets. Differential gene expression analysis revealed significant smoking-associated alterations in cellular responses to influenza infection and hyperoxia exposure. Pathway enrichment indicated heightened immune responses, inflammatory signaling, and cellular survival pathways in smoking-exposed animals. Integration of scRNA-seq and scATAC-seq identified key transcription factors (TFs), including those involved in immune regulation, tissue repair, and chromatin remodeling mediating these responses. Overall, this study underscores the role of chronic cigarette smoke exposure in exacerbating pathways critical to ARDS pathogenesis, providing potential targets for therapeutic intervention.