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) increases susceptibility to acute lung injury, yet how CS reshapes epithelial responses to subsequent insults remains unclear, and the extent to which aberrant epithelial responses are linked to epigenetic changes in vivo is uncertain. In this pilot, proof-of-principle study, we performed paired single-cell RNA-seq and ATAC-seq after chronic CS exposure followed by influenza or hyperoxia, generating joint expression–accessibility maps across epithelial subsets and inferring candidate transcriptional regulators. Influenza and hyperoxia elicited distinct epithelial programs. Prior CS exposure amplified epigenetically regulated inflammatory signaling (cytokine/chemokine modules coupled to ERK/MAPK and PI3K) and suppressed epigenetically regulated reparative and differentiation pathways (γ-secretase/NOTCH). Transcription factor motif enrichment implicated ETS family factors and REL as candidate regulators of the CS-augmented responses in AT2 and ciliated cells. Paired single-cell RNA/ATAC profiling captures injury-conditioned epithelial programs and their epigenetic context after chronic CS exposure, demonstrating feasibility and providing a framework to prioritize targets of exposure-related aberrant responses.