Single-cell transcriptomics identifies altered neutrophil dynamics and accentuated T-cell cytotoxicity in tobacco-flavored e-cigarette-exposed mouse lungs.

Despite the growing public health threat of electronic cigarettes (e-cigs), the cell-specific immune responses to differently flavored e-cig exposure remain poorly understood. To bridge this gap, we characterized the lung immune landscape following acute nose-only exposure to flavored e-cig aerosols in vivo using single-cell RNA sequencing (scRNA seq) in mice. Metal analysis of daily generated aerosols revealed flavor-dependent, day-to-day variation in metal (Ni, Cu, K, and Zn) leaching. scRNA seq profiling of 71,725 lung cells from control and exposed mice revealed pronounced dysregulation of myeloid cell function in menthol (324 differentially expressed genes, DEGs) and tobacco (553 DEGs) flavors, and lymphoid cell dysregulation in fruit-flavor (112 DEGs) e-cig aerosol exposed mouse lung, compared to air controls. Flow cytometry corroborated these findings, showing increased neutrophil frequencies and reduced eosinophil counts in menthol- and tobacco-exposed lungs. Flavored e-cig exposure also increased CD8(+) T-cell proportions, upregulated inflammatory gene expression (Stat4, Il1b, Il1bos, Il1ra, and Cxcl3), and enriched terms like 'Th1 cytokine signaling' and 'NK cell degranulation'. Notably, tobacco-flavored e-cig aerosol exposure increased immature (Ly6G⁻) neutrophils and reduced S100A8 expression, suggesting altered neutrophil activation in vivo. Overall, this study identifies flavor-dependent immune alterations in the lung following acute e-cig aerosol exposure and provides a foundation for future mechanistic studies.