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

E-cigarettes (e-cigs) are a public health concern for young adults due to their rising popularity despite evidence of harmful effects. Yet an extensive study defining the cell-specific immune changes upon exposure to flavored e-cigs remains elusive. To determine the immunological lung landscape upon acute nose-only exposure of C57BL/6J to flavored e-cig aerosols, we performed single-cell RNA sequencing (scRNA seq). Analyses of the levels of metals in the e-cig aerosol generated daily during exposure revealed a flavor-dependent variation in the day-to-day leaching of the levels of metals like Ni, Cu, K and Zn, among others. scRNA profiles of 71,725 cells generated from control and treatment groups (n=2/sex/group) found maximum dysregulation of (a) myeloid cell function in menthol (324 differentially enriched genes (DEG)) and tobacco (553 DEGs) –flavor exposed and (b) lymphoid cell function in fruit (112 DEGs)-flavored e-cig aerosol exposed mouse lungs as compared to air. Flow cytometry analyses identified marked increase in the neutrophil percentage and a decrease in the eosinophil count in menthol and tobacco-flavored e-cig aerosol exposed mouse lungs which corroborated with our scRNA seq data. We further found an: (a) increase in CD8+ T cell percentages, (b) upregulation of inflammatory genes like Stat4 , Il1bos , Il1b , Il1ra and Cxcl3 and (c) enrichment of terms like ‘T-helper cell 1cytokine function’ and ‘NK cell degranulation’ in the lungs of e-cig aerosol exposed mouse when compared to control. Interestingly, the increase in the level of immature neutrophils characterized by Ly6G deficiency and reduction in the S100A8 (marker for neutrophil activation) using immunofluorescence in tobacco-flavored e-cig aerosol exposed mouse lung sections point towards a possible shift in the neutrophil dynamics upon exposure to e-cigs. Overall, this study identifies flavor dependent changes in myeloid and lymphoid-cell mediated responses in the mouse lungs exposed to acute nose-only e-cig aerosols and provides a resource to influence future research in select /specific cell types to understand the immunological implications of long-term use of e-cigs.