Single-cell transcriptomics identifies an increased Ly6G- neutrophil population 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, i.e. increased oxidative stress and immunotoxicity. 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). scRNA profiles of 71,725 cells were generated from control and treatment groups (n=2/sex/group). An increase in the Ly6G- neutrophil population was identified in lungs exposed to tobacco flavored e-cig aerosol. Differential gene expression analyses identified dysregulation of T-cell mediated pro-inflammation (Cct7, Cct8) and stress-response signals (Neurl3, Stap1, Cirbp and Htr2c) in the lungs of mice exposed to e-cig aerosols, with pronounced effects for tobacco flavor. Flow cytometry analyses and cytokine/chemokine assessments within the lungs corroborate the scRNA seq data, demonstrating an increase in the T-cell percentages and levels of T-cell associated cytokine/chemokines in the lungs of tobacco-flavored aerosol exposed mice. Increased levels of Klra4 and Klra8 expression also suggest an enhanced natural killer (NK) cell activity in this mouse group. Interestingly, the increase in the level of Ly6G – (immature) neutrophils upon exposure to tobacco-flavored e-cig aerosols was noticed for female C57BlL6J mice and not their male counterparts which was validated through immunofluorescence staining. Overall, this study identifies sex-specific increase in the percentages of Ly6G- neutrophils that may be responsible for dampened innate immune responses in females and heightened T-cell cytotoxicity in male mouse lungs of tobacco-flavored e-cig aerosol exposed mice.