Figures and data
Flavor-dependent changes in the levels of quantified metals, but no major histological damage on acute exposure to flavored e-cig aerosol in mice.
Schematics showing the exposure profile and experimental design to understand the effect of exposure to differently flavored e-cig aerosols in the lungs from C57BL/6J mice using scRNA seq (A)). Bar Graph showing the levels of metals in the aerosols captured each day of exposure using ICP-MS (B). Lung morphometric changes as observed using H&E staining of lung slices from air, PG:VG and differently flavored e-cig exposed mice. Representative images of n = 2/sex/group at 10X magnification is provided (C).
Table showing the levels of common elements found in the flavored e-liquids and e-cig aerosols as measured using ICP-MS.
scRNA seq analyses reveal maximum changes in the cell states of immune cell population upon exposure to differently flavored e-cig aerosols.
Male and female C57BL/6J mice were exposed to 5-day nose-only exposure to differently flavored e-cig aerosols. The mice were sacrificed after the final exposure and lungs from air (control) and differently flavored e-cig aerosol (fruit, menthol, and tobacco)-exposed mice were used to perform scRNA seq. UMAP plot of 71,725 cells captured during scRNA seq showing the four major cell populations identified from control and experimental mouse lungs (A) and the expression of canonical markers used for identifying stromal (Col3a1), epithelial (Sftpa1), endothelial (Cldn5) and immune (Ptprc) cell populations. The intensity of expression is indicated by the red-yellow coloring (B). The cell frequencies (plotted as counts) of different cell clusters in each sample type showing the sex-dependent variations in the cell composition on exposure to differently flavored e-cig aerosols (C). Group-wise comparison of the UMAPs upon comparing PG:VG (blue), fruit (yellow), menthol (green) and tobacco (red) versus air (air) groups following dimensionality reduction and clustering of scRNA seq data (D). Here, AT1: alveolar type I, AT2: alveolar type II, SMC: smooth muscle cell, gCap: general capillary, aCap: alveolar capillary, and NK: natural killer.
Cellular composition of myeloid cells in air and e-cig aerosol exposed mouse lung reveal sex-specific increase in neutrophil count through scRNA seq but not flow cytometry.
Cell frequencies (represented as counts) of alveolar macrophages (A) and neutrophils (B) across controls and flavored e-cig aerosol exposed mouse lungs were plotted in a sex-specific manner. Representative flow plots (C) and bar graphs (D) showing sex-dependent changes in the percentages of neutrophils (CD45+ CD11b+ Ly6G+) and alveolar macrophages (CD45+ CD11b-SiglecF+) populations in lung digests from mice exposed to differently flavored e-cig aerosols. Data are shown as mean ± SEM (n = 3/sex/group).
Co-immunofluorescence validates the increase of Ly6G– and Ly6G+ neutrophil population in the tobacco flavored e-cig exposed female C57BL/6J mice.
The myeloid cell clusters were subsetted to identify two populations of neutrophils with and without the presence of Ly6G cell marker representing mature and immature neutrophils respectively (Figure S3C). Bar graph showing the sex-specific changes in the cell frequencies (denoted as counts) of Ly6G+ (A) and Ly6G- (B) neutrophils in the lung of tobacco-flavored e-cig aerosol exposed mouse lung as compared to air control. scRNA seq findings were validated by staining the tissue sections from tobacco-flavored e-cig aerosols and control (air) with Ly6G (green) and S100A8 (red, pan-neutrophil marker). Representative images showing the co-immunostaining of Ly6G and S100A8 (shown as yellow puncta) in female (C) and male (D) control and tobacco-flavored e-cig aerosol exposed mice. Here Ly6G+ neutrophils are represented by double staining using yellow arrows, while Ly6G- neutrophils are denoted by red arrows.
scRNA seq analyses and flow cytometry result show flavor-dependent increase in CD8 T cells in lungs of differently flavored e-cig aerosol exposed C57BL/6J mouse.
The cell frequencies (denoted as counts) of CD4 (A) and CD8 (B) T-cells showing the sex-dependent variations in the cellular composition upon exposure to differently flavored e-cig aerosols. Representative flow plots (C) and bar graph (D) showing changes in the mean counts of CD4+ and CD8+ T-cells in the lung tissue digest from male and female mice exposed to differently flavored e-cig aerosols as determined using flow cytometry. Data are shown as mean ± SEM (n = 3/sex/group). *p<0.05, and **p<0.01, per Tukey post hoc two-way ANOVA comparison.
Exposure to fruit flavored e-cig aerosols result in activation of oxidative stress-mediated innate immunity in C57BL/6J mouse lungs.
Male and female C57BL/6J mice were exposed to 5-day nose-only exposure to fruit-flavored e-cig aerosols. The mice were sacrificed after the final exposure and mouse lungs from air (control) and aerosol (fruit-flavored) exposed groups was used to perform scRNA seq. Heatmap and bar plot showing the DESeq2 (i) and GO analyses (ii) results from the significant (p<0.05) DEGs in the myeloid (A) and lymphoid (B) cell cluster from fruit-flavored e-cig aerosol exposed mouse lungs as compared to controls.
Exposure to Menthol flavored e-cig aerosols result in activation of innate immune responses C57BL/6J mouse lungs.
Male and female C57BL/6J mice were exposed to 5-day nose-only exposure to menthol-flavored e-cig aerosols. The mice were sacrificed after the final exposure and mouse lungs from air (control) and aerosol (menthol-flavored) exposed groups was used to perform scRNA seq. Heatmap and bar plot showing the DESeq2 (i) and GO analyses (ii) results from the significant (p<0.05) DEGs in the myeloid (A) and lymphoid (B) cell cluster from menthol-flavored e-cig aerosol exposed mouse lungs as compared to controls.
Exposure to Tobacco flavored e-cig aerosols result in activation of cytolysis and neutrophil chemotaxis in C57BL/6J mouse lungs.
Male and female C57BL/6J mice were exposed to 5-day nose-only exposure to tobacco-flavored e-cig aerosols. The mice were sacrificed after the final exposure and mouse lungs from air (control) and aerosol (tobacco-flavored) exposed groups was used to perform scRNA seq. Heatmap and bar plot showing the DESeq2 (i) and GO analyses (ii) results from the significant (p<0.05) DEGs in the myeloid (A) and lymphoid (B) cell cluster from tobacco-flavored e-cig aerosol exposed mouse lungs as compared to controls.
List of top dysregulated genes on exposure to differently flavored (fruit, menthol and tobacco) e-cig aerosol in C57BL/6J mouse lungs
