Figures and data
E-cig aerosols show flavor-dependent changes in the levels of quantified metals
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) and the nose only exposure system used for performing the mouse experiment (B). The exposure characteristics were assessed by measuring the serum cotinine levels in the blood of exposed and control mice. Data are shown as mean ± SEM (n = 4/group); ns: not significant. **p<0.01, and ***p<0.001 vs air, per one-way ANOVA for multiple comparison (C) and analyzing the levels of metals in the aerosols captured from each day of exposure using ICP-MS (D). Lung morphometric changes as observed using H&E staining of lung slices from air, PGVG and differently flavored e-cig exposed mice. Representative images of n = 2/sex/group at 10X magnification is provided (E) and quantified values for mean linear intercept (Lm) plotted in a sex-specific manner (F)
Table showing the levels of common elements found in the flavored e-liquids and e-cig aerosols in as measured using ICP-MS.
scRNA seq analyses reveals 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 percentage) 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). Bar graphs showing the number of significant DEGs (p <0.01) found to be up (blue) and down (orange)-regulated in each cell cluster in differently flavored e-cig aerosol exposed mouse lungs as compared to air controls as determined by aggregate analyses for both sexes in each group using DESeq2 (D).
DESeq 2 results showing the number of significantly (padj< 0.05) up- or downregulated genes in different cell clusters
Cellular composition of myeloid cells in air and e-cig aerosol exposed mouse lung reveal a unique population of neutrophils.
A total of 14 unique cell clusters were identified from the myeloid cell subset of air and e-cig aerosol exposed mouse lung (A). UMAP plot showing the cell cycle state of different cell types in the myeloid cluster as determined using the Cell cycle scoring in Seurat (B). The heatmap showing the most DEGs in each cluster across the myeloid subset. These differential expressions were used to determine and name the cell types in this cluster. The intensity of expression is indicated by the purple coloring (C). The cell percentages of different cell types in the myeloid cluster showing the sex-dependent variations in the cell composition on exposure to differently flavored e-cig aerosols (D). Flow cytometry results showing sex-dependent changes in the percentages of lung neutrophils (CD45+ CD11b+ Ly6G+) in lung tissue digest (E) and alveolar macrophages (CD45+ CD11b-SiglecF+) in BALF (F) from mice exposed to differently flavored e-cig aerosols. Data are shown as mean ± SEM (n = 3/sex/group). *p<0.05, and ****p<0.0001, per Tukey post hoc two-way ANOVA comparison.
Table showing the cell types identified from myeloid and lymphoid clusters and their abbreviations
Exposure to flavored e-cig aerosols result in dampening of neutrophil-mediated innate immune responses in C57BL/6J mouse lungs.
Heatmap and bar plot showing the DESeq2 and GO analyses results respectively from the DEGs in the myeloid cell cluster from Fruit (A & B), menthol (C&D) and tobacco (E&F)-flavored e-cig aerosol exposed mouse lungs as compared to air controls. Heatmap (G) and bar plot (H) showing the DESeq2 and GO analyses results respectively from the DEGs in the Ly6G-neutrophil cluster from air and flavored e-cig (fruit, menthol and tobacco) aerosol exposed mouse lungs.
Exposure to tobacco-flavored e-cig aerosols results in significant increase in lung T-cell percentages in C57Bl/6J mice.
A total of 10 unique cell clusters were identified from the lymphoid cell subset of air and e-cig aerosol exposed mouse lung (A). The heatmap showing the most DEGs in each cluster across the myeloid subset. These differential expressions were used to determine and name the cell types in this cluster. The intensity of expression is indicated by the purple coloring (B). The cell percentages of different cell types in the lymphoid cluster showing the sex-dependent variations in the cell composition on exposure to differently flavored e-cig aerosols (C). Flow cytometry results showing changes in the percentages of lung CD4+ and CD8+ T-cells in the lung tissue digest (D) from mice exposed to differently flavored e-cig aerosols as depicted in a sex-specific manner. Data are shown as mean ± SEM (n = 3/sex/group). *p<0.05, **p<0.01, and ****p<0.0001, per Tukey post hoc two-way ANOVA comparison.
Exposure to tobacco flavored e-cig aerosols result in activation of T-cytotoxic responses in C57BL/6J mouse lungs.
Heatmap and bar plot showing the DESeq2 and GO analyses results respectively from the DEGs in the lymphoid cell cluster from Fruit (A & B), menthol (C&D) and tobacco (E&F)-flavored e-cig aerosol exposed mouse lungs as compared to air controls.
GO analyses of the commonly dysregulated genes show enrichment of dampened innate immune response upon exposure to flavored e-cig aerosols in C57BL/6J mice.
Heat map showing the sex-dependent average fold changes in the levels of cytokines/chemokines in the lung of differently flavored e-cig aerosol exposed C57Bl/6J mouse lungs as compared to air controls. Data are shown as mean ± SEM (n = 3/sex/group) (A). Heatmap showing the fold changes in the expression of commonly dysregulated genes in the myeloid cluster and lymphoid clusters in mouse lungs exposed to flavored e-cig aerosols as compared to ambient air as determined after DESeq2 analyses (B). CNET plot results showing the pathways regulated by the common DEGs on acute (5-day) exposure to differently flavored fruit, menthol, and tobacco) e-cig aerosols in C57Bl/6J mouse lungs (B).
List of top dysregulated genes on exposure to differently flavored (fruit, menthol and tobacco) e-cig aerosol in C57Bl/6J mouse lungs
