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 C57BL/6J mice.
Schematics showing the exposure profile and experimental design to understand the effects of exposure to differently flavored (fruit, menthol and tobacco) e-cig aerosols in the lungs of C57BL/6J mice using scRNA seq (A). Bar Graph showing the levels of metals as determined by ICP-MS in the aerosols captured daily during exposures using inExpose nose-only inhalation system from Scireq technologies (B). Lung morphometric changes observed using H&E staining of lung slices from air, PG:VG and differently flavored e-cig aerosol exposed mice lungs. 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 transcriptional profile of immune cell population upon exposure to differently flavored e-cig aerosols.
Male and female C57BL/6J mice (n = 2/sex/group) 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 24 major cell clusters 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 black-yellow coloring (B). Group-wise comparison of the UMAPs upon comparing PG:VG (blue), fruit (yellow), menthol (green) and tobacco (red) versus air (grey) groups following dimensionality reduction and clustering of scRNA seq data (C). Bar plot showing the number of significant (p < 0.05) differentially up-(green) and downregulated (red) genes in myeloid and lymphoid clusters in PGVG, fruit-, menthol-and tobacco-flavored e-cig aerosol exposed mouse lungs as compared to air controls. Here, AT1: alveolar type I, AT2: alveolar type II, Fibro: Fibroblast, MΦ: macrophage, 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 lungs reveal increase in neutrophil count through scRNA seq and flow cytometry.
Relative cell frequencies of alveolar macrophages (A) and neutrophils (B) across controls and flavored e-cig aerosol exposed mouse lungs as determined using scRNA seq. Representative flow plots (C) and bar graphs (D) showing sex-dependent changes in the percentages of neutrophils (CD45+ CD11b+ Ly6G+) and alveolar macrophage (CD45+ CD11b-SiglecF+) populations in lung digests from mice exposed to differently flavored e-cig aerosols. Values plotted and written in red on the flow plots are representative of the percentage of each cell population in the total CD45+ cells present in the lung homogenates from treatment and control groups. Data are shown as mean ± SEM (n = 3/sex/group). SE determined using two-way ANOVA with a Tukey post-hoc test for all cell means, to analyze the main effects of sex and treatment and their interaction. The two-way ANOVA results are shown in Supplementary File S3B.
Flow cytometry analyses show significant decrease in the percentage of eosinophils in the lungs of menthol and tobacco-flavored e-cig aerosol exposed C57BL/6J mice.
Representative flow plot (A) and bar graphs (B) showing the changes in the percentages of eosinophils (CD45+ CD11b+/- CD11c− Ly6G− SiglecF+) found in the lungs of differently flavored e-cig aerosol exposed mouse lungs as compared to air controls. Data are shown as mean ± SEM (n = 3/sex/group). *p<0.05, per two-way ANOVA with a Tukey post-hoc test for all cell means, to analyze the main effects of sex and treatment and their interaction. The two-way ANOVA results are shown in Supplementary File S3B. Values plotted and written in red on the flow plots are representative of the percentage of each cell population in the total CD45+ cells present in the lung homogenates from treatment and control groups.
Flow cytometry results show sex-specific flavor-dependent increase in CD8+ T cells in lungs of differently flavored e-cig aerosol exposed C57BL/6J mouse.
Relative cell frequencies of CD4+ (A) and CD8+ (B) T-cells across controls and flavored e-cig aerosol exposed mouse lungs as determined using scRNA seq. Representative flow plots (C) and bar graph (D) showing changes in the mean cell percentages of CD4+ (CD45+ CD11c− Ly6G− CD11b− MHCII− CD4+) and CD8+ (CD45+ CD11c− Ly6G− CD11b− MHCII− CD8+) T-cells in the lung tissue digests from male and female mice exposed to differently flavored e-cig aerosols as determined using flow cytometry. Values plotted and written in red on the flow plots are representative of the percentage of each cell population in the total CD45+ cells present in the lung homogenates from treatment and control groups. Data are shown as mean ± SEM (n = 3/sex/group). *p<0.05, **p<0.01 and ***p<0.001; per two-way ANOVA with a Tukey post-hoc test for all cell means, to analyze the main effects of sex and treatment and their interaction. The two-way ANOVA results are shown in Supplementary File S3B.
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. UMAP showing the 14 distinct cell populations identified upon subsetting and re-clustering the myeloid clusters from scRNA seq dataset from control and e-cig exposed mouse lungs (A). Marker plot showing the differential expression of highly expressed genes in the Ly6G+ and Ly6G-neutrophil cluster. The intensity of expression is indicated by the yellow-blue coloring; black represents nil value for expression for that gene (B). scRNA seq findings for presence of mature (Ly6G+) and immature (Ly6G-) neutrophils were validated by staining the tissue sections from tobacco-flavored e-cig aerosols and control (air) with Ly6G (green) and S100A8 (red, neutrophil activation marker). Representative images showing the co-immunostaining of Ly6G and S100A8 (shown as yellow puncta) (C) with respective quantification of relative fluorescence for Ly6G and S100A8 (D) in control and tobacco-flavored e-cig aerosol exposed mice. Data are shown as mean ± SEM (n = 4/group). SE calculated per Mann-Whitney U test for pairwise comparisons. Here, Neu: neutrophil, AM: alveolar macrophage, RAM: resident AM, pRAM: Proliferating RAM, IM: interstitial macrophage, CM: classical monocyte, NCM: non-classical monocyte, and DC: dendritic cell.
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) up/downregulated 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) up/downregulated 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) up/downregulated 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.
