Single-cell transcriptomics identifies altered neutrophil dynamics and accentuated T-cell cytotoxicity in tobacco-flavored e-cigarette-exposed mouse lungs
- Department of Environmental Medicine, University of Rochester Medical Center, United States
Figures
Figure 1 with 1 supplement
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 (Figure 1—source data 1) as determined by inductively coupled plasma mass spectrometry (ICP-MS) in the aerosols captured daily during exposures using inExpose nose-only inhalation system from Scireq technologies (B). Lung morphometric changes observed using hematoxylin and eosin (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 ×10 magnification are provided (C).
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Figure 1—source data 1
Day-wise levels of Ni, Cu, K, and Zn as determined by inductively coupled plasma mass spectrometry (ICP-MS) in the aerosols captured daily during exposures using the inExpose nose-only inhalation system from Scireq technologies as plotted in Figure 1B.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig1-data1-v1.zip
Figure 1—figure supplement 1
Schematics and characteristics of exposure system used for in vivo experiments.
The nose-only exposure system used for performing the mouse experiment (A). The exposure characteristics were assessed by measuring the serum cotinine levels (Figure 1—figure supplement 1—source data 1) in the blood of exposed and control mice. Data are shown as mean ± SEM (n = 4/group); ns: not significant. **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus air, per one-way ANOVA for multiple comparison (B).
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Figure 1—figure supplement 1—source data 1
Serum cotinine levels in the blood of differently flavored e-cig aerosol exposed and control (air and PG:VG) C57BL/6J mice as plotted in Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig1-figsupp1-data1-v1.zip
Figure 2 with 1 supplement
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. Uniform Manifold Approximation and Projection (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 (gray) 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 (Figure 2—source data 1) in PG:VG, fruit-, menthol-, and tobacco-flavored e-cig aerosol exposed mouse lungs as compared to air controls (D). Here, AT1: alveolar type I, AT2: alveolar type II, Fibro: fibroblast, M: macrophage, SMC: smooth muscle cell, gCap: general capillary, aCap: alveolar capillary, and NK: natural killer.
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Figure 2—source data 1
Number of significant (p < 0.05) differentially up- and downregulated genes in the myeloid and lymphoid clusters in PG:VG, fruit-, menthol-, and tobacco-flavored e-cig aerosol exposed mouse lungs when compared to air controls as plotted in Figure 2D.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig2-data1-v1.zip
Figure 2—figure supplement 1
Quality check of the scRNA seq data generated using 10X Genomics pipeline.
Figure showing the normalized counts, features, and mitochondrial gene percentage in the integrated single-cell data before (A) and after (B) normalization. Cell frequencies of major cell clusters (epithelial, endothelial, stromal, myeloid, and lymphoid) (Figure 2—figure supplement 1—source data 1) in control (air) and exposed (PG:VG, fruit, menthol, and tobacco) lungs as determined by scRNA seq (C). n = 2/sex/group.
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Figure 2—figure supplement 1—source data 1
Cell frequencies of major cell clusters (epithelial, endothelial, stromal, myeloid, and lymphoid) in control (air) and exposed (PG:VG, fruit, menthol, and tobacco) mouse lungs in each sample as determined by scRNA seq after filtering, clustering, and dimensionality reduction as plotted in Figure 2—figure supplement 1C.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig2-figsupp1-data1-v1.zip
Figure 3 with 2 supplements
Cellular composition of myeloid cells in air and e-cig aerosol exposed mouse lungs reveals an 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 (Figure 3—source data 1) 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 (Figure 3—source data 2) 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 1c.
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Figure 3—source data 1
Relative cell frequencies of alveolar macrophages and neutrophils across controls and flavored e-cig aerosol exposed mouse lungs as determined using scRNA seq as plotted in Figure 3A, B.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig3-data1-v1.zip
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Figure 3—source data 2
Values showing the sex-dependent changes in the percentages of macrophages and neutrophils out of total CD45+ cells in lung digests from mice exposed to differently flavored e-cig aerosols as determined using flow cytometry as plotted in Figure 3D, Figure 3—figure supplement 1A, B.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig3-data2-v1.zip
Figure 3—figure supplement 1
Inclusion of PG:VG + Nic group proves that nicotine is not the sole contributor to altered immune response in e-cig aerosol exposed mouse lungs.
Flow cytometry results showing changes in relative percentages of alveolar macrophages (A), neutrophils (B), eosinophils (C), CD4 T cells (D), and CD8 T cells (E) in the lung digests from exposed (PG:VG, PG:VG + Nic, fruit, menthol, and tobacco) and control (air) mice following 5-day acute exposure. The data represented in this figure is an extension of the data represented in Figures 3—5 of the main manuscript with the addition of an extra group (PG:VG + Nic). Data are shown as mean ± SEM (n = 3/sex/group). SE: * p<0.05, ** p <0.01 and **** p<0.0001 as 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.
Figure 3—figure supplement 2
Gating strategy for the flow cytometry-based experiments.
Representative plots showing the gating strategy used to gate for alveolar macrophages, neutrophils, eosinophils, CD4+ and CD8+ T cells using flow cytometry. Data is analyzed using FlowJo software.
Figure 4
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 plots (A) and bar graphs (B) showing the changes in the percentages of eosinophils (CD45+ CD11b+/− CD11c− Ly6G− SiglecF+) (Figure 4—source data 1) 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 1c. 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.
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Figure 4—source data 1
Values showing the sex-dependent changes in the percentages of eosinophils out of total CD45+ cells in lung digests from mice exposed to differently flavored e-cig aerosols as determined using flow cytometry as plotted in Figure 4B, Figure 3—figure supplement 1C.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig4-data1-v1.zip
Figure 5
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 (Figure 5—source data 1) 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 (Figure 5—source data 2) 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 1c.
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Figure 5—source data 1
Relative cell frequencies of CD4 and CD8 T cells across controls and flavored e-cig aerosol exposed mouse lungs as determined using scRNA seq as plotted in Figure 5A, B.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig5-data1-v1.zip
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Figure 5—source data 2
Values showing the sex-dependent changes in the percentages of CD4 and CD8 T cells out of total CD45+ cells in lung digests from mice exposed to differently flavored e-cig aerosols as determined using flow cytometry as plotted in Figure 5D, Figure 3—figure supplement 1D, E.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig5-data2-v1.zip
Figure 6 with 1 supplement
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. Uniform Manifold Approximation and Projection (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) at 20X magnification (C) with respective quantification of relative fluorescence for Ly6G and S100A8 (Figure 6—source data 1) (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.
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Figure 6—source data 1
Mean value of relative fluorescence as determined from 6 to 10 random images captured from tissue sections from control and e-cig aerosol exposed mouse lungs quantitating Ly6G+ and S100A8+ puncta as plotted in Figure 6D.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig6-data1-v1.zip
Figure 6—figure supplement 1
Co-immunofluorescence shows loss of S100A8 positive cells in tobacco-flavored e-cig aerosol exposed mouse lungs.
Co-immunofluorescence results showing single channel staining for DAPI (blue channel), Ly6G (green channel), and S100A8 (red channel) along with the merged images in lung tissue sections from control and tobacco-flavored e-cig aerosol treated mouse lungs at 20X magnification. The above image is an extension of the data presented in Figure 6C, D of the main manuscript.
Figure 7
Exposure to fruit-flavored e-cig aerosols results 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 were 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 differentially expressed genes (DEGs) in the myeloid (A) and lymphoid (B) cell cluster (Figure 7—source data 1) from fruit-flavored e-cig aerosol exposed mouse lungs as compared to controls. Data is representative of n = 2/sex/group.
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Figure 7—source data 1
Z-score table showing significant (p < 0.05) differentially expressed genes (DEGs) and top 10 GO terms associated with the respective genes in the myeloid and lymphoid cluster from mouse lungs exposed to 5-day nose-only exposure to fruit-flavored e-cig aerosol when compared to air control as plotted in Figure 7A(i, ii), B(i, ii).
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig7-data1-v1.zip
Figure 8
Exposure to menthol-flavored e-cig aerosols results in activation of innate immune responses in 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 were 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 differentially expressed genes (DEGs) in the myeloid (A) and lymphoid (B) cell cluster (Figure 8—source data 1) from menthol-flavored e-cig aerosol exposed mouse lungs as compared to controls. Data is representative of n = 2/sex/group.
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Figure 8—source data 1
Z-score table showing significant (p < 0.05) differentially expressed genes (DEGs) and top 10 GO terms associated with the respective genes in the myeloid and lymphoid cluster from mouse lungs exposed to 5-day nose-only exposure to menthol-flavored e-cig aerosol when compared to air control as plotted in Figure 8A(i, ii), B(i, ii).
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig8-data1-v1.zip
Figure 9 with 1 supplement
Exposure to tobacco-flavored e-cig aerosols results 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 were 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 differentially expressed genes (DEGs) in the myeloid (A) and lymphoid (B) cell cluster from (Figure 9—source data 1) tobacco-flavored e-cig aerosol exposed mouse lungs as compared to controls. Data is representative of n = 2/sex/group.
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Figure 9—source data 1
Z-score table showing significant (p < 0.05) differentially expressed genes (DEGs) and top 10 GO terms associated with the respective genes in the myeloid and lymphoid cluster from mouse lungs exposed to 5-day nose-only exposure to tobacco-flavored e-cig aerosol when compared to air control as plotted in Figure 9A(i, ii), B(i, ii).
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig9-data1-v1.zip
Figure 9—figure supplement 1
Exposure to flavored e-cig aerosol results in dysregulated chemokine signaling and T-cell activation.
The levels of pro-inflammatory cytokines/chemokines in the lung digests (Figure 9—figure supplement 1—source data 1) from experimental (PG:VG, PG:VG + Nic, fruit, menthol, and tobacco) and control (air) were assessed using multianalyte assay. The results obtained were plotted as a heatmap with the z-scores represented between the scale of orange (low) to blue (high) (A). Heatmap showing the fold changes in the expression of commonly dysregulated genes in the myeloid and lymphoid clusters (Figure 9—figure supplement 1—source data 2) 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 differentially expressed genes (DEGs) (across all cell types) on acute (5-day) exposure to differently flavored (fruit, menthol, and tobacco) e-cig aerosols in C57BL/6J mouse lungs (C). Data is representative of n = 2/sex/group.
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Figure 9—figure supplement 1—source data 1
Z-score values of the levels of cytokine/chemokine obtained in per mg of protein from mouse lung tissue samples following 5-day nose-only exposure to PG:VG, PG:VG + Nic, fruit, menthol, or tobacco-flavored e-cig aerosols as plotted in Figure 9—figure supplement 1A.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig9-figsupp1-data1-v1.zip
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Figure 9—figure supplement 1—source data 2
Z-score values of the gene expression of commonly dysregulated genes in the mouse lung tissue samples following 5-day nose-only exposure to PG:VG, fruit, menthol, or tobacco-flavored e-cig aerosols as compared to air controls as plotted in Figure 9—figure supplement 1B.
- https://cdn.elifesciences.org/articles/106380/elife-106380-fig9-figsupp1-data2-v1.zip
Tables
Table 1
The levels of common elements found in the flavored e-liquids and e-cig aerosols as measured using inductively coupled plasma mass spectrometry (ICP-MS).
| Element | E-liquid; ppb/mg of e-liquid | E-cig aerosol (mean ± SD); ppb/mg of e-liquid | ||||||
|---|---|---|---|---|---|---|---|---|
| PG:VG | Fruit | Menthol | Tobacco | PG:VG | Fruit | Menthol | Tobacco | |
| S | 75.63 | 68.36 | 95.34 | 79.79 | 70.67 ± 16.94 | 74.05 ± 39.63 | 81.31 ± 27.24 | 91.81 ± 11.67 |
| Ni | 2.09 | 1.63 | 4.73 | 2.84 | 1.04 ± 1.00 | 30.47 ± 14.28 | 83.52 ± 20.53 | 41.73 ± 12.25 |
| Cu | 2.16 | 0.87 | 2.72 | 2.00 | 0.37 ± 0.22 | 1.04 ± 0.48 | 1.85 ± 0.70 | 1.32 ± 0.39 |
| Si | 1.05 | 1.13 | 1.55 | 1.26 | 1.10 ± 0.14 | 1.09 ± 0.35 | 1.32 ± 0.28 | 1.33 ± 0.12 |
| K | 0.80 | 0.84 | 1.21 | 0.67 | 0.94 ± 0.13 | 0.99 ± 0.45 | 1.32 ± 0.28 | 0.51 ± 0.10 |
| Na | 0.39 | 0.40 | 1.10 | 0.63 | 0.65 ± 0.11 | 0.91 ± 0.17 | 1.70 ± 0.37 | 0.79 ± 0.11 |
| W | 0.48 | 0.23 | 0.24 | 0.14 | 0.46 ± 0.20 | 0.30 ± 0.18 | 0.28 ± 0.14 | 0.22 ± 0.05 |
| Zn | 0.32 | 0.36 | 0.68 | 1.94 | 0.18 ± 0.01 | 1.45 ± 0.48 | 2.06 ± 0.48 | 1.71 ± 0.42 |
| Ir | 0.28 | 0.47 | 0.27 | 0.11 | 1.19 ± 0.76 | 0.35 ± 0.29 | 0.19 ± 0.12 | 0.14 ± 0.05 |
| B | 0.14 | 0.02 | 0.02 | 0.02 | 0.07 ± 0.02 | 0.02 ± 0.01 | 0.02 ± 0.01 | 0.02 ± 0.01 |
| Ta | 0.14 | 0.34 | 0.24 | 0.13 | 0.73 ± 0.30 | 0.24 ± 0.09 | 0.18 ± 0.06 | 0.13 ± 0.02 |
| Hf | 0.13 | 0.18 | 0.13 | 0.07 | 0.43 ± 0.19 | 0.14 ± 0.05 | 0.09 ± 0.03 | 0.07 ± 0.01 |
| Mo | 0.08 | 0.01 | 0.01 | 0.01 | 0.02 ± 0.01 | 0.07 ± 0.01 | 0.15 ± 0.04 | 0.06 ± 0.02 |
| Pd | 0.07 | 0.14 | 0.11 | 0.06 | 0.30 ± 0.14 | 0.11 ± 0.06 | 0.07 ± 0.02 | 0.06 ± 0.01 |
| Pt | 0.05 | 0.06 | 0.05 | 0.07 | 0.16 ± 0.07 | 0.09 ± 0.05 | 0.08 ± 0.04 | 0.08 ± 0.03 |
| Zr | 0.04 | 0.01 | 0.02 | 0.02 | 0.02 ± 0.01 | 0.02 ± 0.01 | 0.01 ± 0.00 | 0.01 ± 0.00 |
| Sn | 0.02 | 0.06 | 0.05 | 0.04 | 0.06 ± 0.02 | 0.07 ± 0.02 | 0.03 ± 0.01 | 0.03 ± 0.01 |
| Mg | 0.01 | 0.01 | 0.04 | 0.03 | 0.02 ± 0.00 | 0.12 ± 0.03 | 0.12 ± 0.03 | 0.10 ± 0.02 |
| Al | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 ± 0.00 | 0.01 ± 0.00 | 0.01 ± 0.00 | 0.01 ± 0.00 |
| Co | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 ± 0.00 | 0.02 ± 0.00 | 0.03 ± 0.01 | 0.02 ± 0.00 |
| Nb | 0.01 | 0.00 | 0.00 | 0.00 | 0.01 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
| Ba | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 ± 0.01 | 0.01 ± 0.00 | 0.01 ± 0.00 | 0.01 ± 0.00 |
| Re | 0.01 | 0.01 | 0.01 | 0.02 | 0.06 ± 0.04 | 0.03 ± 0.03 | 0.02 ± 0.01 | 0.02 ± 0.01 |
Table 2
List of top dysregulated genes on exposure to differently flavored (fruit, menthol, and tobacco) e-cig aerosol in C57BL/6J mouse lungs.
| Genes | Gene names | Gene function |
|---|---|---|
| Neurl3 | Neuralized E3 Ubiquitin Protein Ligase 3 | Ubiquitin protein ligase activity |
| Egfem1 | EGF-like and EMI domain containing 1 | Calcium ion-binding activity |
| Stap1 | Signal Transducing Adaptor Family Member 1 | Protein kinase binding and SH3/SH2 adaptor activity |
| Tfec | Transcription Factor EC | Multiple cellular processes including survival, growth, and differentiation |
| Mitf | Melanocyte Inducing Transcription Factor | Critical role in cell differentiation |
| Cirbp | Cold Inducible RNA Binding Protein | Role in cold-induced suppression of cell proliferation |
| Hist1h1c | H1.2 linker histone | Functions in the compaction of chromatin |
| Gmds | GDP-Mannose 4,6-Dehydratase | Coenzyme binding and NADP+ binding |
| Htr2c | 5-Hydroxytryptamine Receptor 2C | G-protein-coupled receptor activity |
| Nfia | Nuclear Factor I A | DNA-binding transcription factor activity |
| Klra8 | killer cell lectin-like receptor | Carbohydrate binding activity. Acts upstream of or within response to virus |
| Trp53i11 | Tumor Protein P53 Inducible Protein 11 | Negative regulation of cell population proliferation |
| Ehd2 | EH Domain Containing 2 | Angiopoietin-like protein 8 regulatory pathway and response to elevated platelet cytosolic Ca2+ |
| Ackr2 | Atypical Chemokine Receptor 2 | Recruitment of effector immune cells to the inflammation site |
| Marcks | Myristoylated Alanine Rich Protein Kinase C Substrate | Involved in cell motility, phagocytosis, membrane trafficking, and mitogenesis |
| Pfkl | Phosphofructokinase, | Protein binding and monosaccharide binding |
| Ramp3 | Receptor Activity Modifying Protein 3 | Signaling receptor activity and coreceptor activity |
| Chrm3 | Cholinergic Receptor Muscarinic 3 | Cellular responses such as adenylate cyclase inhibition, phosphoinositide degeneration, and potassium channel mediation |
| Sftpa1 | Surfactant Protein A1 | Carbohydrate binding and lipid transporter activity |
| Add3 | Adducin 3 | Actin binding and calmodulin binding |
| Hmgb3 | High Mobility Group Box 3 | Important role in maintaining stem cell populations and may be aberrantly expressed in tumor cells |
| Acot1 | Acyl-CoA Thioesterase 1 | Involved in acyl-CoA metabolic process; long-chain fatty acid metabolic process; and very long-chain fatty acid metabolic process |
| H1f0 | H1.0 Linker Histone | Cellular responses to stimuli and Programmed Cell Death |
| Scgb3a2 | Secretoglobin Family 3A Member 2 | Secreted lung surfactant protein |
| Scgb1a1 | Secretoglobin Family 1A Member 1 | Implicated in numerous functions including anti-inflammation, inhibition of phospholipase A2 and the sequestering of hydrophobic ligands |
| Gpam | Glycerol-3-Phosphate Acyltransferase, Mitochondrial | Acyltransferase activity and glycerol-3-phosphate O-acyltransferase activity |
| Cdh11 | Cadherin 11 | Integral membrane proteins that mediate calcium-dependent cell–cell adhesion |
| LDLR | Low-Density Lipoprotein Receptor | Cell surface proteins involved in receptor-mediated endocytosis of specific ligands |
| Myocd | Myocardin | Transcriptional co-activator of serum response factor (SRF) |
Table 3
Gene ontology results showing the top hits from the commonly dysregulated genes in all clusters on exposure to e-cig aerosols.
| GO ID | Ontology | Description | Gene ID | BgRatio | p.adjust |
|---|---|---|---|---|---|
| GO:0045907 | BP | Positive regulation of vasoconstriction | Htr2c/Chrm3/Add3 | 53/23,062 | 0.033815 |
| GO:0019229 | BP | Regulation of vasoconstriction | Htr2c/Chrm3/Add3 | 86/23,062 | 0.042491 |
| GO:1903978 | BP | Regulation of microglial cell activation | Stap1/Ldlr | 16/23,062 | 0.042491 |
| GO:0002683 | BP | Negative regulation of immune system process | Hmgb3/Gpam/Scgb1a1/Stap1/Ldlr | 464/23,062 | 0.042491 |
| GO:0010867 | BP | Positive regulation of triglyceride biosynthetic process | Gpam/Ldlr | 19/23,062 | 0.042491 |
| GO:0042310 | BP | Vasoconstriction | Htr2c/Chrm3/Add3 | 109/23,062 | 0.042491 |
| GO:0046889 | BP | Positive regulation of lipid biosynthetic process | Htr2c/Gpam/Ldlr | 110/23,062 | 0.042491 |
| GO:0010866 | BP | Regulation of triglyceride biosynthetic process | Gpam/Ldlr | 25/23,062 | 0.047046 |
| GO:0001919 | BP | Regulation of receptor recycling | Ldlr/Ramp3 | 29/23,062 | 0.047046 |
| GO:0007271 | BP | Synaptic transmission, cholinergic | Htr2c/Chrm3 | 29/23,062 | 0.047046 |
| GO:0150077 | BP | Regulation of neuroinflammatory response | Stap1/Ldlr | 29/23,062 | 0.047046 |
| GO:0090208 | BP | Positive regulation of triglyceride metabolic process | Gpam/Ldlr | 31/23,062 | 0.047046 |
| GO:0045987 | BP | Positive regulation of smooth muscle contraction | Chrm3/Myocd | 35/23,062 | 0.047046 |
| GO:0097242 | BP | Amyloid-beta clearance | Ldlr/Myocd | 36/23,062 | 0.047046 |
| GO:0040013 | BP | Negative regulation of locomotion | Htr2c/Mitf/Stap1/Myocd | 360/23,062 | 0.047046 |
| GO:0010667 | BP | Negative regulation of cardiac muscle cell apoptotic process | Acot1/Myocd | 37/23,062 | 0.047046 |
| GO:0001881 | BP | Receptor recycling | Ldlr/Ramp3 | 40/23,062 | 0.047046 |
| GO:0010664 | BP | Negative regulation of striated muscle cell apoptotic process | Acot1/Myocd | 40/23,062 | 0.047046 |
| GO:0019432 | BP | Triglyceride biosynthetic process | Gpam/Ldlr | 40/23,062 | 0.047046 |
| GO:0035296 | BP | Regulation of tube diameter | Htr2c/Chrm3/Add3 | 173/23,062 | 0.047046 |
| GO:0097746 | BP | Blood vessel diameter maintenance | Htr2c/Chrm3/Add3 | 173/23,062 | 0.047046 |
| GO:0001774 | BP | Microglial cell activation | Stap1/Ldlr | 42/23,062 | 0.047046 |
| GO:1903725 | BP | Regulation of phospholipid metabolic process | Htr2c/Ldlr | 42/23,062 | 0.047046 |
| GO:0035150 | BP | Regulation of tube size | Htr2c/Chrm3/Add3 | 174/23,062 | 0.047046 |
Additional files
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Supplementary file 1
Detailed account of the analysed dataset and validation cohort for scRNA seq for e-cig exposed mouse lungs.
(a) Table showing the QC parameters used before and after filtering and normalization of scRNA seq data. (b) Variable features with average log2 fold change and p value for genes in each cell cluster identified upon dimensionality reduction and clustering of 71,725 single cells from scRNA seq from treated and control samples. (c) Table showing the two-way ANOVA statistics for the cell frequencies identified through (a) scRNA seq analyses for general clustering using treatment and cell types as independent variables and (b) flow cytometric analyses using treatment and sex as independent variables. (d) (a) DESeq2 results showing the significant (p < 0.05) DEGs and (b) GO results of the DEGs in the myeloid cell cluster from mouse lungs exposed to 5-day nose-only exposure to PG:VG when compared to air control. (e) DESeq2 results showing the significant (p < 0.05) DEGs in the myeloid cell cluster from mouse lungs exposed to 5-day nose-only exposure to fruit-flavored e-cig aerosol when compared to air control. (f) GO analyses results of the (a) upregulated DEGs (log2 fold change >0.5) and (b) downregulated DEGs (log2 fold change <–0.5) in the myeloid cell cluster from mouse lungs exposed to 5-day nose-only exposure to fruit-flavored e-cig aerosols when compared to air control. (g) DESeq2 results showing the significant (p < 0.05) DEGs in the myeloid cell cluster from mouse lungs exposed to 5-day nose-only exposure to menthol-flavored e-cig aerosol when compared to air control. (h) GO analyses results of the (a) upregulated DEGs (log2 fold change >0.5) and (b) downregulated DEGs (log2 fold change <–0.5) in the myeloid cell cluster from mouse lungs exposed to 5-day nose-only exposure to menthol-flavored e-cig aerosols when compared to air control. (i) DESeq2 results showing the significant (p < 0.05) DEGs in the myeloid cell cluster from mouse lungs exposed to 5-day nose-only exposure to tobacco-flavored e-cig aerosols when compared to air control. (j) GO analyses results of the (a) upregulated DEGs (log2 fold change >0.5) and (b) downregulated DEGs (log2 fold change <–0.5) in the myeloid cell cluster from mouse lungs exposed to 5-day nose-only exposure to tobacco-flavored e-cig aerosols when compared to air control. (k) (a) DESeq2 results showing the significant (p < 0.05) DEGs and (b) GO results of the DEGs in the lymphoid cell cluster from mouse lungs exposed to 5-day nose-only exposure to PG:VG when compared to air control. (l) DESeq2 results showing the significant (p < 0.05) DEGs in the lymphoid cell cluster from mouse lungs exposed to 5-day nose-only exposure to fruit-flavored e-cig aerosol when compared to air control. (m) GO analyses results of the (a) upregulated DEGs (log2 fold change >0.5) and (b) downregulated DEGs (log2 fold change <–0.5) in the lymphoid cell cluster from mouse lungs exposed to 5-day nose-only exposure to fruit-flavored e-cig aerosols when compared to air control. (n) DESeq2 results showing the significant (p < 0.05) DEGs in the lymphoid cell cluster from mouse lungs exposed to 5-day nose-only exposure to menthol-flavored e-cig aerosol when compared to air control. (o) GO analyses results of the (a) upregulated DEGs (log2 fold change >0.5) and (b) downregulated DEGs (log2 fold change <–0.5) in the lymphoid cell cluster from mouse lungs exposed to 5-day nose-only exposure to menthol-flavored e-cig aerosols when compared to air control. (p) DESeq2 results showing the significant (p < 0.05) DEGs in the lymphoid cell cluster from mouse lungs exposed to 5-day nose-only exposure to tobacco-flavored e-cig aerosol when compared to air control. (q) GO analyses results of the (a) upregulated DEGs (log2 fold change >0.5) and (b) downregulated DEGs (log2 fold change <–0.5) in the lymphoid cell cluster from mouse lungs exposed to 5-day nose-only exposure to tobacco-flavored e-cig aerosols when compared to air control. Variable features with average log2 fold change and p value for genes in each cell cluster identified upon dimensionality reduction and clustering of myeloid subset. (s) DESeq2 results showing the DEGs in the myeloid cell cluster from mouse lungs exposed to 5-day nose-only exposure to fruit flavored e-cig aerosols when compared to PG:VG exposure. (t) DESeq2 results showing the DEGs in the myeloid cell cluster from mouse lungs exposed to 5-day nose-only exposure to menthol flavored e-cig aerosols when compared to PG:VG exposure. (u) DESeq2 results showing the DEGs in the myeloid cell cluster from mouse lungs exposed to 5-day nose-only exposure to tobacco-flavored e-cig aerosols when compared to PG:VG exposure. (v) DESeq2 results showing the DEGs in the lymphoid cell cluster from mouse lungs exposed to 5-day nose-only exposure to fruit-flavored e-cig aerosol when compared to PG:VG exposure. (w) DESeq2 results showing the DEGs in the lymphoid cell cluster from mouse lungs exposed to 5-day nose-only exposure to menthol-flavored e-cig aerosol when compared to PG:VG exposure. (x) DESeq2 results showing the DEGs in the lymphoid cell cluster from mouse lungs exposed to 5-day nose-only exposure to tobacco-flavored e-cig aerosol when compared to PG:VG exposure.
- https://cdn.elifesciences.org/articles/106380/elife-106380-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/106380/elife-106380-mdarchecklist1-v1.pdf
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Single-cell transcriptomics identifies altered neutrophil dynamics and accentuated T-cell cytotoxicity in tobacco-flavored e-cigarette-exposed mouse lungs
eLife 14:RP106380.
https://doi.org/10.7554/eLife.106380.4
