Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract

Abstract

The factors mediating fatal SARS-CoV-2 infections are poorly understood. Here, we show that cigarette smoke causes a dose-dependent upregulation of angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor, in rodent and human lungs. Using single-cell sequencing data, we demonstrate that ACE2 is expressed in a subset of secretory cells in the respiratory tract. Chronic smoke exposure triggers the expansion of this cell population and a concomitant increase in ACE2 expression. In contrast, quitting smoking decreases the abundance of these secretory cells and reduces ACE2 levels. Finally, we demonstrate that ACE2 expression is responsive to inflammatory signaling and can be upregulated by viral infections or interferon treatment. Taken together, these results may partially explain why smokers are particularly susceptible to severe SARS-CoV-2 infections. Furthermore, our work identifies ACE2 as an interferon-stimulated gene in lung cells, suggesting that SARS-CoV-2 infections could create positive feedback loops that increase ACE2 levels and facilitate viral dissemination.

Keywords: ACE2; COVID-19; Coronavirus; SARS-CoV-2; inflammation; interferon; lung development; smoking.

Graphical abstract

Figure 1

ACE2 Expression in the Lung Is Uncorrelated with Age or Sex (A) ACE2 expression in the lungs of young mice (<26 weeks old) and old mice (>78 weeks old). (B) ACE2 expression in the lungs of young rats (6 weeks old) and old rats (104 weeks old). (C) ACE2 expression in the lungs of female mice and male mice. (D) ACE2 expression in the lungs of female rats and male rats. (E) ACE2 expression in lungs from the GTEx cohort by age. (F) ACE2 expression in lungs from the GTEx cohort by sex. (G) ACE2 expression in lungs from a cohort of organ donors by age. (H) ACE2 expression in lungs from a cohort of organ donors by sex. (I) ACE2 expression in pathologically normal lung tissue from patients from TCGA by age. (J) ACE2 expression in pathologically normal lung tissue from patients from TCGA by sex. Each panel displays log₂-normalized ACE2 expression relative to a control group. Data analyzed in (A) and (C) were from GSE132040. Data analyzed in (B) and (D) were from GSE53960. Data analyzed in (E) and (F) were from www.gtexportal.org. Data analyzed in (G) and (H) were from GSE1643. Data analyzed in (I) and (J) were from https://gdac.broadinstitute.org/. Additional information on the data sources and sample sizes is included in Table S1.

Figure 2

Cigarette Smoke Increases the Expression of ACE2 in Mouse and Human Lungs (A) ACE2 expression in the lungs of mice that were sham-treated or that were exposed to diluted cigarette smoke for 2, 3, or 4 h a day (low, medium, and high smoke exposure, respectively). (B) A diagram showing the approximate locations of tissue samples used in this analysis. Tracheal, large airway epithelial, and small airway epithelial specimens were collected by fiberoptic bronchoscopy as described in their respective publications. Lung resections were collected surgically from various locations. (C) ACE2 expression in human tracheal epithelia analyzed according to smoking history. (D) ACE2 expression in human large airway epithelia analyzed according to smoking history. (E) ACE2 expression in human small airway epithelia analyzed according to smoking history. (F) A volcano plot comparing gene expression in the respiratory epithelia of current smokers and never smokers from (C)–(E). The dotted lines indicate various p value thresholds (e.g., genes located above the 10th percentile have a combined p value greater than 90% of the genes included in the meta-analysis). The location of ACE2 is indicated with a red star. (G) ACE2 expression in the lungs of a cohort of patients undergoing thoracic surgery analyzed according to the number of pack years each patient smoked. (H) ACE2 expression in the lungs of TCGA patients analyzed according to the number of pack years each patient smoked. (I) ACE2 expression in respiratory epithelia collected by fiberoptic bronchoscopy among either current smokers or former smokers. (J) A volcano plot comparing gene expression between current smokers and former smokers. The dotted lines indicate various p value thresholds (e.g., genes located above the 10^th percentile have a combined p value greater than 90% of the genes included in the meta-analysis). The location of ACE2 is indicated with a red star. Each panel displays log₂-normalized ACE2 expression relative to a control group. Data analyzed in (A) were from GSE18344. Data analyzed in (C) were from GSE13933. Data analyzed in (D) were from GSE22047. Data analyzed in (E) were from GSE64614. Data analyzed in (G) were from GSE76925. Data analyzed in (H) were from https://gdac.broadinstitute.org/. Data analyzed in (I) were from GSE79209. Additional information on the data sources and sample sizes are included in Table S1. ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005 (Student’s t test).

Figure 3

ACE2 Is Expressed in Secretory Club and Goblet Cells Along with Alveolar Type 2 Cells in the Mammalian Lung (A) T-SNE clustering of cells from the mouse lung. Cells expressing ACE2 are highlighted in the right panel. (B) Cells in the mouse lung that express various lineage markers (TMEM100 for endothelial cells, EPCAM for epithelial cells, PDGFRA for mesenchymal cells, and PTPRC for immune cells) are highlighted. (C) Cells expressing markers for various epithelial lineages are highlighted: RTKN2 for alveolar type 1 cells, LAMP3 for alveolar type 2 cells, FOXJ1 for ciliated cells, GABRP for both goblet and club cells, MUC5AC for goblet cells, and SCGB1A for club cells. (D) A track plot displaying the expression of ACE2 and several lineage-related genes in different cell populations obtained from Leiden clustering. (E) T-SNE clustering of cells from the human lung. Cells expressing ACE2 are highlighted in the right panel. (F) Cells in the human lung that express various lineage markers (TMEM100 for endothelial cells, EPCAM for epithelial cells, PDGFRA for mesenchymal cells, and PTPRC for immune cells) are highlighted. (G) Cells expressing markers for various epithelial lineages are highlighted: LAMP3 for alveolar type 2 cells, FOXJ1 for ciliated cells, TP63 for basal cells, GABRP for both goblet and club cells, MUC5AC for goblet cells, and SCGB1A1 for club cells. (H) A track plot displaying the expression of ACE2 and several lineage-related genes in different cell populations obtained from Leiden clustering. The gene expression data used in (A)–(D) are from GSE121611. The gene expression data used in (E)–(H) are from GSE122960. Additional information on the data sources and sample sizes are included in Table S1.

Figure 4

Cigarette Smoke Causes the Expansion of ACE2⁺ Secretory Cells (A) T-SNE clustering of the transcriptomes from single cells derived from the airway epithelia of smokers and never smokers. Cells expressing ACE2 are highlighted in the right panel. (B) Cells in the human airway that express various lineage markers (FOXJ1 for ciliated cells, TP63 for basal cells, MUC5AC for goblet cells, and SCGB1A1 for club cells) are highlighted. (C) GO terms enriched among ACE2-correlated transcripts. (D) Dot plots displaying the expression of the top ten differentially expressed marker genes for various airway lineages and for ACE2+ cells. (E) The fraction of cells expressing the indicated marker genes are displayed. FOXJ1 is a marker for ciliated cells, MUC5AC is a marker for goblet cells, and TP63 is a marker for basal cells. (F) The fractions of cells found in each cell type cluster are displayed. (G) The number of counts per ACE2⁺ cell are displayed. (H) The fraction of ACE2⁺ cells co-expressing MUC5AC or that are found within the goblet/club cell cluster are displayed. (I) The 100 top-ranked differentially expressed genes from each cluster in (A) as well as the 100 genes most strongly correlated with ACE2 were used to re-analyze the bulk gene expression data from smokers and non-smokers in Figure 2F. A volcano plot displays the mean expression change of each cell type signature. (J) The same transcriptional signatures as in Figure 4I were used to re-analyze the data from current smokers and former smokers in Figure 2J. A volcano plot displays the mean expression change of each cell type signature. (K) ACE2 expression in mouse tracheal explants undergoing mucociliary differentiation. (L) ACE2 expression in human airway epithelial cells undergoing mucociliary differentiation. (M) ACE2 expression in human airway epithelial cells that underwent mucociliary differentiation in the presence of clean air or cigarette smoke. (N) The same transcriptional signatures as in Figure 4I were used to re-analyze the data from smoke exposure during differentiation from Figure 4M. A volcano plot displaying the mean expression change of each cell type signature is displayed. Data analyzed in (A)–(H) were from GSE131391. Data analyzed in (K) were from GSE75715. Data analyzed in (L) were from GSE39059. Data analyzed in (M) were from GSE135188. Additional information on the data sources and sample sizes are included in Table S1. In (E), (F), and (H), a chi-square test is applied. In (G), a Mann-Whitney U test is applied. In (K)–(M), a Student’s t test is applied. ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005.

Figure 5

ACE2 Is an Interferon-Stimulated Gene that Is Upregulated by Viral Infections (A) ACE2 expression in airway epithelial cells that were infected with influenza. (B) ACE2 expression in airway epithelial cells that were infected with respiratory syncytial virus. (C) ACE2 expression in airway epithelial cells that were infected with SARS. (D) ACE2 expression in airway epithelial cells that were infected with MERS. (E) ACE2 expression in airway epithelial cells that were transfected with the dsRNA mimic poly(I:C). (F) ACE2 expression in human tracheal cells that were cultured in the presence of the indicated cytokine for 24 h. (G) ACE2 expression in human small airway epithelial cells that were cultured in the presence of the indicated cytokine for 24 h. (H) ACE2 expression in airway epithelial cells that were cultured in the presence of IFN-β. Each panel displays log₂-normalized ACE2 expression relative to a control group. Data analyzed in (A) and (B) were from GSE32138. Data analyzed in (C) were from GSE47963. Data analyzed in (D) were from GSE100504. Data analyzed in (E) were from GSE51392. Data analyzed in (H) were from GSE19392. Additional information on the data sources and sample sizes are included in Table S1. ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005 (Student’s t test).