SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells

Abstract

The SARS-CoV-2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID-19 pathogenesis, especially host factors facilitating virus infection and replication. SARS-CoV-2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. While TMPRSS2 is strongly expressed in both tissues, in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. Our data provide a rich resource for future investigations of COVID-19 infection and pathogenesis.

Keywords: FURIN; COVID-19; Human Cell Atlas; epithelial differentiation; respiratory tract.

Figure 1. ACE2 and TMPRSS2 are expressed in specific cell types in lungs and HBECs

1. Sampling location of the surgical lung specimens and human bronchial epithelial cells (HBECs) used in this study. Blue rectangle is zoomed in (B).

2. Overview of the major cell types in the lung and airways.

3. Uniform manifold approximation and projection (UMAP) of primary lung samples single nuclei RNA sequencing. Cell types are color‐coded.

4. Expression values of ACE2 in the cell types of primary lung samples.

5. Expression values of TMPRSS2 in the cell types of primary lung samples.

6. UMAP projections of HBEC single cell RNA sequencing data. Cell types are color‐coded.

7. Expression values of ACE2 in the cell types of HBECs.

8. Expression values of TMPRSS2 in the cell types of HBECs.

Data information: Boxes in box plots indicate the first and third quartile, with the median shown as horizontal lines. Whiskers extend to 1.5 times the inter‐quartile range. Number of patients: Twelve lung samples and four HBEC samples. Each patient is represented as one dot. All individual data points are indicated on the plot.

Figure EV1. Statistical analysis of ACE2 and TMPRSS2 expression differences

1. A–D

   Heatmaps indicating the P‐values of comparisons of expression levels between different cell types in the lung (A, C) and HBECs (B, D).

Data information: Values are obtained from Dunn's post hoc test followed by Benjamini–Hochberg correction for multiple testing.

Figure EV2. Percentage of ACE2‐,TMPRSS2‐, and FURIN‐positive cells

1. A, B

   Percentage of positive cells (defined here as having at least one read of the respective gene) for ACE2 in the lung (A) and HBECs (B).

2. C, D

   Percentage of positive cells (defined here as having at least one read of the respective gene) for TMPRSS2 in the lung (C) and HBECs (D).

3. E, F

   Percentage of positive cells (defined here as having at least one read of the respective gene) for FURIN in the lung (E) and HBECs (F).

Figure EV3. Expression levels of ACE2,TMPRSS2, and FURIN

1. A–C

   ACE2‐ (A), TMPRSS2‐ (B), and FURIN‐ (C) expressing cells in the lung plotted on top of the UMAP coordinates. Expression levels are color‐coded.

2. D–F

   Dot plot indicating expression of ACE2 (D), TMPRSS2 (E), and FURIN (F) across different cell types in the lung. Percentage of expressing cells is size‐coded; mean expression levels are color‐coded.

3. G–I

   ACE2‐ (G), TMPRSS2‐ (H), and FURIN‐ (I) expressing HBECs plotted on top of the UMAP coordinates. Expression levels are color‐coded.

4. J–L

   Dot plot indicating expression of ACE2 (J), TMPRSS2 (K), and FURIN (L) across different cell types in the bronchial epithelium. Percentage of expressing cells is size‐coded; mean expression levels are color‐coded.

Figure EV4. Quantification of ACE2 ⁺/TMPRSS2 ⁺ double‐positive cells

1. A, B

   Overlaps and enrichment statistics for all ACE2 and TMPRSS2 single‐ and double‐positive cells in the lung (A) and HBEC (B) dataset.

Data information: RF: representation factor, enrichment. P: hypergeometric tail probability.

Figure 2. Characterization of transient secretory (secretory3) cells

1. Pseudo‐time trajectory projected onto a UMAP embedding of HBECs. The location of the secretory3 cell type is marked by a red outline. Cell types are color‐coded. Numbers indicate the beginning of each pseudotime trajectory.

2. Expression of goblet (top row) and ciliated cell markers (bottom row) in club, goblet, secretory3, and ciliated cells.

3. Pseudo‐time trajectory projected onto a UMAP embedding of HBECs. Ppseudo‐time values are color‐coded. Numbers indicate the beginning of each pseudotime trajectory.

4. Cell type composition along a binned pseudo‐time axis. The earliest stages are located on the left.

5. Pathway enrichment values for secretory3‐specific marker genes.

Figure 3. FURIN is expressed in ACE2 ⁺ and ACE2 ⁺/TMPRSS2 ⁺ cells

1. Expression values of FURIN in the cell types of primary lung.

2. Overlap of ACE2 ⁺, TMPRSS2 ⁺, and FURIN ⁺ cells in the lung dataset. RF: representation factor, enrichment. P: hypergeometric tail probability. Total number of cells: 39,778.

3. Expression values of FURIN in HBECs.

4. Overlap of ACE2 ⁺, TMPRSS2 ⁺, and FURIN ⁺ cells in the HBEC dataset. Total number of cells: 17,521.

Data information: RF: representation factor, enrichment. P: hypergeometric tail probability. Total number of cells: 17,451. Boxes in box plots indicate the first and third quartile, with the median shown as horizontal lines. Whiskers extend to 1.5 times the inter‐quartile range. Number of patients: Twelve lung samples and four HBEC samples. Each patient is represented as one dot. All individual data points are indicated on the plot.

Figure EV5. Age‐dependent cell type composition

1. A, B

   Cell type composition in the lung (A) and HBEC (B) dataset. Age is color‐coded.

Figure 4. Age, sex, and smoking behavior as possible factors influencing ACE2 expression

1. A

   Metadata of the sample donors.^†

2. B

   Expression of ACE2 in the cell types of the primary lung. History of smoking is color‐coded.

3. C, D

   Expression of ACE2 in HBECs (C) and primary lung (D). Sex is color‐coded.

4. E, F

   Expression of ACE2 in HBECs (E) and primary lung (F). Age is color‐coded.

5. G, H

   ACE2 expression over all cell types versus age in female primary lung cells (G) and primary lung cells of patients aged 40–50 (H). Sex is color‐coded.

Boxes in box plots indicate the first and third quartile, with the median shown as horizontal lines. Whiskers extend to 1.5 times the inter‐quartile range. All individual data points are indicated on the plot. Nine female patients are shown in (G); eight patients between 40 and 50 years are shown in (H). ^†Correction added on 28 April 2020, after first online publication: Smoking was corrected to “No” for the fifth donor sample.