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. 2020 Sep 3;56(3):2001123.
doi: 10.1183/13993003.01123-2020. Print 2020 Sep.

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue

Jennifer A Aguiar  1 , Benjamin J-M Tremblay  1 , Michael J Mansfield  2 , Owen Woody  3 , Briallen Lobb  1 , Arinjay Banerjee  4 , Abiram Chandiramohan  5 , Nicholas Tiessen  5 , Quynh Cao  5 , Anna Dvorkin-Gheva  4 , Spencer Revill  5 , Matthew S Miller  4   6   7 , Christopher Carlsten  8 , Louise Organ  9 , Chitra Joseph  9 , Alison John  9 , Paul Hanson  10 , Richard C Austin  11 , Bruce M McManus  10 , Gisli Jenkins  9 , Karen Mossman  4 , Kjetil Ask  4   5 , Andrew C Doxey  1   5   12 , Jeremy A Hirota  13   4   5   6   12
Affiliations

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue

Jennifer A Aguiar et al. Eur Respir J. .

Abstract

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

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Conflict of interest statement

Conflict of interest: J.A. Aguiar has nothing to disclose. Conflict of interest: B.J-M. Tremblay has nothing to disclose. Conflict of interest: M.J. Mansfield has nothing to disclose. Conflict of interest: O. Woody has nothing to disclose. Conflict of interest: B. Lobb has nothing to disclose. Conflict of interest: A. Banerjee has nothing to disclose. Conflict of interest: A. Chandiramohan has nothing to disclose. Conflict of interest: N. Tiessen has nothing to disclose. Conflict of interest: Q. Cao has nothing to disclose. Conflict of interest: A. Dvorkin-Gheva has nothing to disclose. Conflict of interest: S. Revill has nothing to disclose. Conflict of interest: M.S. Miller has nothing to disclose. Conflict of interest: C. Carlsten has nothing to disclose. Conflict of interest: L. Organ has nothing to disclose. Conflict of interest: C. Joseph has nothing to disclose. Conflict of interest: A. John has nothing to disclose. Conflict of interest: P. Hanson has nothing to disclose. Conflict of interest: R. Austin has a patent US7524826B2 issued to McMaster University and Hamilton Health Sciences Corp., McMaster University. Conflict of interest: B.M. McManus has nothing to disclose. Conflict of interest: G. Jenkins reports grants from AstraZeneca, Biogen, Galecto and GlaxoSmithKline; personal fees from Boehringer Ingelheim, Daewoong, Galapagos, Heptares, Promedior and Roche; grants and personal fees from Pliant; non-financial support from Redx and NuMedii; and is trustee for Action for Pulmonary Fibrosis, outside the submitted work. Conflict of interest: K. Mossman has nothing to disclose. Conflict of interest: K. Ask has nothing to disclose. Conflict of interest: A.C. Doxey has nothing to disclose. Conflict of interest: J.A. Hirota has nothing to disclose.

Figures

FIGURE 1
FIGURE 1
Microarray expression profiles of candidate SARS-CoV-2 receptor genes in upper and lower airways. Normalised log2 expression levels for ACE2 (angiotensin-converting enzyme 2), TMPRSS2, ADAM17, CTSL (cathepsin L1), CD147 and GRP78 genes compared across the upper airway (nasal) and lower airways (trachea, large airway and small airway). CDH1 (E-cadherin) gene expression level is included as a positive control. Statistical values for comparisons for each gene at each airway generation were calculated; those not shown were nonsignificant. *: p<0.05; **: p<0.01; ***: p<0.001.
FIGURE 2
FIGURE 2
Microarray expression profiles of candidate SARS-CoV-2 receptor genes in lower airway epithelial cells, analysed by age and sex. a) Clustered heatmap of log2 expression levels from NCBI Gene Expression Omnibus (GEO) samples (n=504), annotated by age, sex and microarray chip platform. Expression values reflect signal intensities, indicating lowest detected expression of ACE2 (angiotensin-converting enzyme 2) and highest expression of GRP78 and CDH1 (E-cadherin). b and c) Box plots of expression levels separated by b) sex (n=194) and c) smoking status (n=451). d and e) Plots of gene expression levels versus age, with linear regression lines of best fit, for datasets that used either d) the HG-U133 Plus 2 microarray (n=43) or e) the HuGene-1.0-st-v1 microarray (n=181). Correlations were performed separately between platforms because of differences in their age distributions. e) A weak negative correlation (r= −0.20, p=0.015) was detected for ACE2 in the dataset that used the HuGene-1.0-st-v1 microarray.
FIGURE 3
FIGURE 3
Promoter activity for SARS-CoV-2-related genes from the FANTOM5 cap analysis of gene expression (CAGE) dataset. The FANTOM5 CAGE data consist of quantified promoter expression levels across the human genome for 1866 samples from primary cells, cell lines and tissue samples. The FANTOM5 CAGE promoter activity data for several SARS-CoV-2-related genes are shown for samples related to lung, gut, heart and prostate tissues (n=120). Dot sizes are proportional to promoter activity, depicted as log10-transformed normalised transcripts per million (TPM). Notably, angiotensin-converting enzyme 2 (ACE2) is either not expressed or expressed at low levels (<1 TPM in all but one sample) in the airway, including measurements from healthy and cancerous cells.
FIGURE 4
FIGURE 4
Proteomic profiles of candidate SARS-CoV-2 receptor genes in human tissue and airway epithelial cells. a) Intensity values of protein expression from Kim et al. [31] for the genes ACE2 (angiotensin-converting enzyme 2), TMPRSS2, ADAM17, CTSL (cathepsin L1), CD147 and GRP78. CDH1 (E-cadherin) intensity is included as a positive control for expression in airway cells. Intensity values have been log10-transformed to facilitate comparison between candidates with different basal expression levels across tissue types. Grey cells in the heatmap correspond to an untransformed intensity of 0 and represent an undetectable signal. b) Intensity values log10-transformed for visualisation of ACE2, TMPRSS2, ADAM17, CTSL, CD147 and GRP78 proteins in human airway epithelial cells from healthy nonsmokers (n=4; males) grown under air–liquid interface culture conditions [32]. CDH1 intensity is included as a positive control. Counts indicating the number of detected peptides associated with each parent protein are provided. ND: the protein was not detected in this study.
FIGURE 5
FIGURE 5
Immunoblot analysis of ACE2 (angiotensin-converting enzyme 2), TMPRSS2, CD147 and GRP78 protein expression in human airway epithelial cell protein lysates. a) ACE2 with single band for predicted molecular weight of 110 kDa (red box). b) TMPRSS2 with multiple bands including a dominant band at predicted molecular weight of 57 kDa (red box). c) CD147 with a single broad band around predicted molecular weight of 55 kDa (red box). d) GRP78 with a single band at predicted molecular weight of 78 kDa (red box). Lanes 1–3: Calu-3 cells. Lanes 4–6: primary human airway epithelial cells. Lanes 7–9: human bronchial epithelium cell (HBEC)-6KT cell line. All cells grown under submerged monolayer conditions, with n=3 independent passages (Calu-3 or HBEC-6KT) or donor samples (primary human airway epithelial cells; nonsmoker, healthy subjects). For each independent blot of each protein, all of the same samples were run. A total protein loading control is provided to demonstrate protein loaded for each sample.
FIGURE 6
FIGURE 6
Immunohistochemical localisation of ACE2 (angiotensin-converting enzyme 2), TMPRSS2, CD147 and GRP78 protein in human lung tissue. Representative samples from a) a healthy nonsmoker with no underlying chronic airway disease, and b) a smoker with COPD. Black outlines: low magnification (12×) of conducting airways with airway epithelium; scale bars 100 μm. Green outlines: high magnification regions (60×) of conducting airway epithelium that are defined in the low magnification images by green squares; scale bars 50 μm. Red outlines: high magnification regions (50×) of lung tissue away from the airway lumen that are defined in the low magnification images by red squares; scale bars 50 μm. H&E: haematoxylin and eosin. Positive immunohistochemical staining is rust/brown. Total number of independent samples analysed was 49–98.
FIGURE 7
FIGURE 7
Proposed functions of host airway epithelial cell molecules for interaction with SARS-CoV-2. Proteins associated (or suggested to be associated) with host cell entry of SARS-CoV-2 and the activation of the SARS-CoV-2 spike protein (SARS-S) are displayed. Angiotensin-converting enzyme 2 (ACE2) is suggested as the primary SARS-S receptor for viral entry (interaction of ACE2 with SARS-S receptor-binding domain (RBD) leading to endosomal viral uptake), followed by activation of SARS-S via pH-dependent cleavage mediated by cathepsin L1 (CTSL). Secondary methods of viral entry and SARS-S activation are likely to involve proteases (e.g. TMPRSS2 and ADAM17) and/or secondary receptors (CD147 and GRP78). Dashed lines indicate mechanisms that have not been fully validated. CyPA: cyclophilin A; N protein: nucleocapsid protein; ER: endoplasmic reticulum. Adapted from [20] with updates and additional information on candidate host molecules. Figure created with BioRender.com.
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