Gene expressi on and i n si t u prot ei n prof i l i ngof  candi dat e SARS- CoV- 2 recept ors i n humanai rway epi t hel i al  cel l s and l ung t i ssue
Aut horJ enni f er A.  Agui ar,  Benj ami n J - M.  Trembl ay,Mi chael  J .  Mansf i el d,  Owen Woody,  Bri al l enLobb,  Ari nj ay Banerj ee,  Abi ram Chandi ramohan,Ni chol as Ti essen,  Quynh Cao,  AnnaDvorki n- Gheva,  Spencer Revi l l ,  Mat t hew S.Mi l l er,  Chri st opher Carl st en,  Loui se Organ,Chi t ra J oseph,  Al i son J ohn,  Paul  Hanson,Ri chard C.  Aust i n,  Bruce M.  McManus,  Gi sl iJ enki ns,  Karen Mossman,  Kj et i l  Ask,  Andrew C.Doxey,  J eremy A.  Hi rot a
j ournal  orpubl i cat i on t i t l e
European Respi rat ory J ournal
vol ume56
number3
page range2001123
year2020- 09- 03
Publ i sherEuropean Respi rat ory Soci et y
Ri ght s( C)  2020 ERS
Aut hor' s f l agpubl i sher
URLht t p: //i d. ni i . ac. j p/1394/00001563/
doi: info:doi/10.1183/13993003.01123-2020
Creat i ve Commons At t ri but i on- NonCommerci al  4. 0
I nt ernat i onal ( ht t ps: //creat i vecommons. org/l i censes/by- nc/4. 0/)
Gene expression and in situ proteinprofiling of candidate SARS-CoV-2receptors in human airway epithelialcells and lung tissue
Jennifer A. Aguiar1, Benjamin J-M. Tremblay1, Michael J. Mansfield2,Owen Woody3, Briallen Lobb1, Arinjay Banerjee4, Abiram Chandiramohan5,Nicholas Tiessen5, Quynh Cao5, Anna Dvorkin-Gheva4, Spencer Revill5,Matthew S. Miller4,6,7, Christopher Carlsten8, Louise Organ9, Chitra Joseph9,Alison John9, Paul Hanson10, Richard C. Austin11, Bruce M. McManus10,Gisli Jenkins9, Karen Mossman4, Kjetil Ask4,5, Andrew C. Doxey1,5,12 andJeremy A. Hirota1,4,5,6,12
@ERSpublicationsACE2 gene and protein expression is low to absent in airway and alveolar epithelial cells in humanlungs. This study suggests the presence of a mechanism dynamically regulating ACE2 expression inhuman lung or other receptors for SARS-CoV-2. https://bit.ly/3f85R1I
Cite this article as: Aguiar JA, Tremblay BJ-M, Mansfield MJ, et al. Gene expression and in situ proteinprofiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue. Eur Respir J2020; 56: 2001123 [https://doi.org/10.1183/13993003.01123-2020].
ABSTRACTIn December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsiblefor the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serineprotease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicatedin SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 andGRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in therespiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthysubjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sampletypes, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots onmultiple 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 andlow 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 andalveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence ofTMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ proteinexpression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression inhuman lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors forSARS-CoV-2 exist to facilitate initial host cell infection.
This article has supplementary material available from erj.ersjournals.com
Received: 10 April 2020 | Accepted after revision: 1 July 2020
Copyright ©ERS 2020. This version is distributed under the terms of the Creative Commons Attribution Non-Commercial Licence 4.0.
https://doi.org/10.1183/13993003.01123-2020Eur Respir J 2020; 56: 2001123
ORIGINAL ARTICLEINFECTIOUS DISEASE
IntroductionIn 2003, the severe acute respiratory syndrome (SARS) outbreak caused by the SARS coronavirus(SARS-CoV) resulted in 8096 probable cases with 774 confirmed deaths [1, 2]. In patients with SARS,deaths were attributed to acute respiratory distress associated with diffuse bilateral pneumonia and alveolardamage [3]. In December 2019, SARS-CoV-2 emerged, causing the coronavirus disease 2019 (COVID-19)pandemic. SARS-CoV-2 is spreading at a much more rapid rate than SARS-CoV [4–6]. Similar clinicalreports of diffuse bilateral pneumonia and alveolar damage have been reported [7–9]. Severe cases ofSARS-CoV-2 have been associated with infections of the lower respiratory tract, with detection of the virusthroughout this tissue as well as the upper respiratory tract [7–9]. The biological mechanisms that maygovern differences in the number of SARS and COVID-19 cases remain undefined. It is possible thatSARS-CoV-2 possesses distinct molecular mechanisms that affect the virulence through viral proteins,greater susceptibility of host cells to infection, permissivity of host cells to virus replication, or somecombination of these and other potentially unknown factors [10–13]. Understanding SARS-CoV andSARS-CoV-2 virus similarities and differences at the molecular level in the host may provide insights intotransmission, pathogenesis and interventions.
The seminal report identifying the receptor for SARS-CoV used a HEK293 cell over-expression system toidentify angiotensin-converting enzyme 2 (ACE2) as a receptor by co-immunoprecipitation withSARS-CoV spike domain 1 [14]. Subsequently, spike protein of SARS-CoV was identified as the viralinteracting partner of ACE2. Host protease activity by transmembrane serine protease 2 (TMPRSS2)facilitates ACE2 ectodomain cleavage and fusion of SARS-CoV membrane with host cell membrane [15–17]. ADAM17 (ADAM metallopeptidase domain 17, a member of the ADAM (a disintegrin andmetalloprotease domain) family) has also been demonstrated to cleave ACE2 ectodomain, but this was notrequired for SARS-CoV infection [18–20]. Mechanisms of SARS-CoV entry distinct from ACE2 have alsobeen reported and include activation by endosomal cathepsin L and cell surface expression of CD147 (alsoknown as basigin (BSG)) or GRP78 (78-kDa glucose-regulated protein; also known as heat shock proteinfamily A (Hsp70) member 5 (HSPA5)) [21–23]. Each of these receptors was mechanistically interrogatedand results suggested that SARS-CoV could initiate host cell entry and infection using multiplemechanisms. Recent in vitro reports have demonstrated that similar host proteins are involved infacilitating cell entry by SARS-CoV-2, such as ACE2 and TMPRSS2 [5, 24]. Biophysical and structuralevidence strongly supports an interaction of ACE2 with SARS-CoV-2 spike protein, similar to SARS-CoVspike protein [12, 13]. Molecular docking studies have also suggested that SARS-CoV-2 spike protein caninteract with cell surface GRP78 [25]. Indirect evidence for a role of CD147 in SARS-CoV-2 binding hasbeen demonstrated in vitro with the use of an anti-CD147 intervention that prevented virus replication[26]. Furthermore, a clinical study with an anti-CD147 intervention reduced symptoms and duration ofhospital admission for COVID-19 patients [27]. In summary, although there is evidence that SARS-CoV-2and SARS-CoV both utilise ACE2 as a receptor to facilitate virus entry, it is possible that differences inhost entry mechanisms play a role in the large epidemiological differences between the two viruses, whichmay include additional unidentified receptors.
ACE2 and TMPRSS2 were identified as cellular entry determinants for SARS-CoV using mechanisticstudies.TheoriginalreportofinsituhumanlungACE2expressiondescribedpositiveimmunohistochemical staining for alveoli and airway epithelial cells, and immunocytochemical staining inA549 type II alveolar epithelial cells [28]. ACE2 protein expression is also present in the human lungadenocarcinoma cell line Calu-3 [29]. Similar to ACE2, the original report describing the expression ofTMPRSS2 in human respiratory mucosa described expression in airway epithelium and type II alveolarepithelial cells [30]. The specificity of the ACE2 and TMPRSS2 antibodies used for analysis of expressionpatterns in human lung tissues remains to be addressed.
Affiliations:1Dept of Biology, University of Waterloo, Waterloo, ON, Canada.2Genomics and RegulatorySystems Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Japan. 3Faculty ofMathematics, University of Waterloo, Waterloo, ON, Canada.4McMaster Immunology Research Centre,McMaster University, Hamilton, ON, Canada.5Firestone Institute for Respiratory Health – Division ofRespirology, Dept of Medicine, McMaster University, Hamilton, ON, Canada. 6Michael G. DeGroote Institute forInfectious Disease Research, McMaster University, Hamilton, ON, Canada.7Dept of Biochemistry andBiomedical Sciences, McMaster University, Hamilton, ON, Canada. 8Division of Respiratory Medicine, Dept ofMedicine, University of British Columbia, Vancouver, BC, Canada. 9Nottingham NIHR Biomedical ResearchCentre, Respiratory Research Unit, University of Nottingham, Nottingham, UK.10Centre for Heart LungInnovation, University of British Columbia, Vancouver, BC, Canada. 11Division of Nephrology, Dept of Medicine,McMaster University, Hamilton, ON, Canada. 12A.C. Doxey and J.A. Hirota contributed equally to this article aslead authors and supervised the work.
Correspondence: Jeremy A. Hirota, Firestone Institute for Respiratory Health – Division of Respirology, Deptof Medicine, McMaster University, Hamilton, ON, L8N 4A6, Canada. E-mail: hirotaja@mcmaster.ca
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To address the uncertainties related to SARS-CoV-2 receptors in human lung, we performed geneexpression and in situ protein profiling of candidate receptors in human airway epithelial cells and lungtissue. Our computational analysis used publicly available microarray gene expression datasets from airwayepithelial cells of 515 unique subjects, single cell sequencing data from 10 subjects, and the FANTOM5dataset for promoter activities of 74 lung-related cell and tissue types. Our proteomic analysis used datafrom the Human Proteome Map [31] and a dataset from primary human airway epithelial cells grownunder air–liquid interface culture conditions [32]. For our in situ protein profiling, we performedimmunohistochemical analysis of 98 human lung tissue samples. To determine antibody specificity, weperformed immunoblots on protein isolated from Calu-3 cells, primary human airway epithelial cells,primary type II alveolar epithelial cells, the human bronchial epithelium cell (HBEC)-6KT cell line, theA549 type II alveolar epithelial cell line, and HEK cells. Collectively, our data contrast with previousreports, demonstrating rare ACE2 protein expression in the airway epithelium and alveoli of human lung.Our protein expression data are consistent with low ACE2promoter activity in a variety of lung epithelialcell samples and low ACE2 gene expression in both microarray and single cell RNA sequencing(scRNAseq) datasets. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 andGRP78 in the respiratory mucosa. Our data suggest the presence of a mechanism dynamically regulatingACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and/or that alternatereceptors for SARS-CoV-2 exist to facilitate initial host cell infection in lung tissue.
MethodsHuman ethicsProcurement of primary human airway epithelial cells used for immunoblots and lung tissue forimmunohistochemistry was approved by the Hamilton (ON, Canada) integrated Research Ethics Board(HiREB 5099T, 5305T, 11-3559 and 13-523-C). The University of British Columbia (Vancouver, BC,Canada) Research Ethics Office approved heart tissue archives and primary human airway epithelial cellcollection.
Upper and lower airway gene expression analysisPublic microarray experiments using Affymetrix chips (HuGene-1.0-st-v1 and HG-U133 Plus 2) on airwayepithelial cell samples collected from nasal (GSE19190) or bronchial (GSE11906) brushings of healthynonsmokers were obtained from the NCBI Gene Expression Omnibus (GEO) database [33, 34]. Thisresulted in a total of 80 individual samples from the two different experiments that included 11 upperairway samples (nasal n=11) and 69 lower airway samples (trachea n=17, large airway n=17, small airwayn=35). For all dataset samples, raw intensity values and annotation data were downloaded using theGEOquery R package (version 2.52.0) [35] from the Bioconductor project [36]. Probe definition files weredownloaded from Bioconductor and probes were annotated using Bioconductor’s “annotate” package. Allgene expression data were unified into a single dataset that was then normalised by robust multiarrayaverage (RMA) normalisation, and only genes present in both of the Affymetrix platforms (n=16013) werekept for subsequent analyses. Correction of experiment-specific batch effects was performed using theComBat method [37] implemented using the sva R package (version 3.32.1) [38]. RMA-normalisedexpression levels for conventional (ACE2, TMPRSS2, ADAM17 and CTSL (cathepsin L1)) andnon-conventional (CD147 and GRP78) SARS-CoV-2 receptor genes were compared across the four definedairway levels, with CDH1 (E-cadherin) expression level included as a positive control with knownexpression in lung tissue. Gene expression levels were tested for significant differences via pairwiseWilcoxon rank sum tests with Benjamini–Hochberg multiple testing correction using the stats R package(version 3.6.1). Gene expression box plots were generated with the ggplot2 R package (version 3.2.1).
Analysis of curated bronchial epithelial cell brushing datasetA total of 1859 public microarray experiments using Affymetrix chips (HG-U133 Plus 2 andHuGene-1.0-st-v1) on airway epithelial cell samples were selected from the NCBI GEO database. Thesesamples were further filtered by removing individuals with asthma or COPD, resulting in a total of 504individual healthy samples (GSE4302, 28 samples; GSE67472, 43 samples; GSE37147, 159 samples;GSE108134, 274 samples). Within this dataset, sex and/or age information was included for 310 samples;of these, sex data were available for 86 females and 106 males. Smoking status information was alsoprovided for 451 samples, with 260 current smokers, 82 former smokers and 109 never-smokers.
For all dataset samples, raw intensity values and annotation data were downloaded as described. Probedefinition files were retrieved as described. All gene expression data were unified into a single dataset thatwas then RMA-normalised, and only genes present in both of the Affymetrix platforms (n=16105) werekept for subsequent analyses. Correction of experiment-specific batch effects was performed as described.
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Analysis of promoter activity from the FANTOM5 datasetThe FANTOM5 promoterome dataset [39] for the hg38 assembly [40] was used to examine promoteractivity of SARS-CoV-2-related human genes, namely ACE2, TMPRSS2, ADAM17, CTSL, CD147 andGRP78. Using the ZENBU genome browser [41], the nearest cap analysis of gene expression (CAGE) peakupstream and on the same strand as each of the aforementioned genes was extracted and analysed. Thedataset consists of CAGE promoter activity data for 1866 primary cells, cell lines and tissues from humans,and is quantified as normalised transcripts per million (TPM). A subset of FANTOM5 CAGE data (120samples) is presented, considering only samples related to lung, gut, heart and prostate tissues (consistingof 74, 19, 15 and 12 samples, respectively). Normalised TPM values for each CAGE peak, anapproximation for promoter activity, were log10 transformed and separated according to tissue and celltype, and the radius of each point is proportional to these transformed normalised TPM values.
Analysis of protein abundance from proteomic datasetsPublicly available human proteomic data from the datasets of KIM et al. [31] and FOSTER et al. [32] wereused to evaluate SARS-CoV-2 receptor-related protein expression in different human tissues andexperimental conditions. Expression values were extracted from the dataset of KIM et al. [31] for ACE2,TMPRSS2, ADAM17, CTSL, CD147 and GRP78, using CDH1 as a control for airway cells. Data werecreated using the pheatmap package in R (version 1.0.12) and expressed as log10-transformed to facilitatevisualisation. Proteomic data from the dataset of FOSTER et al. [32] consist of bronchial epithelial cellscollected from healthy nonsmokers (n=4; males) and exposed to PBS control vehicle. Intensity values forACE2, TMPRSS2, ADAM17, CD147 and GRP78 were extracted, with CDH1 included as a positive control.Intensity values were determined by the original study authors via normalisation of all detected peptideintensities associated with a given parent protein [32]. Box plots were generated with the ggplot2 R package(version 3.2.1) with intensity values log10-transformed for visualisation purposes.
Analysis of single cell RNA sequencing dataData preprocessed using the Cell Ranger pipeline (10x Genomics) were obtained from GSE135893.Samples from 10 control subjects and 12 idiopathic pulmonary fibrosis patients were downloaded andpost-processed with the Seurat package in R [42]. Cell populations were defined using the markersprovided in the source paper [43]. Cells belonging to the 10 control subjects were used for further analysis.Visualisations of violin plots were created using Seurat.
Primary human airway epithelial cellsThe human lung adenocarcinoma cell line, Calu-3, was grown under culture conditions defined by thesupplier (ATCC HTB-55). Primary human airway epithelial cells isolated via bronchial brushings fromconsented healthy individuals were grown in PneumaCult ExPlus (Stemcell Technologies, Vancouver, BC,Canada) under submerged monolayer culture conditions and used between passages 1 and 4. The humanbronchial epithelial cell line, HBEC-6KT, was grown under submerged monolayer culture conditions inkeratinocyte serum-free media supplemented with epidermal growth factor (0.4 ng·mL−1) and bovinepituitary extract (50 μg·mL−1) [44–47].
ImmunoblotsCell protein was isolated using RIPA lysis buffer (VWR, Mississauga, ON, Canada) supplemented withprotease inhibitor cocktail (Sigma, Oakville, ON, Canada) with quantification performed using Bradfordassay reagents (Bio-Rad, Mississauga, ON, Canada). Immunoblots were performed using stain-free 4–20%pre-cast gradient gels and imaged on a ChemiDoc XRS+ Imaging system (Bio-Rad). For each immunoblot,20 μg of protein was added per lane. ACE2 (MAB933, monoclonal, clone 171606, 2 μg·mL−1 (R&DSystems)),TMPRSS2(HPA035787,polyclonal,0.4 μg·mL−1(AtlasAntibodies)),CD147(ab666,monoclonal, clone MEM-M6/1, 1 μg·mL−1 (Abcam)) and GRP78 (610979, monoclonal, clone 40/BiP,0.25 μg·mL−1 (BD Biosciences), and HPA038845, rabbit polyclonal (Atlas Antibodies)) primary antibodieswere diluted in 5% skimmed milk/TBS with 0.1% Tween-20 and incubated overnight on a rocker at 4°Cwith detection performed the following day using an anti-mouse (ACE2, CD147 and GRP78 (BDBiosciences))oranti-rabbit(TMPRSS2andGRP78(AtlasAntibodies))horseradishperoxidase(HRP)-conjugated secondary antibodies at 1:3000 for 2 h at room temperature (Cell Signaling, Danvers,MA, USA). Visualisation of TMPRSS2, CD147 and GRP78 was performed using Clarity Westernenhanced chemiluminescence (ECL) Substrate, while ACE2 was visualised with Clarity Max ECL Substrate(Bio-Rad). Total protein loading images were collected as a qualitative visualisation of protein loadingbetween sample types [48]. The immunogen for ACE2 primary antibody is mouse myeloma cell lineNS0-derived recombinant human ACE2 Gln18-Ser740 (predicted). The immunogen for TMPRSS2primary antibody is the recombinant protein epitope signature tag antigen sequence, GSPPAIGPYYENHGYQPENPYPAQPTVVPTVYEVHPAQYYPSPVPQYAPRVLTQASNPVVCTQPKSPSGTVCTSKT.
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The immunogen for the CD147 primary antibody is recombinant full-length protein corresponding tohuman CD147. The immunogen for the GRP78 BD Biosciences primary antibody is human BiP/GRP78amino acids 525–628. The immunogen for the GRP78 Atlas Antibodies primary antibody is therecombinant protein epitope signature tag antigen sequence, EKFAEEDKKLKERIDTRNELESYAYSLKNQIGDKEKLGGKLSSEDKETMEKAVEEKIEWLESHQDADIEDFKAKKKELEEIVQPIISKL.
Independent immunoblot analysis (L. Organ, C. Joseph, A. John and G. Jenkins) was performed on A549,HEK and immortalised human bronchial epithelial cells. Equal amounts of protein (20 µg) were loaded onto 4–12%, Bis-Tris gradient gels (NP0326BOX; ThermoFisher) with anti-ACE2 (ab108252, rabbitmonoclonal, clone EPR4435(2), 1/500 dilution of stock antibody; Abcam). A loading control of GAPDHwas used to demonstrate protein loading (ab181603, rabbit monoclonal, EPR16884, 1/10000 dilution ofstock antibody; Abcam). Visualisation was performed with ECL Clarity (Bio-Rad) on a Licor C-DiGit.
ImmunohistochemistryFormalin-fixed paraffin-embedded human lung tissue from non-diseased regions was obtained fromarchived tissue blocks from patients who had undergone lung resection for clinical care. Human hearttissue was from the University of British Columbia Cardiovascular Tissue Registry. Sections 4 μm thickwere cut and stained for ACE2 (15 μg·mL−1), TMPRSS2 (10 μg·mL−1), CD147 (5 μg·mL−1), and GRP78(HPA038845, 1/200 dilution) using the same antibodies used for immunoblot analysis. All staining wasperformed on a Leica Bond RX system with Leica Bond reagents, heat-induced antigen retrieval at pH 6(20 min) with primary antibody incubation for 20 min. Digital slide scanning was performed using anOlympus VS120-L100 Virtual Slide System at 40× magnification with VS-ASW-L100 V2.9 software and aVC50 colour camera, followed by image visualisation with HALO image analysis software.
ResultsCandidate genes important in SARS-CoV-2 infection are detectable at varying levels in humanairway epithelial cells and lung tissueWe performed a targeted analysis of ACE2, TMPRSS2, ADAM17, CTSL, CD147 and GRP78 geneexpression as candidates important for SARS-CoV-2 infection in human airway epithelial cells. Here andthroughout the gene expression analyses, CDH1 (E-cadherin) was used as a control for lung epithelial cellphenotype. We first examined these genes in a curated dataset of upper and lower airway epithelial cellgene expression from the nasal sinus to the 12th generation of airway in the lung (figure 1).
In the upper airways, all candidates were expressed, with the highest level observed for GRP78 and thelowest level observed for ACE2. Analysis along multiple generations of the lower airways (trachea, large(4th–6th generation) and small airways (10th–12th generation)) revealed identical relative expressionpatterns, with ACE2 being the least expressed and GRP78 being the highest expressed. ACE2 geneexpression showed the greatest variability along the upper and lower airways, with greatest expressionobserved in the trachea samples and the lowest expression in the small airway (figure 1).
Expression level
ACE2TMPRSS2ADAM17
AirwayNasalTracheaLargeSmall
CTSLCD147GRP78CDH1
** ** ******
FIGURE 1 Microarray expression profiles of candidate SARS-CoV-2 receptor genes in upper and lowerairways. 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 positivecontrol. Statistical values for comparisons for each gene at each airway generation were calculated; those notshown were nonsignificant. *: p<0.05; **: p<0.01; ***: p<0.001.
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Following our observation of consistent expression along the upper and lower airways of candidate genesimportant in SARS-CoV-2 infection, we determined whether sex or age affected gene expression levels inhealthyindividualsusinga curateddatasetofbronchialbrushingsfrom504healthy subjects(supplementary table S1). The expression levels for the candidate genes in healthy subjects paralleled thepatterns observed in the smaller survey of upper airways, trachea, large and small airways (figure 2).
Expression level
ACE2TMPRSS2 ADAM17
SexFemaleMale
CTSLCD147GRP78
Age >40
Expression level
Age >40
SexChipACE2
ADAM17
TMPRSS2
Expression level
20 30 40
ACE2TMPRSS2ADAM17CTSLCD147GRP78CDH1
Expression level
TMPRSS2 ADAM17
Smoking statusCurrentFormer
CTSLCD147GRP78CDH1
Female
HG-U133 Plus 2
HuGene-1.0-st-v1
20 30 40 50 60
20 30 40 50 60
20 30 40 50Age years
Age years
20 30 40 50 60
20 30 40 50 60
10.020 30 40 50 60
5.0506070
ACE2e)
TMPRSS2ADAM17CTSLCD147GRP78CDH1
Expression level
8.5506070506070506070506070506070506070
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 microarraychip platform. Expression values reflect signal intensities, indicating lowest detected expression of ACE2 (angiotensin-converting enzyme 2) andhighest 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-U133Plus 2 microarray (n=43) or e) the HuGene-1.0-st-v1 microarray (n=181). Correlations were performed separately between platforms because ofdifferences in their age distributions. e) A weak negative correlation (r=−0.20, p=0.015) was detected for ACE2 in the dataset that used theHuGene-1.0-st-v1 microarray.
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Median ACE2 gene expression was the lowest, while GRP78 gene expression was the highest (figure 2a).No gene candidate demonstrated sex dependence for expression levels (figure 2b). ACE2, TMPRSS2,CD147 and GRP78 were elevated in current smokers relative to never-smokers (figure 2c andsupplementary table S2). CTSL was reduced in current smokers relative to never-smokers. No microarraychip-dependent effects were observed for relationships between sex or smoking status and gene expression.For quantitative analyses related to age and gene expression, our curated database was divided into datasetsthat used either the HG-U133 Plus 2 or HuGene-1.0-st-v1 microarray due to differences in agedistributions. In the HuGene-1.0-st-v1 dataset (n=181), which included a greater proportion of olderindividuals (>50 years), we observed reduced ACE2 gene expression with age (figure 2e; p<0.05).
Promoter activity data of each of the candidate genes important in SARS-CoV-2 binding and infection wereextracted and analysed from the FANTOM5 dataset, which includes 1866 primary cells, cell lines, and tissuesample types (figure 3). We selected all sample formats that included “lung”, “nasal”, “airway” or “olfactory”,to identify lung-specific sample types. Gut, heart and prostate tissue samples were analysed as controls.Consistent with our observed gene expression analysis along the upper and lower airways, normalised TPMvalues for each CAGE peak demonstrated that CD147 promoter activity was elevated relative to ACE2promoter activity across airway epithelial cells and lung tissue samples. CTSL promoter activity was thelowest of all candidate genes, which contrasted with the modest expression observed at the gene level(figure 2a). Both microarray gene expression analysis and promoter activity were consistent with results ofcandidate gene expression in a scRNAseq dataset of 10 healthy subjects (supplementary figure S1).
Collectively, our gene expression analysis of the upper and lower airways of healthy males and females ofdiverse ages suggests that ACE2 gene expression is low relative to all other candidate SARS-CoV-2 receptorgenes analysed in human airway epithelial cells. Furthermore, we observe no sex-dependent orage-dependent expression patterns of any candidates at the gene level, although smoking status did havean impact on gene expression levels.
In vitro and in situ protein profiling reveals distinct expression patterns for candidates importantin SARS-CoV-2 infectionAnalysis of transcriptional data may not be indicative of in situ protein expression levels [49]. To extendour gene expression observations, we mined publicly available proteomic data from whole lung andprimary human airway epithelial cell cultures and performed in vitro immunoblots on human airwayepithelial cell lysates and in situ protein immunohistochemistry on human lung tissue using the sameantibodies for each method.
The Human Proteome Map is a publicly available resource that includes select adult and fetal tissues andcirculating immune cell populations [31]. Using this resource, we examined protein expression of ACE2,TMPRSS2, ADAM17, CTSL, CD147 and GRP78. In human lung tissue homogenate, ACE2 was notdetected, while being detected in heart, gut and testes, known positive control tissues (figure 4a). The rankorder of the remaining molecules in human lung tissue homogenate was: GRP78>CD147>CTSL>ADAM17>TMPRSS2. Human lung tissue homogenate is a heterogeneous population of cells, precludingthe ability to associate protein expression to a given cell type. We therefore interrogated a publiclyavailable proteomic dataset derived from primary human airway epithelial cells grown under air–liquidinterface culture conditions [32], examining the same candidates. Again, ACE2 protein expression was notdetectable (figure 4b). CD147, GRP78 and CTSL were expressed with multiple peptide counts, whileTMPRSS2 and ADAM17 were only marginally expressed with low peptide counts. Collectively, twoproteomic datasets from distinct lung sample formats provide complementary and consistent expressionprofiles of candidate molecules important in SARS-CoV-2 infection.
To localise the in situ expression of the candidate molecules of interest at the protein level, we pursuedimmunohistochemical analysis paired with immunoblot validation of the specificity of the selectedantibodies for recognition of proteins of the predicted molecular weight. An anti-ACE2 antibody detectedonly a single band in Calu-3 cells at the predicted molecular weight of ACE2 protein (∼110 kDa) (figure 5a,lanes 1–3). The anti-ACE2 antibody required the use of a super-sensitive ECL solution. No ACE2 proteinwas detected in primary airway epithelial cells or the HBEC-6KT cell line, despite confirmation of proteinloading (figure 5a, lanes 4–9, protein loading shown underneath main blot). Independent immunoblottingwith a distinct anti-ACE2 primary antibody was performed, with a single band observed in HEK cells, butnot in immortalised human bronchial epithelial cells or A549 cells (supplementary figure S2).
An anti-TMPRSS2 antibody detected multiple bands in all airway epithelial cell samples, with a dominantband at the predicted molecular weight of ∼57 kDa (figure 5b). These patterns were conserved across allcell types that were analysed.
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An anti-CD147 antibody detected a single band in all airway epithelial cell samples, with a dominant bandat the predicted molecular weight of ∼55 kDa (figure 5c). The immunoblot bands were consistent with theheavy glycosylation of CD147 [50].
An anti-GRP78 antibody (BD Biosciences 610979) detected a single band in all airway epithelial cellsamples, with a dominant band at the predicted molecular weight of ∼78 kDa (figure 5d).
The immunoblots using anti-ACE2, anti-CD147 and anti-GRP78 demonstrated a single band of predictedmolecular weight, suggesting that observed immunohistochemical staining should be specific to the protein
Airway
Log10 (TPM)
Cell state
NormalCancerous
1001000
GutHeartProstate
TMPRSS2
ADAM17
Tissue source
FIGURE 3 Promoter activity for SARS-CoV-2-related genes from the FANTOM5 cap analysis of geneexpression (CAGE) dataset. The FANTOM5 CAGE data consist of quantified promoter expression levels acrossthe human genome for 1866 samples from primary cells, cell lines and tissue samples. The FANTOM5 CAGEpromoter activity data for several SARS-CoV-2-related genes are shown for samples related to lung, gut,heartandprostatetissues(n=120).Dotsizesareproportionaltopromoteractivity,depictedaslog10-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, includingmeasurements from healthy and cancerous cells.
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of interest based on the target epitope, as both methods detect denatured proteins [51]. The sameanti-ACE2 and anti-CD147 antibodies were validated for immunohistochemistry. The anti-TMPRSS2 wasused for immunohistochemistry, although the multiple bands observed by immunoblot caution thespecificity of any observed in situ staining. Attempts to optimise anti-GRP78 antibody application forimmunohistochemistry were unsuccessful, requiring additional antibody interrogation with HPA038845
Adult adrenalAdult colonAdult oesophagusAdult frontal cortexAdult gallbladderAdult heartAdult kidneyAdult liverAdult lungAdult ovaryAdult pancreasAdult prostateAdult rectumAdult retinaAdult spinal cordAdult testisAdult urinary bladderB-cellsCD4 cellsCD8 cellsMonocytesNK cellsPlacentaPlateletsFetal brainFetal gutFetal heartFetal liverFetal ovaryFetal testis
Log10-transformedprotein expression
ACE2(count = 0) 
Log10-transformed protein intensity
CDH1ACE2TMPRSS2 ADAM17CTSLCD147GRP78
TMPRSS2(count = 2) ADAM17(count = 1) CTSL(count = 4) CD147(count = 7) GRP78(count = 47) CDH1(count = 13) 
FIGURE 4 Proteomic profiles of candidate SARS-CoV-2 receptor genes in human tissue and airway epithelialcells. a) Intensity values of protein expression from KIM et al. [31] for the genes ACE2 (angiotensin-convertingenzyme 2), TMPRSS2, ADAM17, CTSL (cathepsin L1), CD147 and GRP78. CDH1 (E-cadherin) intensity isincluded as a positive control for expression in airway cells. Intensity values have been log10-transformed tofacilitate comparison between candidates with different basal expression levels across tissue types. Grey cellsin 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 GRP78proteins in human airway epithelial cells from healthy nonsmokers (n=4; males) grown under air–liquidinterface culture conditions [32]. CDH1 intensity is included as a positive control. Counts indicating thenumber of detected peptides associated with each parent protein are provided. ND: the protein was notdetected in this study.
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(AtlasAntibodies),whichwassuitableforbothimmunoblottingandimmunohistochemistry(supplementary figure S3).
ACE2 immunohistochemistry revealed only select staining in rare cells in the airways and the alveoli of all98 human lung samples analysed, which included healthy subjects and those with chronic lung diseases(figure 6). A single healthy human sample contained one positive airway epithelial cell with additionalpositive staining in the peripheral lung in cells with type II alveolar epithelial cell morphology (figure 6a).
150 kDa
ACE2 (110 kDa)
100 kDa
75 kDa
CD147 (55 kDa)GRP78 (78 kDa)
50 kDa
b)TMPRSS2 (57 kDa)
75 kDa100 kDa
75 kDa
50 kDa37 kDa
25 kDa
15 kDa
FIGURE 5 Immunoblot analysis of ACE2 (angiotensin-converting enzyme 2), TMPRSS2, CD147 and GRP78protein expression in human airway epithelial cell protein lysates. a) ACE2 with single band for predictedmolecular weight of 110 kDa (red box). b) TMPRSS2 with multiple bands including a dominant band atpredicted molecular weight of 57 kDa (red box). c) CD147 with a single broad band around predictedmolecular 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: humanbronchial epithelium cell (HBEC)-6KT cell line. All cells grown under submerged monolayer conditions, withn=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. Atotal protein loading control is provided to demonstrate protein loaded for each sample.
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A representative image of a sample from a smoker with COPD (figure 6b) shows no ACE2 proteinstaining in the airway epithelium and a rare positive cell in sub-basement membrane tissue. Quantificationof positive pixel count for ACE2 staining normalised to total tissue pixel count revealed no differencesbetween healthy nonsmokers and tobacco smokers (supplementary figure S4). Lung microvasculature andhuman heart tissue had positive staining (supplementary figures S5 and S6), consistent with previouslydescribed reports for ACE2 protein staining patterns [52, 53].
TMPRSS2 immunohistochemistry revealed diffuse staining in the airway epithelium and in immune cellsin the lung periphery, with greater staining in smokers with COPD (figure 6). These observations wereconsistent in the majority of the 98 human samples examined.
CD147 immunohistochemistry revealed strong membrane-restricted staining in the airway epithelium anddiffuse staining in immune cells in the lung periphery (figure 6). CD147 displayed greater staining in smokerswith COPD. These observations were consistent in the majority of the 98 human samples examined.
GRP78 immunohistochemistry revealed diffuse staining in airway and alveolar epithelium and in immunecells in the lung periphery (figure 6; 49 samples). No qualitative differences in GRP78 staining wereobserved between healthy subjects and smokers with COPD.
Collectively, our in vitro and in situ protein profiling is consistent with our gene expression analysis, withCD147 and GRP78 protein expression dominant over TMPRSS2 and ACE2. Additional examples ofstaining in lung tissue are provided in supplementary figure S7. ACE2 protein expression is rare in humanlung tissue and found in select cells in both healthy individuals and those with chronic lung diseases.
a)b)HealthyTobacco smoker
H&EACE2TMPRSS2CD147GRP78
FIGURE 6 Immunohistochemical localisation of ACE2 (angiotensin-converting enzyme 2), TMPRSS2, CD147and GRP78 protein in human lung tissue. Representative samples from a) a healthy nonsmoker with nounderlying chronic airway disease, and b) a smoker with COPD. Black outlines: low magnification (12×) ofconducting 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 lumenthat are defined in the low magnification images by red squares; scale bars 50 μm. H&E: haematoxylin andeosin. Positive immunohistochemical staining is rust/brown. Total number of independent samples analysedwas 49–98.
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TMPRSS2 and CD147 protein expression are potentiated in individuals with a history of tobacco smokingand a diagnosis of COPD.
DiscussionThe global COVID-19 pandemic that emerged in late 2019 is caused by SARS-CoV-2. The possible hostreceptor(s) for SARS-CoV-2 have not been exhaustively surveyed in human lung tissue at the gene andprotein level. Understanding the expression levels and localisation of candidate SARS-CoV-2 receptors inhost lung tissue may provide insights into therapeutic interventions that might reduce disease spread, viralreplication or disease pathology. To address this knowledge gap, we performed gene expression, proteomicprofiling at the tissue and cell level, and in situ protein profiling of candidate receptors in human airwayepithelial cells and lung tissue (summarised in figure 7). Collectively, our data demonstrate rare ACE2protein expression in human airway epithelial cells in vitro and in situ. Our protein expression data areconsistent with low ACE2 promoter activity in a panel of lung epithelial cell samples and low ACE2 geneexpression in bronchial epithelial cells (microarray) and lung cells (scRNAseq). We present confirmatoryevidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells andconfirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa. Our datasuggest that for ACE2 to be an integral receptor for SARS-CoV-2, mechanisms are likely to exist thatdynamically regulate expression in human lung, perhaps in periods of SARS-CoV-2 infection [54]. It isalso possible that alternate receptors for SARS-CoV-2 are important in initial host cell infection.
Using a curated microarray gene expression dataset generated from bronchial brushings of 504 healthysubjects that considers the limitations of merging multiple datasets from distinct sources, we observed thatsex did not correlate with gene expression of any candidate host molecule involved in SARS-CoV-2
SARS-CoV-2 spike protein
Virion enters host cell via endosome
Infection
RBD of spike protein binds to ACE2 receptor
pH-dependent cleavage of spike protein by CTSL activates spike protein
Cleavage of ACE2 receptor by 
TMPRSS2 or ADAM17 suggested to facilitate uptake of virion into host cell for activation
CD147 complexes with CyPA which has been shown to bind to SARS-CoVN protein
GRP78 translocates from ER to plasma membrane during cellular stress
Fusion of viral envelope with host cell membrane
PathogenesisCleavage of ACE2 
receptor by 
ADAM17 suggested to facilitate shedding of ACE2 and pathogenesis
RBD of spike protein binds to ACE2 receptor and is activated by the protease TMPRSS2
RBD of spike protein binds to GRP78 
receptor
FIGURE 7 Proposed functions of host airway epithelial cell molecules for interaction with SARS-CoV-2. Proteins associated (or suggested to beassociated) with host cell entry of SARS-CoV-2 and the activation of the SARS-CoV-2 spike protein (SARS-S) are displayed. Angiotensin-convertingenzyme 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). Secondarymethods of viral entry and SARS-S activation are likely to involve proteases (e.g. TMPRSS2 and ADAM17) and/or secondary receptors (CD147 andGRP78). Dashed lines indicate mechanisms that have not been fully validated. CyPA: cyclophilin A; N protein: nucleocapsid protein; ER: endoplasmicreticulum. Adapted from [20] with updates and additional information on candidate host molecules. Figure created with BioRender.com.
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infection and that ACE2 and TMPRSS2 were the lowest expressed genes of interest examined. In onedataset, ACE2 gene expression modestly decreased with age, although protein level confirmation was notpossible. The low level of ACE2 and TMPRSS2 gene expression in bulk bronchial epithelial cell geneexpression samples suggests low levels of cells expressing both of these genes within this lung tissue. Weconfirm that tobacco smoking is associated with elevated ACE2 gene expression levels in bronchialepithelial cell samples [55], although we were unable to confirm this with immunohistochemistry analysisof protein on human lung samples.
Advances in transcriptomics have enabled scRNAseq that has identified unique and rare cell types inhuman lung that may have importance in health and disease [56, 57]. scRNAseq provides an opportunityto look at transcriptional profiles in subsets of cell populations, which may isolate a cell signal from a bulksample. We therefore utilised scRNAseq data from healthy human lung samples as a parallel approach.The resolution of scRNAseq for subpopulations of epithelial cells revealed low or absent expression ofACE2 gene in all populations examined, whereas CD147 and GRP78 were present in all populations. Ourresults are consistent with current publicly available data that discuss the presence of rare ACE2/TMPRSS2-positive cells [54]. Using lung samples from eight individuals (four HIV and active tuberculosisdouble positive, two HIV positive and tuberculosis negative, and two double negative controls), ZIEGLERet al. [54] have reported in humans that only 0.8% of type II alveolar epithelial cells expressed both ACE2and TMPRSS2 genes. Further analysis of ciliated cells found that 5.3% of these cells expressed both ACE2and TMPRSS2 genes. In vitro models with SARS-CoV are consistent with this finding, as ciliated cells arepreferentiallytargetedbythiscoronavirus[58].Mostintriguingis thatACE2andTMPRSS2gene-expressing cells were only identified in the HIV and tuberculosis double positive samples. Theseobservations were replicated in the upper airways, with only a rare population of secretory epithelial cells(0.3% of this population) co-expressing ACE2 and TMPRSS2. The reported scRNAseq results areconsistent with a focused analysis looking at only ACE2 gene expression in a variety of lung cell types [59].Importantly, these elegant transcriptomic analyses confirm our observations in bulk tissue microarraydatasets.
Consortium-based publicly available datasets represent another parallel approach to confirm our data. Wehave used the FANTOM5 dataset containing CAGE promoter activation data for 1866 primary cells, celllines and tissue samples from humans [39] to examine the level of promoter activity for each candidateSARS-CoV-2 receptor gene. The FANTOM5 CAGE data provide an additional and complementaryapproach to quantifying gene expression, since a given gene’s shared promoter can yield multipletranscripts at different expression levels, as well as being partially independent of any given transcript’shalf-life in the cell. In general, the promoter activity of ACE2 in airway-related tissues is low or absent;only a single sample originating from an adult lung yielded a normalised CAGE promoter expression level>1 TPM, while expression was observed in gut cells, consistent with known patterns of ACE2 expression[60]. Consistent with the microarray data, CD147 promoter activity is elevated relative to ACE2 acrossairway-related cells and tissues, although the relatively low CTSL promoter activity is incongruent withmodest levels of gene expression.
The expression of genes does not always correlate with protein expression [49]. With this in mind, weperformed combination proteomic analyses with immunoblot analyses. For our immunoblots, we used thehuman Calu-3 adenocarcinoma cell line, as this cell line is susceptible and permissive to SARS-CoV-2infection and expresses ACE2, an observation we confirm [24, 29]. We also used primary human airwayepithelial cells and the bronchial epithelial cell line (HBEC-6KT). We performed immunoblots for ACE2and TMPRSS2 as these have been highlighted as interacting with SARS-CoV-2, while we probed CD147 asrecent pre-clinical and clinical studies have provided proof of concept for this as a candidate SARS-CoV-2receptor [26, 27]. Lastly, GRP78 was dominantly expressed throughout transcriptomic studies and wasselected as a positive control, as previous expression has been confirmed in human airway epithelial cells[61]. Cathepsin L was excluded from the present analysis due to low promoter activity (figure 3), whileADAM17 was excluded as the proposed function in coronavirus infections is via ACE2 [5, 24], which wasincluded in analysis. Immunoblot analysis with all antibodies revealed dominant bands of predictedmolecular weight, with the anti-TMPRSS2 polyclonal antibody revealing additional minor bands in all cellsamples examined. The identity of these other bands remains unclear and suggests downstreamimmunohistochemical analysis may be confounded by the specificity of this antibody. In contrast,antibodies for ACE2, CD147 and GRP78 were specific and could be used for immunohistochemistrywithout concerns of specificity. Interestingly, ACE2 protein could only be detected with a super-sensitiveECL solution and only in Calu-3 cells, suggesting absent protein in primary human airway epithelial cellsand the HBEC-6KT cell line. Our data are consistent with previous immunoblots of primary humanairway epithelial cells grown under submerged monolayer conditions using the same primary antibody,where ACE2 protein was absent, and only expressed under air–liquid interface culture conditions [62]. The
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observation that CD147 and GRP78 are also expressed in Calu-3 cells encourages further interrogationinto these host proteins, as they may contribute to function of ACE2 and TMPRSS2 in SARS-CoV-2binding and fusion in this cell type. Collectively, the profiling of antibodies by immunoblot of airwayepithelial cells revealed distinct band patterns demonstrative of antibody specificity for ACE2, CD147 andGRP78, and to a lesser extent for TMPRSS2.
Immunohistochemical analysis has been performed for localisation of ACE2 and TMPRSS2 in humanlung [28, 30]. The observation of positive staining in human lung tissue for these proteins was notaccompanied by companion immunoblot or complementary approaches to define the specificity of theantibody used [51]. In the absence of determination of antibody specificity, the historical data presentedshould be interpreted with caution. To address the issue of antibody specificity for immunohistochemicalstaining, we used the same antibodies we validated by immunoblot and confirmed findings usingproteomics as an orthogonal, antibody independent, approach. We again focused on ACE2 and TMPRSS2asthesearecandidateproteinsimportantforSARS-CoV-2infectionofhostcells.Ourimmunohistochemical staining patterns of ACE2 were consistent with transcriptional profiling andimmunoblots with only one out of 98 human samples demonstrating rare staining in the airway andalveolar epithelium. Positive ACE2 staining in heart tissues and areas of lung microvasculature suggest ourstaining protocol was successful. These results directly contrast with those reported using antibodies thatlacked validation for specificity [28, 30]. TMPRSS2 was expressed more frequently across all samplesexamined, with variability in the airway epithelium associated with history of smoking and/or COPDstatus. In contrast, CD147 expression was observed in airway epithelium of all samples. Similar toTMPRSS2, elevated CD147 expression was associated with history of smoking and/or COPD status,consistent with previous reports [50]. Our original GRP78 antibody selected for immunoblotting was notvalidated for immunohistochemistry. We therefore performed confirmatory GRP78 immunoblotting andimmunohistochemistry with an additional antibody (HPA038845) and provided demonstration ofexpression in human airway epithelial cells in vitro and in situ. Importantly, it is well established thatGRP78 resides in the endoplasmic reticulum (ER) under normal physiological conditions, where it acts asan ER-resident molecular chaperone to facilitate correct protein folding. However, under conditions of ERstress, including viral infection, a portion of ER-resident GRP78 relocates to the cell surface, where it mayact as a viral co-receptor [63, 64]. The presence of cell surface GRP78 has been reported in atheroscleroticplaques [65], prostate cancer [66] and kidney [67]. However, there are currently no commercially availableanti-GRP78 antibodies that bind specifically to cell surface GRP78 and dual immunofluorescence is usedto show co-localisation of cell surface GRP78 with an established surface receptor [65]. This drawbackprecludes our ability to perform accurate cell surface GRP78 immunohistochemistry on lung tissue tointerrogate this concept further in the context of SARS-CoV-2 receptors. Utilisation of prostate cancerpatient-derived GRP78 auto-antibodies that are specific for cell surface GRP78 [65] may be suitable onlung tissue for assessment of cell susceptibility to SARS-CoV-2 infection by GRP78.
Our study has several limitations that have not already been addressed. Our observation of differences ingene expression between upper and lower airways and along the airway tree were not corroborated at theprotein level. It remains possible that entirely different protein expression profiles for the candidatemolecules examined exist in the upper airway, presenting a different environment for SARS-CoV-2interaction with the respiratory mucosa. Nasal pharyngeal swabs are capable of detecting SARS-CoV-2virus [68] and this anatomical region is probably important for subsequent infection in the lower airways[8, 69]. Related to this potential temporality of effect, it is possible that SARS-CoV-2 induces theexpression of receptors on host cells following infection [54]. Our study is also limited by examiningcandidate molecules important in SARS-CoV-2 infection under basal conditions, in the absence of viral orenvironmental stimuli that may regulate gene transcription and protein translation.
SARS-CoV-2 infection and transmission has caused the global COVID-19 pandemic. An understanding ofthe receptors used by SARS-CoV-2 for host cell infection and the parallel characterisation in humansamples is required to inform development of intervention strategies aimed at mitigating COVID-19. Ourdata demonstrate rare ACE2 protein expression in human airway epithelial cells in vitro and in situ,consistent with low ACE2 promoter activity and ACE2 gene expression in bronchial epithelial cells. Wepresent confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airwayepithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratorymucosa. Due to the overwhelming evidence that the SARS virus interacts with ACE2, there are likely to bealternate mechanisms regulating ACE2 in the respiratory mucosa in the context of SARS-CoV-2 infection,and/or perhaps other co-receptors, beyond what is expressed under basal conditions at the protein level.
Acknowledgements: We would like to acknowledge Mary Jo Smith from the McMaster Immunology Research CentreCore Histology facility (McMaster University, Hamilton, ON, Canada) for her timely and professional expertise withantibody staining for immunohistochemistry. We would like to thank Sam Wadsworth (Aspect Biosystems, Vancouver,
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BC, Canada) and John McDonough (Yale University, New Haven, CT, USA) for their intellectual discussions aroundCD147 and airway epithelial cell biology. We would like to thank Charles Plessy (Okinawa Institute of Science andTechnology, Onna, Japan) for his suggestion to analyse the FANTOM5 data. We would like to thank all of the personaland professional support from the authors’ respective families (Candace Lee Bastedo, Julianne Meiko Hirota and HanaLee Hirota) and research institutes and, most importantly, the frontline healthcare workers during the COVID-19pandemic.
Conflict of interest: J.A. Aguiar has nothing to disclose. B.J-M. Tremblay has nothing to disclose. M.J. Mansfield hasnothing to disclose. O. Woody has nothing to disclose. B. Lobb has nothing to disclose. A. Banerjee has nothing todisclose. A. Chandiramohan has nothing to disclose. N. Tiessen has nothing to disclose. Q. Cao has nothing to disclose.A. Dvorkin-Gheva has nothing to disclose. S. Revill has nothing to disclose. M.S. Miller has nothing to disclose.C. Carlsten has nothing to disclose. L. Organ has nothing to disclose. C. Joseph has nothing to disclose. A. John hasnothing to disclose. P. Hanson has nothing to disclose. R. Austin has a patent US7524826B2 issued to McMasterUniversity and Hamilton Health Sciences Corp., McMaster University. B.M. McManus has nothing to disclose.G. Jenkins reports grants from AstraZeneca, Biogen, Galecto and GlaxoSmithKline; personal fees from BoehringerIngelheim, Daewoong, Galapagos, Heptares, Promedior and Roche; grants and personal fees from Pliant; non-financialsupport from Redx and NuMedii; and is trustee for Action for Pulmonary Fibrosis, outside the submitted work.K. Mossman has nothing to disclose. K. Ask has nothing to disclose. A.C. Doxey has nothing to disclose. J.A. Hirotahas nothing to disclose.
Support statement: This work was supported by start-up funds from J.A. Hirota and a CIHR grant from K. Mossman.J.A. Hirota is supported by the Canada Research Chairs program and an Ontario Early Researcher Award. A.C. Doxeyis supported by NSERC and an Ontario Early Researcher Award. Funding information for this article has beendeposited with the Crossref Funder Registry.
References1Bauch CT, Lloyd-Smith JO, Coffee MP, et al. Dynamically modeling SARS and other newly emerging respiratoryillnesses: past, present, and future. Epidemiology 2005; 16: 791–801.
2Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases(NCIRD). SARS Basics Fact Sheet. www.cdc.gov/sars/about/fs-sars.html Date last updated: 6 December 2017.
3Peiris JSM, Yuen KY, Osterhaus ADME, et al. The severe acute respiratory syndrome. N Engl J Med 2003; 349:2431–2441.
4Munster VJ, Koopmans M, van Doremalen N, et al. A novel coronavirus emerging in China – key questions forimpact assessment. N Engl J Med 2020; 382: 692–694.
5Zhou P, Lou YX, Wang XG, et al. A pneumonia outbreak associated with a new coronavirus of probable batorigin. Nature 2020; 579: 270–273.
6Cascella M, Rajnik M, Cuomo A, et al. Features, Evaluation and Treatment Coronavirus (COVID-19). TreasureIsland, StatPearls Publishing, 2020.
7Gu J, Korteweg C. Pathology and pathogenesis of severe acute respiratory syndrome. Am J Pathol 2007; 170:1136–1147.
8Zou L, Ruan F, Huang M, et al. SARS-CoV-2 viral load in upper respiratory specimens of infected patients.N Engl J Med 2020; 382: 1177–1179.
9Wölfel R, Corman VM, Guggemos W, et al. Virological assessment of hospitalized patients with COVID-2019.Nature 2020; 581: 465–469.
10Lu R, Zhao X, Li J, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications forvirus origins and receptor binding. Lancet 2020; 395: 565–574.
11Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and otherlineage B betacoronaviruses. Nat Microbiol 2020; 5: 562–569.
12Yan R, Zhang Y, Li Y, et al. Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2.Science 2020; 367: 1444–1448.
13Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science 2020; 367: 1260–1263.
14Li W, Moore MJ, Vasllieva N, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARScoronavirus. Nature 2003; 426: 450–454.
15Glowacka I, Bertram S, Muller MA, et al. Evidence that TMPRSS2 activates the severe acute respiratory syndromecoronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response.J Virol 2011; 85: 4122–4134.
16Shulla A, Heald-Sargent T, Subramanya G, et al. A transmembrane serine protease is linked to the severe acuterespiratory syndrome coronavirus receptor and activates virus entry. J Virol 2011; 85: 873–882.
17Chen YW, Lee MS, Lucht A, et al. TMPRSS2, a serine protease expressed in the prostate on the apical surfaceof luminal epithelial cells and released into semen in prostasomes, is misregulated in prostate cancer cells.Am J Pathol 2010; 176: 2986–2996.
18Haga S, Nagata N, Okamura T, et al. TACE antagonists blocking ACE2 shedding caused by the spike protein ofSARS-CoV are candidate antiviral compounds. Antiviral Res 2010; 85: 551–555.
19Iwata M, Silva Enciso JE, Greenberg BH. Selective and specific regulation of ectodomain shedding ofangiotensin-converting enzyme 2 by tumor necrosis factor alpha-converting enzyme. Am J Physiol Cell Physiol2009; 297: C1318–C1329.
20Heurich A, Hofmann-Winkler H, Gierer S, et al. TMPRSS2 and ADAM17 cleave ACE2 differentially and onlyproteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spikeprotein. J Virol 2014; 88: 1293–1307.
21Simmons G, Gosalia DN, Rennekamp AJ, et al. Inhibitors of cathepsin L prevent severe acute respiratorysyndrome coronavirus entry. Proc Natl Acad Sci USA 2005; 102: 11876–11881.
https://doi.org/10.1183/13993003.01123-202015
INFECTIOUS DISEASE | J.A. AGUIAR ET AL.
22Chen Z, Mi L, Xu J, et al. Function of HAb18G/CD147 in invasion of host cells by severe acute respiratorysyndrome coronavirus. J Infect Dis 2005; 191: 755–760.
23Chu H, Chan CM, Zhang X, et al. Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 bothcan utilize GRP78 for attachment onto host cells. J Biol Chem 2018; 293: 11709–11726.
24Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and isblocked by a clinically proven protease inhibitor. Cell 2020; 181: 271–280.
25Ibrahim IM, Abdelmalek DH, Elshahat ME, et al. COVID-19 spike–host cell receptor GRP78 binding siteprediction. J Infect 2020; 80: 554–562.
26Wang K, Chen W, Zhou YS, et al. SARS-CoV-2 invades host cells via a novel route: CD147-spike protein. bioRxiv2020; preprint [https://doi.org/10.1101/2020.03.14.988345].
27Bian H, Zheng ZH, Wei D, et al. Meplazumab treats COVID-19 pneumonia: an open-labelled, concurrentcontrolled add-on clinical trial. medRxiv 2020; preprint [https://doi.org/10.1101/2020.03.21.20040691].
28Hamming I, Timens W, Bulthuis MLC, et al. Tissue distribution of ACE2 protein, the functional receptor forSARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol 2004; 203: 631–637.
29Tseng CTK, Tseng J, Perrone L, et al. Apical entry and release of severe acute respiratory syndrome-associatedcoronavirus in polarized Calu-3 lung epithelial cells. J Virol 2005; 79: 9470–9479.
30Bertram S, Heurich A, Lavender H, et al. Influenza and SARS-coronavirus activating proteases TMPRSS2 andHAT are expressed at multiple sites in human respiratory and gastrointestinal tracts. PLoS One 2012; 7: e35876.
31Kim MS, Pinto SM, Getnet D, et al. A draft map of the human proteome. Nature 2014; 509: 575–581.
32Foster MW, Gwinn WM, Kelly FL, et al. Proteomic analysis of primary human airway epithelial cells exposed tothe respiratory toxicant diacetyl. J Proteome Res 2017; 16: 538–549.
33Giovannini-Chami L, Marcet B, Moreilhon C, et al. Distinct epithelial gene expression phenotypes in childhoodrespiratory allergy. Eur Respir J 2012; 39: 1197–1205.
34Raman T, O’Connor TP, Hackett NR, et al. Quality control in microarray assessment of gene expression in humanairway epithelium. BMC Genomics 2009; 10: 493.
35Davis S, Meltzer PS. GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor.Bioinformatics 2007; 23: 1846–1847.
36Huber W, Carey VJ, Gentleman R, et al. Orchestrating high-throughput genomic analysis with Bioconductor. NatMethods 2015; 12: 115–121.
37Johnson WE, Li C, Rabinovic A. Adjusting batch effects in microarray expression data using empirical Bayesmethods. Biostatistics 2007; 8: 118–127.
38Leek JT, Johnson WE, Parker HS, et al. The sva package for removing batch effects and other unwanted variationin high-throughput experiments. Bioinformatics 2012; 28: 882–883.
39Forrest ARR, Kawaji H, Rehli M, et al. A promoter-level mammalian expression atlas. Nature 2014; 507: 462–470.
40Abugessaisa I, Noguchi S, Hasegawa A, et al. FANTOM5 CAGE profiles of human and mouse reprocessed forGRCh38 and GRCm38 genome assemblies. Sci Data 2017; 4: 170107.
41Severin J, Lizio M, Harshbarger J, et al. Interactive visualization and analysis of large-scale sequencing datasetsusing ZENBU. Nat Biotechnol 2014; 32: 217–219.
42Butler A, Hoffman P, Smibert P, et al. Integrating single-cell transcriptomic data across different conditions,technologies, and species. Nat Biotechnol 2018; 36: 411–420.
43Habermann AC, Gutierrez AJ, Bui LT, et al. Single-cell RNA sequencing reveals profibrotic roles of distinctepithelial and mesenchymal lineages in pulmonary fibrosis. Sci Adv 2020; 6: eaba1972.
44Huff RD, Rider CF, Yan D, et al. Inhibition of ABCC4 potentiates combination beta agonist and glucocorticoidresponses in human airway epithelial cells. J Allergy Clin Immunol 2018; 141: 1127–1130.e5.
45Hirota JA, Gold MJ, Hiebert PR, et al. The nucleotide-binding domain, leucine-rich repeat protein 3inflammasome/IL-1 receptor I axis mediates innate, but not adaptive, immune responses after exposure toparticulate matter under 10 μm. Am J Respir Cell Mol Biol 2015; 52: 96–105.
46Hirota JA, Marchant DJ, Singhera GK, et al. Urban particulate matter increases human airway epithelial cellIL-1β secretion following scratch wounding and H1N1 influenza A exposure in vitro. Exp Lung Res 2015; 41:353–362.
47Ramirez RD, Sheridan S, Girard L, et al. Immortalization of human bronchial epithelial cells in the absence ofviral oncoproteins. Cancer Res 2004; 64: 9027–9034.
48Gürtler A, Kunz N, Gomolka M, et al. Stain-Free technology as a normalization tool in Western blot analysis.Anal Biochem 2013; 433: 105–111.
49Vogel C, Marcotte EM. Insights into the regulation of protein abundance from proteomic and transcriptomicanalyses. Nat Rev Genet 2012; 13: 227–232.
50Jouneau S, Khorasani N, De Souza P, et al. EMMPRIN (CD147) regulation of MMP-9 in bronchial epithelial cellsin COPD. Respirology 2011; 16: 705–712.
51Uhlen M, Bandrowski A, Carr S, et al. A proposal for validation of antibodies. Nat Methods 2016; 13: 823–827.
52Donoghue M, Hsieh F, Baronas E, et al. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2)converts angiotensin I to angiotensin 1–9. Circ Res 2000; 87: E1–E9.
53Burrell LM, Risvanis J, Kubota E, et al. Myocardial infarction increases ACE2 expression in rat and humans.Eur Heart J 2005; 26: 1142–1143.
54Ziegler CGK, Allon SJ, Nyquist SK, et al. SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in humanairway epithelial cells and is detected in specific cell subsets across tissues. Cell 2020; 181: 1016–1035.
55Leung JM, Yang CX, Tam A, et al. ACE-2 expression in the small airway epithelia of smokers and COPD patients:implications for COVID-19. Eur Respir J 2020; 55: 2000688.
56Schiller HB, Montoro DT, Simon LM, et al. The human lung cell atlas: a high-resolution reference map of thehuman lung in health and disease. Am J Respir Cell Mol Biol 2019; 61: 31–41.
57Plasschaert LW, Žilionis R, Choo-Wing R, et al. A single-cell atlas of the airway epithelium reveals the CFTR-richpulmonary ionocyte. Nature 2018; 560: 377–381.
58Sims AC, Baric RS, Yount B, et al. Severe acute respiratory syndrome coronavirus infection of human ciliatedairway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs. J Virol 2005; 79:15511–15524.
https://doi.org/10.1183/13993003.01123-202016
INFECTIOUS DISEASE | J.A. AGUIAR ET AL.
59Qi F, Qian S, Zhang S, et al. Single cell RNA sequencing of 13 human tissues identify cell types and receptors ofhuman coronaviruses. Biochem Biophys Res Commun 2020; 526: 135–140.
60Hikmet F, Méar L, Edvinsson Å, et al. The protein expression profile of ACE2 in human tissues. Mol Syst Biol2020; 16: e9610.
61Aksoy MO, Kim V, Cornwell WD, et al. Secretion of the endoplasmic reticulum stress protein, GRP78, into theBALF is increased in cigarette smokers. Respir Res 2017; 18: 78.
62Jia HP, Look DC, Shi L, et al. ACE2 receptor expression and severe acute respiratory syndrome coronavirusinfection depend on differentiation of human airway epithelia. J Virol 2005; 79: 14614–14621.
63Gonzalez-Gronow M, Selim MA, Papalas J, et al. GRP78: a multifunctional receptor on the cell surface. AntioxidRedox Signal 2009; 11: 2299–2306.
64Zhang Y, Liu R, Ni M, et al. Cell surface relocalization of the endoplasmic reticulum chaperone and unfoldedprotein response regulator GRP78/BiP. J Biol Chem 2010; 285: 15065–15075.
65Crane ED, Al-Hashimi AA, Chen J, et al. Anti-GRP78 autoantibodies induce endothelial cell activation andaccelerate the development of atherosclerotic lesions. JCI Insight 2018; 3: e99363.
66Al-Hashimi AA, Lebeau P, Majeed F, et al. Autoantibodies against the cell surface-associated chaperone GRP78stimulate tumor growth via tissue factor. J Biol Chem 2017; 292: 21180–21192.
67Van Krieken R, Mehta N, Wang T, et al. Cell surface expression of 78-kDa glucose-regulated protein (GRP78)mediates diabetic nephropathy. J Biol Chem 2019; 294: 7755–7768.
68Wang W, Xu Y, Gao R, et al. Detection of SARS-CoV-2 in different types of clinical specimens. JAMA 2020; 323:1843–1844.
69Hou YJ, Okuda K, Edwards CE, et al. SARS-CoV-2 reverse genetics reveals a variable infection gradient in therespiratory tract. Cell 2020; 182: 429–446.
https://doi.org/10.1183/13993003.01123-202017
INFECTIOUS DISEASE | J.A. AGUIAR ET AL.