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

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

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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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  1. Version published to 10.1183/13993003.01123-2020
  2. ScreenIT

    SciScore for 10.1101/2020.04.07.030742: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIRB: Human ethics: Procurement of primary human airway epithelial cells used for immunoblots and lung tissue for immunohistochemistry was approved by Hamilton integrated Research Ethics Board (HiREB 5099T, 5305T, 11-3559 and 13-523-C).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableWithin this dataset, sex and age information was included for 310 samples with 86 females/106 males.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Monoclonal - Clone 171606 – 2μg/ml), TMPRSS2 (Atlas Antibodies - HPA035787 – Polyclonal – 0.4μg/ml), CD147 (Abcam – ab666
    TMPRSS2
    suggested: (Sigma-Aldrich Cat# HPA035787, RRID:AB_2674782)
    Monoclonal – Clone – 40/BiP – 0.25μg/ml) primary antibodies were diluted in 5% skim milk/Tris buffered saline with 0.1% Tween-20 and incubated overnight on a rocker at 4°C with detection performed the following day using an anti-mouse-HRP (ACE2, CD147, GRP78) or anti-rabbit-HRP (
    anti-mouse-HRP
    suggested: (Kindle Biosciences Cat# R1005, RRID:AB_2800463)
    anti-rabbit-HRP
    suggested: None
    The immunogen for the CD147 primary antibody is recombinant full-length protein corresponding to human CD147.
    CD147
    suggested: None
    The immunogen for the GRP78 primary antibody is human BiP/GRP78 amino acids 525-628.
    GRP78
    suggested: None
    Rabbit monoclonal – EPR16884 – 1/10000 dilution of stock antibody).
    EPR16884
    suggested: (Abcam Cat# ab204276, RRID:AB_2889198)
    Four-micron thick sections were cut and stained for ACE2 (15μg/ml), TMPRSS2 (10μg/ml), and CD147 (5μg/ml) using the same antibodies used for immunoblot analysis.
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Independent immunoblot analysis (L.O, C.J. A.J. and G.J) were performed on A549, HEK, and immortalized human bronchial epithelial cells.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    HEK
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    Upper and lower airway gene expression analysis: Public microarray experiments using Affymetrix chips (HuGene-1.0-st-v1 and HG-U133 Plus 2) on airway epithelial cell samples collected from nasal (GSE19190) or bronchial (GSE11906) brushings of healthy, non-smokers were obtained from the NCBI Gene Expression Omnibus (GEO) database [31, 32].
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    For all dataset samples, raw intensity values and annotation data were downloaded using the GEOquery R package (version 2.52.0)[33] from the Bioconductor project [34].
    Bioconductor
    suggested: (Bioconductor, RRID:SCR_006442)
    Correction of experiment-specific batch effects was performed using the ComBat method[35] implemented using the sva R package (version 3.32.1)[36].
    ComBat
    suggested: (ComBat, RRID:SCR_010974)
    Gene expression box plots were generated with the ggplot2 R package (version 3.2.1).
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    HG-U133 Plus 2 and HuGene-1.0-st-v1) on airway epithelial cell samples were selected from the NCBI GEO database.
    NCBI GEO
    suggested: None
    Samples from 10 control subjects and 12 IPF patients were downloaded and post-processed with Seurat package in R[40].
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Using a curated microarray gene expression dataset generated from bronchial brushings of 504 healthy subjects that considers the limitations of merging multiple datasets from distinct sources, we observed that sex did not correlate with gene expression of any candidate host molecule involved in SARS-CoV-2 infection and that ACE2 and TMPRSS2 were the lowest expressed genes of interest examined. In one dataset, ACE2 gene expression modestly decreased with age, although protein level confirmation was not possible. The low level of ACE2 and TMPRSS2 gene expression in bulk bronchial epithelial cell gene expression samples suggests low levels of cells expressing both of these genes within this lung tissue. Advances in transcriptomics have enabled scRNAseq that has identified unique and rare cell types in human lung that may have importance in health and disease[53, 54]. scRNAseq provides an opportunity to look at transcriptional profiles in subsets of cell populations, which may isolate a cell signal from a bulk sample. We therefore utilized 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 of ACE2 gene in all populations examined, whereas CD147 and GRP78 were present in all populations. Our results are consistent with current publicly available data that discuss the presence of a rare ACE2/TMPRSS2 positive cells[52]. Using lung samples from eight individuals (fou...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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  3. Version published to 10.1101/2020.04.07.030742v2 on bioRxiv
  4. Version published to 10.1101/2020.04.07.030742v1 on bioRxiv