Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract

  1. Miguel E. Ortiz Bezara
  2. Andrew Thurman
  3. Alejandro A. Pezzulo
  4. Mariah R. Leidinger
  5. Julia A. Klesney-Tait
  6. Philip H. Karp
  7. Ping Tan
  8. Christine Wohlford-Lenane
  9. Paul B. McCray
  10. David K. Meyerholz

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Abstract

Background

Zoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors.

Materials and methods

We systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19.

Findings

Our results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19.

Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19.

Interpretation

These results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19.

Research in context

Evidence before this study

Previous studies of ACE2 mRNA transcript abundance in the human respiratory tract have suggested a possible association between ACE2 expression and age, sex, and the presence of comorbidities. However, these studies have provided conflicting results, as well as a lack of protein validation. Previous ACE2 protein studies have been limited by a paucity of lung tissue samples and reports that have produced contradictory results.

Added value of this study

Using a combination of single-cell RNA sequencing and immunohistochemistry, we describe ACE2 expression in the human respiratory tract. Staining protocols were optimized and validated to show consistent apical localization and avoid non-specific staining. We show ACE2 protein is found in subsets of airway cells and is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development for COVID-19, respectively. We show age, sex, and comorbidities do not increase ACE2 protein expression in the human respiratory tract.

Implications of all the available evidence

ACE2 protein abundance does not correlate with risk factors for severe clinical outcomes, but in some cases showed an inversed relationship. Features driving COVID-19 susceptibility and severity are complex, our data suggests factors other than ACE2 protein abundance as important determinants of clinical outcomes.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.22.056127: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethics: Studies on human tissues were approved by the institutional review board of the University of Iowa (Iowa IRB #199507432).
    Consent: Informed consent was obtained for all the tissues included in the study.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variable(15 chronic comorbidities and 14 controls) with a broad range of ages (0·5 – 71 years) and both sexes were represented (13 female and 16 male).

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies anti-ACE2 (1:100
    anti-ACE2
    suggested: (Enzo Life Sciences Cat# ALX-804-722-C100, RRID:AB_11180102)
    Secondary antibodies anti-mouse Alexa568 (for ACE2) and anti-rabbit Alexa488 (for TMPRSS2) were applied at a concentration of 1:600 for 1 hour at room temperature.
    anti-mouse
    suggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160)
    anti-rabbit
    suggested: None
    TMPRSS2
    suggested: None
    Software and Algorithms
    SentencesResources
    Analysis of single cell RNA sequencing data: Single cell RNA sequencing data sets were accessed from Gene Expression Omnibus (GEO) series GSE121600 (27) and GSE122960 (26).
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    All analyses were performed using R package Seurat version 3.1.1 (34).
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)
    Statistical analyses: Statistical analyses for group comparisons and tissue scoring data were performed using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.04.22.056127 on bioRxiv