ACE2 expression is elevated in airway epithelial cells from older and male healthy individuals but reduced in asthma

  1. Peter A.B. Wark
  2. Prabuddha S. Pathinayake
  3. Gerard Kaiko
  4. Kristy Nichol
  5. Ayesha Ali
  6. Ling Chen
  7. Erika N. Sutanto
  8. Luke W. Garratt
  9. Sukhwinder S. Sohal
  10. Wenying Lu
  11. Mathew S. Eapen
  12. Christopher Oldmeadow
  13. Nathan Bartlett
  14. Andrew Reid
  15. Punnam Veerati
  16. Alan C.‐Y. Hsu
  17. Kevin Looi
  18. Thomas Iosifidis
  19. Stephen M. Stick
  20. Philip M. Hansbro
  21. Anthony Kicic

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Abstract

Background and objective

COVID‐19 is complicated by acute lung injury, and death in some individuals. It is caused by SARS‐CoV‐2 that requires the ACE2 receptor and serine proteases to enter AEC. We determined what factors are associated with ACE2 expression particularly in patients with asthma and COPD.

Methods

We obtained lower AEC from 145 people from two independent cohorts, aged 2–89 years, Newcastle ( n = 115) and Perth ( n = 30), Australia. The Newcastle cohort was enriched with people with asthma ( n = 37) and COPD ( n = 38). Gene expression for ACE2 and other genes potentially associated with SARS‐CoV‐2 cell entry was assessed by qPCR, and protein expression was confirmed with immunohistochemistry on endobronchial biopsies and cultured AEC.

Results

Increased gene expression of ACE2 was associated with older age ( P = 0.03) and male sex ( P = 0.03), but not with pack‐years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 expression was lower in asthma patients ( P = 0.01). Gene expression of furin, a protease that facilitates viral endocytosis, was also lower in patients with asthma ( P = 0.02), while ADAM‐17, a disintegrin that cleaves ACE2 from the surface, was increased ( P = 0.02). ACE2 protein expression was also reduced in endobronchial biopsies from asthma patients.

Conclusion

Increased ACE2 expression occurs in older people and males. Asthma patients have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism and may in part explain susceptibility factors and why asthma patients are not over‐represented in those with COVID‐19 complications.

Article activity feed

  1. Version published to 10.1111/resp.14003
  2. ScreenIT

    SciScore for 10.1101/2020.07.26.20162248: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The study was approved by Hunter New England Human Research Ethics Committee (Reference No 05/08/10/3.09) and all participants provided written informed consent.
    Consent: Those with recent chest infection were excluded from the study and written consent was then obtained from parents or guardians of children and adult participants.
    Randomizationnot detected.
    BlindingAntigen staining was quantified by blinded counting of the number of positively stained epithelial cells as a percentage of total epithelial cells.
    Power Analysisnot detected.
    Sex as a biological variableThey included children undergoing elective surgery for non-respiratory-related conditions (n = 14; 2.4-6.8 years of age; 7 males) and adult volunteers (n = 16; 25.5-57.1 years of age; 5 males).

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies included anti-ACE2 (Abcam; ab15348, 1:800), anti-acetylated tubulin (Sigma; 6-11B-1), and anti-E-cadherin (Cell Signaling; 4A2).
    anti-ACE2
    suggested: (Abcam Cat# ab15348, RRID:AB_301861)
    anti-acetylated tubulin
    suggested: (Santa Cruz Biotechnology Cat# sc-23950, RRID:AB_628409)
    anti-E-cadherin
    suggested: (Cell Signaling Technology Cat# 14472, RRID:AB_2728770)
    Anti-rabbit and anti-mouse fluorophore-conjugated (Alexa Fluor 488 and Alexa Fluor 594) secondary antibodies were incubated with tissue sections for 1 hour at room temperature.
    Anti-rabbit
    suggested: None
    anti-mouse fluorophore-conjugated
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical Analysis: Data were analysed using Stata software version 15 (StataCorp, College Station, TX, USA) or with Graphpad prism 8.
    Stata
    suggested: (Stata, RRID:SCR_012763)
    StataCorp
    suggested: (Stata, RRID:SCR_012763)
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)
    A total of 200ng mRNA from each sample was used to synthesise cDNA using high-capacity cDNA reverse transcription kits (Applied Biosystems™). qPCR was performed using a Quanstudio(tm) 7 Flex as per manufacturer’s instructions using TaqMan® gene expression assays (ThemoFisher Scientific, Australia) and normalized to the18s housekeeping gene(52) (Table 2).
    ThemoFisher Scientific
    suggested: None

    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:
    Our study has some weaknesses. We examined specimens of AECs from a relatively large cohort, who were recruited to study chronic airways disease and were enriched for subjects with moderate to severe asthma and COPD. Our numbers overall and with asthma though were not large enough to give a clear answer with multivariate analysis to define if this was independent of age and sex, and it is likely we would need to have included younger people with asthma. All of our subjects were on ICS and had required either GINA step 3 or above treatment. The majority, 28/39(72%) had evidence of type 2 high airway inflammation, with either atopy or elevated airway eosinophils. We had relatively few participants with other diseases, such as hypertension, diabetes and cardiovascular disease and relatively few on ACE inhibitors, meaning we cannot make any reliable predictions about these groups. Similarly, we had relatively few current smokers, though a larger number of former smokers, and the effect may have been confounded by the fact that all these smokers had been selected because of chronic airways disease. ACE2 expression and associated proteases are but one factor that will be associated with susceptibility to infection. Similarly, the risk of severe disease or the development of complications from COVID-19 will be more complex than receptor expression alone for the virus. We present the largest pathology study that looks at ACE2 expression from lower airway bronchial epithelial cells en...

    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.

  3. ScreenIT

    SciScore for 10.1101/2020.07.26.20162248: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementThe study was approved by Hunter New England Human Research Ethics Committee (Reference No 05/08/10/3.09) and all participants provided written informed consent.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variableResults: Increased gene expression of ACE2 was associated with older age (p=0.02) and male sex (p=0.03), but not pack-years smoked.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Statistical Analysis Data were analysed using Stata software version 15 (StataCorp, College Station, TX, USA) or with Graphpad prism 8.
    Stata
    suggested: (Stata, SCR_012763)
          <div style="margin-bottom:8px">
        <div><b>StataCorp</b></div>
        <div>suggested: (Stata, <a href="https://scicrunch.org/resources/Any/search?q=SCR_012763">SCR_012763</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Graphpad</b></div>
        <div>suggested: (GraphPad, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000306">SCR_000306</a>)</div>
      </div>
    </td></tr></table>

    Data from additional tools added to each annotation on a weekly basis.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2020.07.26.20162248 on medRxiv