ACE2 expression and localization are regulated by CFTR: implications beyond cystic fibrosis

  1. Valentino Bezzerri
  2. Valentina Gentili
  3. Martina Api
  4. Alessia Finotti
  5. Chiara Papi
  6. Anna Tamanini
  7. Debora Olioso
  8. Martina Duca
  9. Erika Tedesco
  10. Sara Leo
  11. Monica Borgatti
  12. Sonia Volpi
  13. Paolo Pinton
  14. Giulio Cabrini
  15. Roberto Gambari
  16. Francesco Blasi
  17. Giuseppe Lippi
  18. Alessandro Rimessi
  19. Roberta Rizzo
  20. Marco Cipolli

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Abstract

As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis (CF) could be considered a comorbidity for coronavirus disease 2019 (COVID-19) 1 . Instead, CF seems to constitute an advantage in COVID-19 infection 2-5 .

To clarify whether host factors expressed by the CF epithelia may influence COVID-19 progression, we investigated the expression of SARS-CoV-2 receptor and coreceptors in primary airway epithelial cells. We found that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by cystic fibrosis transmembrane conductance regulator (CFTR) channels. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral infection in CF cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin (IL)-6 in healthy donor-derived primary airway epithelial cells but a very weak response in primary CF cells. Collectively, these data support the hypothesis that CF condition is unfavorable for SARS-CoV-2 infection.

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  1. ScreenIT

    SciScore for 10.1101/2021.11.19.469220: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Human samples: All human samples were obtained and analyzed in accordance with the Declaration of Helsinki after written consent was obtained.
    IRB: All protocols were approved by the Ethics Committee of the Azienda Ospedaliera Universitaria Integrata (Verona, Italy), approval No. 2917CESC.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Membranes were probed with primary anti-human ACE2 rabbit polyclonal antibody (ab15348, Abcam, Cambridge, UK),
    anti-human ACE2
    suggested: (Abcam Cat# ab15348, RRID:AB_301861)
    rabbit monoclonal antibody (ab109131, Abcam), or anti-CD13 rabbit monoclonal antibody (ab108382, Abcam) in TBS/T with 5% BSA or mouse IgG2b anti-CFTR NBD2 (antibody 596, Cystic Fibrosis North American Foundation, Chapel Hill, NC, 1:2500 dilution) in 5% nonfat dried milk and incubated overnight at 4°C.
    anti-CD13
    suggested: (Abcam Cat# ab108382, RRID:AB_10890797)
    mouse IgG2b
    suggested: (Miltenyi Biotec Cat# 130-095-596, RRID:AB_2857898)
    NBD2
    suggested: None
    After washing, membranes were incubated with mouse anti-rabbit IgG-horseradish peroxidase-coupled secondary antibody (sc2357, Santa Cruz Biotechnology, Dallas, TX, USA, dilution 1:2000) or donkey anti-mouse IgG-horseradish peroxidase-coupled secondary antibody (RnD Systems) diluted in TBS/T for 90 minutes at room temperature.
    anti-rabbit IgG-horseradish
    suggested: None
    anti-mouse IgG-horseradish
    suggested: None
    Blots were then reprobed with monoclonal anti-β-actin-peroxidase clone AC-15 antibody (a3854, Merck, Saint Louis, MO, dilution 1:5000) in TBS/T for 90 minutes.
    anti-β-actin-peroxidase
    suggested: None
    After membrane stripping, a β-actin monoclonal antibody (Sigma Aldrich, St. Louis, MO, USA) was used to ensure equal loading of samples.
    β-actin
    suggested: None
    Subsequently, a double immunofluorescence procedure was performed, and the cells were incubated overnight at 4°C with a mouse anti-CFTR (antibody 570, cod.
    anti-CFTR
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, stable expression of short hairpin RNAs (shRNAs) against CFTR was induced in Calu-3 cells by transfecting the Sleeping Beauty transposon vector pT2/si-PuroV2, generating a CFTR knockout cell line (SH3).
    Calu-3
    suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
    As previously described, viral titer was determined by plaque assay in Vero E6 cells30.
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    Briefly, stable expression of short hairpin RNAs (shRNAs) against CFTR was induced in Calu-3 cells by transfecting the Sleeping Beauty transposon vector pT2/si-PuroV2, generating a CFTR knockout cell line (SH3).
    pT2/si-PuroV2
    suggested: None
    Software and Algorithms
    SentencesResources
    Gels were transferred to nitrocellulose membrane (Thermo Fisher Scientific) using Trans Blot Turbo (Bio-Rad Laboratories).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)

    Results from OddPub: Thank you for sharing your data.

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.11.19.469220v1 on bioRxiv