Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction

  1. Guillaume Beucher
  2. Marie-Lise Blondot
  3. Alexis Celle
  4. Noémie Pied
  5. Patricia Recordon-Pinson
  6. Pauline Esteves
  7. Muriel Faure
  8. Mathieu Métifiot
  9. Sabrina Lacomme
  10. Denis Dacheux
  11. Derrick R. Robinson
  12. Gernot Längst
  13. Fabien Beaufils
  14. Marie-Edith Lafon
  15. Patrick Berger
  16. Marc Landry
  17. Denis Malvy
  18. Thomas Trian
  19. Marie-Line Andreola
  20. Harald Wodrich

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

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  1. Version published to 10.1073/pnas.2202370119
  2. ScreenIT

    SciScore for 10.1101/2021.05.28.446159: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Mice experiments have been performed in the conventional animal facilities of the University of Bordeaux (France) (approval number of B-33-036-917), with the approval of institutional guidelines determined by the local Ethical Committee of the University of Bordeaux and in conformity with the Ministry for Higher Education and Research and the French Committee of Genetic Engineering (approval number n °17621 -V5-2018112201234223).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The following primary antibodies and IF dilutions were used in this study; mouse monoclonal Ab anti-SARS-CoV-2-N clone 3G9 (this study, 1:500), rabbit monoclonal Ab anti-human Cytokeratin 5 (Abcam, ab52635, 1:200), rabbit polyclonal Ab anti-human Acetylated tubulin (Cell Signaling, D20G3, 1:200), rabbit polyclonal Ab anti-human ACE2 (Abcam, ab15348, 1:50)
    anti-SARS-CoV-2-N
    suggested: None
    anti-human Cytokeratin 5 ( Abcam ,
    suggested: None
    anti-human Acetylated tubulin ( Cell Signaling , D20G3
    suggested: None
    anti-human ACE2
    suggested: (Abcam Cat# ab15348, RRID:AB_301861)
    ab15348
    suggested: None
    The following secondary antibodies were used in this study; cross absorbed Donkey anti-mouse Alexa Fluor 488 or 647 (Life technologies, A212020/A31571, 1:300) and cross absorbed Donkey anti-rabbit Alexa Fluor 594 (Life technologies, A31573, 1:300) as well as Alexa-Fluor 594 labeled phalloidin (Invitrogen, 1:500)
    anti-mouse
    suggested: (Molecular Probes Cat# A-31571, RRID:AB_162542)
    A212020/A31571
    suggested: None
    anti-rabbit
    suggested: (Molecular Probes Cat# A-31573, RRID:AB_2536183)
    A31573
    suggested: (Molecular Probes Cat# A-31573, RRID:AB_2536183)
    Experimental Models: Cell Lines
    SentencesResources
    Viruses and cell lines: Vero E6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS) and gentamicin (50μg/mL) at 37°C in a humidified CO2 incubator.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Image processing was done using Image J software.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)

    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 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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 24, 32, 35 and 28. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

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  3. Version published to 10.1101/2021.05.28.446159v1 on bioRxiv