Neutrophil extracellular traps induce the epithelial-mesenchymal transition: implications in post-COVID-19 fibrosis

  1. Laura Pandolfi
  2. Sara Bozzini
  3. Vanessa Frangipane
  4. Elena Percivalle
  5. Ada De Luigi
  6. Martina Bruna Violatto
  7. Gianluca Lopez
  8. Elisa Gabanti
  9. Luca Carsana
  10. Maura D’Amato
  11. Monica Morosini
  12. Mara De Amici
  13. Manuela Nebuloni
  14. Tommaso Fossali
  15. Riccardo Colombo
  16. Laura Saracino
  17. Veronica Codullo
  18. Massimiliano Gnecchi
  19. Paolo Bigini
  20. Fausto Baldanti
  21. Daniele Lilleri
  22. Federica Meloni

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Abstract

The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19.

Neutrophils activated with PMA (PMA-Neu), a stimulus known to induce NETs formation, induce both EMT and cell death in the lung epithelial cell line, A549. Notably, NETs isolated from PMA-Neu induce EMT without cell damage. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs. Thus, we tested in an in vitro alveolar model the hypothesis that virus-induced NET may drive EMT. Co-culturing A549 at air-liquid interface with alveolar macrophages, neutrophils and SARS-CoV2, we demonstrated a significant induction of the EMT in A549 together with high concentration of NETs, IL8 and IL1β, best-known inducers of NETosis. Lung tissues of COVID-19 deceased patients showed that epithelial cells are characterized by increased mesenchymal markers. These results show for the first time that NETosis plays a major role in triggering lung fibrosis in COVID-19 patients.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.09.374769: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Study approval: Research and data collection protocols were approved by the Institutional Review Boards (Comitato Etico di Area 1) (prot. 20100005334) and by IRCCS Policlinico San Matteo Foundation Hospital (
    Consent: Written informed consent was obtained by all conscious patients.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After electrophoresis, the gels were transferred to polyvinylidene difluoride membranes (Millipore), therefore blocked (5% no fat milk in 0.1% Tween 20 TBS) and incubated with the primary Ab (1:1000 in TBST + 2% BSA; overnight at 4 °C or 2 h at room temperature): anti-E-Cadherin [M168] (ab76055, Abcam), anti-α-SMA [E184] (ab32575, Abcam), and anti-β-Actin (MAB1501R, Chemicon).
    anti-β-Actin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV2: A wild strain of SARS-CoV-2 was isolated in VERO E6 cell line from a nasal swab of a COVID-19 infected patient.
    VERO E6
    suggested: None
    EMT induction (A549 + neutrophils/NETs): To study the induction of EMT on A549 by PMA-Neu/NETs, 0.2 × 106 A549 were cultured on 12-well plate for 24 h.
    A549
    suggested: None
    Software and Algorithms
    SentencesResources
    All analyses were carried out with a GraphPad Prism 6.0 statistical program (GraphPad software, San Diego, CA, USA).
    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 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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2020.11.09.374769 on bioRxiv