Neutrophil extracellular traps in COVID-19

  1. Yu Zuo
  2. Srilakshmi Yalavarthi
  3. Hui Shi
  4. Kelsey Gockman
  5. Melanie Zuo
  6. Jacqueline A. Madison
  7. Christopher Blair
  8. Andrew Weber
  9. Betsy J. Barnes
  10. Mikala Egeblad
  11. Robert J. Woods
  12. Yogendra Kanthi
  13. Jason S. Knight

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Abstract

In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have the potential to propagate inflammation and microvascular thrombosis — including in the lungs of patients with acute respiratory distress syndrome. We now report that sera from patients with COVID-19 have elevated levels of cell-free DNA, myeloperoxidase-DNA (MPO-DNA), and citrullinated histone H3 (Cit-H3); the latter 2 are specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute-phase reactants, including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.30.20086736: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This study complied with all relevant ethical regulations, and was approved by the University of Michigan Institutional Review Board (HUM00179409), which waived the requirement for informed consent given the discarded nature of the samples.
    Consent: This study complied with all relevant ethical regulations, and was approved by the University of Michigan Institutional Review Board (HUM00179409), which waived the requirement for informed consent given the discarded nature of the samples.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4°C with anti-human myeloperoxidase antibody (Bio-Rad 0400-0002), diluted to a concentration of 1 µg/ml in coating buffer (Cell Death kit).
    anti-human myeloperoxidase
    suggested: (Bio-Rad Cat# 0400-0002, RRID:AB_617350)
    The plate was washed five times, and then incubated for 90 minutes at room temperature with 10x anti-DNA antibody (HRP-conjugated; from the Cell Death kit) diluted 1:100 in blocking buffer.
    anti-DNA
    suggested: None
    Software and Algorithms
    SentencesResources
    Data analysis was with GraphPad Prism software version 8.
    GraphPad Prism
    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. ScreenIT

    SciScore for 10.1101/2020.04.09.20059626: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This study complied with all relevant ethical regulations, and was approved by the University of Michigan Institutional Review Board (HUM00179409), which waived the requirement for informed consent given the discarded nature of the samples.
    Consent: This study complied with all relevant ethical regulations, and was approved by the University of Michigan Institutional Review Board (HUM00179409), which waived the requirement for informed consent given the discarded nature of the samples.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationAuthentication: Neutrophil preparations were at least 95% pure as confirmed by nuclear morphology.

    Table 2: Resources

    Antibodies
    SentencesResources
    First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4°C with anti-human MPO antibody (Bio-Rad 0400-0002), diluted to a concentration of 1 µg/ml in coating buffer (Cell Death kit).
    anti-human MPO
    suggested: None
    The plate was washed five times, and then incubated for 90 minutes at room temperature with 10x anti-DNA antibody (HRP-conjugated; from the Cell Death kit) diluted 1:100 in blocking buffer.
    anti-DNA
    suggested: None
    The primary antibody was against neutrophil elastase (Abcam 21595, diluted 1:100), and the FITC-conjugated secondary antibody was from SouthernBiotech (4052-02, diluted 1:250).
    neutrophil elastase (Abcam 21595
    suggested: (Abcam Cat# ab21595, RRID:AB_446409)
    Software and Algorithms
    SentencesResources
    Data analysis was with GraphPad Prism software version 8.
    GraphPad Prism
    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: We detected the following sentences addressing limitations in the study:
    While our study certainly has limitations including the use of serum (plasma is typically preferred by our group and others for measurement of NETs) and a relatively small sample size, we hope it will ignite further research into the role of neutrophil effector functions in the complications of COVID-19. As a first step, future studies should investigate the predictive power of circulating NETs in well-phenotyped longitudinal cohorts. Furthermore, given the dichotomy we found here between MPO-DNA and Cit-H3, investigators should be encouraged to continue to include diverse markers of NETosis in future studies. As we await definitive antiviral and immunologic solutions to the current pandemic, we posit that anti-neutrophil therapies may be part of a personalized strategy for some individuals affected by COVID-19 who are at risk for progression to respiratory failure.

    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. Version published to 10.1172/jci.insight.138999
  4. Version published to 10.1101/2020.04.30.20086736 on medRxiv
  5. Version published to 10.1101/2020.04.09.20059626 on medRxiv