Blunted Fas signaling favors RIPK1-driven neutrophil necroptosis in critically ill COVID-19 patients

  1. Tiziano A. Schweizer
  2. Srikanth Mairpady Shambat
  3. Clément Vulin
  4. Sylvia Hoeller
  5. Claudio Acevedo
  6. Markus Huemer
  7. Alejandro Gomez-Mejia
  8. Chun-Chi Chang
  9. Jeruscha Baum
  10. Sanne Hertegonne
  11. Eva Hitz
  12. Daniel A. Hofmaenner
  13. Philipp K. Buehler
  14. Holger Moch
  15. Reto A. Schuepbach
  16. Silvio D. Brugger
  17. Annelies S. Zinkernagel

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Abstract

Critically ill COVID-19 patients are characterized by a severely dysregulated cytokine profile and elevated neutrophil counts, which are thought to contribute to disease severity. However, to date it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the tightly regulated cell death program of neutrophils. We show that in a subpopulation of neutrophils, canonical apoptosis was skewed towards rapidly occurring necroptosis. This phenotype was characterized by abrogated caspase-8 activity and increased RIPK1 levels, favoring execution of necroptosis via the RIPK1-RIPK3-MLKL axis, as further confirmed in COVID-19 biopsies. Moreover, reduction of sFas-L levels in COVID-19 patients and hence decreased signaling to Fas directly increased RIPK1 levels and correlated with disease severity. Our results suggest an important role for Fas signaling in the regulation of cell death program ambiguity via the ripoptosome in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.19.436166: (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 the local ethics committee of the Canton of Zurich, Switzerland (Kantonale Ethikkommission Zurich BASEC ID 2020 – 00646) and is registered at clinicaltrials.gov (ClinicalTrials.gov Identifier: NCT04410263).
    Consent: The non-COVID-19 thrombus section was obtained from a patient with a general consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    purified anti-human CD95 (FAS) blocking antibody (10 μg/ml, clone A16086F, Biolegend) or respective DMSO and H2O control were added prior to incubation.
    anti-human CD95
    suggested: None
    Next, staining with anti-Fas-L BV421 (NOK-1) from Biolegend or anti-RIPK1 AF488 (Polyclonal) from Bioss Antibodies was carried out in Fix/Perm solution B for 30 min at 4°C.
    anti-Fas-L
    suggested: None
    anti-RIPK1
    suggested: None
    Software and Algorithms
    SentencesResources
    Lactate dehydrogenase release measurement: Supernatants were incubated 1:2 with the substrate solution of the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) for 30 min in the dark in a 96-well plate (Greiner), after which the stop solution was added.
    CytoTox
    suggested: None
    Images were processed using ImageJ software (Schneider et al., 2012).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Differences between two groups were calculated using either unpaired t-test, Mann-Whitney, paired t-test or Wilcoxon signed-rank test in Prism (GraphPad).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Correlation of clinical parameters was computed using non-parametric Spearman correlation in Prism.
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04410263RecruitingMicrobiota in COVID-19 Patients for Future Therapeutic and P…

    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. Version published to 10.1101/2021.03.19.436166 on bioRxiv