SARS-CoV-2 engages inflammasome and pyroptosis in human primary monocytes

  1. André C. Ferreira
  2. Vinicius Cardoso Soares
  3. Isaclaudia G. de Azevedo-Quintanilha
  4. Suelen da Silva Gomes Dias
  5. Natalia Fintelman-Rodrigues
  6. Carolina Q. Sacramento
  7. Mayara Mattos
  8. Caroline S. de Freitas
  9. Jairo R. Temerozo
  10. Lívia Teixeira
  11. Eugenio Damaceno Hottz
  12. Ester A. Barreto
  13. Camila R. R. Pão
  14. Lohanna Palhinha
  15. Milene Miranda
  16. Dumith Chequer Bou-Habib
  17. Fernando A. Bozza
  18. Patrícia T. Bozza
  19. Thiago Moreno L. Souza

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Abstract

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocyte death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with 2019 coronavirus disease (COVID-19). Here, we show that SARS-CoV-2 engages inflammasome and triggers pyroptosis in human monocytes, experimentally infected, and from patients under intensive care. Pyroptosis associated with caspase-1 activation, IL-1ß production, gasdermin D cleavage, and enhanced pro-inflammatory cytokine levels in human primary monocytes. At least in part, our results originally describe mechanisms by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb to control severe COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.25.20182055: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Ethics statement: Experimental procedures involving human cells from healthy donors were performed with samples obtained after written informed consent and were approved by the Institutional Review Board (IRB) of the Oswaldo Cruz Foundation/Fiocruz (Rio de Janeiro, RJ, Brazil) under the number 397-07.
    IRB: The National Review Board approved the study protocol (CONEP 30650420.4.1001.0008), and informed consent was obtained from all participants or patients’ representatives.
    Randomizationnot detected.
    BlindingAfter 3 to 5 days, the cytopathic effects were scored in at least 10 replicates per dilution by independent readers, who were blind with respect to source of the supernatant.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The membranes were blocked with 5 % albumin diluted in Tris-buffered saline containing 0.05 % of Tween 20 for 2 hours at room temperature and incubated with the specific primary antibodies (Cell Signaling Technology), to detect pro-caspase-1 and cleaved-caspase-1, after overnight incubation at 4 °C.
    cleaved-caspase-1
    suggested: None
    After washing, membranes were incubated with secondary antibodies (IRDye® 800CW Goat-anti-Mouse and IRDye® 680LT Goat anti-Rabbit IgG Antibody, LI-COR, Lincoln) for 30 minutes at RT.
    anti-Rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero cells were incubated at 37°C in 5 % CO2 atmosphere.
    Vero
    suggested: None
    SARS-CoV-2 was isolated and expanded on Vero E6 cells from a nasopharyngeal swab of a confirmed case from Rio de Janeiro, Brazil.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    The antiviral Lopinavir/ritonavir (4:1 proportion) was pruchased from AbbVie (Ludwingshafen, Germany).
    AbbVie
    suggested: (AbbVie, RRID:SCR_010484)
    ELISA assays were purchased from R&D Bioscience.
    R&D Bioscience
    suggested: (UMD Bioprocess Scale-Up Facility, RRID:SCR_012703)
    Around 10,000 gated events were acquired using FACSCalibur and the analysis was performed using the CellQuest software.
    FACSCalibur
    suggested: None
    CellQuest
    suggested: (BD CellQuest Pro, RRID:SCR_014489)
    Acquisition of data was set to count a total of 10,000 events, and the FLOWJO software package was used to analyze the data.
    FLOWJO
    suggested: (FlowJo, RRID:SCR_008520)
    The protein bands were visualized by digital fluorescence (Odyssey® CLx Imaging System), and protein density was analyzed by the ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

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

    IdentifierStatusTitle
    NCT04468087Not yet recruitingAntiviral Agents Against COVID-19 Infection

    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

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  2. Version published to 10.1038/s41420-021-00428-w
  3. Version published to 10.1101/2020.08.25.20182055v2 on medRxiv
  4. Version published to 10.1101/2020.08.25.20182055v1 on medRxiv