Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients

  1. Tamara S. Rodrigues
  2. Keyla S.G. de Sá
  3. Adriene Y. Ishimoto
  4. Amanda Becerra
  5. Samuel Oliveira
  6. Leticia Almeida
  7. Augusto V. Gonçalves
  8. Debora B. Perucello
  9. Warrison A. Andrade
  10. Ricardo Castro
  11. Flavio P. Veras
  12. Juliana E. Toller-Kawahisa
  13. Daniele C. Nascimento
  14. Mikhael H.F. de Lima
  15. Camila M.S. Silva
  16. Diego B. Caetite
  17. Ronaldo B. Martins
  18. Italo A. Castro
  19. Marjorie C. Pontelli
  20. Fabio C. de Barros
  21. Natália B. do Amaral
  22. Marcela C. Giannini
  23. Letícia P. Bonjorno
  24. Maria Isabel F. Lopes
  25. Rodrigo C. Santana
  26. Fernando C. Vilar
  27. Maria Auxiliadora-Martins
  28. Rodrigo Luppino-Assad
  29. Sergio C.L. de Almeida
  30. Fabiola R. de Oliveira
  31. Sabrina S. Batah
  32. Li Siyuan
  33. Maira N. Benatti
  34. Thiago M. Cunha
  35. José C. Alves-Filho
  36. Fernando Q. Cunha
  37. Larissa D. Cunha
  38. Fabiani G. Frantz
  39. Tiana Kohlsdorf
  40. Alexandre T. Fabro
  41. Eurico Arruda
  42. Renê D.R. de Oliveira
  43. Paulo Louzada-Junior
  44. Dario S. Zamboni

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Abstract

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1β, and IL-18. Although participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease are unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and is active in COVID-19 patients. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of postmortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that inflammasomes participate in the pathophysiology of the disease, indicating that these platforms might be a marker of disease severity and a potential therapeutic target for COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.05.20168872: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Peripheral blood mononuclear cells isolation: Whole blood was collected from healthy donors (Ethics Committee Protocol from the Clinical Hospital of Ribeirão Preto-USP: CAAE, n° 06825018.2.3001.5440) in tubes containing EDTA (BD Vacutainer CPTTM), according to the manufacturer’s instructions.
    IACUC: Minimally invasive autopsies were approved by the FMRP/USP Ethical Committee (protocol #4.089.567).
    Consent: Written informed consent was obtained from recruited patients.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableThe patients’ outcomes were divided into 3 categories (37 patients in total, 16 women and 21 men): death (n = 10 individuals, a total of 25 samples), mild recovery (patients that were hospitalized but did not require mechanical ventilation; n = 17 individuals, a total of 53 samples) and critical recovery (patients that required mechanical ventilation at the UCI and recovered; n = 10 individuals, a total of 27 samples).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Controls were collected either before the COVID-19 pandemic or tested negative for COVID-19 using RT-PCR and/or serology (specific IgM and IgG antibodies) (Asan Easy Test.COVID-19 IgM/IgG kits, Asan Pharmaceutical Co.).
    IgG
    suggested: None
    Secondary antibodies used were goat anti-rabbit 488 (Invitrogen,1:3000) and goat anti-rabbit 594 (Life Technologies,1:3000).
    anti-rabbit
    suggested: None
    Sequential immunoperoxidase labeling and erasing: Tissue sections from paraffin-embedded lung fragments obtained from COVID-19 fatal cases were tested by immunohistochemistry (IHC) using anti-SARS-CoV-2 polyclonal antibody for in situ detection of SARS-CoV-2.
    anti-SARS-CoV-2
    suggested: None
    Sequential immunoperoxidase labeling and erasing (SIMPLE) was then performed to determine additional markers after SARS-CoV-2 immune stain, using antibodies to CD14 (Abcam, 1:100 dilution), NLRP3 (Cell Signaling, 1:100 dilution) (Glass et al., 2009).
    CD14
    suggested: None
    NLRP3
    suggested: None
    After the incubation with primary antibody, the slides were incubated with immune-peroxidase polymer anti-mouse visualization system (SPD-125, Spring Bioscience, Biogen) and then with the chromogen substrate AEC peroxidase system kit (SK-4200, Vector Laboratories, Burlingame, CA).
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Viral stock was propagated under BSL3 conditions in Vero E6 cells, cultured in Dulbecco minimal essential medium (DMEM) supplemented with heat-inactivated fetal bovine serum (10%) and antibiotics/antimycotics (Penicillin 10,000 U/mL; Streptomycin 10,000 μg/mL).
    Vero E6
    suggested: None
    The supernatant was stored at -80°C, and the virus titration was performed in Vero cells using standard limiting dilution to confirm the 50% tissue culture infectious dose (TCID50).
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    The supernatant was collected and LDH release was measured using CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Winsconsin, USA) following the manufacturer’s instructions.
    CytoTox
    suggested: None
    Sequential immunoperoxidase labeling and erasing (SIMPLE) was then performed to determine additional markers after SARS-CoV-2 immune stain, using antibodies to CD14 (Abcam, 1:100 dilution), NLRP3 (Cell Signaling, 1:100 dilution) (Glass et al., 2009).
    SIMPLE
    suggested: (SIMPLE, RRID:SCR_009389)
    After the incubation with primary antibody, the slides were incubated with immune-peroxidase polymer anti-mouse visualization system (SPD-125, Spring Bioscience, Biogen) and then with the chromogen substrate AEC peroxidase system kit (SK-4200, Vector Laboratories, Burlingame, CA).
    Biogen
    suggested: (Biogen Idec, RRID:SCR_003790)
    These statistical procedures and graph plots were performed with GraphPad Prism 8.4.2 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    These analyses were performed in R (version 4.0.2) using RStudio (version 1.3.1056), and are detailed as R Markdown object at the supplementary material.
    RStudio
    suggested: (RStudio, RRID:SCR_000432)

    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. Version published to 10.1084/jem.20201707
  3. Version published to 10.1101/2020.08.05.20168872 on medRxiv