Fatal cytokine release syndrome by an aberrant FLIP/STAT3 axis

  1. Chiara Musiu
  2. Simone Caligola
  3. Alessandra Fiore
  4. Alessia Lamolinara
  5. Cristina Frusteri
  6. Francesco Domenico Del Pizzo
  7. Francesco De Sanctis
  8. Stefania Canè
  9. Annalisa Adamo
  10. Francesca Hofer
  11. Roza Maria Barouni
  12. Andrea Grilli
  13. Serena Zilio
  14. Paolo Serafini
  15. Evelina Tacconelli
  16. Katia Donadello
  17. Leonardo Gottin
  18. Enrico Polati
  19. Domenico Girelli
  20. Ildo Polidoro
  21. Piera Amelia Iezzi
  22. Domenico Angelucci
  23. Andrea Capece
  24. Ying Chen
  25. Zheng-Li Shi
  26. Peter J. Murray
  27. Marco Chilosi
  28. Ido Amit
  29. Silvio Bicciato
  30. Manuela Iezzi
  31. Vincenzo Bronte
  32. Stefano Ugel

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Abstract

Inflammatory responses rapidly detect pathogen invasion and mount a regulated reaction. However, dysregulated anti-pathogen immune responses can provoke life-threatening inflammatory pathologies collectively known as cytokine release syndrome (CRS), exemplified by key clinical phenotypes unearthed during the SARS-CoV-2 pandemic. The underlying pathophysiology of CRS remains elusive. We found that FLIP, a protein that controls caspase-8 death pathways, was highly expressed in myeloid cells of COVID-19 lungs. FLIP controlled CRS by fueling a STAT3-dependent inflammatory program. Indeed, constitutive expression of a viral FLIP homolog in myeloid cells triggered a STAT3-linked, progressive, and fatal inflammatory syndrome in mice, characterized by elevated cytokine output, lymphopenia, lung injury, and multiple organ dysfunctions that mimicked human CRS. As STAT3-targeting approaches relieved inflammation, immune disorders, and organ failures in these mice, targeted intervention towards this pathway could suppress the lethal CRS inflammatory state.

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  1. Version published to 10.1038/s41418-021-00866-0
  2. ScreenIT

    SciScore for 10.1101/2021.05.04.21256298: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: All the patients (and/or initially their families) provided written informed consent before sampling and for the use of their clinical and biological data.
    IACUC: This study was approved by the local ethical committee (protocol 17963; principal investigator, Vincenzo Bronte; ClinicalTrials.gov identifier NCT04438629).
    Sex as a biological variableAll genetically transgenic mice and their respective controls were gender and age-matched (typically 3–10 weeks) and both males and females were used in this study.
    RandomizationMice were assigned randomly to experimental groups.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    Germ free C57BL/6 mice were originally purchased from Charles River Laboratories Inc.,
    C57BL/6
    suggested: None
    CD45.1+congenic mice (B6.SJL-PtrcaPepcb/BoyJ) and LySM-CRE mice (B6.129P2-Lyz2tm1(cre)Ifo/J) were purchased from Jackson Laboratories; Rosa26.vFLIP mice were a gift from Dr. Ethel Cesarman (Weill Cornell Medicine, NY, USA).
    CD45.1+congenic
    suggested: None
    B6.SJL-PtrcaPepcb/BoyJ
    suggested: None
    LySM-CRE
    suggested: None
    B6.129P2-Lyz2tm1(cre)Ifo/J
    suggested: RRID:IMSR_JAX:004781)
    Rosa26.vFLIP
    suggested: None
    These mice were obtained by crossing ROSA26.vFLIP knock-in mice with mice expressing Cre recombinase under control of the endogenous Lyz2 promoter.
    ROSA26.vFLIP knock-in
    suggested: None
    Software and Algorithms
    SentencesResources
    Germ free C57BL/6 mice were originally purchased from Charles River Laboratories Inc.,
    Charles River Laboratories
    suggested: (Charles River Laboratories, RRID:SCR_003792)
    Statistical analyses were performed using Graph Pad Prism (version 8.0.2).
    Graph Pad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Despite the caveats linked to species-specific profiles, our findings revealed some conserved genetic features of lung-infiltrating myeloid cells between vFLIP mice and COVID-19 patients. Indeed, neutrophil subsets characterized by the expression of S100-family genes, type I interferon response genes (ISG15/ISG20) and chemokines (Ccl3/CCL3), as well as macrophages expressing proliferation-associated gene signatures (Mki67, Top2a) showed similarities among species. The shared leukocyte subsets showed a higher expression of inflammatory response-associated genes and were more prone to STAT3 therapy. In summary, in this study we demonstrated the pivotal role of FLIP-expressing myeloid cells to stimulate directly a lethal inflammatory status, by fueling an aberrant STAT3-dependent signaling pathway. Moreover, we substantiated the therapeutic effectiveness of STAT3 on-target strategy to mitigate uncontrolled inflammation and acute disease, which serve as a foundation for the development of more accurate and evidence-based therapies to control CRS disorders, as well as severe clinical aspects of the ongoing COVID-19 pandemic crisis.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04438629RecruitingEvaluation of Immune Response in COVID-19 Patients

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 24 and 26. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.
    • Thank you for including a protocol registration statement.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2021.05.04.21256298v1 on medRxiv