Altered increase in STAT1 expression and phosphorylation in severe COVID‐19

  1. Hector Rincon‐Arevalo
  2. Arman Aue
  3. Jacob Ritter
  4. Franziska Szelinski
  5. Dmytro Khadzhynov
  6. Daniel Zickler
  7. Luisa Stefanski
  8. Andreia C. Lino
  9. Sixten Körper
  10. Kai‐Uwe Eckardt
  11. Hubert Schrezenmeier
  12. Thomas Dörner
  13. Eva V. Schrezenmeier

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Abstract

The interferon pathway, a key antiviral defense mechanism, is being considered as a therapeutic target in COVID‐19. Both, substitution of interferon and JAK/STAT inhibition to limit cytokine storms have been proposed. However, little is known about possible abnormalities in STAT signaling in immune cells during SARS‐CoV‐2 infection. We investigated downstream targets of interferon signaling, including STAT1, STAT2, pSTAT1 and 2, and IRF1, 7 and 9 by flow cytometry in 30 patients with COVID‐19, 17 with mild, and 13 with severe infection. We report upregulation of STAT1 and IRF9 in mild and severe COVID‐19 cases, which correlated with the IFN‐signature assessed by Siglec‐1 (CD169) expression on peripheral monocytes. Interestingly, Siglec‐1 and STAT1 in CD14+ monocytes and plasmablasts showed lower expression among severe cases compared to mild cases. Contrary to the baseline STAT1 expression, the phosphorylation of STAT1 was enhanced in severe COVID‐19 cases, indicating a dysbalanced JAK/STAT signaling that fails to induce transcription of interferon stimulated response elements (ISRE). This abnormality persisted after IFN‐α and IFN‐γ stimulation of PBMCs from patients with severe COVID‐19. Data suggest impaired STAT1 transcriptional upregulation among severely infected patients may represent a potential predictive biomarker and would allow stratification of patients for certain interferon‐pathway targeted treatments.

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  1. Version published to 10.1002/eji.202149575
  2. ScreenIT

    SciScore for 10.1101/2021.08.13.21262006: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: All participants or their legal representatives gave written informed consent according to the approval of the ethics committee at the Charité University Hospital, Berlin (EA2/066/20 Pa-Covid-19 and University of Ulm (CAPSID trial (115/20 and 488/20) [16, 17]. 2.2.
    IRB: All participants or their legal representatives gave written informed consent according to the approval of the ethics committee at the Charité University Hospital, Berlin (EA2/066/20 Pa-Covid-19 and University of Ulm (CAPSID trial (115/20 and 488/20) [16, 17]. 2.2.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For flow cytometric analysis, the following fluorochrome-labeled antibodies were used: BUV395 anti-CD14 (BD, clone M5E2, 1:50)
    anti-CD14
    suggested: None
    Quantitative analysis was done using the following intracellular fluorochrome-labeled antibodies: PE anti-STAT1 (BD, clone 1/Stat1, 1:10), FITC anti-pSTAT1 (BD, clone 4a, 3:20), AF647 anti-pSTAT2 (R&D Systems, clone 1021D, 1:5), PE anti-IRF1 (BD, clone 20/IRF-1, 1:20), AF647 anti-IRF7 (BD, clone K47-671, 1:10).
    anti-STAT1
    suggested: None
    anti-pSTAT1
    suggested: (Fluidigm Cat# 3153003A, RRID:AB_2811248)
    anti-pSTAT2
    suggested: None
    anti-IRF1
    suggested: None
    anti-IRF7
    suggested: None
    For IRF9 analysis an unconjugated IRF9 antibody (Thermo Fisher,
    IRF9
    suggested: None
    Recombinant DNA
    SentencesResources
    Analytical methods and flow cytometry: Intracellular phenotyping of STAT1, pSTAT1, pSTAT2, IRF1 and IRF7 levels in B and T cells was conducted as previously published [18].
    pSTAT1
    suggested: None
    pSTAT2
    suggested: None
    Software and Algorithms
    SentencesResources
    Study participants: Peripheral blood samples (EDTA anti-coagulated, BD vaccutainer system, BD Diagnostics, Franklin Lakes, NJ, USA) from 20 healthy controls and 30 COVID patients were analyzed, 17 with mild (WHO 8-point ordinal scale 1 and 2) and 13 with a severe course of the disease (WHO 8-point ordinal scale ≥ 4) [15].
    BD Diagnostics
    suggested: None
    l analysis: Flow cytometry data were analysed using FACSDiva software (Becton Dickinson, Franklin Lakes, NJ, USA) and FlowJo (version 10, TreeStar, Ashland, OR, USA).
    FACSDiva
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    For graphical and statistical analysis, GraphPad Prism (version 7.00, GraphPad Software, La Jolla, CA, USA) was used.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04433910CompletedA Clinical Trial of Convalescent Plasma Compared to Best Sup…

    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 page 14. 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.
    • No protocol registration statement was detected.

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

    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 10.1101/2021.08.13.21262006v1 on medRxiv