Surface proteins of SARS-CoV-2 drive airway epithelial cells to induce interferon-dependent inflammation

  1. Gautam Anand
  2. Alexandra M. Perry
  3. Celeste L. Cummings
  4. Emma St. Raymond
  5. Regina A. Clemens
  6. Ashley L. Steed

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Abstract

SARS-CoV-2, the virus that has caused the COVID-19 pandemic, robustly activates the host immune system in critically ill patients. Understanding how the virus engages the immune system will facilitate the development of needed therapeutic strategies. Here we demonstrate both in vitro and in vivo that the SARS-CoV-2 surface proteins Spike (S) and Envelope (E) activate the key immune signaling interferon (IFN) pathway in both immune and epithelial cells independent of viral infection and replication. These proteins induce reactive oxidative species generation and increases in human and murine specific IFN-responsive cytokines and chemokines, similar to their upregulation in critically ill COVID-19 patients. Induction of IFN signaling is dependent on canonical but discrepant inflammatory signaling mediators as the activation induced by S is dependent on IRF3, TBK1, and MYD88 while that of E is largely MYD88 independent. Furthermore, these viral surface proteins, specifically E, induced peribronchial inflammation and pulmonary vasculitis in a mouse model. Finally we show that the organized inflammatory infiltrates are dependent on type I IFN signaling, specifically in lung epithelial cells. These findings underscore the role of SARS-CoV-2 surface proteins, particularly the understudied E protein, in driving cell specific inflammation and their potential for therapeutic intervention.

Author Summary

SARS-CoV-2 robustly activates widespread inflammation, but we do not understand mechanistically how the virus engages the immune system. This knowledge will facilitate the development of critically needed therapeutic strategies to promote beneficial immune responses will dampening harmful inflammation. Here we demonstrate that SARS-CoV-2 surface proteins spike and envelope alone activated innate cell function and the interferon signaling pathway. This activation occurred in both immune and epithelial cells, and mechanistic studies demonstrated dependence on known key inflammatory signaling mediators, IRF3, TBK1, and MYD88. In animal studies, we showed that these viral surface proteins induce epithelial cell IFN-dependent lung pathology, reminiscent to acute COVID-19 pulmonary infection. These findings underscore the need for further investigation into the role of SARS-CoV-2 surface proteins, particularly the understudied E protein, in driving cell specific inflammation.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.14.422710: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Ethics Statement: All animal protocols used in this study were approved by the Washington University’s Animal Studies Committee (19-0768), which approved these methods.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAdult (8-16 week-old male and female) mice were anesthetized with isoflurane and intranasally administered 10µg of protein E, S, or water in 50µl total volume (25 µl per nare).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For the IFN-depleting experiments, mice were injected intraperitoneally with 2mg antibody in 500µl volume (anti-Ifnar or isotype control) 6 days prior and 0.5mg antibody in 500ul volume 2 days prior to intranasal administration of protein E.
    anti-Ifnar
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: The cell lines A549-Dual (adenocarcinoma human alveolar basal epithelial cells) and RAW-Lucia ISG (RAW-mouse macrophages) (InvivoGen, San Diego, CA, USA) were cultured in DMEM (Sigma-Aldrich, St Louis, Mo, USA).
    A549-Dual
    suggested: RRID:CVCL_5I73)
    The test media for A549 and THP-1 cells excluded Zeocin and Blasticidin from their respective growth media.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    THP-1
    suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)
    Reporter cell assays: A549-Dual, THP1-Dual, THP1-Dual KO and RAW-Lucia ISG cells stably express an interferon regulatory factor-inducible Lucia luciferase reporter construct.
    RAW-Lucia ISG
    suggested: RRID:CVCL_X596)
    Software and Algorithms
    SentencesResources
    Dot arrays were quantified for pixel density with ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistics: GraphPad Prism (San Diego, CA, USA) version 7.02 software was used to perform all statistical analyses as described.
    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: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.
    • No funding statement was detected.
    • 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 10.1101/2020.12.14.422710 on bioRxiv