SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation

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SARS-CoV-2 infection causes broad-spectrum immunopathological disease, exacerbated by inflammatory co-morbidities. A better understanding of mechanisms underpinning virus-associated inflammation is required to develop effective therapeutics. Here we discover that SARS-CoV-2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through activation of cytoplasmic RNA-ensors RIG-I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing, or by inhibiting downstream signalling pathways. SARS-CoV-2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS-CoV-2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation-associated COVID-19.


  • SARS-CoV-2 activates RNA sensors and consequent inflammatory responses in lung epithelial cells

  • Epithelial RNA sensing responses drive pro-inflammatory macrophage activation

  • Exogenous inflammatory stimuli exacerbate responses to SARS-CoV-2 in both eplithelial cells and macrophages

  • Immunomodulators inhibit RNA sensing responses and consequent macrophage inflammation

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  1. SciScore for 10.1101/2020.12.23.424169: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants.
    Consent: The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Cells were then incubated with 1μg/ml CR3009 SARS-CoV-2 cross-reactive antibody (a kind gift from Dr. Laura McCoy,) in permeabilisation buffer for 30 min at room temperature, washed once and incubated with secondary Alexa Fluor 488-Donkey-anti-Human IgG (Jackson Labs).
    488-Donkey-anti-Human IgG
    suggested: None
    A blocking step was carried out for 1h at room temperature with 10% goat serum/1%BSA in PBS. Nucleocapsid (N) proten detection was performed by primary incubation with human anti-N antibody (Cr3009, 1ug/ml) for 18h, and washed thoroughly in PBS.
    suggested: None
    suggested: None
    Primary antibodies were detected by labelling with with secondary anti-human AlexaFluor488 and anti-rabbit AlexaFluor546 conjugates (Jackson Immuno Research) for 1h.
    suggested: None
    Experimental Models: Cell Lines
    Vero.E6 were provided by NIBSC, Beas2B (ATCC CRL-9609) and Hulec5a (ATCC CRL-3244) were obtained from ATCC, and Detroit 562 (ATCC CCL-138) were a kind gift from Dr Caroline Weight (UCL).
    suggested: ATCC Cat# CRL-3244, RRID:CVCL_0A11)
    THP-1 cells were cultured in RPMI (Gibco) supplemented with 10 heat-inactivated FBS (Labtech)
    suggested: None
    Caco-2 and Calu-3 cells were stimulated for 24 h with media containing TLR4 agonist Lipopolysaccharide (LPS) (Peprotech), the TLR3 agonist poly I:C (Peprotech) or the TLR7 agonist R837 (Invivogen), using the concentration stated on each figure.
    suggested: None
    Virus culture and infection: SARS-CoV-2 strain BetaCoV/Australia/VIC01/2020 (NIBSC) was propagated by infecting Caco-2 cells at MOI 0.01 TCID50/cell, in DMEM supplemented with 2% FBS at 37°C.
    suggested: None
    Virus titres were determined by 50% tissue culture infectious dose (TCID50) on Vero.E6 cells.
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    All samples were acquired on a BD Fortessa X20 or LSR II using BD FACSDiva software.
    BD FACSDiva
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    Data was analysed using FlowJo v10 (Tree Star).
    suggested: (FlowJo, RRID:SCR_008520)
    Image analysis: NF-κB, IRF3, IL6 and GAPDH raw image channels were pre-processed using a batch rolling ball background correction in FIJI imagej software package (Schindelin et al., 2012) prior to quantification.
    suggested: (ImageJ, RRID:SCR_003070)
    Statistical analysis: Statistical analysis was performed using GraphPad Prism.
    GraphPad Prism
    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 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 29. 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.

    About SciScore

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