Serum Amyloid P inhibits single stranded RNA-induced lung inflammation, lung damage, and cytokine storm in mice

  1. Tejas R. Karhadkar
  2. Darrell Pilling
  3. Richard H. Gomer

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Abstract

SARS-CoV-2 is a single stranded RNA (ssRNA) virus and contains GU-rich sequences distributed abundantly in the genome. In COVID-19, the infection and immune hyperactivation causes accumulation of inflammatory immune cells, blood clots, and protein aggregates in lung fluid, increased lung alveolar wall thickness, and upregulation of serum cytokine levels. A serum protein called serum amyloid P (SAP) has a calming effect on the innate immune system and shows efficacy as a therapeutic for fibrosis in animal models and clinical trials. Here we show that aspiration of the GU-rich ssRNA oligonucleotide ORN06 into mouse lungs induces all of the above COVID-19-like symptoms. Men tend to have more severe COVID-19 symptoms than women, and in the aspirated ORN06 model, male mice tended to have more severe symptoms than female mice. Intraperitoneal injections of SAP starting from day 1 post ORN06 aspiration attenuated the ORN06-induced increase in the number of inflammatory cells and formation of clot-like aggregates in the mouse lung fluid, reduced ORN06-increased alveolar wall thickness and accumulation of exudates in the alveolar airspace, and attenuated an ORN06-induced upregulation of the inflammatory cytokines IL-1β, IL-6, IL-12p70, IL-23, and IL-27 in serum. SAP also reduced D-dimer levels in the lung fluid. In human peripheral blood mononuclear cells, SAP attenuated ORN06-induced extracellular accumulation of IL-6. Together, these results suggest that aspiration of ORN06 is a simple model for both COVID-19 as well as cytokine storm in general, and that SAP is a potential therapeutic for diseases with COVID-19-like symptoms and/or a cytokine storm.

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  1. Version published to 10.1371/journal.pone.0245924
  2. ScreenIT

    SciScore for 10.1101/2020.08.26.269183: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: The Texas A&M University Animal Use and Care Committee approved the protocol.
    RandomizationThree fields of view were chosen randomly for imaging.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableMouse model of lung damage and cytokine storm: 5-week old 20 g male and female C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were given an oropharyngeal aspiration of 1 mg/kg (or less where indicated) ORN06/LyoVec (a ssRNA oligonucleotide with 6 UGU repeats complexed with a lipid which protects the ssRNA from degradation; henceforth referred to as ORN06) (InvivoGen, San Diego, CA) in 50 µl phosphate-buffered saline (PBS), or an oropharyngeal aspiration of an equal volume of PBS as a control, following (55, 56).

    Table 2: Resources

    Antibodies
    SentencesResources
    Slides were incubated with monoclonal antibodies overnight at 4°C using anti-CD3 (100202, clone 17A2, BioLegend, San Diego, CA) to detect T-cells, anti-CD11b (101202, clone M1/70, BioLegend) to detect blood and inflammatory macrophages, anti-CD11c (M100-3, clone 223H7, MBL International, Woburn, MA) to detect alveolar macrophages and dendritic cells, anti-CD45 (103102, clone 30-F11, BioLegend) for total leukocytes, anti-Ly6G (127602, clone 1A8, BioLegend) to detect neutrophils, with isotype-matched irrelevant rat antibodies as controls, diluted to 5 µg/ml in PBS containing 2% (w:v
    anti-CD3
    suggested: (BioLegend Cat# 100202, RRID:AB_312659)
    anti-CD11c
    suggested: (MBL International Cat# M100-6, RRID:AB_842946)
    Slides were then washed in PBS, with 6 changes of PBS over 30 minutes, and then incubated with 1 µg/ml biotin-conjugated F(ab’)2-donkey anti-rat antibodies (Novus Biologicals, Littleton, CO) in PBS-BSA for 30 minutes at room temperature.
    anti-rat
    suggested: None
    Lung sections were also assessed for the presence of immune cells using anti-CD11b, anti-CD11c, anti-CD45, anti-Ly6G, or anti-Mac2 (clone M3/38; BioLegend) antibodies with isotype-matched irrelevant rat antibodies as controls.
    anti-CD11b
    suggested: (Thermo Fisher Scientific Cat# 88-7774-75, RRID:AB_476399)
    anti-CD45
    suggested: None
    anti-Ly6G
    suggested: None
    anti-Mac2
    suggested: (Fluidigm Cat# 3153026B, RRID:AB_2814900)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse model of lung damage and cytokine storm: 5-week old 20 g male and female C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were given an oropharyngeal aspiration of 1 mg/kg (or less where indicated) ORN06/LyoVec (a ssRNA oligonucleotide with 6 UGU repeats complexed with a lipid which protects the ssRNA from degradation; henceforth referred to as ORN06) (InvivoGen, San Diego, CA) in 50 µl phosphate-buffered saline (PBS), or an oropharyngeal aspiration of an equal volume of PBS as a control, following (55, 56).
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    Image quantification was done using Image J (NIH, Bethesda,
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    Statistics: Statistical analyses with t tests or 1-way ANOVA with the indicated post-test were done using GraphPad Prism 7 (GraphPad, San Diego, CA).
    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: 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.

  3. Version published to 10.1101/2020.08.26.269183v1 on bioRxiv