SARS-CoV-2 Spike S1 glycoprotein is a TLR4 agonist, upregulates ACE2 expression and induces pro-inflammatory M 1 macrophage polarisation

  1. Mohamed M. Aboudounya
  2. Mark R. Holt
  3. Richard J. Heads

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Abstract

Background and aims

TLR4 is an important innate immune receptor that recognizes bacterial LPS, viral proteins and other pathogen associated molecular patterns (PAMPs). It is expressed on tissue-resident and immune cells. We previously proposed a model whereby SARS-CoV-2 activation of TLR4 via its spike glycoprotein S1 domain increases ACE2 expression, viral loads and hyperinflammation with COVID-19 disease [1]. Here we test this hypothesis in vitro and demonstrate that the SARS-CoV-2 spike S1 domain is a TLR4 agonist in rat and human cells and induces a pro-inflammatory M1 macrophage phenotype in human THP-1 monocyte-derived macrophages.

Methods

Adult rat cardiac tissue resident macrophage-derived fibrocytes (rcTMFs) were treated with either bacterial LPS or recombinant SARS-CoV-2 spike S1 glycoprotein. The expression of ACE2 and other inflammatory and fibrosis markers were assessed by immunoblotting. S1/TLR4 co-localisation/binding was assessed by immunocytochemistry and proximity ligation assays on rcTMFs and human HEK-293 HA-TLR4-expressing cells. THP-1 monocytes were differentiated into M1 or M2 macrophages with LPS/IFNγ, S1/IFNγ or IL-4 and RNA was extracted for RT-qPCR of M1/M2 markers and ACE2.

Results

TLR4 activation by spike S1 or LPS resulted in the upregulation of ACE2 in rcTMFs as shown by immunoblotting. Likewise, spike S1 caused TLR4-mediated induction of the inflammatory/wound healing marker COX-2 and concomitant downregulation of the fibrosis markers CTGF and Col3a1, similar to LPS. The specific TLR4 TIR domain signalling inhibitor CLI-095 (Resatorvid®), blocked the effects of spike S1 and LPS, confirming that spike S1 is a TLR4 agonist and viral PAMP (VAMP). ACE2 expression was also inhibited by the dynamin inhibitor Dynasore®, suggesting ACE2 expression is mediated by the alternative endosomal/β-interferon pathway. Confocal immunofluorescence microscopy confirmed 1:1 stoichiometric spike S1 co-localisation with TLR4 in rat and human cells. Furthermore, proximity ligation assays confirmed spike S1 and TLR4 binding in human and rat cells. Spike S1/IFN-γ treatment of THP-1-derived macrophages induced pro-inflammatory M 1 polarisation as shown by an increase in IL-1β and IL-6 mRNA.

Conclusions

These results confirm that TLR4 is activated by the SARS-CoV-2 spike protein S1 domain and therefore TLR4 may be a receptor/accessory factor for the virus. By binding to and activating TLR4, spike S1 caused upregulation of ACE2, which may facilitate viral entry into cells. In addition, pro-inflammatory M 1 macrophage polarisation via TLR4 activation, links TLR4 activation by spike S1 to inflammation. The clinical trial testing of CLI-095 (Resatorvid®) and other TLR4 antagonists in severe COVID-19, to reduce both viral entry into cells and hyperinflammation, is warranted. Our findings likely represent an important development in COVID-19 pathophysiology and treatment, particularly regarding cardiac complications and the role of macrophages.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.11.455921: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    interferon gamma (IFNγ) (SRP3058) and human Interleukin-4 (IL-4) (I4269) were from Sigma-Aldrich, UK; Primary antibodies were as follows: anti-CTGF goat polyclonal antibody (sc-14939), and anti-COL3A1 goat polyclonal antibody (sc-8781) were from Santa Cruz Biotechnology,
    IL-4
    suggested: None
    anti-CTGF
    suggested: (Santa Cruz Biotechnology Cat# sc-14939, RRID:AB_638805)
    anti-COL3A1
    suggested: (Santa Cruz Biotechnology Cat# sc-8781, RRID:AB_638604)
    Secondary antibodies were as follows: polyclonal rabbit anti-goat Immunoglobulins/HRP (P0160) and polyclonal swine anti-rabbit Immunoglobulins/HRP (P0217) were from Dako, DK; Cy™3-conjugated
    anti-goat
    suggested: None
    P0160
    suggested: None
    anti-rabbit
    suggested: (Agilent Cat# P0217, RRID:AB_2728719)
    The corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako, Denmark), diluted at 1:2500 in PBSTM were added for approx.
    HRP)-conjugated secondary antibodies ( Dako , Denmark)
    suggested: None
    The antibody concentration varied and depended on the antibody used, but it was usually at 1:200 or similar for the primary antibodies (i.e. TLR4) and 1:400 for ACE2.
    TLR4
    suggested: None
    ACE2
    suggested: None
    Primary antibodies used were mouse anti-human TLR4 and rabbit anti-spike S1 with the appropriate secondary antibodies (anti-rabbit-minus and anti-mouse-plus PLA probes) from the kit and red detection reagents. 293-hTLR4-HA cells: 293-hTLR4-HA cells (InvivoGen, USA) are HEK 293 cells stably transfected with the hTLR4a gene fused at the 3’ end to an influenza haemagglutinin (HA) tag.
    anti-human TLR4
    suggested: None
    anti-spike
    suggested: None
    anti-rabbit-minus
    suggested: None
    anti-mouse-plus PLA
    suggested: None
    HA
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Primary antibodies used were mouse anti-human TLR4 and rabbit anti-spike S1 with the appropriate secondary antibodies (anti-rabbit-minus and anti-mouse-plus PLA probes) from the kit and red detection reagents. 293-hTLR4-HA cells: 293-hTLR4-HA cells (InvivoGen, USA) are HEK 293 cells stably transfected with the hTLR4a gene fused at the 3’ end to an influenza haemagglutinin (HA) tag.
    293-hTLR4-HA
    suggested: RRID:CVCL_Y394)
    HEK 293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    293 cells express very low levels of endogenous TLR4.
    293
    suggested: NCI-DTP Cat# NCI-293TT, RRID:CVCL_1D85)
    THP-1 cells and differentiation: The human monocytic cell line THP-1 was routinely maintained in RPMI 1640 (1x) growth medium containing 10% of heat-inactivated FBS, 4.5 mg/ml D-glucose, 2mM L-glutamine, 10mM HEPES, 1mM pyruvate, 0.05 mM 2-mercaptoethanol, and 1% Penicillin/streptomycin.
    THP-1
    suggested: None
    Software and Algorithms
    SentencesResources
    Phorbol 12-myristate 13-acetate (PMA) (1201/1) was from Bio-Techne, UK; Cytokines:
    Cytokines
    suggested: None
    Statistical analysis was performed and graphs were produced using GraphPad Prism 8, either using a Kruskal Wallis test, Wilcoxon Signed Rank Test, or Repeated Measures One-way ANOVA with G-G correction followed by Tukey’s multiple comparisons post hoc test as appropriate to the experiment and as indicated in the figure legends.
    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 25. 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.
    • No funding statement was detected.
    • 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.

  2. Version published to 10.1101/2021.08.11.455921v1 on bioRxiv