SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

  1. Shahanshah Khan
  2. Mahnoush S Shafiei
  3. Christopher Longoria
  4. John W Schoggins
  5. Rashmin C Savani
  6. Hasan Zaki

  • Curated by eLife

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    Evaluation Summary:

    The identification of a novel role of the spike protein expressed on SARS-CoV-2 in directly evoking the host inflammatory responses has a substantial impact in understanding the molecular mechanism of COVID-19 pathogenesis, which may have implication for development of new therapeutics. The elegant analytic approach conducted herein justifies the major conclusions of this work though several additional steps can be made to validate these claims.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

The pathogenesis of COVID-19 is associated with a hyperinflammatory response; however, the precise mechanism of SARS-CoV-2-induced inflammation is poorly understood. Here, we investigated direct inflammatory functions of major structural proteins of SARS-CoV-2. We observed that spike (S) protein potently induced inflammatory cytokines and chemokines, including IL-6, IL-1β, TNFα, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and nucleocapsid (N) proteins. When stimulated with extracellular S protein, human and mouse lung epithelial cells also produced inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly were non-inflammatory, but elicited an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-κB pathway in a MyD88-dependent manner. Further, such an activation of the NF-κB pathway was abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein-induced IL-6, TNF-α, and IL-1β in wild-type, but not Tlr2-deficient mice. Notably, upon recognition of S protein, TLR2 dimerizes with TLR1 or TLR6 to activate the NF-κB pathway. Taken together, these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.

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  1. Version published to 10.7554/elife.68563 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    In this manuscript, Khan et al. investigated the roles of SARS-CoV-2 proteins on activation of immune cells. The authors found that the macrophage cell lines such as human THP-1 cells and mouse RAW 274.7 cells with recombinant viral proteins, and found that only spike proteins (S1 and S2) could potently activated macrophages to produce pro-inflammatory cytokines and chemokines.

    Strengths:

    It was Intriguing that only spike proteins (S1 and S2) could potently activate macrophages to produce pro-inflammatory cytokines and chemokines. The authors also observed that direct contact of macrophages with spike protein transfected epithelial cells, that mimic viral infection, resulted in the activation of macrophages. Detail analyses showed that spike proteins were recognized by Toll-like receptor 2 to activate NF-kB signaling. In vivo mouse experiments further supported the in vitro experiments. This study revealed a pathogenic of the SARS-CoV-2 spike proteins that is directly activating the host inflammatory responses, which therefore may have a profound impact in understanding a novel aspect of the cytokine signaling that is involved critically in the COVID-19 pathogenesis unveiled for the first time.

    Weaknesses:

    Recent report by Shirato and Kizaki (Heliyon 7(2021) e06187: 10.1016/j.heliyon.2021.e06187) showed that SARS-CoV-2 spike protein can stimulate macrophages (RAW 264.7 cells and THP-1 cells) to produce pro-inflammatory cytokines via TLR4-dependent manner. This is likely to contradict this study. The authors must thoroughly argue these controversial observations.

    We are grateful to the reviewer for finding our study impactful. We also appreciate the reviewer’s suggestion to discuss why our findings are inconsistent with other studies. In fact, three recent studies attempted to explore the role of SARS-CoV-2 structural proteins in inflammatory responses (Shirato K, Heliyon, 2021; Zhao Y, Cell Res, 2021; Zheng M, Nat Immunol, 2021). While two studies demonstrated that the Spike protein is responsible for triggering inflammation (Shirato K, Heliyon, 2021; Zhao Y, Cell Res, 2021), the other study found that the E protein is inflammatory (Zheng M, Nat Immunol, 2021). Further, as they tried to identify the cellular sensor for S and E proteins, the first two studies demonstrated that S protein is sensed by TLR4, which contradicts our findings. Notably, Dr. Kanneganti’s group nicely showed that Tlr2-/- macrophages do not produce cytokines during SARS-CoV2 infection, while Tlr4-/- macrophages are responsive. These data are consistent with our finding that TLR2 but not TLR4 is the sensor for S protein. It is intriguing that the findings of all these studies are partially consistent and partially contradictory. We believe that the discrepant results of other studies are possibly due to contamination of bacterial pattern molecules. Indeed, we noticed that other studies used recombinant proteins generated in E. coli. Thus, it is possible that those recombinant proteins were contaminated by LPS and other bacterial PAMPs, which may activate the TLR pathways. Since the inception of our studies, we were concerned about the possible contamination of recombinant SARS-CoV2 proteins. Therefore, throughout the study, we used recombinant proteins generated in mammalian cells (HEK293T cells). We used three different S proteins – S1, S2, and S tri from two different commercial sources (RayBiotech and R&D), and we found that all S proteins triggered the inflammatory response (Figure 1- Figure supplement 1E). Further, both S subunit and S-tri are sensed by TLR2, but not TLR4 (Figure 5E and 5F). We have included a discussion on this concern in Page 16.

  3. eLife

    Reviewer #3 (Public Review):

    Khan and colleagues evaluate the ability of purified components of the SARS-CoV-2 virus to induce inflammatory responses in macrophages and epithelial cells. They observe that the spike protein drives a TLR2-dependent inflammatory response both in vitro and in vivo. There also appears to be a potential crosstalk between epithelial cells and macrophages in response to the spike protein, however the specifics of this interaction remain unresolved.

  4. eLife

    Evaluation Summary:

    The identification of a novel role of the spike protein expressed on SARS-CoV-2 in directly evoking the host inflammatory responses has a substantial impact in understanding the molecular mechanism of COVID-19 pathogenesis, which may have implication for development of new therapeutics. The elegant analytic approach conducted herein justifies the major conclusions of this work though several additional steps can be made to validate these claims.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  5. eLife

    Reviewer #2 (Public Review):

    Overzealous inflammation is a significant clinical concern in COVID-19 patients. However, mechanisms underlying this hyperinflammatory response is unclear. In the current work by Khan and colleagues, the team investigates the role of the SARS-CoV-2 spike protein in driving inflammation and demonstrates the role of TLR2 in this mechanism. Here, the team identifies a range of inflammatory cytokines and chemokines generated following treatment of human and mouse cells with the S protein. Interestingly, the team did not observe any impacts on interferon signaling, suggesting a disconnect between the cytokine/chemokine response and interferon production. Likewise, this appears to be spike-protein dependent in the research team's hands. Biochemical studies suggest that the spike protein induces NF-kB signaling through a TLR2/MyD88 dependent mechanism. In general, the studies were well conducted and the data presented support the overall conclusions of the study. However, there are a few limitations to the work noted. These include questions associated with TLR2 heterodimer formation, the use of A549 cells that are refractory to SARS-CoV-2 infection as a model system, and clear data linking the mouse studies that are critical to the mechanism back to the human studies which lack specific assessments of TLR2/MyD88.

  6. eLife

    Reviewer #1 (Public Review):

    In this manuscript, Khan et al. investigated the roles of SARS-CoV-2 proteins on activation of immune cells. The authors found that the macrophage cell lines such as human THP-1 cells and mouse RAW 274.7 cells with recombinant viral proteins, and found that only spike proteins (S1 and S2) could potently activated macrophages to produce pro-inflammatory cytokines and chemokines.

    Strengths:

    It was Intriguing that only spike proteins (S1 and S2) could potently activate macrophages to produce pro-inflammatory cytokines and chemokines. The authors also observed that direct contact of macrophages with spike protein transfected epithelial cells, that mimic viral infection, resulted in the activation of macrophages. Detail analyses showed that spike proteins were recognized by Toll-like receptor 2 to activate NF-kB signaling. In vivo mouse experiments further supported the in vitro experiments. This study revealed a pathogenic of the SARS-CoV-2 spike proteins that is directly activating the host inflammatory responses, which therefore may have a profound impact in understanding a novel aspect of the cytokine signaling that is involved critically in the COVID-19 pathogenesis unveiled for the first time.

    Weaknesses:

    Recent report by Shirato and Kizaki (Heliyon 7(2021) e06187: 10.1016/j.heliyon.2021.e06187) showed that SARS-CoV-2 spike protein can stimulate macrophages (RAW 264.7 cells and THP-1 cells) to produce pro-inflammatory cytokines via TLR4-dependent manner. This is likely to contradict this study. The authors must thoroughly argue these controversial observations.

  7. ScreenIT

    SciScore for 10.1101/2021.03.16.435700: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All studies were approved by the Institutional Animal Care and Use Committee (IACUC) and were conducted in accordance with the IACUC guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAll experimental groups were conducted with age and sex-matched male and female mice.
    Cell Line AuthenticationContamination: Each cell lines were confirmed free from mycoplasma contamination by testing with mycoplasma detection kit (Sigma).

    Table 2: Resources

    Antibodies
    SentencesResources
    The membranes were immunoblotted with antibodies against Phospho-NF-κB p65 (3033, Cell Signaling), Phospho-IkBa (9246, Cell Signaling), IkBa (4812, Cell Signaling), Phospho-ERK (4370, Cell Signaling), ERK (4695, Cell Signaling), Phospho-JNK (4668, Cell Signaling), Phospho-AKT (4060, Cell Signaling), AKT (9272, Cell Signaling), Phospho-STAT3 (9145, Cell Signaling), Flag®M2 (F1804, Sigma-Aldrich), SARS-CoV-2 S (GTX632605, GeneTex) and ß-actin (A2228, Sigma).
    Phospho-NF-κB p65
    suggested: (Cell Signaling Technology Cat# 3033, RRID:AB_331284)
    Phospho-IkBa (9246
    suggested: None
    Phospho-ERK (4370
    suggested: None
    ERK
    suggested: None
    Phospho-JNK (4668
    suggested: None
    Phospho-AKT (4060, Cell Signaling), AKT
    suggested: None
    Phospho-STAT3 (9145
    suggested: (Cell Signaling Technology Cat# 9145, RRID:AB_2491009)
    GTX632605
    suggested: None
    ß-actin
    suggested: (RayBiotech Cat# 168-10656, RRID:AB_2885189)
    Cells were then incubated with Fc block CD16/32 monoclonal antibody (14-0161-82, eBioscience) and stained with primary antibody SARS-CoV-2 (GTX632604, GeneTex) for 30 min on ice.
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    Experimental Models: Cell Lines
    SentencesResources
    THP1 (ATCC, CRL-TIB-202) cells were cultured in Roswell Park Memorial Institute (
    THP1
    suggested: None
    cDNA constructs and transient transfection: At 50 - 60 % confluency, HEK293T and A549 cells were transfected with GFP-Flag (VB200507-2985cmv) or SARS-CoV-2 S-Flag (VB200507-2984jyv) (1.5 μg/ml) constructs using Lipofectamine 3000 reagent (Invitrogen) according to manufacturer’s instructions, and confirmed by observing GFP under fluorescence microscope and western blot analysis of SARS-CoV-2 S and Flag proteins.
    A549
    suggested: None
    RNA was isolated and measured for the expression inflammatory genes by real-time PCR Co-culture of macrophages and epithelial cells: HEK293T-GFP and HEK293T-SARS-CoV-2 S or A549-GFP and A549-SARS-CoV-2 S cells were cultured with THP-1 macrophage-like cells in a ratio of 1:2 (macrophages were twice in number to epithelial cells).
    A549-SARS-CoV-2 S
    suggested: None
    Real-time PCR: Epithelial cells, BMDMs, and THP-1 macrophage-like cells, RAW264.7 cells were lysed in TRIzol™ reagent (Invitrogen).
    RAW264.7
    suggested: CLS Cat# 400319/p462_RAW-2647, RRID:CVCL_0493)
    ELISA: BMDMs and THP-1 macrophage-like cells were lysed in ice-cold RIPA buffer supplemented with complete protease inhibitor and phosphatase inhibitor cocktails (Roche)
    THP-1
    suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)
    Western blot: THP-1 macrophage-like cells, BMDM, HEK293T and A549 cells were lysed in ice-cold RIPA lysis buffer containing complete protease inhibitor and phosphatase inhibitor cocktails (Roche), resolved by SDS-PAGE, and transferred onto a PVDF membrane.
    HEK293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: C57BL6/J (WT), Myd88−/−, Tlr2−/−, and Tlr4−/− mice (all C57BL6/J strain), purchased from Jackson Laboratory were used in this study.
    C57BL6/J
    suggested: None
    Myd88−/−
    suggested: None
    Tlr4−/−
    suggested: None
    Inflammatory response of SARS-CoV-2 S protein in mice: WT and Tlr2−/− mice were intraperitoneally injected with S1 and S2 subunits of SARS-CoV-2 S protein at equal concentration (1ug each/mouse).
    Tlr2−/−
    suggested: None
    Software and Algorithms
    SentencesResources
    Flow cytometric data were analyzed by FlowJo software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Data were analyzed by Prism8 (GraphPad Software) and statistical significance was determined by two-tailed unpaired Student’s t test.
    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 did not find any issues relating to the usage of bar graphs.

    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.
    • 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

    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.

  8. Version published to 10.1101/2021.03.16.435700v1 on bioRxiv