Induction of Exaggerated Cytokine Production in Human Peripheral Blood Mononuclear Cells by a Recombinant SARS-CoV-2 Spike Glycoprotein S1 and Its Inhibition by Dexamethasone

  1. Olumayokun A. Olajide
  2. Victoria U. Iwuanyanwu
  3. Izabela Lepiarz-Raba
  4. Alaa A. Al-Hindawi

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Abstract

An understanding of the pathological inflammatory mechanisms involved in SARS-CoV-2 virus infection is necessary in order to discover new molecular pharmacological targets for SARS-CoV-2 cytokine storm. In this study, the effects of a recombinant SARS-CoV-2 spike glycoprotein S1 was investigated in human peripheral blood mononuclear cells (PBMCs). Stimulation of PBMCs with spike glycoprotein S1 (100 ng/mL) resulted in significant elevation in the production of TNFα, IL-6, IL-1β and IL-8. However, pre-treatment with dexamethasone (100 nM) caused significant reduction in the release of these cytokines. Further experiments revealed that S1 stimulation of PBMCs increased phosphorylation of NF-κB p65 and IκBα, and IκBα degradation. DNA binding of NF-κB p65 was also significantly increased following stimulation with spike glycoprotein S1. Treatment of PBMCs with dexamethasone (100 nM) or BAY11-7082 (1 μM) resulted in inhibition of spike glycoprotein S1-induced NF-κB activation. Activation of p38 MAPK by S1 was blocked in the presence of dexamethasone and SKF 86002. CRID3, but not dexamethasone pre-treatment, produced significant inhibition of S1-induced activation of NLRP3/caspase-1. Further experiments revealed that S1-induced increase in the production of TNFα, IL-6, IL-1β and IL-8 was reduced in the presence of BAY11-7082 and SKF 86002, while CRID3 pre-treatment resulted in the reduction of IL-1β production. These results suggest that SARS-CoV-2 spike glycoprotein S1 stimulated PBMCs to release pro-inflammatory cytokines through mechanisms involving activation of NF-κB, p38 MAPK and NLRP3 inflammasome. It is proposed that the clinical benefits of dexamethasone in COVID-19 are possibly due to its anti-inflammatory activity in reducing SARS-CoV-2 cytokine storm.

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  1. Version published to 10.1007/s10753-021-01464-5
  2. Version published to 10.1101/2021.02.03.429536v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.02.03.429536: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The following antibodies were used: rabbit anti-phospho-p65 (Cell Signalling Technology), rabbit anti-phospho-IκBα (Santa Cruz Biotechnology), rabbit total IκBα (Santa Cruz Biotechnology), rabbit anti-phospho-p38 (Cell Signalling Technology) and rabbit anti-NLRP3 (Abcam) antibodies.
    anti-phospho-p65
    suggested: None
    anti-phospho-IκBα
    suggested: None
    anti-phospho-p38 (Cell Signalling Technology)
    suggested: None
    anti-NLRP3 (Abcam)
    suggested: (Abcam Cat# ab214185, RRID:AB_2819003)
    Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature.
    anti-rabbit HRP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    2.9 Co-culture of human A549 epithelial cells and PBMCs: Human A549 lung epithelial cells were co-cultured with PBMCs using the transwell system (0.4 μm porous membrane; Corning).
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Software and Algorithms
    SentencesResources
    Statistical analysis were conducted using the GraphPad Prism software.
    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: 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.

  4. Version published to 10.1101/2021.02.03.429536v1 on bioRxiv