Variable Induction of Pro-Inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

  1. Weiming Ouyang
  2. Tao Xie
  3. Hui Fang
  4. Chunling Gao
  5. Tzanko Stantchev
  6. Kathleen A. Clouse
  7. Kun Yuan
  8. Tongzhong Ju
  9. David M. Frucht

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Abstract

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.

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  1. Version published to 10.3390/ijms22147540
  2. ScreenIT

    SciScore for 10.1101/2021.05.26.445843: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Human peripheral blood mononuclear cells: Leukapheresis blood packs from healthy donors were obtained under an institutional review board-approved protocol at the National Institutes of Health.
    Consent: All donors provided written informed consent, and anonymized samples were provided to our laboratory.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    anti-S1 neutralizing antibody (Catalog# 40591-MM43, lot HB14AP2001) and anti-S1 non-neutralizing antibody (Catalog # 40591-MM42, lot HB14AP0701) were also purchased from Sino Biological (Vendor #2).
    anti-S1
    suggested: None
    anti-S1 non-neutralizing
    suggested: None
    Following 3 washes with PBST, 100 μL of HRP-labelled anti-human IgG Fc antibody (Catalog# 31413, Thermo Fisher) was added to each well and incubated for 1 hour.
    anti-human IgG
    suggested: (Thermo Fisher Scientific Cat# 31413, RRID:AB_429693)
    To assess the blockade of spike protein binding to ACE2 by soluble ACE2 and anti-S1 neutralizing antibodies, 400 ng/mL of S1-Fc protein was preincubated with 2 μg/mL of ACE2 or 1 μg/mL of anti-S1 antibodies at room temperature for 20 minutes before incubation with plate-bound ACE2.
    ACE2 by soluble ACE2
    suggested: None
    anti-S1 neutralizing antibodies, 400
    suggested: None
    ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    Flow cytometry data were analyzed using FlowJo (Tree Star).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.05.26.445843v1 on bioRxiv