A zebrafish model of COVID-19-associated cytokine storm syndrome reveals differential proinflammatory activities of Spike proteins of SARS-CoV-2 variants of concern

  1. Sylwia D. Tyrkalska
  2. Alicia Martínez-López
  3. Ana B. Arroyo
  4. Francisco J. Martínez-Morcillo
  5. Sergio Candel
  6. Diana García-Moreno
  7. Pablo Mesa-del-Castillo
  8. María L. Cayuela
  9. Victoriano Mulero

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Abstract

The sudden and unexpected appearance of the COVID-19 pandemic turned the whole world upside down in a very short time. One of the main challenges faced has been to understand COVID-19 patient heterogeneity, as a minority develop life-threatening hyperinflammation, the so-called cytokine storm syndrome (CSS). Using the unique advantages of the zebrafish model we report here the proinflammatory role of Spike (S) proteins from different SARS-CoV-2 variants of concern after injection into the hindbrain ventricle, a cavity filled with cerebrospinal fluid to which immune cells can be easily recruited and that mimics the alveolar environment of the human lung. We found that wild type/Wuhan variant S1 (S1WT) protein promoted neutrophil and macrophage recruitment, local and systemic hyperinflammation, emergency myelopoiesis, and hemorrhages. In addition, S1γ protein was more proinflammatory and S1δ was less proinflammatory than S1WT and, strikingly, S1β promoted delayed and long-lasting inflammation. Pharmacological inhibition of the canonical inflammasome robustly alleviated S1 protein-induced inflammation and emergency myelopoiesis. In contrast, genetic inhibition of angiotensin-converting enzyme 2 strengthened the proinflammatory activity of S1, and the administration of angiopoietin (1-7) fully rescued S1-induced hyperinflammation and hemorrhages. These results shed light into the mechanisms orchestrating the COVID-19-associated CSS and the host immune response to different SARS-CoV-2 S protein variants.

Highlights

  • S proteins of SARS-CoV-2 promote hyperinflammation, neutrophilia, monocytosis and hemorrhages in zebrafish.

  • S protein effects in zebrafish are mediated via the canonical inflammasome and the Ace2/Angiopoietin (1-7) axis.

  • Delta S1 is less proinflammatory than wild type S1 and fails to induce emergency myelopoiesis in zebrafish.

  • Naïve and primed human white blood cells are unable to respond to S proteins.

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  1. ScreenIT

    SciScore for 10.1101/2021.12.05.471277: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Human PBMC, neutrophils and synovial fluid cell culture and treatments: All the experiments and procedures were performed as approved by the Ethical Clinical Research Committee of The University Hospital Virgen de la Arrixaca (approval number #5/2021).
    Sex as a biological variableThe synovial fluid -obtained from a 13-year-old female with recent onset of oligoarticular juvenile idiopathic arthritis -was composed of 87% mononuclear cells and 13% neutrophils.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    The lines Tg(mpx:eGFP)i114 (Renshaw et al., 2006), Tg(mfap4:mCherry-F)ump6 (Phan et al., 2018), Tg(NFkB-RE:eGFP)sh235 referred to as nfkb:eGFP (Kanther et al., 2011),
    Tg(mpx:eGFP)i114
    suggested: None
    Tg(mfap4:mCherry-F)ump6
    suggested: None
    Tg(NFkB-RE:eGFP)sh235 referred to as nfkb:eGFP
    suggested: None
    Tg(gata1a:DsRed)sd2 (Traver et al., 2003), Tg(fli1:EGFP)y1 (Delov et al., 2014) and casper (mitfaw2/w2; mpv17a9/a9) (White et al., 2008) were previously described.
    Tg(gata1a:DsRed)sd2
    suggested: None
    Tg(fli1:EGFP)y1
    suggested: None
    Software and Algorithms
    SentencesResources
    The number of neutrophils or macrophages was determined by counting visually and the fluorescence intensity was obtained and analyzed with ImageJ (FIJI) software (Schindelin et al., 2012).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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: We detected the following sentences addressing limitations in the study:
    Therefore, this model circumvents the limitations and discrepancies obtained with human and mouse macrophages concerning the immunogenic and proinflammatory properties of SARS-CoV-2 S protein and shed light into the COVID-19 associated CSS. Thus, we observed that S protein not only induced the local and systemic expression of genes encoding proinflammatory mediators, but also NFκB, neutrophilia and monocytosis. To the best of our knowledge, this is the first study showing that S protein is able to induce a dramatic neutrophilia and monocytosis, which are both involved in the pathogenesis of COVID-19 (Mei et al., 2020; Narasaraju et al., 2020). Although the inflammasome has been found to participate in COVID-19, a mechanistic understanding of its involvement in COVID-19 progression remains unclear (Vora et al., 2021). Clinical trials with Anakinra, a modified human IL-1 receptor antagonist approved to treat rheumatoid arthritis, provided mixed results on patient benefits (Vora et al., 2021). We found that pharmacological inhibition of caspase-1, the effector of the canonical inflammasome, strongly alleviated the proinflammatory activity of S protein in zebrafish larvae. This effect was observed at two different levels: (i) the dampening of gene encoding proinflammatory mediators used in our study as a surrogate of the COVID-19-associated CSS, and (ii) the alleviation of neutrophilia. However, no differences in neutrophil recruitment to S protein was found between untreated and...

    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.
    • 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.

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  2. Version published to 10.1101/2021.12.05.471277 on bioRxiv