The SARS-CoV-2 receptor-binding domain facilitates neutrophil transepithelial migration and nanoparticle uptake in the mice airways

  1. Elena L. Bolkhovitina
  2. Julia D. Vavilova
  3. Andrey O. Bogorodskiy
  4. Yuliya A. Zagryadskaya
  5. Ivan S. Okhrimenko
  6. Alexander M. Sapozhnikov
  7. Valentin I. Borshchevskiy
  8. Marina A. Shevchenko

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Abstract

SARS-CoV-2-induced infection is still dangerous. Mouse models are convenient to the investigation of virus-activated immune response mechanisms. However, mice are not proper model organisms to study COVID-19 due to decreased interaction affinity between the SARS-CoV-2 receptor-binding domain (RBD) and mouse angiotensin-converting enzyme 2 (ACE2) compared with human ACE2. In the present study, we propose a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system. To mimic the effects of RBD– ACE2 high-affinity interaction, mice received the ACE2 inhibitor MLN-4760. To simulate virus loading, we applied 100 nm particles suspended in the solution of RBD via the oropharyngeal route to mice. In this model, MLN-4760 application enhanced neutrophil egress from the bone marrow to the bloodstream and RBD attracted neutrophils to the luminal side of the conducting airway epithelium. By contrast, inert 100 nm particles were not potent to stimulate neutrophil recruitment to the conducting airway mucosa. Using this model, and by altering the dosage of the ACE2 inhibitor, nanoparticles, and RBD, one can adapt it to investigate different COVID-19 states characterized with mild or severe airway inflammation.

Statement

This study presents a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system and investigates immune cell-model virus particle interactions in the conducting airway mucosa.

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  1. ScreenIT

    SciScore for 10.1101/2022.04.12.488042: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All animal experiments were performed in concordance with the Guide for the Care and Use of Laboratory Animals under a protocol approved by the Institutional Animal Care and Use Committee at Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences (protocol numbers 302/2020, 319/2021).
    Sex as a biological variableAnimals and ethics statement: C57BL/6 mice were purchased from Pushchino Animal Facility of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; female (18-20 g, 8-12 weeks).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Then the following antibodies (all from Miltenyi Biotec) were used: anti-mouse Ly6G–VioBlue (130-119-902), anti-mouse CD11b–VioGreen (130-113-811), anti-mouse FcεR1–PE (130-118-896), anti-mouse CD172–PE-Vio770 (130-123-154), anti-mouse CD45–APC-Vio770 (130-110-800).
    anti-mouse Ly6G–VioBlue
    suggested: None
    anti-mouse CD11b–VioGreen
    suggested: None
    anti-mouse FcεR1–PE
    suggested: None
    anti-mouse
    suggested: (Miltenyi Biotec Cat# 130-123-154, RRID:AB_2802004)
    anti-mouse CD45–APC-Vio770
    suggested: None
    The following antibodies and dilutions were used: anti-mouse CD11b-APC (BioLegend, 101212, dilution 1:50), anti-mouse CD169-AlexaFluor488 (BioLegend, 142419, dilution 1:50), anti-mouse Ly6G-AlexaFluor647 (BioLegend, 127609, dilution 1:50).
    anti-mouse CD11b-APC
    suggested: (SouthernBiotech Cat# 1560-11, RRID:AB_2794902)
    anti-mouse CD169-AlexaFluor488
    suggested: None
    For isotype-control staining APC-conjugated Rat IgG2b, κ (BioLegend, 400611) and Rat IgG2a, κ (BioLegend, 400525) The specimens were incubated with antibodies overnight then washed with 0.3% Triton X-100 in PBS and stained against actin by incubation for 1 h with Phalloidin-Atto425 (Sigma-Aldrich, 66939, dilution 1:50).
    Rat IgG2a
    suggested: (BioLegend Cat# 400525, RRID:AB_2864283)
    stained against actin
    suggested: (Beta Cell Biology Consortium Cat# AB1644, RRID:AB_10014592)
    Experimental Models: Organisms/Strains
    SentencesResources
    Animals and ethics statement: C57BL/6 mice were purchased from Pushchino Animal Facility of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; female (18-20 g, 8-12 weeks).
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    The images were created using Imaris version 9.8 software (Oxford Instruments).
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    Finally, the images were processed using Adobe Photoshop CS version 5 (Adobe Systems, Mountain View, CA).
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)
    The differences between two groups were analyzed with the Mann– Whitney test using GraphPad Prism software (GraphPad Software, San Diego, CA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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.

    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 10.1101/2022.04.12.488042v1 on bioRxiv