Targeting neutrophils extracellular traps (NETs) reduces multiple organ injury in a COVID-19 mouse model

  1. Flavio P. Veras
  2. Giovanni F. Gomes
  3. Bruna M. S. Silva
  4. Diego B. Caetité
  5. Cicero J. L. R. Almeida
  6. Camila Meirelles S. Silva
  7. Ayda H. Schneider
  8. Emily S. Corneo
  9. Caio S. Bonilha
  10. Sabrina S. Batah
  11. Ronaldo Martins
  12. Eurico Arruda
  13. Alexandre T. Fabro
  14. José C. Alves-Filho
  15. Thiago M. Cunha
  16. Fernando Q. Cunha

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Abstract

Background

COVID-19 is characterized by severe acute lung injury, which is associated with neutrophil infiltration and the release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment.

Methods

Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. SARS-CoV-2-infected K18-hACE2 mice were performed for clinical sickness scores and lung pathology. Moreover, the levels of NETs were assessed and lung injuries were by histopathology and TUNEL assay. Finally, the injury in the heart and kidney was assessed by histopathology and biochemical-specific markers.

Results

DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potentially deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro , which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I.

Conclusions

Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.

Article activity feed

  1. Version published to 10.1186/s12931-023-02336-2
  2. Rapid Reviews Infectious Diseases

    Narasaraju Teluguakula

    Review 1: "Targeting Neutrophils Extracellular Traps (NETs) Reduces Multiple Organ Injury in a COVID-19 Mouse Model"

    Both reviewers agree that this study may have important implications for DNase as a therapeutic agent for COVID-19. Reviewers point out the small sample size and short follow-up period as potential limitations.

  3. Rapid Reviews Infectious Diseases

    Kimberly Martinod

    Review 2: "Targeting Neutrophils Extracellular Traps (NETs) Reduces Multiple Organ Injury in a COVID-19 Mouse Model"

    Both reviewers agree that this study may have important implications for DNase as a therapeutic agent for COVID-19. Reviewers point out the small sample size and short follow-up period as potential limitations.

  5. ScreenIT

    SciScore for 10.1101/2022.04.27.489676: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The manipulation of these animals was performed in Biosafety Levels 3 (BSL3) facility and the study was approved by Ethics Committee on the Use of Animals of the Ribeirão Preto Medical School, University of São Paulo (#066/2020).
    Sex as a biological variableDNase I treatment in SARS-CoV-2 experimental infection: Male K18-hACE2 mice, aged 8 weeks, were infected with 2×104 PFU of SARS-CoV-2 (in 40 μL) by intranasal route.
    RandomizationA total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope ScanScope (Olympus) and Leica.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After blocking with IHC Select Blocking Reagent (Millipore, cat. 20773-M) for 2 hours at room temperature (RT), the following primary antibodies were incubated overnight at 4°C: rabbit polyclonal anti-myeloperoxidase (anti-MPO; Thermo Fisher Scientific; cat.
    anti-myeloperoxidase
    suggested: None
    anti-MPO
    suggested: None
    The slides were then washed with TBS-T (Tris-Buffered Saline with Tween 20) and incubated with secondary antibodies alpaca anti-rabbit IgG AlexaFluor 488 (Jackson ImmunoReseacher; Cat. 615-545-215; 1:1000) and alpaca anti-rabbit IgG AlexaFluor 594 (Jackson ImmunoReseacher; Cat. 611-585-215; 1:1000).
    anti-rabbit IgG
    suggested: (Biorbyt Cat# orb14385, RRID:AB_10735740)
    Cells were then stained with Fixable Viability Dye eFluor 780 (eBioscience; cat. 65–0865-14; 1:3,000) and monoclonal antibodies specific for CD45 (BioLegend; clone 30-F11; cat. 103138; 1:200), CD11b (BD Biosciences; clone M1/70; cat. 553311) and Ly6G (Biolegend; clone 1A8; cat. 127606) for 30 min at 4°C.
    CD45
    suggested: (BioLegend Cat# 103138, RRID:AB_2563061)
    CD11b
    suggested: None
    Ly6G
    suggested: (BioLegend Cat# 127606, RRID:AB_1236494)
    Experimental Models: Cell Lines
    SentencesResources
    Human alveolar basal epithelial A549 cells (5×104) were maintained in DMEM and cultured with purified NETs (10 ng/ml) pretreated, or not, with DNase I (0.5 mg/ml; Pulmozyme, Roche).
    A549
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    K18-hACE2 mice: K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from The Jackson Laboratory and were bred in the Centro de Criação de Animais Especiais (Ribeirão Preto Medical School/University of São Paulo).
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    DNase I treatment in SARS-CoV-2 experimental infection: Male K18-hACE2 mice, aged 8 weeks, were infected with 2×104 PFU of SARS-CoV-2 (in 40 μL) by intranasal route.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    Images were analyzed with Fiji by Image J.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    Neutrophils isolation and NETs purification: Peripheral blood samples were collected from healthy controls by venipuncture and the neutrophil population was isolated by Percoll density gradient (GE Healthcare; cat. 17-5445-01).
    GE Healthcare
    suggested: (GE Healthcare, RRID:SCR_000004)
    Apoptosis assay: Lung tissue were harvested for detection of apoptotic cells in situ with Click-iT Plus TUNEL Assay Alexa Fluor 488, according to the manufacturer’s instructions (Thermo Fisher Scientific; cat. C10617).
    Thermo Fisher Scientific
    suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
    Data were collected on a FACSVerse (BD Biosciences) and analyzed with FlowJo (TreeStar).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analyses and graph plots were performed and built with GraphPad Prism 9.3.1 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.

    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.

  6. Version published to 10.1101/2022.04.27.489676v1 on bioRxiv