Targeting Neutrophils Extracellular Traps (NETs) reduces multiple organ injury in a COVID-19 mouse model
This article has been Reviewed by the following groups
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- Evaluated articles (Rapid Reviews Infectious Diseases)
Abstract
COVID-19 is characterized by severe acute lung injury, which is associated with neutrophils infiltration and 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. Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. 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 potential 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. 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
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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.
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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.
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Strength of evidence
Reviewers: N Teluguakula (Adichunchanagiri Institute of Medical Sciences) | 📗📗📗📗◻️
Kimberly M (KU Leuven) | 📒📒📒◻️◻️ -
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
Ethics IRB: 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 variable 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. Randomization A total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope ScanScope (Olympus) and Leica. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Reso… 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
Ethics IRB: 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 variable 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. Randomization A total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope ScanScope (Olympus) and Leica. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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-myeloperoxidasesuggested: Noneanti-MPOsuggested: NoneThe 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 IgGsuggested: (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. CD45suggested: (BioLegend Cat# 103138, RRID:AB_2563061)CD11bsuggested: NoneLy6Gsuggested: (BioLegend Cat# 127606, RRID:AB_1236494)Experimental Models: Cell Lines Sentences Resources 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). A549suggested: NoneExperimental Models: Organisms/Strains Sentences Resources 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/Jsuggested: 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-hACE2suggested: RRID:IMSR_GPT:T037657)Software and Algorithms Sentences Resources Images were analyzed with Fiji by Image J. Fijisuggested: (Fiji, RRID:SCR_002285)Image Jsuggested: (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 Healthcaresuggested: (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 Scientificsuggested: (Thermo Fisher Scientific, RRID:SCR_008452)Data were collected on a FACSVerse (BD Biosciences) and analyzed with FlowJo (TreeStar). FlowJosuggested: (FlowJo, RRID:SCR_008520)Statistical analyses and graph plots were performed and built with GraphPad Prism 9.3.1 software. GraphPad Prismsuggested: (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.
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