Gasdermin-D activation by SARS-CoV-2 triggers NET and mediate COVID-19 immunopathology

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1. Version published to 10.1186/s13054-022-04062-5

   Jul 7, 2022
2. 

   ScreenIT

   Jan 28, 2022

   SciScore for 10.1101/2022.01.24.22269768: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: The exclusion criteria were defined as diarrhea resulting in dehydration; pregnancy or lactation; metastatic cancer or immunosuppressive chemotherapy and inability to understand the consent form (Lopes et al., 2021). IRB: The study was approved by the National Ethics Committee, Brazil (CONEP, CAAE: 30248420.9.0000.5440).
   Sex as a biological variable	Eight-week-old male K18-hACE2 mice were infected with 2×104 PFU of SARS-CoV-2 (in 40 μL) by the intranasal route as previously described (Oladunni et al, 2020).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Authentication: The dataset generated by authors is publicly available at …

   SciScore for 10.1101/2022.01.24.22269768: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: The exclusion criteria were defined as diarrhea resulting in dehydration; pregnancy or lactation; metastatic cancer or immunosuppressive chemotherapy and inability to understand the consent form (Lopes et al., 2021). IRB: The study was approved by the National Ethics Committee, Brazil (CONEP, CAAE: 30248420.9.0000.5440).
   Sex as a biological variable	Eight-week-old male K18-hACE2 mice were infected with 2×104 PFU of SARS-CoV-2 (in 40 μL) by the intranasal route as previously described (Oladunni et al, 2020).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Authentication: The dataset generated by authors is publicly available at https://covid19-balf.cells.ucsc.edu/.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The slides were overnight stained with the following antibodies: a) Mouse anti-myeloperoxidase (MPO, 2C7, Abcam, cat. ab25989, 1:500); B) Rabbit anti-GSDMD-NT (Abcam, cat. ab215203).	anti-myeloperoxidase ( MPO suggested: (Antibodies-Online Cat# ABIN235893, RRID:AB_10764910) anti-GSDMD-NT suggested: None
   Next, the samples were incubated with alpaca anti-mouse IgG Alexa Fluor 488 (Jackson ImmunoResearch, cat. 615-545-214, 1:1000) or alpaca anti-rabbit IgG Alexa Fluor 594 (Jackson ImmunoResearch, cat. 611-585-215, 1:1000) secondary antibodies for 30 minutes.	anti-mouse IgG suggested: (Jackson ImmunoResearch Labs Cat# 615-545-214, RRID:AB_2721889) anti-rabbit IgG suggested: (Jackson ImmunoResearch Labs Cat# 611-585-215, RRID:AB_2721875)
   tenofovir disoproxil fumarate) – CAS 202138-50-9 - (10 μM); neutralizing anti-hACE2 antibody - αACE2 - RheaBiotech; Cat.	anti-hACE2 suggested: None
   The membranes were incubated overnight at 4°C under mild agitation in 5% nonfat dry milk in 1X TBS-T buffer containing the following primary antibodies: mouse anti-Caspase-1 (p20) (mAb (Bally-1); Adipogen; AG-20B-0048-C100; 1:1000), mouse anti-Caspase-4 (MBL cat.	anti-Caspase-1 suggested: None anti-Caspase-4 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Vero-E6 cells were cultured in DMEM high glucose supplemented with 10% fetal bovine serum (FBS; HyClone, Logan,Utah) and 100 U/mL penicillin, and 1% μg/mL streptomycin (P/S Corning; cat. 30-002-CI), 1% glutamine (Corning; cat. 15718008), 1% hepes (Corning; cat.25-060-CI), and 1% fungizone (Gibco; cat. 15290-018) at 37°C in the 5% CO2 atmosphere.	Vero-E6 suggested: None
   Plaque Reduction Neutralization Test using disulfiram against SARS-CoV-2: A PRNT (Plaque Reduction Neutralization Test) was performed in VERO E6 cells on a cell-monolayer in 24-well plates to evaluate the effect of disulfiram on SARS-CoV-2 infection.	VERO E6 suggested: None
   Cytopathic effect of SARS-CoV-2 infected neutrophils on A549 and HUVECcell lines by flow cytometry: Neutrophils were isolated from healthy controls (1×106) incubated or not with disulfiram (10 μM) for 1 h.	A549 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mouse infection and treatment: To evaluate the effects of Disulfiram in vivo, we infected the K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) (McCray et al., 2007; Oladunni et al., 2020; Bao et al., 2020).	B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: RRID:IMSR_JAX:034860)
   Eight-week-old male K18-hACE2 mice were infected with 2×104 PFU of SARS-CoV-2 (in 40 μL) by the intranasal route as previously described (Oladunni et al, 2020).	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
   Software and Algorithms
   Sentences	Resources
   The blood cells were then resuspended in Hank’s balanced salt solution (Corning; cat. 21-022-CV), and the neutrophil population was isolated by Percoll (GE Healthcare; cat. 17-5445-01) density gradient (72%, 63%, 54%, and 45%).	GE Healthcare suggested: (GE Healthcare, RRID:SCR_000004)
   This fluid was mixed 1:1 with 0.1 M dithiothreitol (Thermo Fisher Scientific; cat.	Thermo Fisher Scientific suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
   All images acquired were analyzed using Fiji by ImageJ.	Fiji suggested: (Fiji, RRID:SCR_002285) ImageJ suggested: (ImageJ, RRID:SCR_003070)
   The acquisition of the cells was performed in a flow cytometer (FACS Verse) and the analyses were made using the FlowJo software (FlowCytometry Analysis Software v10).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Statistical analyses and graph plots were performed with GraphPad Prism 8.4.2 software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your data.

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   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).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33, 24, 26, 27, 29 and 30. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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.

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

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3. 

   Version published to 10.1101/2022.01.24.22269768v1 on medRxiv

   Jan 25, 2022