SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release

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   ScreenIT

   Mar 15, 2021

   SciScore for 10.1101/2021.03.06.21252796: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: All summary data used in this work are publicly available, together with a description of relevant participant consent and ethical approval secured in the original investigation.
   Randomization	Mendelian randomization is a form of instrumental variable analysis that exploits the random allocation of alleles at meiosis to draw causal inferences using observational data by attempting to emulate randomization procedures that would be adopted in a clinical trial.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Monocytes were then blocked …

   SciScore for 10.1101/2021.03.06.21252796: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: All summary data used in this work are publicly available, together with a description of relevant participant consent and ethical approval secured in the original investigation.
   Randomization	Mendelian randomization is a form of instrumental variable analysis that exploits the random allocation of alleles at meiosis to draw causal inferences using observational data by attempting to emulate randomization procedures that would be adopted in a clinical trial.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Monocytes were then blocked for 30 min with PBS + 5% FBS, washed twice and then stained with unconjugated primary antibodies for ASC (1:200, mouse or rabbit), NLRP3 (1:200, goat), AIM2 (1:200, mouse), GSDMD (1:200, mouse), pyrin (1:200, rabbit), dsRNA (J2, mouse) (1:500) or SARS-CoV-2 nucleocapsid protein (1:500, rabbit) for 2 h, followed by 3 washes with PBS + 3% FBS.	ASC suggested: None NLRP3 suggested: None AIM2 suggested: None GSDMD suggested: None SARS-CoV-2 nucleocapsid protein suggested: (Bioss Cat# bsm-41414M, RRID:AB_2848129)
   After washing with 1x Annexin V buffer, cells were blocked for 10 min with anti-CD32 (1:100) in PBS + 3% FBS, and then stained for 15 min on ice with a cocktail of antibodies to identify lymphocyte and myeloid cell subsets (all 1:200 except CD19 BV650, CD123 PerCP-Cy5.5 and CD56 APC-Cy7, 1:100)	CD123 suggested: (BD Biosciences Cat# 557939, RRID:AB_2802162) PerCP-Cy5.5 suggested: (Thermo Fisher Scientific Cat# A14940, RRID:AB_2534378) CD56 suggested: None APC-Cy7 suggested: (StressMarq Biosciences Cat# SMC-100B-APCCY7, RRID:AB_2820311)
   Purified monocytes and an A549 cell line overexpressing ACE2 were blocked with anti-CD32, then stained with primary antibodies for ACE2 (1:100) for 15 min on ice.	anti-CD32 suggested: None ACE2 suggested: None
   Cells were stained with primary antibodies for dsRNA (J2, mouse) (1:500), then stained with secondary antibody (donkey anti-mouse conjugated with AlexaFluor 647, at 1:500) and anti-CD14 PE-Cy7.	J2 suggested: None anti-mouse suggested: None anti-CD14 suggested: None
   The viral inoculum was treated with 10 μg/ml of antibody (isotype control mAb114, anti-Spike C1A-H12, or anti-Spike C1A-B12), or 10% HD or COVID-19 patient pooled plasma (heat inactivated or not; Ig-depleted or not, as indicated) before infection with SARS-CoV-2 for 30 min at room temperature.	anti-Spike C1A-H12 suggested: None anti-Spike C1A-B12 suggested: None
   C1A-B12 and C1A-H12, two SARS-CoV-2 Spike-targeting human monoclonal antibodies, were produced as previously described (49). qRT-PCR: RNA extracted from HD monocytes (stimulated or not with LPS (100 ng/ml for 16 h), or COVID-19 patient monocytes was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and the cDNA was analyzed by qRT-PCR using the Sso Fast™ EvaGreen® Supermix (BioRad) (30 sec at 95°C, 40 cycles (3 sec at 95°C; 3 sec at 54 °C) for both ACE2 and BSG (CD147)) using a CFX96 Touch Real-Time PCR Detection System (BioRad)	C1A-B12 suggested: None BSG suggested: None CD147 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Monocyte treatment and culture: Purified monocytes and THP-1 cells (ATCC) as controls, were cultured in RPMI + 10% FBS + 1% Pen/Strep for 30 min at 37°C in the presence or absence of 20 µM nigericin.	THP-1 suggested: None
   Purified monocytes and an A549 cell line overexpressing ACE2 were blocked with anti-CD32, then stained with primary antibodies for ACE2 (1:100) for 15 min on ice.	A549 suggested: None
   Software and Algorithms
   Sentences	Resources
   GSDMD was measured in the same samples using the Human GSDMD ELISA kit (MyBiosource) following the manufacturer’s instructions, and LDH activity was measured using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega).	CytoTox suggested: None
   Cells were acquired using a FACS Canto II or LSR II and data were analyzed using FlowJo Version 10.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Uncorrelated single-nucleotide polymorphisms (SNPs) (r2 < 0.001 in European ancestry individuals in the 1000 Genomes Project, Phase 3 release) were associated with whole-blood RNA expression of GSDMD and other immune genes at genome-wide significance (P<5×10−8) from the eQTLGen consortium (22).	1000 Genomes Project suggested: (1000 Genomes Project and AWS, RRID:SCR_008801)
   CrossMap (68) was used to convert genomic positions from hg38 (as reported in the COVID-19 GWAS) to hg19 using the UCSC liftover chain file to ensure both the exposure and outcome datasets were reported on the same genome assembly.	CrossMap suggested: (CrossMap, RRID:SCR_001173)
   Statistical Analysis: Statistical analysis was performed using GraphPad Prism V7.0.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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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 did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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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.
   • Thank you for including a protocol registration statement.

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   Version published to 10.1101/2021.03.06.21252796 on medRxiv

   Mar 8, 2021