Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

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   ScreenIT

   Nov 30, 2021

   SciScore for 10.1101/2021.11.23.469755: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: In brief, CD34+ HSPCs were purified from human umbilical cord blood (CB) acquired from healthy donors under informed consent from the Department of Gynecology and Obstetrics, Aarhus University Hospital,
   Sex as a biological variable	Individuals for whom there were no cryopreserved peripheral blood mononuclear cells (PBMCs) at baseline, who were pregnant, breastfeeding or had serum total bilirubin x3 above upper limit of normal were excluded from the study.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   (Purified anti-human CD304 (Neuropilin-1), clone 12C2, BioLegend Cat#354502) …

   SciScore for 10.1101/2021.11.23.469755: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: In brief, CD34+ HSPCs were purified from human umbilical cord blood (CB) acquired from healthy donors under informed consent from the Department of Gynecology and Obstetrics, Aarhus University Hospital,
   Sex as a biological variable	Individuals for whom there were no cryopreserved peripheral blood mononuclear cells (PBMCs) at baseline, who were pregnant, breastfeeding or had serum total bilirubin x3 above upper limit of normal were excluded from the study.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   (Purified anti-human CD304 (Neuropilin-1), clone 12C2, BioLegend Cat#354502) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) antibody for 15 minutes prior to stimulation with TLR7 (2.5 μg/mL R837) agonist for 4 hrs in 200 uL, after which the culture volume was topped up to 1 mL.	anti-human CD304 suggested: (BioLegend Cat# 354502, RRID:AB_2564475) Neuropilin-1 suggested: (BioLegend Cat# 354502, RRID:AB_2564475) TLR7 suggested: None
   To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.	anti-human IFNAR2 suggested: (PBL Assay Science Cat# 21385-1, RRID:AB_387828) anti-human IFN suggested: None IgG1 suggested: (BioLegend Cat# 400102, RRID:AB_2891079)
   As secondary antibodies, peroxidase-conjugated donkey-anti-rabbit and donkey-anti-mouse was used (Jackson Immuno Research 711-036-152 and 715-036-150).	and donkey-anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: Calu-3 epithelial lung cancer cells (kindly provided by Laureano de le Vega, Dundee University, Scotland, UK) and human lung adenocarcinoma epithelial A549 cells expressing hACE2 (kindly provided by Brad Rosenberg, Icahn School of Medicine at Mount Sinai, New York, USA) were grown as a monolayer in DMEM10 (Dulbecco’s minimal essential medium, DMEM, Life Technologies, supplemented with 10% (v/v) hiFCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin.	A549 suggested: None
   Virus was propagated using VeroE6 cells expressing human TMPRSS2 (41).	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.	Calu-3 suggested: None
   Limiting dilution assay: To determine the amount of infectious virus in cell culture supernatant or generated virus stocks, a limiting dilution assay was performed. 2×104 VeroE6-TMPRRS2 cells were seeded in 50 μL DMEM5 in a 96 well plate.	VeroE6-TMPRRS2 suggested: None
   Recombinant DNA
   Sentences	Resources
   Cell lines: Calu-3 epithelial lung cancer cells (kindly provided by Laureano de le Vega, Dundee University, Scotland, UK) and human lung adenocarcinoma epithelial A549 cells expressing hACE2 (kindly provided by Brad Rosenberg, Icahn School of Medicine at Mount Sinai, New York, USA) were grown as a monolayer in DMEM10 (Dulbecco’s minimal essential medium, DMEM, Life Technologies, supplemented with 10% (v/v) hiFCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin.	hACE2 suggested: RRID:Addgene_1786)
   Software and Algorithms
   Sentences	Resources
   Fluorescent intensity was measured with a BD LSR-Fortessa X-20, using BD FACSDiva	BD FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
   Software, gates were set using fluorescent minus one (FMO) controls and data were analyzed with Flow-Jo software.	Flow-Jo suggested: (FlowJo, RRID:SCR_008520)
   Protein levels were quantified using the Human DuoSet ELISAs for IL-6, IL-8, TNFα, CXCL10 (R&D Systems) or the Human IFN-a pan ELISA kit (Mabtech 3425-1M-6, detecting IFN-a subtypes 1/13, 2, 4, 5, 6, 7, 8, 19, 14, 16 and 17), according to the manufacturer’s instructions on a Synergy SynergyHTX multi-mode platereader (BioTek) using the Gen5 version 3.04 program.	Gen5 suggested: (Gen5, RRID:SCR_017317)
   The raw data were processed using the nSOLVER 4.0 software (NanoString Technologies) for Dhigh and Dlow separately to ensure proper normalization of each dataset.	nSOLVER suggested: None
   Data were plotted using Prism 8.2.0 (GraphPad, La Jolla, CA, USA) and R software version 3.5.1 with the following packages installed: ggplot2, circlize, dendextend, ComplexHeatmap and RColorBrewer.	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798) ggplot2 suggested: (ggplot2, RRID:SCR_014601) ComplexHeatmap suggested: (ComplexHeatmap, RRID:SCR_017270)
   Reactome pathway overrepresentation analysis: To assign pathways to the gene clusters identified in pDCs from Dhigh and Dlow 48 hrs after SARS-CoV-2 exposure using unsupervised hierarchical cluster analysis on the NanoString nCounter data, we utilized the Reactome Pathway Browser version 3.7, database release 75 (https://reactome.org/PathwayBrowser); a comprehensive web-based resource for curated human pathways.	Reactome Pathway Browser suggested: None
   Disease pathways were excluded from the analyses and we used UniProt as the source of entities (maximum pathway size was 400).	UniProt suggested: (UniProtKB, RRID:SCR_004426)
   To determine correlation between IFNα production by pDCs and time of exposure to SARS-CoV-2, as well as to compare gene expression changes in Dhigh and Dlow after 4 and 48 hrs after exposure to SARS-CoV-2, and determine correlation between pDC frequency and disease severity, simple linear regression analysis were performed using GraphPad Prism.	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 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: 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.
   • No protocol registration statement was detected.

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

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   Version published to 10.1101/2021.11.23.469755v1 on bioRxiv

   Nov 24, 2021