RAGE engagement by SARS-CoV-2 enables monocyte infection and underlies COVID-19 severity

Version published to 10.1016/j.xcrm.2023.101266

Nov 1, 2023

SciScore for 10.1101/2022.05.22.492693: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

Antibodies
Sentences	Resources
After blocking with 3% albumin (Sigma-Aldrich) and primary antibody incubation RAGE (ab3611, Abcam), ACE2 (XXX), ADAM17 (ab2051, Abcam)), TMPRSS2 (ab109131, Abcam), the membranes were incubated with an anti-rabbit peroxidase-conjugated secondary antibody (GE healthcare).	ACE2 suggested: None ADAM17 suggested: (Abcam Cat# ab2051, RRID:AB_302796) TMPRSS2 suggested: (Abcam Cat# ab109131, RRID:AB_10863728) anti-rabbit peroxidase-conjugated secondary suggested: None
500 μg of protein lysate was incubated with Anti-6X His tag® antibody [HIS.H8] (ab18184, Abcam) overnight at 4°C, anti-Mouse IgG (Invitrogen) was used …

SciScore for 10.1101/2022.05.22.492693: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

Antibodies
Sentences	Resources
After blocking with 3% albumin (Sigma-Aldrich) and primary antibody incubation RAGE (ab3611, Abcam), ACE2 (XXX), ADAM17 (ab2051, Abcam)), TMPRSS2 (ab109131, Abcam), the membranes were incubated with an anti-rabbit peroxidase-conjugated secondary antibody (GE healthcare).	ACE2 suggested: None ADAM17 suggested: (Abcam Cat# ab2051, RRID:AB_302796) TMPRSS2 suggested: (Abcam Cat# ab109131, RRID:AB_10863728) anti-rabbit peroxidase-conjugated secondary suggested: None
500 μg of protein lysate was incubated with Anti-6X His tag® antibody [HIS.H8] (ab18184, Abcam) overnight at 4°C, anti-Mouse IgG (Invitrogen) was used as isotype control.	Anti-6X suggested: (Abcam Cat# ab18184, RRID:AB_444306) anti-Mouse IgG suggested: None
P4417-100TAB-Sigma-Aldrich) plus 1% Bovine Serum Albumin (BSA) (Cat.A9647-500G-Sigma-Aldrich) and 0,02% NP-40 alternative (Cat.492016-100ML) for 1h at room temperature prior to overnight incubation at 4°C with primary antibody 1:100 (6xHisTag clone#HIS.H8 Cat.ab18184-Abcam or SARS-CoV-2 spike polyclonal antibody, GeneTex).	6xHisTag suggested: None
Cells were collected and stained using primary RAGE antibody 1:100 (PA5-24787, Thermo Scientific) for FACS analysis.	RAGE suggested: (Thermo Fisher Scientific Cat# PA5-24787, RRID:AB_2542287) PA5-24787 suggested: (Thermo Fisher Scientific Cat# PA5-24787, RRID:AB_2542287)
Experimental Models: Cell Lines
Sentences	Resources
1 × 105 THP-1 cells were seeded on a 24-well plate in their culture medium.	THP-1 suggested: None
THP1 and Monocytes infection with SARS-CoV-2: THP1 cells were plated at 5×105 cell/ml in 48-well plates in 200 μl of RPMI-1640 supplemented with 1% fetal bovine serum (FBS) (Euroclone).	THP1 suggested: None
Cell culture supernatants were collected 24, 48, 72 and 144 h post-infection and stored at – 80°C until the determination of the viral titers by a plaque-forming assay in Vero cells.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
The following day, cells were pretreated or not with 2μM Azeliragon (Cat.S6415-Selleckchem) for 30 minutes before adding 100 ng/mL of Sars-CoV-2 spike protein (RBD, HisTag) (Cat. ZO3483-1-GenScript) or infected using Heat-inactivated SARS-CoV-2 (VR-1986HK, ATCC) at 4 TCID50/mL for 2h at 37°C 5%CO2.	VR-1986HK suggested: None
Software and Algorithms
Sentences	Resources
Sequencing: Different library types were pooled at different ratios based on their targeted reads per cell and the nanomolarity of the library pools was confirmed using the Agilent Bioanalyzer 2100.	Agilent Bioanalyzer suggested: None
Separately for the two selected categories of disease severity (mild vs severe/critical), the pseudo-bulk counts were then fitted with a generalised linear model using the EdgeR package, to identify those genes characterised by a well-defined decreasing or increasing trend of the expression over the sample time-points.	EdgeR suggested: (edgeR, RRID:SCR_012802)
Among these sets, the GO:0050786 genelist was then expanded using the Cytoscape ‘stringApp’ (81) in order to identify among the nearest neighbours with confidence score > 0.7 the ones showing the highest absolute FC values in Myeloid cells.	Cytoscape suggested: (Cytoscape, RRID:SCR_003032)
The GSEA has been done with the clusterProfiler library (82, 83), using gene lists ranked by the FDR of the differential analysis and the sign of the logFC.	GSEA suggested: (SeqGSEA, RRID:SCR_005724)
The gating strategy and the relative analysis were performed with FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)

Results from OddPub: Thank you for sharing your code and data.

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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RAGE engagement by SARS-CoV-2 enables monocyte infection and underlies COVID-19 severity

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