Primary macrophages exhibit a modest inflammatory response early in SARS-CoV-2 infection

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   ScreenIT

   Feb 8, 2022

   SciScore for 10.1101/2022.02.02.478897: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	Monocytes derived from a 54-year-old, HBV-, HCV- and HIV-negative, African American male.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   African green monkey kidney cells (Vero E6; ATCC CRL-1586) were maintained in DMEM, 10% FCS, 1% non-essential amino acids (NEAA) and 1% penicillin-streptomycin (P/S)	Vero suggested: ATCC Cat# CRL-1586, RRID:CVCL_0574)
   Human epithelial colorectal adenocarcinoma cells (Caco-2; ATCC HTB-37) and human lung cancer cells (Calu-3; ATCC HTB-55) were maintained in DMEM, 20% FCS, 1% NEAA and 1% P/S.	Caco…

   SciScore for 10.1101/2022.02.02.478897: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	Monocytes derived from a 54-year-old, HBV-, HCV- and HIV-negative, African American male.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   African green monkey kidney cells (Vero E6; ATCC CRL-1586) were maintained in DMEM, 10% FCS, 1% non-essential amino acids (NEAA) and 1% penicillin-streptomycin (P/S)	Vero suggested: ATCC Cat# CRL-1586, RRID:CVCL_0574)
   Human epithelial colorectal adenocarcinoma cells (Caco-2; ATCC HTB-37) and human lung cancer cells (Calu-3; ATCC HTB-55) were maintained in DMEM, 20% FCS, 1% NEAA and 1% P/S.	Caco-2 suggested: None
   Briefly, plasmids, encoding internal virus segments from indicated viruses and PR8 HA and NA, were co-transfected into 293-T cells alongside pCAGGs vectors expressing the polymerase and NP proteins were then co-cultured with MDCK cells.	MDCK suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
   The inoculum was added to macrophages, Calu-3, Caco-2 or Vero E3 cells and incubated at 37 °C for 1h.	Calu-3 suggested: None Vero E3 suggested: None
   The inoculum was then removed and cells maintained as described above. 6, 24 or 72h post infection (hpi), the culture supernatants were collected and quantified by TCID50 assay on Vero E6 cells by the Spearman–Karber method [35] or qPCR for the SARS-CoV-2 Envelope gene (E).	Vero E6 suggested: RRID:CVCL_XD71)
   Recombinant DNA
   Sentences	Resources
   Briefly, plasmids, encoding internal virus segments from indicated viruses and PR8 HA and NA, were co-transfected into 293-T cells alongside pCAGGs vectors expressing the polymerase and NP proteins were then co-cultured with MDCK cells.	pCAGGs suggested: RRID:Addgene_127347)
   Software and Algorithms
   Sentences	Resources
   Immunospots were imaged with the Azure c600 Gel Imaging System (Azure Biosystems, USA) and data (Figure S1) were analysed as spot intensities using Image J (Laboratory for Optical and Computational Instrumentation, USA).	Image J suggested: (ImageJ, RRID:SCR_003070)
   All data analyses and preparation of graphs were carried out with GraphPad Prism version 8.01	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   1 (GraphPad Software, San Diego, CA).	GraphPad 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: We detected the following sentences addressing limitations in the study:

   We caveat that the cells we worked with here may reflect a more dominant M2 macrophage population and the observed phenotypes may differ in tissue-resident macrophages. Nevertheless, our findings are aligned with reports that macrophages are unlikely to drive the initial wave of pro-inflammatory cytokines upon SARS-CoV-2 infection [29, 31]. In addition, the substantial increase in induction of proinflammatory cytokines upon IFN-stimulation suggests that macrophages may participate in secondary or ensuing waves of inflammatory responses leading to ARDS in severe COVID-19 patients. Interestingly, we found a significant increase of the subgenomic E gene in the cell lysate of IFN-treated MDM but infection was still abortive as we found no new infectious particles in the supernatant (by TCID50 assay and qPCR for the E gene; Fig 2G). Like others [56], we found that pre-treatment of MDM with IFN-α resulted in induction of ACE2 mRNA (Fig 2F). ACE2 is the receptor that SARS-CoV-2 uses to infect epithelial cells of lung alveoli and also an ISG [57]. However, a previous report that the ACE2 isoform upregulated by interferon is non-functional for SARS-CoV-2 entry [57], so our results might be explained by other changes to the cells that render them more infectable by the virus.

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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 10.1101/2022.02.02.478897 on bioRxiv

   Feb 4, 2022