High expression of angiotensin-converting enzyme-2 (ACE2) on tissue macrophages that may be targeted by virus SARS-CoV-2 in COVID-19 patients

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   ScreenIT

   Jul 20, 2020

   SciScore for 10.1101/2020.07.18.210120: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: The final data were analyzed using the Kaluza Flow Cytometry Analysis Software (Beckman Coulter, Brea, CA, USA). 2.6. Immunohistochemistry: Female NOD/LtJ mice (aged 3 weeks) and NOD-scid mice (aged 5–6 weeks) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained under pathogen-free conditions, according to a protocol approved by the institutional Animal Care Committee (ACC).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	PBMC isolation: To determine the expression of ACE2 on different types of immune cells, human buffy coat blood units (N = 6; mean age of 42 ± 13.89; age range …

   SciScore for 10.1101/2020.07.18.210120: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: The final data were analyzed using the Kaluza Flow Cytometry Analysis Software (Beckman Coulter, Brea, CA, USA). 2.6. Immunohistochemistry: Female NOD/LtJ mice (aged 3 weeks) and NOD-scid mice (aged 5–6 weeks) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained under pathogen-free conditions, according to a protocol approved by the institutional Animal Care Committee (ACC).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	PBMC isolation: To determine the expression of ACE2 on different types of immune cells, human buffy coat blood units (N = 6; mean age of 42 ± 13.89; age range from 27 to 58 years old; 3 males and 3 females) were purchased from the New York Blood Center (New York, NY, USA).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The purified CD4+ T cells were seeded at 1 × 105 cells/well in the anti-CD3 monoclonal antibody (mAb) (10 μg/mL, BD Pharmingen, Franklin Lakes, NJ, USA) precoated 96-well tissue culture-treated plate, in the presence of soluble anti-CD28 mAb (1 μg/mL, BD Pharmingen, Franklin Lakes, NJ, USA), interleukin (IL)-6 (10 ng/mL, Biolegend, San Diego, CA, USA), IL-1β (10 ng/mL, Biolegend, San Diego, CA, USA), transforming growth factor (TGF)-β1 (1 ng/mL, Biolegend, San Diego, CA, USA), IL-23 (10 ng/mL, Biolegend, San Diego, CA, USA), penicillin-streptomycin (10 μg/mL, Sigma, Saint Louis, MO, USA), the neutralizing antibodies anti-IL-4 mAb (10 μg/mL, BD Pharmingen, Franklin Lakes, NJ, USA) and anti-IFN-γ mAb (10 μg/mL, BD Pharmingen, Franklin Lakes, NJ, USA), in X-VIVO 15 serum-free medium (200 μl per well), at 37 °C, 5% CO2 conditions.	anti-CD3 suggested: None anti-CD28 suggested: (Bio X Cell Cat# BE0015-1, RRID:AB_1107624) TGF)-β1 (1 ng/mL suggested: None IL-23 suggested: None anti-IL-4 suggested: None anti-IFN-γ suggested: None
   The antibodies FITC-conjugated anti-human Hsp60, PB-conjugated anti-human CD3, and APC-conjugated anti-IL17A mAbs were purchased from Biolegend (San Diego, CA, USA).	anti-human suggested: None anti-human Hsp60 suggested: None anti-human CD3 suggested: None anti-IL17A suggested: None
   The antibodies PE-Cy7-conjugated anti-human CD11c mAb, PE-Cy7-conjugated anti-human CD56 mAb, BV-421-conjugated anti-human CD127 mAb, PE-conjugated anti-RORγT, and BV 421-conjugated anti-RORγT mAbs were purchased from BD (Franklin Lakes, NJ, USA).	anti-RORγT suggested: None
   Tissue sections were initially immunostained with ACE2 rabbit polyclonal antibody (Abcam, Cambridge, MA, USA) at 1:100 dilution for 2 hours at room temperature.	ACE2 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   The final data were analyzed using the Kaluza Flow Cytometry Analysis Software (Beckman Coulter, Brea, CA, USA). 2.6. Immunohistochemistry: Female NOD/LtJ mice (aged 3 weeks) and NOD-scid mice (aged 5–6 weeks) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained under pathogen-free conditions, according to a protocol approved by the institutional Animal Care Committee (ACC).	NOD-scid suggested: None
   Tissue sections from NOD/LtJ mice (3 weeks old) were utilized for immunohistochemistry including lung tissue sections (n = 4), small intestine tissue sections (n = 4), liver tissue sections (n = 4), and kidney tissue sections (n = 4).	NOD/LtJ suggested: None
   Software and Algorithms
   Sentences	Resources
   Finally, the slides were covered by using mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and photographed with a Nikon A1R confocal microscope on a Nikon Eclipse Ti2 inverted base, using NIS Elements Version 4.60 software. 2.7. Statistics: Statistical analyses were performed with GraphPad Prism 8 (version 8.0.1) software.	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: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04299152	Not yet recruiting	Stem Cell Educator Therapy Treat the Viral Inflammation in C…

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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 page 32. 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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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   Version published to 10.1101/2020.07.18.210120v1 on bioRxiv

   Jul 20, 2020