Macrophages govern antiviral responses in human lung tissues protected from SARS-CoV-2 infection

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   ScreenIT

   Jul 21, 2021

   SciScore for 10.1101/2021.07.17.452554: (What is this?)

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

   Table 1: Rigor

   Ethics

   IACUC: Animal work: Institutional approvals: All animal experiments described in this study were performed in accordance with protocols (number 1930) that were reviewed and approved by the Institutional Animal Care and Use and Committee of Princeton University (#1930) and Boston University (PROTO202000020). Euthanasia Agents: Briefly, mice were anesthetized using isoflurane (2.5%), placed into a body conforming animal mold (BCAM) (InVivo Analytics) and imaged within 5 min of injection.

   Sex as a biological variable

   Heterozygous K18-hACE2 C57BL/6J mice of both sexes (strain 034860, 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from The Jackson Laboratory and maintained at the NEIDL at Boston …

   SciScore for 10.1101/2021.07.17.452554: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Animal work: Institutional approvals: All animal experiments described in this study were performed in accordance with protocols (number 1930) that were reviewed and approved by the Institutional Animal Care and Use and Committee of Princeton University (#1930) and Boston University (PROTO202000020). Euthanasia Agents: Briefly, mice were anesthetized using isoflurane (2.5%), placed into a body conforming animal mold (BCAM) (InVivo Analytics) and imaged within 5 min of injection.
   Sex as a biological variable	Heterozygous K18-hACE2 C57BL/6J mice of both sexes (strain 034860, 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from The Jackson Laboratory and maintained at the NEIDL at Boston University.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells and antibodies: VeroE6 cells and AAV-293 cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (Bio-Techne, R&D systems, Minneapolis, MN, USA) and 1% (v/v) Penicillin Streptomycin (Thermo Scientific, Waltham, MA, USA).	Penicillin Streptomycin suggested: None
   The following anti-mouse antibodies were used for flow cytometry: from BioLegend (San Diego, CA, USA): CD45-PE-Cy7 clone 30-F11, CD45-PE-Dazzle5 clone 30-F11.	CD45-PE-Dazzle5 suggested: None
   The following anti-human antibodies were used for flow cytometry: from BD Biosciences (San Jose, CA, USA): CD45-V500 clone HI30, CD34-FITC clone 581, CD8-FITC clone G42-8, CD11c-allophycocyanin clone B-ly6; from BioLegend: CD56-allophycocyanin-Cy7 clone HCD56, CD33-PerCP-Cy5.5 clone WM53, CD11c-Alexa Fluor 700 clone 3.9, CD123-eFluor450 clone 6H6, CD14-PE-eFluor610 clone 61D3, CD3-FITC clone SK7, CD19-allophycocyanin clone HIB19, HLA-DR-BV510 clone L243, CD3-BV605 clone UCHT1, CD20-BV650 clone 2H7, CD16-BV117 clone B73.1, CD45-BV785 clone H130, CD8-FITC clone RPA-T8, CD33-PE clone WM53, CD14-PerCP-Cy5.5 clone HCD14, CD45RA-PE-Cy7 clone HI100, CD56-allophycocyanin clone QA17A16, CD4-Alexa Fluor 700 clone SK3; from Thermo Scientific: CD14-Alexa 700 clone Tuk4, CD3-PE-Cy5.5 clone 7D6, CD4-PE clone RPA-T4, CD19-PacBlue clone SJ25C1, CD16-PE-TexasRed clone 3G8.	anti-human suggested: (SouthernBiotech Cat# 9590-09, RRID:AB_2796984) CD34-FITC suggested: None CD8-FITC suggested: None CD11c-allophycocyanin suggested: None CD56-allophycocyanin-Cy7 suggested: None CD33-PerCP-Cy5.5 suggested: None CD123-eFluor450 suggested: None CD14-PE-eFluor610 suggested: None CD3-FITC suggested: None CD19-allophycocyanin suggested: None HLA-DR-BV510 suggested: None CD3-BV605 suggested: None CD20-BV650 suggested: None CD16-BV117 suggested: None CD45-BV785 suggested: None CD33-PE suggested: (SouthernBiotech Cat# 9590-09, RRID:AB_2796984) CD14-PerCP-Cy5.5 suggested: None CD45RA-PE-Cy7 suggested: None CD56-allophycocyanin suggested: None CD3-PE-Cy5.5 suggested: None CD4-PE suggested: None CD19-PacBlue suggested: None
   The following primary antibodies were used for immunochemistry: anti-human CD31 clone JC/70A (Biocare Medical, Pacheco, CA, USA), anti-mouse CD31 clone D8V9E (Cell Signaling Technology, Danvers, MA, USA), ACE2 clone EPR34435 (Abcam, Waltham, MA, USA), polyclonal SFTPC (Seven Hills Bioreagents, Cincinnati, OH, USA), anti-human CD68 clone KP1 (LS Bio, Seattle, WA, USA), anti-human CD61 clone ARC0460 (Thermo Scientific), anti-human CD4 clone SP35 and anti-human CD8 clone SP57 (Roche, Basel, Switzerland), and anti-human CD20 clone L26 Dako Omnis (Agilent, Santa Clara, CA, USA).	anti-human CD31 suggested: None anti-mouse CD31 suggested: None ACE2 suggested: None anti-human CD68 suggested: None anti-human CD61 suggested: None anti-human CD4 suggested: None anti-human CD8 suggested: None anti-human CD20 suggested: None
   The secondary antibody used in this study included HRP Goat anti-Rabbit IgG (H&L) (Vector Laboratories, Burligame, CA, USA).	anti-Rabbit IgG suggested: None
   The following anti-SARS-CoV-2 antibodies were used for immunohistochemistry: rabbit polyclonal anti-SARS-CoV Nucleoprotein (Novus Biological, Littleton, CO, USA), mouse monoclonal anti-SARS-CoV-2 Spike clone 2B3E5 (This antibody was used in this study as clone E7U60, which was the pre-production clone ID of clone 2B3E5; Cell Signaling Technology).	anti-SARS-CoV-2 suggested: None anti-SARS-CoV Nucleoprotein suggested: None
   Purification of human CD34+ cells were assessed by flow cytometry using an anti-human CD34-FITC antibody (clone 581, BD Biosciences).	anti-human CD34-FITC suggested: None
   Flow cytometry fluorophore compensation for antibodies was performed using an AbC™ Anti-Mouse Bead Kit (ThermoFisher Scientific).	Anti-Mouse suggested: None
   Quantification of peripheral human chimerism in HNFL mice: 2-4×106 PBMCs of human or murine origin were isolated as described above and stained for 1 h at 4°C in the dark with an antibody cocktail targeting human(h)CD45, mouse CD45, hCD3, hCD4, hCD8, hCD16, hCD19, hCD11c, hCD56 and hCD14.	CD45 suggested: None hCD16 suggested: None hCD14 suggested: None
   Antibody staining and flow cytometry analysis of NRG-L splenocytes and fLX: 2-4×106 splenocytes or fetal lung cells of human or murine origins were isolated as described above and stained for 1 h at 4°C in the dark with an antibody cocktail targeting mouse CD45, human(h)CD45, hCD19, hCD3, hCD33, hCD11c, hCD56, hCD68, hCD123, hCD14.	hCD68 suggested: None hCD123 suggested: None
   After blocking, 25 µL of antibody cocktail targeting hCD3, hCD20, hCD16, hHLA-DR, hCD45, hCD8, hCD4, hCD33, hCD45RA, hCD56, hCD14, mCD45, and containing a LIVE/DEAD viability dye (ThermoFisher Scientific) was added to each sample and incubated in the dark for 30 min at 4°C.	hCD20 suggested: None hHLA-DR, hCD45, hCD8, hCD4, hCD33, hCD45RA suggested: None hCD56 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Our P2 working stock of the virus was prepared by infecting Vero E6 cells with the P1 stock, at a multiplicity of infection (MOI) of 0.1.	Vero E6 suggested: RRID:CVCL_XD71)
   AAV-293 cells (Agilent) at 50% confluency in 15 cm dishes were transfected via the calcium phosphate method with 22.5 µg XR8 (NGVB, Indianapolis, IN), 7.5 µg pHelper (Agilent), 7.5 µg of pAB269-TBG-FLT3 LG-BGH per plate.	AAV-293 suggested: NCBI_Iran Cat# C548, RRID:CVCL_6871)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mouse models and housing: NOD Rag1-/- IL2Rgnull mice (NOD.Cg-Rag1tm1MomIl2rgtm1Wjl/SzJ, were obtained from the Jackson Laboratory, catalog number 007799).	NOD Rag1-/- IL2Rgnull suggested: RRID:BCBC_1261) NOD.Cg-Rag1tm1MomIl2rgtm1Wjl/SzJ suggested: None
   NRG-Flk2-/- (NRGF) mice (NOD.Cg-Rag1tm1Mom Flt3tm1Irl Il2rgtm1Wjl/J) were generated as described previously (Douam et al., 2018) and are available at The Jackson Laboratory (Bar Harbor, ME, USA) (catalog number 033127).	NRG-Flk2-/- suggested: None NOD.Cg-Rag1tm1Mom Flt3tm1Irl Il2rgtm1Wjl/J suggested: RRID:IMSR_JAX:033127)
   Heterozygous K18-hACE2 C57BL/6J mice of both sexes (strain 034860, 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from The Jackson Laboratory and maintained at the NEIDL at Boston University.	C57BL/6J suggested: RRID:MGI:3589388)
   Recombinant DNA
   Sentences	Resources
   Generation of AAV-Flt3LG: The pAB269 AAV backbone containing AAV2 ITRs was kindly provided by Markus Grompe (OHSU, Oregon, USA).	pAB269 suggested: None
   The FLT3 was PCR amplified from pAL119-FLT3L (Addgene, item #21910), and the TBG was amplified from pX602-AAV-TBG:NLS-SaCas9-NLS-HA-OLLAS-bGHpA;U6::BsaI-sgRNA (Addgene, item #61593).	pAL119-FLT3L suggested: RRID:Addgene_21910)
   Software and Algorithms
   Sentences	Resources
   Flow cytometry data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   An unstained fLX section was used to create an autofluorescence signature that was subsequently removed from multispectral images using InForm software version 2.4.8 (Akoya Biosciences).	InForm suggested: (inForm, RRID:SCR_019155)
   Each sample, comprising 100 μg peptides, was TMT-labeled with TMTPro 16plex reagents (ThermoFisher Scientific: # A44520) as per the manufacturer’s protocol.	ThermoFisher Scientific suggested: None
   The MaxQuant output files designated ‘‘Phospho(STY)sites’’ and ‘‘proteinGroups’’ were filtered to remove entries that were either entirely mouse, or in the case of completely homologous peptides, had annotations of both mouse and human.	MaxQuant suggested: (MaxQuant, RRID:SCR_014485)
   Raw FASTQ files were quality-checked with FastQC v0.11.7.	FastQC suggested: (FastQC, RRID:SCR_014583)
   Reads were found to be excellent quality and were aligned to the combined human (GRCh38, Ensembl 101) and mouse (GRCm38, Ensembl 101) genomes with STAR v2.7.1a followed by quantification with featureCounts v1.6.2.	STAR suggested: (STAR, RRID:SCR_004463) featureCounts suggested: (featureCounts, RRID:SCR_012919)
   Quality was checked with MultiQC v1.6.	MultiQC suggested: (MultiQC, RRID:SCR_014982)
   DESeq2 result were imported into Ingenuity Pathway Analysis (IPA; Service curated by Qiagen; Access provided through the Boston University Genome Science Institute) (Kramer et al., 2014), and a canonical pathway enrichment analysis was performed using the default settings and the same differential expression thresholds as before (shrunken log2 fold change > 2 or < -2 and FDR-adjusted p-value < 0.01).	DESeq2 suggested: (DESeq, RRID:SCR_000154) Ingenuity Pathway Analysis suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
   All statistical tests and graphical depictions of results were performed using GraphPad Prism version 9.0.1 software (GraphPad Software, La Jolla, CA).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Data deposition: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner repository with the dataset identifier PXD025851.	PRIDE suggested: (Pride-asap, RRID:SCR_012052)
   Transcriptomic data generated during this study are available through the National Center for Biotechnology Information Gene Expression Omnibus (GEO) under series accession no. GSE180063.	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   To enhance our understanding of the cellular and molecular mechanisms driving protective immune responses in the respiratory tract of asymptomatic and mild COVID-19 patients, investigations need to go beyond human patient studies due to their inherent limitations. Immunodeficient mice engrafted with fLX have been previously reported to be highly permissive to coronavirus infection (Wahl et al., 2021). Such in vivo platforms carry substantial advantages over other in vivo models (including humans) in a way that enables investigations into coronavirus infection within a human lung environment over time and in controlled experimental settings. However, given our common understanding that the quality of immune responses during SARS-CoV-2 infection defines disease outcome, the lack of significant human hematopoietic reconstitution in these models has precluded their use for investigating human immune responses against coronavirus infection. Here, we report that NRG-L mice singly engrafted with fLX are highly susceptible to SARS-CoV-2 infection. Inoculated fLX were prone to extensive inflammation, which associated with severe histopathological manifestations of disease. In sharp contrast, co-engraftment of fLX and human HSC in HNFL mice resulted in protection against SARS-CoV-2 infection, as well as limited inflammation and histopathology. Protection associated with the induction of a superior ISG response in HNFL mice as compared to NRG-L mice, which was dominated by the upregulat...

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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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 68, 70, 71, 72 and 73. 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.
   • 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 10.1101/2021.07.17.452554 on bioRxiv

   Jul 19, 2021