Neuropilin-1 Mediates SARS-CoV-2 Infection in Bone Marrow-derived Macrophages
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Abstract
SARS-CoV-2 infection in human can cause medical complications across various tissues and organs. Despite of the advances to understanding the pathogenesis of SARS-CoV-2, its tissue tropism and interactions with host cells have not been fully understood. Existing clinical data have suggested possible SARS-CoV-2 infection in human skeleton system. In the present study, we found that authentic SARS-CoV-2 could efficiently infect human and mouse bone marrow-derived macrophages (BMMs) and alter the expression of macrophage chemotaxis and osteoclast-related genes. Importantly, in a mouse SARS-CoV-2 infection model that was enabled by the intranasal adenoviral (AdV) delivery of human angiotensin converting enzyme 2 (hACE2), SARS-CoV-2 was found to be present in femoral BMMs as determined by in situ immunofluorescence analysis. Using single-cell RNA sequencing (scRNA-Seq), we characterized SARS-CoV-2 infection in BMMs. Importantly, SARS-CoV-2 entry on BMMs appeared to be dependent on the expression of neuropilin-1 (NRP1) rather than the widely recognized receptor ACE2. It was also noted that unlike brain macrophages which displayed aging-dependent NRP1 expression, BMMs from neonatal and aged mice had constant NRP1 expression, making BMMs constantly vulnerable target cells for SARS-CoV-2. Furthermore, it was found that the abolished SARS-CoV-2 entry in BMM-derived osteoclasts was associated with the loss of NRP1 expression during BMM-to-osteoclast differentiation. Collectively, our study has suggested that NRP1 can mediate SARS-CoV-2 infection in BMMs, which precautions the potential impact of SARS-CoV-2 infection on human skeleton system.
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SciScore for 10.1101/2021.04.14.439793: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Cell culture: All experiments including human and animal tissues were approved by the ethics committee of the Shanghai Jiao Tong University Affiliated Shanghai Sixth People’s Hospital.
IACUC: All protocols were approved by the Institutional Animal Care and Use Committees of Guangzhou Medical University.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: All cells were confirmed by PCR to be free of mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources Cells were then incubated with a rabbit anti-SARS-CoV nucleocapsid protein polyclonal … SciScore for 10.1101/2021.04.14.439793: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Cell culture: All experiments including human and animal tissues were approved by the ethics committee of the Shanghai Jiao Tong University Affiliated Shanghai Sixth People’s Hospital.
IACUC: All protocols were approved by the Institutional Animal Care and Use Committees of Guangzhou Medical University.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: All cells were confirmed by PCR to be free of mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources Cells were then incubated with a rabbit anti-SARS-CoV nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labelled goat anti-rabbit secondary antibody (Cat. No.: 111-035-144, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA). anti-SARS-CoV nucleocapsid proteinsuggested: (Creative Diagnostics Cat# DMAB8869, RRID:AB_2392503)anti-rabbitsuggested: (Proteintech Cat# SA00006-2, RRID:AB_2651036)After permeabilizing in 0.1% Triton X-100 in PBS for 5 min, cells were incubated with 3% BSA-PBS for 30 min to block nonspecific antibody binding and then incubated with SARS-CoV-2 nucleocapsid protein antibody (Sino Biological), followed by incubating with Alexa Fluor 488 (Thermo). SARS-CoV-2 nucleocapsid proteinsuggested: (Bioss Cat# bsm-41414M, RRID:AB_2848129)BMMs were labelled by F4/80 antibody (Abcam), followed by incubating with Cy3-labeled Goat Anti-Rat IgG(H+L) (Beyotime). Anti-Rat IgG(H+Lsuggested: NoneMembranes were incubated with primary antibodies, including actin JLA20 antibody (Developmental Studies Hybridoma Bank), actinsuggested: NoneExperimental Models: Cell Lines Sentences Resources Vero E6 cells (derived from African Green monkey kidney) were grown in (DMEM, Thermo) supplemented with 10% FBS (Thermo) and 1% P/S (Thermo). Vero E6suggested: NoneProduction and infection of SARS-CoV-2 pseudovirus: HEK293T cells were transfected with psPAX, plentiv2-Tdtomato and plasmid that carried SARS-Cov-2 S gene or empty vector using Lipofectamine 3000 (Thermo)34 HEK293Tsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources SARS-CoV-2 infection in Balb/c mouse: Ad5-hACE2 transduced Balb/c mouse model were described previously25. Balb/csuggested: NoneSoftware and Algorithms Sentences Resources Fiji (National Institutes of Health) was employed to analyze and assemble images. Fijisuggested: (Fiji, RRID:SCR_002285)Then the feature-barcode matrices were obtained by aligning reads to the mouse genome (GRCm38 Ensemble: version 92) using CellRanger v3.1.0 CellRangersuggested: (SCIGA, RRID:SCR_021002)Statistical analysis: The data were graphed and statistically analysed using GraphPad Prism 8.0. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: Thank you for sharing your 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.
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