Macrophages only sense infectious SARS-CoV-2 when they express sufficient ACE2 to permit viral entry, where rapid cytokine responses then limit viral replication

  1. Larisa I Labzin
  2. Keng Yih Chew
  3. Kathrin Eschke
  4. Xiaohui Wang
  5. Tyron Esposito
  6. Claudia J Stocks
  7. James Rae
  8. Ralph Patrick
  9. Helen Mostafavi
  10. Brittany Hill
  11. Teodor E. Yordanov
  12. Caroline L Holley
  13. Stefan Emming
  14. Svenja Fritzlar
  15. Francesca L. Mordant
  16. Daniel P. Steinfort
  17. Kanta Subbarao
  18. Christian M. Nefzger
  19. Anne K Lagendijk
  20. Emma Gordon
  21. Robert Parton
  22. Kirsty R. Short
  23. Sarah L. Londrigan
  24. Kate Schroder

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Abstract

Macrophages are key cellular contributors to COVID-19 pathogenesis. Whether SARS-CoV-2 can enter macrophages, replicate and release new viral progeny remains controversial. Similarly, whether macrophages need to sense replicating virus to drive cytokine release is also unclear. Macrophages are heterogeneous cells poised to respond to their local microenvironment, and accordingly, the SARS-CoV-2 entry receptor ACE2 is only present on a subset of macrophages at sites of human infection. Here, we use in vitro approaches to investigate how SARS-CoV-2 interacts with ACE2-negative and ACE2-positive human macrophages and determine how these macrophage populations sense and respond to SARS-CoV-2. We show that SARS-CoV-2 does not replicate within ACE2-negative human macrophages and does not induce pro-inflammatory cytokine expression. By contrast, ACE2 expression in human macrophages permits SARS-CoV-2 entry, replication, and virion release. ACE2-expressing macrophages sense replicating virus to trigger pro-inflammatory and anti-viral programs that limit virus release. These combined findings resolve several controversies regarding macrophage-SARS-CoV-2 interactions and identify a signaling circuit by which macrophages sense SARS-CoV-2 cell entry and respond by restricting viral replication.

One sentence summary

Lack of macrophage ACE2 expression precludes SARS-CoV-2 entry and sensing, while ACE2-expressing macrophages sense intramacrophage SARS-CoV-2 replication to induce rapid anti-viral responses that limit new virion release.

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  1. Version published to 10.1101/2022.03.22.485248v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2022.03.22.485248: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Studies using primary human cells were approved by the University of Queensland Human Medical Research Ethics Committee.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    THP-1 cells (TIB-202; ATCC) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), 2 mM GlutaMAX (Life Technologies) and 50 U/ml penicillin–streptomycin (Life Technologies).
    THP-1
    suggested: None
    Calu-3 cells purchased from ATCC (HTB-55) were maintained in Minimal Essential Media (Invitrogen), containing 10% heat-inactivated foetal bovine serum (Cytiva), 50 U/ml penicillin and streptomycin (Life Technologies Australia), and were seeded at 300 000 cells per well in a 12-well plate 48 h prior to experiments.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    HEK-293T cells were transfected with the expression vectors according to the manufacturer’s protocol with PEI 2500 (BioScientific) and transduced target THP-1 cells were selected with puromycin (1 μg/mL) after 24 h and used for assays after 72 h.
    HEK-293T
    suggested: None
    Virus was grown on Vero E6 TMPRSS2 cells for 48 h in DMEM with 2% FBS, and cell debris was cleared by centrifugation at 500G for 5 minutes at room temperature.
    Vero E6 TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    For Calu3 cells, virus was added to cells to give a total volume of 500 μL of RPMI 1640 with 2% FBS (HMDM and THP-1) or MEM with 2% FBS (Calu3) per well.
    Calu3
    suggested: None
    For studies involving SARS-CoV-2 infection of BAL macrophages, the SARS-CoV-2 isolate hCoV-19/Australia/VIC01/2020 (kindly provided by the Victorian Infectious Diseases Reference Laboratory) was grown in Vero cells for 72 h in serum-free MEM with 1 μg/ml TPCK trypsin.
    Vero
    suggested: None
    Recombinant DNA
    SentencesResources
    Lentiviral transduction: A lentiviral construct containing human ACE2 (Addgene 155295), or mScarlet (Addgene 85044) was cloned into pLV-CMV-MCS-IRES-Puro-Sin (48) and packaged into lentivirus in HEK-293T cells by means of third generation lentiviral packaging plasmids (
    pLV-CMV-MCS-IRES-Puro-Sin
    suggested: None
    The Mpro expression vector was generated by cloning the Mpro PCR product into a modified pEF6 plasmid, with an HA-tag N-terminal of the multiple cloning site, by standard restriction digest cloning techniques.
    pEF6
    suggested: RRID:Addgene_70765)
    Software and Algorithms
    SentencesResources
    Statistical Analysis: Statistics were calculated using GraphPad Prism using tests indicated in figure legends.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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).

    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.

    About SciScore

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  3. Version published to 10.1101/2022.03.22.485248v1 on bioRxiv