CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses

  1. Sallieu Jalloh
  2. Judith Olejnik
  3. Jacob Berrigan
  4. Annuurun Nisa
  5. Ellen L. Suder
  6. Hisashi Akiyama
  7. Maohua Lei
  8. Sita Ramaswamy
  9. Sanjay Tyagi
  10. Yuri Bushkin
  11. Elke Mühlberger
  12. Suryaram Gummuluru

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Abstract

Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169-mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and subgenomic RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral genomic and subgenomic RNA in CD169 + macrophages elicited a pro-inflammatory cytokine expression (TNFα, IL-6 and IL-1β) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response.

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  1. Version published to 10.1371/journal.ppat.1010479
  2. ScreenIT

    SciScore for 10.1101/2022.03.29.486190: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: This research has been determined to be exempt by the Institutional Review Board of the Boston University Medical Center since it does not meet the definition of human subjects research, since all human samples were collected in an anonymous fashion and no identifiable private information was collected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: All cell lines are routinely tested for mycoplasma contamination and confirmed negative.

    Table 2: Resources

    Antibodies
    SentencesResources
    For CD169 blocking experiments, primary MDMs from 3 different donors were pre-incubated with 20 μg/ml anti-CD169 antibody (HSn 7D2, Novus Biologicals) or IgG1k (P3.6.2.8.1, eBioscience) for 30 min at 4°C prior to infection.
    anti-CD169
    suggested: (Thermo Fisher Scientific Cat# MA1-16891, RRID:AB_568734)
    This is followed by secondary staining for 30 min at 4°C with APC-conjugated mouse anti-His antibody (BioLegend, #362605, 1:50) or isotype control.
    anti-His
    suggested: (BioLegend Cat# 362605, RRID:AB_2715818)
    The cells were incubated overnight at 4°C with a rabbit antibody directed against the SARS-CoV nucleocapsid protein (Rockland; 1:1000 dilution in 5% goat serum), which cross-reacts with the SARS-CoV-2 nucleocapsid protein, as previously described (99).
    SARS-CoV nucleocapsid protein
    suggested: (Creative Diagnostics Cat# DMAB8869, RRID:AB_2392503)
    SARS-CoV-2 nucleocapsid protein
    suggested: None
    The cells were washed four times in PBS and incubated with goat anti-rabbit antibody conjugated with AlexaFluor594 for 1 hour at room temperature (Invitrogen; 1:200 dilution in blocking reagent). 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was used at 200 ng/ml for nuclei staining.
    anti-rabbit
    suggested: None
    For dsRNA staining (61), anti-dsRNA (Pan-Enterovirus Reagent, clone 9D5, Light Diagnostics, Millipore) antibody was used 1:2 overnight and anti-mouse-AF488 (Invitrogen) 1:200 dilution as secondary antibody with DAPI.
    anti-dsRNA ( Pan-Enterovirus Reagent , clone 9D5 , Light Diagnostics , Millipore )
    suggested: None
    anti-dsRNA ( Pan-Enterovirus Reagent ,
    suggested: None
    anti-mouse-AF488
    suggested: None
    Flow cytometry: To examine cell surface expression of CD169 or ACE2 in transduced THP1 or primary MDMs, approximately 0.5x106 cells were harvested with CellStripper (Corning), stained with Zombie-NIR (BioLegend, #423105, 1:250) followed by staining for 30 min at 4°C with the following antibodies; Alexa647-conjugated mouse anti-CD169 antibody (BioLegend, #346006, 1:50), Alexa647-conjugated
    ACE2
    suggested: (Enzo Life Sciences Cat# BML-SA445, RRID:AB_2273641)
    mouse anti-ACE2 antibody (R&D systems, 1:200), or unconjugated goat anti-ACE2 polyclonal antibody (R&D systems, #AF933, 1:200) followed by Alexa488-conjugated chicken anti-goat antibody (Invitrogen, #A-21467, 1:100).
    anti-goat
    suggested: (Molecular Probes Cat# A-21467, RRID:AB_141893)
    Immunoblot Analysis: To assess expression of endogenous or transduced proteins, cell lysates containing 30- 40 µg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and the membranes were probed with the following antibodies: mouse anti- TMPRSS2 (Santa Cruz, #515727, 1:1000), mouse anti-Cathepsin-L (Santa Cruz, #32320, 1:1000), goat anti-ACE-2 (R&D systems, #AF933, 1;1000), rabbit anti-STING (Cell Signaling, #13647, 1:1000), rabbit anti-MAVS (Thermo Fisher, #PA5-17256, 1:1000), mouse anti-RIG-I (AdipoGen, #20B-0009, 1:1000)
    anti- TMPRSS2 ( Santa Cruz , #515727
    suggested: None
    anti-Cathepsin-L ( Santa Cruz , #32320
    suggested: None
    anti-ACE-2
    suggested: None
    anti-STING ( Cell Signaling , #13647
    suggested: (Cell Signaling Technology Cat# 13647, RRID:AB_2732796)
    anti-MAVS
    suggested: (Thermo Fisher Scientific Cat# PA5-17256, RRID:AB_10979584)
    anti-RIG-I ( AdipoGen , #20B-0009
    suggested: None
    Specific staining was visualized with secondary antibodies, goat anti-mouse-IgG-DyLight 680 (Thermo Scientific, #35518, 1:20000), goat anti-rabbit-IgG-DyLight 800 (Thermo Scientific, #SA5-35571, 1:20000), or a donkey anti-goat-IgG-IR-Dye 800 (Licor, #926-32214, 1:20000).
    anti-mouse-IgG-DyLight 680
    suggested: None
    anti-rabbit-IgG-DyLight
    suggested: None
    #SA5-35571
    suggested: (Thermo Fisher Scientific Cat# SA5-35571, RRID:AB_2556775)
    anti-goat-IgG-IR-Dye
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    THP1 cells (ATCC) were maintained in RPMI/1640 (Gibco) containing 10% FBS and 1% pen/strep (50)
    THP1
    suggested: None
    To generate HEK293T/ACE2+, THP1/ACE2+ and THP1/CD169+/ACE2+ cells, HEK293T, THP1 or THP1/CD169 cells were transduced with pLenti-ACE2-IRES-puro lentivector and cultured in puromycin-containing media (2 μg/ml).
    THP1/CD169
    suggested: None
    SARS-CoV-2 titer was determined in Vero E6 cells by tissue culture infectious dose 50 (TCID50) assay using the Spearman Kärber algorithm.
    Vero E6
    suggested: None
    Infection: For RNA analysis, 1x106 cells (THPI/PMA, MDMs, HEK293T) were seeded in 12-well plates.
    HEK293T
    suggested: None
    S binding: To evaluate SARS-CoV-2 S binding to various THP1 monocytes expressing different surface receptors, approximately 0.25x106 cells from parental THP1 or those expressing wt CD169, mutant CD169 (R116A), ACE2, or both wt CD169 and ACE2 were incubated for 30 min at 4 °C with 2 μg of spike glycoprotein (stabilized) from Wuhan-Hu-1 SARS- CoV-2 containing a C-terminal Histidine Tag, recombinant from HEK293F cells (BEI resources, #NR-52397).
    HEK293F
    suggested: None
    Recombinant DNA
    SentencesResources
    For ACE2 cloning, the NotI-XhoI fragment from pLenti-ACE2- IRES-puro was inserted into the LV-3’LTR backbone.
    pLenti-ACE2- IRES-puro
    suggested: None
    For cloning CD169 into LV-3’LTR vector, a BglII-AgeI fragment from LNC-CD169 was inserted into LV-3’LTR vector.
    LV-3’LTR
    suggested: None
    HIV-1 packaging plasmid psPAX2 and VSV-G expression constructs have been previously described (39).
    psPAX2
    suggested: RRID:Addgene_12260)
    VSV-G
    suggested: RRID:Addgene_138479)
    All lentiviral vectors (pLKO.1) expressing shRNAs used for knockdown of host proteins were purchased from Sigma
    pLKO.1
    suggested: RRID:Addgene_13425)
    Software and Algorithms
    SentencesResources
    Data analysis was performed using FlowJo software (FlowJo).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    63x oil immersion objective; numerical aperture 1.4) controlled by Metamorph image acquisition software (Molecular Devices, San Jose, CA)
    Metamorph
    suggested: None
    Statistics: All the statistical analysis was performed using GraphPad Prism 9.
    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 10.1101/2022.03.29.486190v2 on bioRxiv
  4. Version published to 10.1101/2022.03.29.486190v1 on bioRxiv