Severe COVID-19 patients have impaired plasmacytoid dendritic cell-mediated control of SARS-CoV-2-infected cells

  1. Manon Venet
  2. Margarida Sa Ribeiro
  3. Elodie Décembre
  4. Alicia Bellomo
  5. Garima Joshi
  6. Marine Villard
  7. David Cluet
  8. Magali Perret
  9. Rémi Pescamona
  10. Helena Paidassi
  11. Thierry Walzer
  12. Omran Allatif
  13. Alexandre Belot
  14. Sophie Assant
  15. Emiliano Ricci
  16. Marlène Dreux

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Abstract

Type I and III interferons (IFN-I/λ) are key antiviral mediators against SARS-CoV-2 infection. Here, we demonstrate that plasmacytoid dendritic cells (pDCs) are the predominant IFN-I/λ source following their sensing of SARS-CoV-2-infected cells. Mechanistically, this short-range sensing by pDCs requires sustained integrin-mediated cell adhesion with infected cells. In turn, pDCs restrict viral spread by an IFN-I/λ response directed toward SARS-CoV-2-infected cells. This specialized function enables pDCs to efficiently turn-off viral replication, likely via a local response at the contact site with infected cells. By exploring the pDC response in SARS-CoV-2 patients, we further demonstrate that pDC responsiveness inversely correlates with the severity of the disease. The pDC response is particularly impaired in severe COVID-19 patients. Overall, we propose that pDC activation is essential to control SARS-CoV-2-infection. Failure to unfold this response could be key to understand severe cases of COVID-19.

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  1. Version published to 10.1101/2021.09.01.21262969v2 on medRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.09.01.21262969: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Other reagents included LPS, TLR3 agonist (Poly(I:C); LMW) and TLR7 agonist (R848 and Imiquimod) (Invivogen); TLR7 antagonist (IRS661, 5’-TGCTT GCAAGCTTGCAAGCA-3’ synthesized on a phosphorothionate backbone; MWG Biotech); mouse ant αL integrin (clone 38; Antibodies Online); mouse anti-ICAM-1 (Clone LB-2 ; BD Bioscience): Arp2/3 complex inhibitor I (CK-666 ; Merck Millipore) ; Fc Blocking solution (MACS Miltenyi Biotec); Golgi-Plug, cytoperm/cytofix and permeabilization-wash solutions (BD Bioscience); IFNα and IFNλ1/2/3 ELISA kit (PBL Interferon Source); IL6 and TNFα ELISA kit (Affymetrix, eBioscience); 96-well format transwell chambers (Corning); IL6 and IFNλ by U-PLEX Custom Human Cytokine assay (Meso Scale Diagnostics, Rockville, MD) ; 96-Well Optical-Bottom Plates (Thermo Fisher Scientific); cell-labeling solution using CellTraceTM Violet Cell Proliferation Kit (Life Technologies ref # C34557, C34571), Live/Dead Fixable Dead Cell Stain Near-IR (Life Technologies ref #10119); Fixable Viability Dye eFluor 450 (Life Technologies); Zombie Aqua and Zombie Green Fixable Viability Kits (Biolegend); FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend); cDNA synthesis and qPCR kit (Life Technologies) ; poly-L-lysin (P6282, Sigma-Aldrich).
    TLR7 agonist (R848
    suggested: None
    anti-ICAM-1
    suggested: None
    TNFα
    suggested: None
    poly-L-lysin (P6282
    suggested: None
    After surface staining and fixation with cytoperm/cytofix solution (BD Bioscience) for 20 minutes at 4°C, IFNα, IL6, IFNλ 1, and TNFα were stained by a 45-minute incubation at 4°C with antibodies diluted in permeabilization buffer (BD Bioscience ; antibodies are listed in the Key resource table).
    IL6
    suggested: None
    When indicated, the cocultures were treated with an anti-αL integrin blocking antibody at 10mg/mL.
    anti-αL integrin blocking
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell Lines and Primary Cell Cultures: SARS-CoV-2-infected cells included the human alveolar basal epithelial cell lines, Calu-3 cells (ATCC HTB-55), A549 cells (ATCC CCL-185) and NCI-H358 cells (ATCC CRL-5807), Huh7.5.1 cells (Dreux et al., 2012) and HEK-293 cells (ATCC CRL-1573).
    A549
    suggested: None
    HEK-293
    suggested: ATCC Cat# CRL-1573, RRID:CVCL_0045)
    The A549 cells, NCI-H358 cells and HEK-293 cells were transduced to stably express the human angiotensin converting enzyme 2 (ACE2; accession number: NM_021804) using a lentiviral vector, as previously described (Dreux et al., 2012; Rebendenne et al., 2021).
    NCI-H358
    suggested: None
    The cells were infected at MOI 0.01, 0.1, 0.02 and 0.5 for, respectively, NCI- H358-ACE2, Huh7.5.1, A549-ACE2 and Calu-3 cells for 48 hours maximum prior to collection of the cells and their SNs for coculture.
    Huh7.5.1
    suggested: RRID:CVCL_E049)
    Calu-3
    suggested: None
    Briefly, 10-fold serial dilutions of icSARS-CoV-2-mNG-containing supernatants were added to 2x10e4 Vero cells seeded in 96-well plates and fixed 24 hours post-infection.
    Vero
    suggested: None
    Live imaging of coculture with spinning-disc confocal microscopy analysis: A549-ACE2 cells were infected with icSARS-CoV-2-mNG for 48 hours prior to coculture with pDCs.
    A549-ACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    PBMCs were freshly isolated by Ficoll-Hypaque density centrifugation followed by washing in pDC/PMBC culture medium (i.e., RPMI 1640 Medium supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, non-essential amino acids, 1 mM sodium pyruvate and 10 mM Hepes).
    pDC/PMBC
    suggested: None
    Software and Algorithms
    SentencesResources
    Other reagents included LPS, TLR3 agonist (Poly(I:C); LMW) and TLR7 agonist (R848 and Imiquimod) (Invivogen); TLR7 antagonist (IRS661, 5’-TGCTT GCAAGCTTGCAAGCA-3’ synthesized on a phosphorothionate backbone; MWG Biotech); mouse ant αL integrin (clone 38; Antibodies Online); mouse anti-ICAM-1 (Clone LB-2 ; BD Bioscience): Arp2/3 complex inhibitor I (CK-666 ; Merck Millipore) ; Fc Blocking solution (MACS Miltenyi Biotec); Golgi-Plug, cytoperm/cytofix and permeabilization-wash solutions (BD Bioscience); IFNα and IFNλ1/2/3 ELISA kit (PBL Interferon Source); IL6 and TNFα ELISA kit (Affymetrix, eBioscience); 96-well format transwell chambers (Corning); IL6 and IFNλ by U-PLEX Custom Human Cytokine assay (Meso Scale Diagnostics, Rockville, MD) ; 96-Well Optical-Bottom Plates (Thermo Fisher Scientific); cell-labeling solution using CellTraceTM Violet Cell Proliferation Kit (Life Technologies ref # C34557, C34571), Live/Dead Fixable Dead Cell Stain Near-IR (Life Technologies ref #10119); Fixable Viability Dye eFluor 450 (Life Technologies); Zombie Aqua and Zombie Green Fixable Viability Kits (Biolegend); FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend); cDNA synthesis and qPCR kit (Life Technologies) ; poly-L-lysin (P6282, Sigma-Aldrich).
    BD Bioscience)
    suggested: None
    Thermo Fisher Scientific)
    suggested: None
    Data were processed with nSolver software (NanoString Technologies, Seattle, WA), which included assessment of the quality of the runs, and combined, normalized, and analyzed in nSolver and Excel.
    Excel
    suggested: None
    Flow cytometric analysis was performed using a BD LSR Fortessa 4L and the data were analyzed using Flow Jo software (Tree Star).
    Flow Jo
    suggested: (FlowJo, RRID:SCR_008520)
    The quantification of mNeonGreen fluorescence intensity of infected cells and the duration of contacts between pDCs and infected cells were performed using Image J software package (http://rsb.info.nih.gov/ij).
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    The set of Figures was prepared using PRISM software.
    PRISM
    suggested: (PRISM, RRID:SCR_005375)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 60. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2021.09.01.21262969v1 on medRxiv