Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection

  1. Martina Severa
  2. Roberta A. Diotti
  3. Marilena P. Etna
  4. Fabiana Rizzo
  5. Stefano Fiore
  6. Daniela Ricci
  7. Marco Iannetta
  8. Alessandro Sinigaglia
  9. Alessandra Lodi
  10. Nicasio Mancini
  11. Elena Criscuolo
  12. Massimo Clementi
  13. Massimo Andreoni
  14. Stefano Balducci
  15. Luisa Barzon
  16. Paola Stefanelli
  17. Nicola Clementi
  18. Eliana M. Coccia

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Abstract

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure.

Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro , asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum.

This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo . These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients.

Author summary

SARS-CoV-2 pandemic has resulted in millions of infections and deaths worldwide, yet the role of host innate immune responses in COVID-19 pathogenesis remains only partially characterized. Innate immunity represents the first line of host defense against viruses. Upon viral recognition, the secretion of type I and III interferons (IFN) establishes the cellular state of viral resistance, and contributes to induce the specific adaptive immune responses. Moving from in vitro evidences on the protective role played by plasmacytoid dendritic cells (pDC)-released type I IFN in the early phase of SARS-CoV-2 infection, here we characterized ex vivo the pDC phenotype and the balance between anti-viral and pro-inflammatory cytokines of COVID-19 patients stratified according to disease severity. Our study confirms in COVID-19 the crucial and protective role of pDC/type I IFN axis, whose deeper understanding may contribute to the development of novel pharmacological strategies and/or host-directed therapies aimed at boosting pDC response since the early phases of SARS-CoV-2 infection.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.17.440278: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: were enrolled and provided written informed consent.
    Sex as a biological variableIn particular, for this study six hospitalized COVID-19 [CP; 2 females/4 males; median age ±Standard Deviation (SD) 51.5 ± 23.3 yrs.]
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The purity of the recovered cells was greater than 95% as assessed by flow cytometry analysis with anti-BDCA4 (Miltenyi biotech) or anti-CD14 (BD Biosciences) monoclonal antibodies.
    anti-BDCA4
    suggested: None
    anti-CD14
    suggested: None
    Flow cytometry analyses: Monoclonal antibodies anti-Lineage cocktail (Lin), PD-L1, CD80, CD86, HLA-DR, CD123, CXCR-3, CXCR-4, CD62-L and CCR7 as well as IgG1 or IgG2a isotype controls were purchased from BD Biosciences, while BDCA4 from Miltenyi Biotech.
    anti-Lineage
    suggested: None
    PD-L1
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    CD80
    suggested: None
    CD86
    suggested: None
    HLA-DR
    suggested: (BD Biosciences Cat# 341068, RRID:AB_2264695)
    CD123
    suggested: None
    CXCR-3
    suggested: None
    CXCR-4
    suggested: None
    CD62-L
    suggested: None
    CCR7
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Virus production: Vero E6 (Vero C1008, clone E6-CRL-1586; ATCC) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with non-essential amino acids (NEAA, 1x), penicillin/streptomycin (P/S, 100 U/mL), HEPES buffer (10 mM) and 10% (v/v) Fetal bovine serum (FBS).
    Vero C1008
    suggested: ATCC Cat# CRL-1586, RRID:CVCL_0574)
    A clinical isolate hCoV-19/Italy/UniSR1/2020 (GISAID accession ID: EPI_ISL_413489) was isolated and propagated in Vero E6 cells, and viral titer was determined by 50% tissue culture infective dose (TCID50) and plaque assay for confirming the obtained titer.
    Vero E6
    suggested: None
    Calu-3 cells were seeded into 12-wells plates to reach confluence.
    Calu-3
    suggested: None
    Flow cytometry analyses: Monoclonal antibodies anti-Lineage cocktail (Lin), PD-L1, CD80, CD86, HLA-DR, CD123, CXCR-3, CXCR-4, CD62-L and CCR7 as well as IgG1 or IgG2a isotype controls were purchased from BD Biosciences, while BDCA4 from Miltenyi Biotech.
    PD-L1
    suggested: None
    Recombinant DNA
    SentencesResources
    In the mixed cell population of PBMC, pDC were considered as those cells in live/single FvDye-Lineage-CD123+BDCA4+HLADR+ gate (S3A Fig).
    pDC
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were analyzed by Flow Jo software v.10.7 (BD Biosciences).
    Flow Jo
    suggested: (FlowJo, RRID:SCR_008520)
    RNA isolation and quantitative real time PCR analysis: Total RNA was isolated by Trizol® Reagent (Invitrogen, Thermo Fischer Scientific), quantified using a Nanodrop2000 spectrophotometer and quality assessed with an established cut-off of ~1.8 for 260/280 absorbance ratio.
    Thermo Fischer Scientific
    suggested: None

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.04.17.440278 on bioRxiv