Human surfactant protein D facilitates SARS-CoV-2 pseudotype binding and entry in DC-SIGN expressing cells, and downregulates spike protein induced inflammation

  1. Nazar Beirag
  2. Chandan Kumar
  3. Taruna Madan
  4. Mohamed H. Shamji
  5. Roberta Bulla
  6. Daniel Mitchell
  7. Valarmathy Murugaiah
  8. Martin Mayora Neto
  9. Nigel Temperton
  10. Susan Idicula-Thomas
  11. Praveen M. Varghese
  12. Uday Kishore

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Abstract

Lung surfactant protein D (SP-D) and Dendritic cell-specific intercellular adhesion molecules-3 grabbing non-integrin (DC-SIGN) are pathogen recognising C-type lectin receptors. SP-D has a crucial immune function in detecting and clearing pulmonary pathogens; DC-SIGN is involved in facilitating dendritic cell interaction with naïve T cells to mount an anti-viral immune response. SP-D and DC-SIGN have been shown to interact with various viruses, including SARS-CoV-2, an enveloped RNA virus that causes COVID-19. A recombinant fragment of human SP-D (rfhSP-D) comprising of α-helical neck region, carbohydrate recognition domain, and eight N-terminal Gly-X-Y repeats has been shown to bind SARS-CoV-2 Spike protein and inhibit SARS-CoV-2 replication by preventing viral entry in Vero cells and HEK293T cells expressing ACE2. DC-SIGN has also been shown to act as a cell surface receptor for SARS-CoV-2 independent of ACE2. Since rfhSP-D is known to interact with SARS-CoV-2 Spike protein and DC-SIGN, this study was aimed at investigating the potential of rfhSP-D in modulating SARS-CoV-2 infection. Coincubation of rfhSP-D with Spike protein improved the Spike Protein: DC-SIGN interaction. Molecular dynamic studies revealed that rfhSP-D stabilised the interaction between DC-SIGN and Spike protein. Cell binding analysis with DC-SIGN expressing HEK 293T and THP- 1 cells and rfhSP-D treated SARS-CoV-2 Spike pseudotypes confirmed the increased binding. Furthermore, infection assays using the pseudotypes revealed their increased uptake by DC-SIGN expressing cells. The immunomodulatory effect of rfhSP-D on the DC-SIGN: Spike protein interaction on DC-SIGN expressing epithelial and macrophage-like cell lines was also assessed by measuring the mRNA expression of cytokines and chemokines. RT-qPCR analysis showed that rfhSP-D treatment downregulated the mRNA expression levels of pro-inflammatory cytokines and chemokines such as TNF-α, IFN-α, IL-1β, IL- 6, IL-8, and RANTES (as well as NF-κB) in DC-SIGN expressing cells challenged by Spike protein. Furthermore, rfhSP-D treatment was found to downregulate the mRNA levels of MHC class II in DC expressing THP-1 when compared to the untreated controls. We conclude that rfhSP-D helps stabilise the interaction between SARS- CoV-2 Spike protein and DC-SIGN and increases viral uptake by macrophages via DC-SIGN, suggesting an additional role for rfhSP-D in SARS-CoV-2 infection.

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  1. Version published to 10.3389/fimmu.2022.960733
  2. ScreenIT

    SciScore for 10.1101/2022.05.16.491949: (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.
    BlindingMolecular docking: Tripartite complex models of DC-SIGN tetramer, Spike trimer and rfhSP-D trimer were predicted through blind molecular docking using ZDOCK module of Discovery Studio 2021.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The cells were incubated for 30-min with mouse anti-human DC-SIGN antibodies to detect DC-SIGN and rabbit anti-SARS-CoV-2 Spike antibodies.
    anti-human DC-SIGN
    suggested: None
    anti-SARS-CoV-2 Spike antibodies .
    suggested: None
    Next, cells were washed and incubated with a staining buffer containing Alexa Fluor 647 conjugated goat anti-mouse antibody (Abcam), Alexa fluor 488 conjugated goat anti-rabbit antibody (Abcam), and Hoechst (Invitrogen, Life Technologies).
    anti-mouse
    suggested: None
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK 293T cells were transiently transfected with a plasmid expressing human DC-SIGN (HG10200-UT; Sino Biological), using Promega FuGENE™ HD Transfection Reagent (Fisher Scientific).
    HEK 293T
    suggested: None
    Next day, the cells were washed and cultured in the presence of hygromycin to select DC-SIGN expressing HEK-293T cells (DC HEK) (Thermo Fisher Scientific).
    HEK-293T
    suggested: None
    THP-1 cells were induced to express DC-SIGN surface molecules by the treatment with PMA (10□ng/mL) in combination with IL-4 (1000□units/mL) and incubated for 72 h (43).
    THP-1
    suggested: None
    Briefly, HEK 293Tcells were cultured in growth media to 70-80% confluence at 37°C under 5% v/v CO2.
    HEK 293Tcells
    suggested: None
    Quantitative qRT-PCR Analysis: DC-HEK and DC-THP-1 cells (0.5 × 106) were seeded overnight in growth medium.
    DC-THP-1
    suggested: None
    Recombinant DNA
    SentencesResources
    Expression and purification of soluble tetrameric DC-SIGN: The pT5T construct expressing tetrameric form of human DC-SIGN was transformed into Escherichia coli BL21 ((λDE3)
    pT5T
    suggested: None
    Cells were co-transfected using FuGENE® HD Transfection Reagent (Promega) with Opti-MEM® diluted plasmids (450 ng of pCAGGS-SARS-CoV-2 spike, 500ng of p8.91-lentiviral vector and 750 ng of pCSFLW).
    pCAGGS-SARS-CoV-2
    suggested: None
    p8.91-lentiviral
    suggested: None
    pCSFLW
    suggested: None
    Software and Algorithms
    SentencesResources
    The primer BLAST software (Basic Local Alignment Search Tool) was used to design primer sequences as listed in Table 1.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    Molecular dynamics (MD) simulation: MD simulations for the complexes B, C1 and C2 were performed using GROMACS v2020.6 (44).
    GROMACS
    suggested: (GROMACS, RRID:SCR_014565)
    Statistical analysis: Graphs were generated using GraphPad Prism 8.0 software.
    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

    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.

  3. Version published to 10.1101/2022.05.16.491949v1 on bioRxiv