Human surfactant protein D facilitates SARS-CoV-2 pseudotype binding and entry in DC-SIGN expressing cells, and downregulates spike protein induced inflammation
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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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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
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding Molecular 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 Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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-SIGNsuggested: Noneanti-SARS-CoV-2 Spike antibodies .suggested: NoneNext, cells were washed and incubated with a staining buffer containing Alexa Fluor 647 … 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
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding Molecular 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 Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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-SIGNsuggested: Noneanti-SARS-CoV-2 Spike antibodies .suggested: NoneNext, 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-mousesuggested: Noneanti-rabbitsuggested: NoneExperimental Models: Cell Lines Sentences Resources 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 293Tsuggested: NoneNext 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-293Tsuggested: NoneTHP-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-1suggested: NoneBriefly, HEK 293Tcells were cultured in growth media to 70-80% confluence at 37°C under 5% v/v CO2. HEK 293Tcellssuggested: NoneQuantitative qRT-PCR Analysis: DC-HEK and DC-THP-1 cells (0.5 × 106) were seeded overnight in growth medium. DC-THP-1suggested: NoneRecombinant DNA Sentences Resources 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) pT5Tsuggested: NoneCells 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-2suggested: Nonep8.91-lentiviralsuggested: NonepCSFLWsuggested: NoneSoftware and Algorithms Sentences Resources The primer BLAST software (Basic Local Alignment Search Tool) was used to design primer sequences as listed in Table 1. BLASTsuggested: (BLASTX, RRID:SCR_001653)Molecular dynamics (MD) simulation: MD simulations for the complexes B, C1 and C2 were performed using GROMACS v2020.6 (44). GROMACSsuggested: (GROMACS, RRID:SCR_014565)Statistical analysis: Graphs were generated using GraphPad Prism 8.0 software. GraphPad Prismsuggested: (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.
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