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.
Keywords: COVID-19; SARS-CoV-2; collectins; cytokine response; innate immune system; rfhSP-D.
【저자키워드】 COVID-19, SARS-CoV-2, innate immune system, collectins, cytokine response, rfhSP-D., 【초록키워드】 Treatment, viruses, Macrophage, ACE2, immune response, Cytokines, macrophages, spike, T cells, SARS-COV-2 infection, cytokine, chemokines, viral entry, chemokine, Spike protein, Anti-viral, Replication, innate immune system, Protein, RT-qPCR, pathogen, cells, pseudotype, SARS-CoV-2 spike protein, SARS- CoV-2, IL-8, immune function, Pathogens, MHC class II, pro-inflammatory cytokines, receptors, RNA virus, epithelial, SARS-CoV-2 replication, dendritic cell, binding, TNF-α, NF-κB, IL-1β, innate immune, Interaction, Analysis, cytokine response, DC-SIGN, IFN-α, independent of, cell surface receptor, fragment of, cell interaction, domain, help, SIGN, Vero cells, pro-inflammatory cytokine, cell line, carbohydrate, surface receptor, mRNA expression, mRNA levels, infection assays, naïve T cells, HEK293T cells, N-terminal, RANTES, MHC class, mRNA level, Vero Cell, immunomodulatory effect, controls, Cell, THP-1, independent, HEK 293T, shown, involved, eight, treated, increase, cause, expressing, downregulated, downregulate, the Spike, HEK293T cell, IL- 6, infection assay, inhibit SARS-CoV-2, intercellular, modulating, naïve T cell, 【제목키워드】 Inflammation, Human, Protein, cells, pseudotype, binding, facilitate, expressing, downregulate,