Human Surfactant Protein D as an Innate Immune Surveillance Molecule

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonSurfactant protein D (SP-D) belongs to the family called Collectins (collagen-containing lectins). It has a primary structure characterised by an N-terminal cysteine-rich region, triple-helical collagen region composed of Gly-X-Y repeats (where X and Y can be any amino acid), an a-helical coiled-coil neck region, and a C-terminal carbohydrate recognition domain (CRD). The primary subunit oligomerizes to form a trimeric structure that can further acquire a cruciform organization and multimers (dodecamers). SP-D is a potent innate immune molecule whose presence in the lungs as well as at a range mucosal surfaces and extrapulmonary tissues allows immune surveillance against pathogens, apoptotic/necrotic cells, allergens, and cancer cells. A recombinant fragment of human SP-D (rfhSP-D) composed of 8 Gly-X-Y repeats, neck and CRD region, expressed in E. coli, is well known to act against a range of pathogen and allergen challenge in vitro, in vivo and ex vivo. In this thesis, we have examined the interaction between rfhSP-D and two subtypes (pH1N1 and H3N2) of Influenza A Virus (IAV) (Chapter 3). rfhSP-D interacted with haemagglutinin, neuraminidase and matrix protein 1; inhibited their infectivity against lung epithelia cell lines; and suppressed the cytokine storm induced by the viral challenge. The protective role of rfhSP-D against IAV as an entry inhibitor was further validated by the use of pseudotyped lentiviruses containing haemagglutinin and neuraminidase of the two IAV subtypes. Continuing with the theme of innate immune surveillance by SP-D against lung, pancreatic, ovarian, and prostate cancers, we extended on recent studies to breast cancer using HER2 over-expressing (SKBR3), triple-positive (BT474) and triple-negative (BT20) breast cancer cell lines (Chapter 4). rfhSP-D induced apoptosis at 24 h in SKBR3 and BT747 cells (but not in BT20) via intrinsic apoptosis pathway. However, this protective effect of rfhSP-D was fully negated by the presence of hyaluronic acid, a major extracellular matrix component within the tumour microenvironment (Chapter 5). This thesis highlights the therapeutic potential of rfhSP-D in influenza A Virus infection as well as breast cancer and merits pre-clinical trials in murine models

C4b Binding Protein Acts as an Innate Immune Effector Against Influenza A Virus

C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation

A self-amplifying RNA vaccine provides protection in a murine model of bubonic plague

Mice were immunized with a combination of self-amplifying (sa) RNA constructs for the F1 and V antigens of Yersinia pestis at a dose level of 1 μg or 5 μg or with the respective protein sub-units as a reference vaccine. The immunization of outbred OF1 mice on day 0 and day 28 with the lowest dose used (1 μg) of each of the saRNA constructs in lipid nanoparticles protected 5/7 mice against subsequent sub-cutaneous challenge on day 56 with 180 cfu (2.8 MLD) of a 2021 clinical isolate of Y. pestis termed 10-21/S whilst 5/7 mice were protected against 1800cfu (28MLD) of the same bacteria on day 56. By comparison, only 1/8 or 1/7 negative control mice immunized with 10 μg of irrelevant haemagglutin RNA in lipid nanoparticles (LNP) survived the challenge with 2.8 MLD or 28 MLD Y. pestis 10-21/S, respectively. BALB/c mice were also immunized with the same saRNA constructs and responded with the secretion of specific IgG to F1 and V, neutralizing antibodies for the V antigen and developed a recall response to both F1 and V. These data represent the first report of an RNA vaccine approach using self-amplifying technology and encoding both of the essential virulence antigens, providing efficacy against Y. pestis. This saRNA vaccine for plague has the potential for further development, particularly since its amplifying nature can induce immunity with less boosting. It is also amenable to rapid manufacture with simpler downstream processing than protein sub-units, enabling rapid deployment and surge manufacture during disease outbreaks

Human properdin modulates macrophage: Mycobacterium bovis BCG interaction via thrombospondin repeats (TSR) 4 and 5

Mycobacterium tuberculosis can proficiently enter macrophages and diminish complement activation on its cell surface. Within macrophages, the mycobacterium can suppress macrophage apoptosis and survive within the intracellular environment. Previously, we have shown that complement regulatory proteins such as factor H may interfere with pathogen–macrophage interactions during tuberculosis infection. In this study, we show that Mycobacterium bovis BCG binds properdin, an upregulator of the complement alternative pathway. TSR4+5, a recombinant form of thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an inhibitor of the alternative pathway, was also able to bind to M. bovis BCG. Properdin and TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Quantitative real-time PCR revealed elevated pro-inflammatory responses (TNF-α, IL-1β, and IL-6) in the presence of properdin or TSR4+5, which gradually decreased over 6 h. Correspondingly, anti-inflammatory responses (IL-10 and TGF-β) showed suppressed levels of expression in the presence of properdin, which gradually increased over 6 h. Multiplex cytokine array analysis also revealed that properdin and TSR4+5 significantly enhanced the pro-inflammatory response (TNF-α, IL-1β, and IL-1α) at 24 h, which declined at 48 h, whereas the anti-inflammatory response (IL-10) was suppressed. Our results suggest that properdin may interfere with mycobacterial entry into macrophages via TSR4 and TSR5, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. This study offers novel insights into the non-complement related functions of properdin during host–pathogen interactions in tuberculosis.MA-A has been supported by the Ministry of Higher Education, Malaysia and the Universiti Sains Malaysia

Human Surfactant Protein D Facilitates SARS-CoV-2 Pseudotype Binding and Entry in DC-SIGN Expressing Cells, and Downregulates Spike protein Induced Inflammation

Copyright © 2022 Beirag, Kumar, Madan, Shamji, Bulla, Mitchell, Mayora Neto, Murugaiah, Temperton, Idicula-Thomas, Varghese and Kishore. 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 as an antigen-presenting cell 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 55 SIGN expressing epithelial and macrophage-like cell lines was also assessed by measuring the mRNA expression of cytokines and chemokines. The 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 of 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

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

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

5-methylcytosine (m⁵C) RNA modification controls the innate immune response to virus infection by regulating type I interferons

5-methylcytosine (m5C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m5C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m5C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant noncoding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I–mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m5C methylation controls antiviral innate immunity through modulating the m5C methylome of ncRNAs and their expression