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RationaleCOVID-19 is an acute infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Human surfactant protein D (SP-D) is known to interact with spike protein of SARS-CoV, but its immune-surveillance against SARS-CoV-2 is not known.ObjectiveThis study aimed to examine the potential of a recombinant fragment of human SP-D (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2.MethodsrfhSP-D interaction with spike protein of SARS-CoV-2 and hACE-2 receptor was predicted via docking analysis. The inhibition of interaction between spike protein and ACE-2 by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was studied by measuring the expression of RdRp gene of the virus using qPCR.Measurements and Main ResultsIn-silico interaction studies indicated that three amino acid residues in the RBD of spike of SARS-CoV-2 were commonly involved in interacting with rfhSP-D and ACE-2. Studies using clinical samples of SARS-CoV-2 positive cases (asymptomatic, n=7 and symptomatic, n=8 and negative controls n=15) demonstrated that treatment with 5μM rfhSP-D inhibited viral replication by ~5.5 fold and was more efficient than Remdesivir (100 μM). Approximately, a 2-fold reduction in viral infectivity was also observed after treatment with 5μM rfhSP-D.ConclusionsThese results conclusively demonstrate that the calcium independent rfhSP-D mediated inhibition of binding between the receptor binding domain of the S1 subunit of the SARS-CoV-2 spike protein and human ACE-2, its host cell receptor, and a significant reduction in SARS-CoV-2 infection and replication in-vitro.

Copyright: © 2021 by the authors. Alzheimer’s disease is a type of dementia characterized by problems with short-term memory, cognition, and difficulties with activities of daily living. It is a progressive, neurodegenerative disorder. The complement system is an ancient part of the innate immune system and comprises of more than thirty serum and membrane-bound proteins. This system has three different activating pathways and culminates into the formation of a membrane attack complex that ultimately causes target cell lysis (usually pathogens) The complement system is involved in several important functions in the central nervous system (CNS) that include neurogenesis, synaptic pruning, apoptosis, and neuronal plasticity. Here, we discuss how the complement system is involved in the effective functioning of CNS, while also contributing to chronic neuroinflammation leading to neurodegenerative disorders such as Alzheimer’s disease. We also discuss potential targets in the complement system for stopping its harmful effects via neuroinflammation and provide perspective for the direction of future research in this field.

Data Availability Statement: Not applicable. Copyright: © 2021 by the authors. Nanoparticles are efficient drug delivery vehicles for targeting specific organs as well as systemic therapy for a range of diseases, including cancer. However, their interaction with the immune system offers an intriguing challenge. Due to the unique physico-chemical properties, carbon nanotubes (CNTs) are considered as nanocarriers of considerable interest in cancer diagnosis and therapy. CNTs, as a promising nanomaterial, are capable of both detecting as well as delivering drugs or small therapeutic molecules to tumour cells. In this study, we coupled a recombinant fragment of human surfactant protein D (rfhSP-D) with carboxymethyl-cellulose (CMC) CNTs (CMC-CNT, 10–20 nm diameter) for augmenting their apoptotic and immunotherapeutic properties using two leukemic cell lines. The cell viability of AML14.3D10 or K562 cancer cell lines was reduced when cultured with CMC-mwCNT-coupled-rfhSP-D (CNT + rfhSP-D) at 24 h. Increased levels of caspase 3, 7 and cleaved caspase 9 in CNT + rfhSP-D treated AML14.3D10 and K562 cells suggested an involvement of an intrinsic pathway of apoptosis. CNT + rfhSP-D treated leukemic cells also showed higher mRNA expression of p53 and cell cycle inhibitors (p21 and p27). This suggested a likely reduction in cdc2-cyclin B1, causing G2/M cell cycle arrest and p53-dependent apoptosis in AML14.3D10 cells, while p53-independent mechanisms appeared to be in operation in K562 cells. We suggest that CNT + rfhSP-D has therapeutic potential in targeting leukemic cells, irrespective of their p53 status, and thus, it is worth setting up pre-clinical trials in animal models Funding: This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (15-NAN-3664-02).

Data Availability Statement: The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author. Copyright © 2021 Awoyemi, Motta-Mejia, Zhang, Kouser, White, Kandzija, Alhamlan, Cribbs, Tannetta, Mazey, Redman, Kishore and Vatish. Syncytiotrophoblast derived Extracellular Vesicles (STBEV) from normal pregnancy (NP) have previously been shown to interact with circulating monocytes and B cells, and induce pro-inflammatory cytokine release. Early-onset preeclampsia (EOPE) is associated with an exacerbated inflammatory response, yet there is little data regarding late-onset PE (LOPE) and immune function. Here, using a macrophage/monocyte cell line THP-1, we investigated the inflammatory potential of STBEV, comprising medium/large-STBEV (>200nm) and small-STBEV (

Data Availability Statement: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Copyright © 2021 Hsieh, Beirag, Murugaiah, Chou, Kuo, Kao, Madan, Kishore and Wang. Human SP-D is a potent innate immune molecule whose presence at pulmonary mucosal surfaces allows its role in immune surveillance against pathogens. Higher levels of serum SP-D have been reported in the patients with severe acute respiratory syndrome coronavirus (SARS-CoV). Studies have suggested the ability of human SP-D to recognise spike glycoprotein of SARS-CoV; its interaction with HCoV-229E strain leads to viral inhibition in human bronchial epithelial (16HBE) cells. Previous studies have reported that a recombinant fragment of human SP-D (rfhSP-D) composed of 8 Gly-X-Y repeats, neck and CRD region, can act against a range of viral pathogens including influenza A Virus and Respiratory Syncytial Virus in vitro, in vivo and ex vivo. In this context, this study was aimed at examining the likely protective role of rfhSP-D against SARS-CoV-2 infection. rfhSP-D showed a dose-responsive binding to S1 spike protein of SARS-CoV-2 and its receptor binding domain. Importantly, rfhSP-D inhibited interaction of S1 protein with the HEK293T cells overexpressing human angiotensin converting enzyme 2 (hACE2). The protective role of rfhSP-D against SARS-CoV-2 infection as an entry inhibitor was further validated by the use of pseudotyped lentiviral particles expressing SARS-CoV-2 S1 protein; ~0.5 RLU fold reduction in viral entry was seen following treatment with rfhSP-D (10 µg/ml). These results highlight the therapeutic potential of rfhSP-D in SARS-CoV-2 infection and merit pre-clinical studies in animal models. Ministry of Science and Technology; Headquarters of University Advancement at the National Cheng Kung University

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.

Surfactant protein D (SP-D), expressed in respiratory epithelium, is important for modulating both innate and adaptive immunity against inhaled allergens. This chapter evaluates the key immunomodulatory role of SP-D in allergic airway inflammatory events. Studies have shown that SP-D ameliorated various cellular mechanisms implicated in pulmonary allergic reactions, including allergic eosinophilia through induction of apoptosis and clearance of activated eosinophils, and inhibition of basophils activation. In addition, blocking allergen-specific IgE synthesis, T cell proliferation, and hindrance to the binding of allergen-IgE complex to B lymphocytes are also brought about by SP-D. In vitro, a recombinant fragment of human SP-D, composed of homotrimeric neck and C-type lectin domains (rfhSP-D), binds to allergens derived from Aspergillus fumigatus, house dust mite and grass pollen in a calcium-dependent manner and inhibits specific IgE interaction with allergens. Murine models of hypersensitivity reactions, when treated with rfhSP-D, showed reduced levels of IgE, clearance of peripheral and pulmonary eosinophilia, and Th2 polarisation to Th1 type. SP-D knock-out mice accordingly show hypereosinophilia and predominantly exaggerated Th2 response following allergen challenge; these features can be ameliorated by rfhSP-D treatment. The current state of the filed appears to suggest that rfhSP-D is an excellent therapeutic immunomodulator in airway allergic diseases.

Surfactant proteins, SP-A and SP-D, are hydrophillic C-type lectins that are involved in innate immune responses at the mucosal surfaces, especially in the lungs, against various pathogens including viruses, bacteria and fungi. Both proteins have characteristic N-terminal triple-helical collagen region followed by trimeric neck and C-type lectin domains. Several in vivo and in vitro studies have shown that SP-A and SP-D can bind, mostly via their C-type lectin domains, to a range of pathogens displaying different carbohydrate molecular patterns; they show greater affinity towards microbe-associated ligands such as mannose and glucose. Oligomerizations of these collectins have been found to be a critical feature of its function for efficient host defence such as pathogen neutralization and opsonisation. SP-A and SP-D can also bind and activate macrophages for enhanced phagocytosis and reactive oxygen species-mediated destruction of pathogens, and influence pro-inflammatory cytokine production. Studies on gene knock-out mice and patient samples have revealed that SP-A or SP-D levels in the lungs can render susceptibility or resistance to microbial challenge and subsequent pathogenesis. This present chapter summarises the biological activities of SP-A and SP-D in preventing dissemination of infectious microbes inside the host.

Mammalian collectins play key roles in innate immunity, health and disease. These have been well studied in humans and other mammalian species, but much has still to be learned about the bovine collectins and the role they may play in the health and disease in cattle which have significant economic implications to the agricultural and food industries. This chapter focuses on the bovine collectins, which are important innate immune molecules and play a key role in immunoregulation and protection against pathogens. Like other mammalian collectins, they all have a related structure comprised of an N-terminal triple-helical collagen-like region and a ligand binding C-terminal C-type lectin domain. The bovine collectins are able to recognise and bind to complex glycoconjugates, particularly on the surface of microbes, neutralising infection, enhancing clearance by phagocytes and modulating immune response. Here, we will review the bovine collectins, surfactant proteins A (SP-A) and D (SP-D), mannose binding lectins (MBL-A and MBL-C), conglutinin (CGN), collectin 43 (CL-43) and collectin 46 (CL-46), and their role in bovine immunity and disease. We will particularly focus on the structural, functional and anti-microbial roles of these collectins with an additional focus on genetic polymorphisms in their genes and predisposition to infectious diseases in the bovine host. In the absence of specific data on the bovine host, findings from studies with other mammalian collectins will be discussed with respect to bovine health and disease.

Human SP-D is a potent innate immune molecule whose presence at pulmonary mucosal surfaces allows immune surveillance role against pulmonary pathogens. Higher levels of serum SP-D have been reported in patients with severe acute respiratory syndrome coronavirus-1 (SARS-CoV). Studies have suggested the ability of human SP-D to recognise spike glycoprotein of SARS-CoV; its interaction with HCoV-229E strain leads to viral inhibition in human bronchial epithelial (16HBE) cells. Previous studies have reported that a recombinant fragment of human SP-D (rfhSP-D) composed of 8 Gly-X-Y repeats, neck and CRD region, can act against a range of viral pathogens including influenza A Virus and Respiratory Syncytial Virus in vitro, in vivo and ex vivo models. In this context, this study was aimed at examining the likely protective role of rfhSP-D against SARS-CoV-2 infection. rfhSP-D showed a dose-responsive binding to S1 spike protein of SARS-CoV-2 and its receptor binding domain. Importantly, rfhSP-D inhibited interaction of S1 protein with the HEK293T cells overexpressing Angiotensin Converting Enzyme 2. The protective role of rfhSP-D against SARS-CoV-2 infection as an entry inhibitor was further validated by the use of pseudotyped lentiviral particles expressing SARS-CoV-2 S1 protein; ~0.5 RLU fold reduction in viral entry was seen following rfhSP-D treatment (10 μg/ml). The results highlight the therapeutic potential of rfhSP-D in SARS-CoV-2 infection and merits pre-clinical studies in murine models.

Data Availability Statement: The datasets presented in this study can be found in online repositories. The names of the repositories and accession number(s) can be found in the article/Supplementary Material (https://www.frontiersin.org/articles/10.3389/fimmu.2020.600660/full#h11). Copyright © 2021 Thakur, Sathe, Kundu, Biswas, Gautam, Alkahtani, Idicula-Thomas, Sirdeshmukh, Kishore and Madan. Surfactant protein-D (SP-D), a member of the collectin family has been shown to induce apoptosis in cancer cells. SP-D is composed of an N-terminal collagen-like domain and a calcium-dependent carbohydrate recognition domain (CRD). Recently, we reported that a recombinant fragment of human SP-D (rfhSP-D), composed of homotrimeric CRD region, induced intrinsic apoptotic pathway in prostate cancer cells. Here, we analyzed the membrane interactome of rfhSP-D in an androgen-independent prostate cancer cell line, PC3, by high resolution mass spectrometry and identified 347 proteins. Computational analysis of PPI network of this interactome in the context of prostate cancer metastasis and apoptosis revealed Glucose Regulated Protein of 78 kDa (GRP78) as an important binding partner of rfhSP-D. Docking studies suggested that rfhSP-D (CRD) bound to the substrate-binding domain of glycosylated GRP78. This was further supported by the observations that human recombinant GRP78 interfered with the binding of rfhSP-D to anti-SP-D polyclonal antibodies; GRP78 also significantly inhibited the binding of recombinant full-length human SP-D with a monoclonal antibody specific to the CRD in a dose-dependent manner. We conclude that the interaction with rfhSP-D is likely to interfere with the pro-survival signaling of GRP78. ICMR- National Institute for Research in Reproductive Health ICMR-NIRRH (Accession no. 921); ICMR- National Institute for Research in Reproductive Health ICMR-NIRRH-JRF; ICMR- National Institute for Research in Reproductive Health ICMR-SRF; Researchers Supporting Project (RSP-2020/26) King Saud University, Riyadh.

Copyright © 2020 Murugaiah, Agostinis, Varghese, Belmonte, Vieni, Alaql, Alrokayan, Khan, Kaur, Roberts, Madan, Bulla and Kishore. Human surfactant protein D (SP-D) belongs to the family of collectins that is composed of a characteristic amino-terminal collagenous region and a carboxy-terminal C-type lectin domain. Being present at the mucosal surfaces, SP-D acts as is a potent innate immune molecule and offers protection against non-self and altered self-such as pathogens, allergens, and tumour. Here, we examined the effect of a recombinant fragment of human SP-D (rfhSP-D) on a range of breast cancer lines. Breast cancer has four molecular subtypes characterised by varied expression of oestrogen (ER), progesterone (PR) and EGF receptors (HER2). The cell viability of HER2 over-expressing (SKBR3) and triple-positive (BT474) breast cancer cell lines (but not of triple-negative cell line (BT20), was reduced following rfhSP-D treatment at 24h. Upregulation of p21/p27 cell cycle inhibitors and p53 phosphorylation (Ser15) in rfhSP-D-treated BT474 and SKBR3 cell lines signified G2/M cell cycle arrest. Cleaved caspase 9 and 3 were detected in rfhSP-D- treated BT474 and SKBR3 cells, suggesting an involvement of intrinsic apoptosis pathway. However, rfhSP-D-induced apoptosis was nullified in the presence of hyaluronic acid whose increased level in breast tumor microenvironment is associated with malignant tumor progression and invasion. rfhSP-D bound to solid-phase HA and promoted tumor cell proliferation. rfhSP-D-treated SKBR3 cells in the presence of hyaluronic acid showed decreased transcriptional levels of p53 when compared to SKBR3 cells treated with rfhSP-D only. Thus, hyaluronic acid appears to negate the anti-tumorigenic properties of rfhSP-D against HER2-over-expressing and triple-positive breast cancer cells. King Saud University, Riyadh, International Scientific Partnership Program (ISPP) ISPP-145; Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy, RC20/16 and 5MILLE15D.

With limited therapeutic options and associated severe adverse effects, fungal infections are a serious threat to human health. Innate immune response mediated by pattern recognition proteins is integral to host defense against fungi. A soluble pattern recognition protein, Surfactant protein D (SP-D), plays an important role in immune surveillance to detect and eliminate human pathogens. SP-D exerts its immunomodulatory activity via direct interaction with several receptors on the epithelial cells lining the mucosal tracts, as well as on innate and adaptive immune cells. Being a C-type lectin, SP-D shows calcium- and sugar-dependent interactions with several glycosylated ligands present on fungal cell walls. The interactome includes cell wall polysaccharides such as 1,3-β-D-glucan, 1,6-β-D-glucan, Galactosaminogalactan Galactomannan, Glucuronoxylomannan, Mannoprotein 1, and glycosylated proteins such as gp45, gp55, major surface glycoprotein complex (gpA). Recently, binding of a recombinant fragment of human SP-D to melanin on the dormant conidia of Aspergillus fumigatus was demonstrated that was not inhibited by sugars, suggesting a likely protein-protein interaction. Interactions of the ligands on the fungal spores with the oligomeric forms of full-length SP-D resulted in formation of spore-aggregates, increased uptake by phagocytes and rapid clearance besides a direct fungicidal effect against C. albicans. Exogenous administration of SP-D showed significant therapeutic potential in murine models of allergic and invasive mycoses. Altered susceptibility of SP-D gene-deficient mice to various fungal infections emphasized relevance of SP-D as an important sentinel of anti-fungal immunity. Levels of SP-D in the serum or lung lavage were significantly altered in the murine models and patients of fungal infections and allergies. Here, we review the cell wall ligands of clinically relevant fungal pathogens and allergens that are recognized by SP-D and their impact on the host defense. Elucidation of the molecular interactions between innate immune humoral such as SP-D and fungal pathogens would facilitate the development of novel therapeutic interventions.

Copyright © 2020 Murugaiah, Varghese, Saleh, Tsolaki, Alrokayan, Khan, Collison, Sim, Nal, Al-Mohanna and Kishore. The complement system is an ancient innate immune defence mechanism that can recognise molecular patterns on the invading pathogens. Factor H, as an inhibitor of the alternative pathway, down-regulates complement activation on the host cell surface. Locally synthesised factor H at the site of infection/injury, including lungs, can act as a pattern recognition molecule without involving complement activation. Here, we report that factor H, a sialic acid binder, interacts with influenza A virus (IAV) and modulates IAV replication, as observed by an upregulation of matrix protein 1 (M1) expression in H3N2-infected A549 cells, while downregulating M1 in H1N1 subtype-infected cells. Far-western blot revealed that factor H binds hemagglutinin (HA, ~70kDa), neuraminidase (NA, ~60kDa), and M1 (~25kDa). IAV-induced transcriptional levels of IFN-α, TNF-α, IL-12, IL-6, IFN-α, while RANTES were reduced following factor H treatment for the H1N1 subtype at 6 h post-infection. However, for the H3N2 subtype, mRNA levels of these pro-inflammatory cytokines were enhanced. Recombinant form of vaccinia virus complement control protein (VCP), which like factor H, contains CCP modules and has complement-regulatory activity, mirrored the results obtained with factor H. Both factor H (25%) and VCP (45%) were found to reduce luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles. Factor H (50%) and VCP (30%) enhanced the luciferase reporter activity for H3N2, suggesting an entry inhibitory role of factor H and VCP against H1N1, but not H3N2. Thus, factor H can modulate IAV infection and inflammatory response independent of its complement-related functions. King Saud University, Riyadh, International Scientific Partnership Programme (ISPP) ISPP-145.

Surfactant protein A (SP-A) and D (SP-D) are hydrophilic collagenous C-type lectins, which were originally discovered in the lungs associated with surfactant phospholipids. It was later shown that the two proteins, unlike hydrophobic surfactant proteins, SP-B and SP-C, are keenly involved in protecting lungs against insults from pathogens, allergens, apoptotic, and necrotic cells (1). Two aspects became clear in subsequent years that (i). SP-A and SP-D have extra-pulmonary existence; and (ii). They can manipulate immune cells, and thus, regulate inflammatory responses (2). Although there have been a constant debate about their candidate receptor(s)—there are several reported so far (1). Much of the immunological studies, beyond interaction with surfactant system and pathogens, have been followed up toward SP-D. It has become apparent that SP-D is an innate immune surveillance molecule at the mucosal surfaces, which can act as a bridge between innate and adaptive immunity. The role of SP-D in modulating antigen presentation, helper T cell polarization and B cell differentiation and class switching (3) are few neat examples. This volume comprises 14 papers that extend our knowledge on SP-A and SP-D, and their roles in infection, inflammation and cancer. A consistent theme discussed by several contributors is the differential role of two forms of SP-A in oxidative stress and lung innate immunity (Thorenoor, Umstead et al.; Nalian et al.; Thorenoor, Kawasawa et al.; Wang et al.). In humans, there are two SP-A variants differing in the collagen region, SP-A1 and SP-A2, encoded by SFTPA1 and SFTPA2, respectively, and produced by the alveolar type II cells in the lung. Importantly, SP-A1 and SP-A2 seem to differentially bind to phagocytic, but not to non-phagocytic cells (Thorenoor, Umstead et al.). SP-A1 and SP-A2 differentially bind and regulate neonatal and adult human alveolar macrophages (AMs) (Thorenoor, Umstead et al.). AMs from transgenic mice expressing human SP-A1 and SP-A2 exhibit differential expression of cell surface proteins (Thorenoor, Kawasawa et al.) Rodents express only one SP-A variant; thus, Nalian et al. have compared the rodent and human SP-A with respect to structural determinants of the function. The data infers that mouse SP-A is a functional hybrid of human SP-A1 and SP-A2. Particularly striking in this regard is the differential response in the two sexes. Humanized transgenic (hTG) male and female mice, carrying both SP-A1/SP-A2 (6A2/1A0, co-expressed) and SP-A-gene deficient mice were exposed to filtered air (FA) or ozone (O3), and miRNA levels were measured in isolated AMs (Thorenoor, Kawasawa et al.). The AM miRNome of co-expressed females was significantly downregulated in response to ozone induced oxidative stress. Several of the validated miRNA targets were involved in pro-inflammatory response, anti-apoptosis, cell cycle, cellular growth, and proliferation (Thorenoor, Kawasawa et al.). Continuing with this

Mycobacterium tuberculosis (M.tb) infection results in approximately 1.3 million human deaths each year. M.tb resides primarily inside macrophages, and maintains persistent infection. In response to infection and inflammation, platelet activating factor C-16 (PAF C16), a phospholipid compound, is released by various cells including neutophils and monocytes. We have recently shown that PAF C-16 can directly inhibit the growth of two representative non-pathogenic mycobacteria, Mycobacterium bovis BCG and Mycobacterium smegmatis (M. smegmatis), by damaging the bacterial cell membrane. Here, we have examined the effect of PAF C-16 on M. smegmatis residing within macrophages, and identified mechanisms involved in their growth inhibitory function. Our results demonstrated that exogenous PAF C-16 inhibited the growth of M. smegmatis inside phagocytic cells of monocytic cell line, THP1; this effect was partially blocked by PAF receptor antagonists, suggesting the involvement of PAF receptor-mediated signalling pathways. Arachidonic acid, a downstream metabolite of PAF C-16 signalling pathway, directly inhibited the growth of M. smegmatis in vitro. Moreover, the inhibition of phospholipase C and phospholipase A2 activities, involved in PAF C-16 signalling pathway, increased survival of intracellular M. smegmatis. Interestingly, we also observed that inhibition of inducible nitric oxide synthase (iNOS) enzyme and antibody-mediated neutralization of TNF-α partially mitigated the intracellular growth inhibitory effect of PAF C-16. Use of a number of PAF C-16 structural analogues, including Lyso-PAF, 2-O-methyl PAF, PAF C-18 and Hexanolamino PAF, revealed that the presence of acetyl group (CH3CO) at sn-2 position of the glycerol backbone of PAF is important for the intracellular growth inhibition activity against M. smegmatis. Taken together, these results suggest that exogenous PAF C-16 treatment inhibits intracellular M. smegmatis growth, at least partially, in a nitric oxide and TNF-α dependent manner.

Surfactant Protein D (SP-D), a pattern recognition innate immune molecule, has been implicated in the immune surveillance against cancer. A recent report showed an association of decreased SP-D expression in human prostate adenocarcinoma with an increased Gleason score and severity. In the present study, the SP-D expression was evaluated in primary prostate epithelial cells (PrEC) and prostate cancer cell lines. LNCaP, an androgen dependent prostate cancer cell line, exhibited significantly lower mRNA and protein levels of SP-D than PrEC and the androgen independent cell lines (PC3 and DU145). A recombinant fragment of human SP-D, rfhSP-D, showed a dose and time dependent binding to prostate cancer cells via its carbohydrate recognition domain. This study, for the first time, provides evidence of significant and specific cell death of tumor cells in rfhSP-D treated explants as well as primary tumor cells isolated from tissue biopsies of metatstatic prostate cancer patients. Viability of PrEC was not altered by rfhSP-D. Treated LNCaP (p53+/+) and PC3 (p53 −/−) cells exhibited reduced cell viability in a dose and time dependent manner and were arrested in G2/M and G1/G0 phase of the cell cycle, respectively. rfhSP-D treated LNCaP cells showed a significant upregulation of p53 whereas a significant downregulation of pAkt was observed in both PC3 and LNCaP cell lines. The rfhSP-D-induced apoptosis signaling cascade involved upregulation of Bax:Bcl2 ratio, cytochrome c and cleaved products of caspase 7. The study concludes that rfhSP-D induces apoptosis in prostate tumor explants as well as in androgen dependent and independent prostate cancer cells via p53 and pAkt pathways. ICMR-NIRRH

Background: In pregnancy, excessive inflammation and break down of immunologic tolerance can contribute to miscarriage. Endothelial cells (ECs) are able to orchestrate the inflammatory processes by secreting pro-inflammatory mediators and bactericidal factors by modulating leakiness and leukocyte trafficking, via the expression of adhesion molecules and chemokines. The aim of this study was to analyse the differences in the phenotype between microvascular ECs isolated from decidua (DECs) and ECs isolated from human skin (ADMECs). Methods: DECs and ADMECs were characterized for their basal expression of angiogenic factors and adhesion molecules. A range of immunological responses was evaluated, such as vessel leakage, reactive oxygen species (ROS) production in response to TNF-&alpha, stimulation, adhesion molecules expression and leukocyte migration in response to TNF-&alpha, and IFN-&gamma, stimulation. Results: DECs produced higher levels of HGF, VEGF-A and IGFBP3 compared to ADMECs. DECs expressed adhesion molecules, ICAM-2 and ICAM-3, and a mild response to TNF-&alpha, was observed. Finally, DECs produced high levels of CXCL9/MIG and CXCL10/IP-10 in response to IFN-&gamma, and selectively recruited Treg lymphocytes. Conclusion: DEC phenotype differs considerably from that of ADMECs, suggesting that DECs may play an active role in the control of immune response and angiogenesis at the foetal-maternal interface.

Effective prophylactic strategy against the current epidemic of sexually transmitted HIV-1 infection requires understanding of the innate gatekeeping mechanisms at the genital mucosa. Surfactant protein D (SP-D), a member of the collectin family of proteins naturally present in the vaginal tract, is a potential HIV-1 entry inhibitor at the cellular level. Human EpiVaginal tissues compartmentalized in culture inserts were apically exposed to HIV-1 and/or a recombinant fragment of human SP-D (rfhSP-D) and viral passage was assessed in the basal chamber containing mononuclear leukocytes. To map the gene signature facilitating or resisting the transepithelial viral transfer, microarray analysis of the HIV-1 challenged EpiVaginal tissues was performed in the absence or presence of rfhSP-D. Mucosal biocompatibility of rfhSP-D was assessed ex vivo and in the standard rabbit vaginal irritation model. The passage of virus through the EpiVaginal tissues toward the underlying target cells was associated with a global epithelial gene signature including differential regulation of genes primarily involved in inflammation, tight junctions and cytoskeletal framework. RfhSP-D significantly inhibited HIV-1 transfer across the vaginal tissues and was associated with a significant reversal of virus induced epithelial gene signature. Pro-inflammatory NF-κB and mTOR transcripts were significantly downregulated, while expression of the tight junctions and cytoskeletal genes was upheld. In the absence of virus, rfhSP-D directly interacted with the EpiVaginal tissues and upregulated expression of genes related to structural stability of the cell and epithelial integrity. There was no increment in the viral acquisition by the PBMCs present in basal chambers wherein, the EpiVaginal tissues in apical chambers were treated with rfhSP-D. The effective concentrations of rfhSP-D had no effect on lactobacilli, epithelial barrier integrity and were safe on repeated applications onto the rabbit vaginal mucosa. This pre-clinical safety data, coupled with its efficacy of restricting viral passage via reversal of virus-induced gene expression of the vaginal barrier, make a strong argument for clinical trials of rfhSP-D as a topical anti-HIV microbicide. The authors thank HIV Research Trust, UK for providing scholarship to Hrishikesh Pandit and Brigham and Women’s Hospital (BWH) for permitting to visit and work in Fichorova lab. The work was partly supported by Medical Innovation Fund (Project no. 2011-16850) of Indian Council of Medical Research (ICMR), New Delhi, India. We are grateful to Director, NIRRH for providing financial support as the Institutional Grant and all the Institutional facilities for experimentation (Accession no. 618). The authors thank HIV Research Trust, UK for providing scholarship to Hrishikesh Pandit to work at the Fichorova's Laboratory. This study was partly supported by Medical Innovation Fund (Project no. 2011-16850) of Indian Council of Medical Research (ICMR), New Delhi, India. We are grateful to Director, NIRRH for providing financial support via the Institutional Grant and the Institutional facilities (Accession no. 618).

The complement system is an important humoral immune surveillance mechanism against tumours. However, many malignant tumours are resistant to complement mediated lysis. Here, we report secretion of complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma multiforme (GBM) patients. We investigated whether the secreted FHR5 exhibited functional activity similar to factor H, including inhibition of complement mediated lysis, acting as a co-factor for factor I mediated cleavage of C3b, and decay acceleration of C3 convertase. Immunoblotting analysis of primary GBM cells (B30, B31 and B33) supernatant showed the active secretion of FHR5, but not of Factor H. ELISA revealed that the secretion of soluble GBM-FHR5 by cultured GBM cells increased in a time-dependent manner. Primary GBM-FHR5 inhibited complement mediated lysis, possessed co-factor activity for factor I mediated cleavage and displayed decay acceleration of C3 convertase. In summary, we detected the secretion of FHR5 by primary GBM cells B30, B31 and B33. The results demonstrated that GBM-FHR5 shares biological function with FH as a mechanism primary GBM cells potentially use to resist complement mediated lysis. peerReviewed

Surfactant protein D (SP-D), a C-type collagen containing lectin (collectin), is expressed in the mucosal secretion of the lung and contribute to the innate host defence against a variety of pathogens, including influenza A virus (IAV). SP-D has been shown to inhibit haemagglutination activity and infectivity of IAV, in addition to reducing neuraminidase (NA) activity. SP-D exhibits a strong anti-IAV activity by virtue of its carbohydrate recognition domain (CRD) binding to carbohydrate pattern (N-linked mannosylated) on NA and hemagglutinin (HA) of the IAV. Here, we demonstrate that a recombinant fragment of human SP-D (rfhSP-D), containing homotrimeric neck and CRD regions, acts as an entry inhibitor of IAV and down-regulates M1 expression considerably in A549 cells infected with pH1N1 as well as H3N2 IAV strains at 2h treatment. In addition, rfhSP-D down-regulated mRNA levels of TNF-α, IFN-a, IFN-b, IL-6 and RANTES production, as judged by qPCR, particularly during the initial stage of IAV infection of A549 cell line. rfhSP-D also interfered with IAV infection of Madin-Darby canine kidney (MDCK) cells through HA1 binding, as confirmed by luciferase reporter assay and far western blotting. Furthermore, rfhSP-D was found to reduce luciferase reporter activity of MDCK cells transduced with H1+N1 pseudotyped lentiviral particles in a dose-dependent manner, where 50% of reduction was observed with 10 μg/ml rhfSP-D. Thus, binding of rfhSP-D to HA1 and reduction in luciferase reporter activity are suggestive of a critical role of rfhSP-D in mediating the inhibition of IAV infectivity and that of pesudotyped lentivirus as an entry inhibitor. Multiplex cytokine array revealed that rfhSP-D treatment of IAV challenged A549 cells led to a dramatic suppression of some of the key pro-inflammatory cytokines and chemokines in the virus challenged A549 cells. In the case of pH1N1, soluble factors such as TNF-a, IFN- a, IL-10, IL-12 (p40), VEGF, GM-CSF and eotaxin were considerably suppressed by rfhSP51 D treatment at 24h. However, these suppressive effects of IL-10, VEGF, eotaxin and IL-12 (p40) were not so evident in the case of H3N2 strain at the secreted protein level, with the exception of TNF-a, IFN-a, and GM-CSF. These data seem to suggest that the extent of immunomodulatory effect of SP-D on host cells can vary considerably in a strain-specific manner. Thus, rfhSP-D treatment can downregulate pro-inflammatory milieu encouraged by IAV via aberrant inflammatory cell recruitment leading to cell death and other possible long term immune defects and lung damage.

Copyright: © 2018 Kaur, Riaz, Murugaiah, Varghese, Singh and Kishore. Human Surfactant Protein (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53 mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated apoptosis pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53mt), MiaPaCa-2 (p53mt), and Capan-2 (p53wt) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of proapoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h, and subsequent, apoptosis of the cells. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan2 (~35%). Our studies appear to suggest that rfhSP-D can be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc. Surfactant protein D (SP-D), a C-type lectin and pattern-recognition soluble factor, plays an important role in immune surveillance to detect and eliminate human pulmonary pathogens. SP-D has been shown to protect against infections with the most ubiquitous airborne fungal pathogen, Aspergillus fumigatus, but the fungal surface component(s) interacting with SP-D is unknown. Here, we show that SP-D binds to melanin pigment on the surface of A. fumigatus dormant spores (conidia). SP-D also exhibited an affinity to two cell-wall polysaccharides of A. fumigatus, galactomannan (GM) and galactosaminogalactan (GAG). The immunolabeling pattern of SP-D was punctate on the conidial surface and was uniform on germinating conidia, in accordance with the localization of melanin, GM, and GAG. We also found that the collagen-like domain of SP-D is involved in its interaction with melanin, whereas its carbohydrate-recognition domain recognized GM and GAG. Unlike un-opsonized conidia, SP-D- opsonized conidia were phagocytosed more efficiently and stimulated the secretion of proinflammatory cytokines by human monocyte-derived macrophages. Furthermore, SP-D/ mice challenged intranasally with wildtype conidia or melanin ghosts (i.e. hollow melanin spheres) displayed significantly reduced proinflammatory cytokines in the lung compared with wildtype mice. In summary, SP-D binds to melanin present on the dormant A. fumigatus conidial surface, facilitates conidial phagocytosis, and stimulates the host immune response.

Mycobacterium tuberculosis can proficiently enter phagocytes and diminish complement 23 activation on its cell surface. Within phagocytes, the mycobacterium can suppress 24 macrophage apoptosis and survive the intracellular environment. Complement regulatory 25 proteins such as factor H may facilitate pathogen-macrophage interactions during 26 tuberculosis infection. In this study, we show that M. bovis BCG binds properdin, an up27 regulator of the complement alternative pathway. TSR4+5, a recombinant form of 28 thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an 29 inhibitor of the alternative pathway, was also able to bind M. bovis BCG. Properdin and 30 TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a 31 dose-dependent manner. Quantitative real-time PCR assays using transcripts from the THP-1 32 cells revealed elevated pro-inflammatory responses (TNF-α, IL-1β and IL-6) in the presence 33 of properdin and TSR4+5, which gradually decreased over 6 hours. Correspondingly, anti34 inflammatory responses (IL-10, TGF-β and IL-12) showed suppressed levels of expression in 35 the presence of properdin, which gradually increased over 6 hours. Multiplex cytokine array 36 analysis further revealed that properdin and TSR4+5 significantly enhanced the pro37 inflammatory response (TNF-α, IL-1β and IL-1α) at 24 hours, which declined at 48 hours, 38 whereas the anti-inflammatory response (IL-10 and IL-12) was suppressed. Our results 39 suggest that properdin may interfere with mycobacterial entry into macrophages involving 40 TSR4 and TSR5, particularly during the initial stages of infection, thus, affecting the 41 extracellular survival of the pathogen. This study offers novel insights into non-complement 42 related functions of properdin, which may be independent of other complement proteins 43 during host-pathogen interactions in tuberculosis. Thus, human properdin modulates 44 macrophage-M. bovis BCG interaction via TSR4+5. Properdin residing in the granules of 45 neutrophils is secreted upon stimulation and may also be produced by other cell types such as 3 monocytes, bone marrow progenitor cell lines and T cells. The local production 46 of properdin 47 may be crucial for recruitment at sites of infection and in the control of M. tuberculosis 48 infection.

International audience; Development of nanoparticles as tissue-specific drug delivery platforms can be consid erably influenced by the complement system because of their inherent pro-inflammatory and tumorigenic consequences. The complement activation pathways, and its recognition subcomponents, can modulate clearance of the nanoparticles and subsequent inflammatory response and thus alter the intended translational applications. Here, we report, for the first time, that human properdin, an upregulator of the complement alternative pathway, can opsonize functionalized carbon nanotubes (CNTs) via its thrombospondin type I repeat (TSR) 4 and 5. Binding of properdin and TSR4+5 is likely to involve charge pattern/ polarity recognition of the CNT surface since both carboxymethyl cellulose-coated carbon nanotubes (CMC-CNT) and oxidized (Ox-CNT) bound these proteins well. Properdin enhanced the uptake of CMC-CNTs by a macrophage cell line, THP-1, mounting a robust pro-inflammatory immune response, as revealed by qRT-PCR, multiplex cytokine array, and NF-κB nuclear translocation analyses. Properdin can be locally synthesized by immune cells in an inflammatory microenvironment, and thus, its interaction with nanoparticles is of considerable importance. In addition, recombinant TSR4+5 coated on the CMC-CNTs inhibited complement consumption by CMC-CNTs, suggesting that nanoparticle decoration with TSR4+5, can be potentially used as a complement inhibitor in a number of pathological contexts arising due to exaggerated complement activation.

Complement factor H has been extensively studied since its discovery 50 years ago, and its role in the complement system is quite well established. It has another role, however, as a binding protein for the regulatory peptide adrenomedullin. Part of this role appears to be protection of adrenomedullin from proteolytic degradation. The binding interaction is unusual and merits further investigation. Adrenomedullin has potential therapeutic uses in diseases affecting the vasculature, and factor H has been administered with adrenomedullin in some animal models of disease.

C1q is the first recognition subcomponent of the complement classical pathway, which acts towards the clearance of pathogens and apoptotic cells. C1q is also known to modulate a range of functions of immune and non-immune cells, including their involvement in placental development and sensorial synaptic pruning. We have recently shown that C1q can promote tumour by encouraging their adhesion, migration and proliferation in addition to angiogenesis and metastasis. In this study, we have examined the role of C1q in the microenvironment of malignant pleuric mesothelioma (MPM), a rare form of cancer commonly associated with exposure to asbestos. We found that C1q was highly expressed in all MPM histotypes, particularly in epithelioid rather than in sarcomatoid histotype. C1q avidly bound high and low molecular weight hyaluronic acid via its globular domain. C1q bound to hyaluronic acid was able to induce adhesion and proliferation of mesothelioma cells via enhancement of ERK1/2, SAPK/JNK and p38 phosphorylation; however, it did not activate the complement cascade. Consistent with the modular organisation of the globular domain, we demonstrated that C1q may bind to hyaluronic acid through ghA module, whereas it may interact with human mesothelioma cells through the ghC. In conclusion, C1q highly expressed in MPM binds to hyaluronic acid and enhances the tumour growth promoting cell adhesion and proliferation. These data can help develop novel diagnostic markers and molecular targets for MPM.

Anti-phospholipid syndrome (APS) is a complex autoimmune disease, associated with recurrent venous and arterial thrombosis in various tissues. APS is associated with specific antibodies against plasma beta-2 glycoprotein 1 (β2-GP1), and these antibodies react with β2-GP1 bound to negatively charged phospholipids (e.g. cardiolipin) on cell membranes. Some APS patients also have autoantibodies to complement factor H (FH), a homologue of β2-GP1, which also binds to anionic phospholipids. β2-GP1 has earlier been shown to inhibit the intrinsic (contact) activated blood coagulation pathway, promoted by anionic phospholipids. Here we examine whether FH could have similar anti-thrombotic properties. In vitro experiments with surface-bound phospholipids and human plasma, in the presence of FH, confirm this hitherto unreported property of FH.

Bradykinin (BK) is one of the most potent vasodilator agonists known and belongs to the kinin family of proinflammatory peptides. BK induces its activity via two G protein–coupled receptors: BK receptor 1 (B1R) and BK receptor 2. Although BK receptor 2 is constitutively expressed on endothelial cells (ECs), B1R is induced by IL-1β. The C1q receptor, receptor for the globular heads of C1q (gC1qR), which plays a role in BK generation, is expressed on activated ECs and is also secreted as soluble gC1qR (sgC1qR). Because sgC1qR can bind to ECs, we hypothesized that it may also serve as an autocrine/paracrine signal for the induction of B1R expression. In this study, we show that gC1qR binds to ECs via a highly conserved domain consisting of residues 174–180, as assessed by solid-phase binding assay and deconvolution fluorescence microscopy. Incubation of ECs (24 h, 37°C) with sgC1qR resulted in enhancement of B1R expression, whereas incubation with gC1qR lacking aa 174–180 and 154–162 had a diminished effect. Binding of sgC1qR to ECs was through surface-bound fibrinogen and was inhibited by anti-fibrinogen. In summary, our data suggest that, at sites of inflammation, sgC1qR can enhance vascular permeability by upregulation of B1R expression through de novo synthesis, as well as rapid translocation of preformed B1R.

Complement protein C1q is the first recognition subcomponent of the complement classical pathway that plays a vital role in the clearance of immune complexes, pathogens, and apoptotic cells. C1q also has a homeostatic role involving immune and non-immune cells; these functions not necessarily involve complement activation. Recently, C1q has been shown to be expressed locally in the microenvironment of a range of human malignant tumors, where it can promote cancer cell adhesion, migration, and proliferation, without involving complement activation. C1q has been shown to be present in the ascitic fluid formed during ovarian cancers. In this study, we have examined the effects of human C1q and its globular domain on an ovarian cancer cell line, SKOV3. We show that C1q and the recombinant globular head modules induce apoptosis in SKOV3 cells in a time-dependent manner. C1q expression was not detectable in the SKOV3 cells. Exogenous treatment with C1q and globular head modules at the concentration of 10 µg/ml induced apoptosis in approximately 55% cells, as revealed by immunofluorescence microscopy and FACS. The qPCR and caspase analysis suggested that C1q and globular head modules activated tumor necrosis factor (TNF)-α and upregulated Fas. The genes of mammalian target of rapamycin (mTOR), RICTOR, and RAPTOR survival pathways, which are often overexpressed in majority of the cancers, were significantly downregulated within few hours of the treatment of SKOV3 cells with C1q and globular head modules. In conclusion, C1q, via its globular domain, induced apoptosis in an ovarian cancer cell line SKOV3 via TNF-α induced apoptosis pathway involving upregulation of Bax and Fas. This study highlights a potentially protective role of C1q in certain cancers.

International audience

C1q is the recognition subunit of the classical pathway of the complement system and a major connecting link between classical pathway-driven innate immunity and IgG- or IgM-mediated acquired immunity. The basic structural subunit of C1q is composed of an N-terminal triple-helical collagen-like region and a C-terminal heterotrimeric globular head domain (gC1q) that is made up of individual A, B, and C chains. Recent crystallographic studies have revealed that the gC1q domain, which is the main target-binding region of C1q, has a compact and spherical heterotrimeric assembly, held together by both electrostatic and nonpolar interactions, with quasi-3-fold symmetry. A characteristic feature of the gC1q domain is the presence of a exposed Ca(2+) located near the apex. We have investigated, using theoretical and experimental approaches, the role of Ca(2+) in the electrostatic stability and target-binding properties of the native C1q as well as recombinant monomeric forms of the C-terminal regions of the A, B, and C chains. Here, we report that Ca(2+) primarily influences the target recognition properties of C1q toward IgG, IgM, C-reactive protein, and pentraxin 3. At pH 7.4, the loss of Ca(2+) leads to changes in the direction of electric moment from coaxial (where the putative C-reactive protein-binding site is located) to perpendicular to the molecular axis (toward the most likely IgG-binding site), which appears important for target recognition by C1q and subsequent complement activation.

This investigation was aimed to study the effect of 2,3-dimercapto-1-propanesulfonic acid (DMPS) on mercuric chloride (HgCl2)-induced alterations in urinary excretion of various carnitine fractions including free carnitine (FC), acylcarnitine (AC), and total carnitine (TC). Different groups of Wistar male rats were treated with HgCl2 at the doses of 0.1, 0.5, 1.0, 2.0, and 3.0 mg/kg body weight, and the animals were sacrificed at 24 hours following HgCl2 injection. A separate batch of animals received HgCl2 (2 mg/kg) with or without DMPS (100 mg/kg) and sacrificed at 24 or 48 hours after dosing. Administration of HgCl2 resulted in statistically significant and dose-dependent increase in the urinary excretion of FC, AC, and TC in rats. However, the ratio of urinary AC:FC was significantly decreased by HgCl2. Pretreatment with DMPS offered statistically significant protection against HgCl2-induced alterations in various urinary carnitine fractions in rats.

Gram-negative bacteria can bind complement protein C1q in an antibody-independent manner and activate classical pathway via their lipopolysaccharides (LPS). Earlier studies have implicated the collagen-like region of human C1q in binding LPS. In recent years, a number of C1q target molecules, previously considered to interact with collagen-like region of C1q, have been shown to bind via the globular domain (gC1q). Here we report, using recombinant forms of the globular head regions of C1q A, B and C chains, that LPS derived from Salmonella typhimurium interact specifically with the B-chain of the gC1q domain in a calcium-dependent manner. LPS and IgG-binding sites on the gC1q domain appear to be overlapping and this interaction can be inhibited by a synthetic C1q inhibitor, suggesting common interacting mechanisms.

Classical complement pathway is an important innate immune mechanism, which is usually triggered by binding of C1q to immunoglobulins, pentraxins and other target molecules. Although the activation of the classical pathway is crucial in the host defence, its undesirable and uncontrolled activation can lead to tissue damage. Thus, understanding the molecular basis of complement activation and its inhibition are of great biomedical importance. Recently, we proposed a mechanism for target recognition and classical pathway activation by C1q, which is likely governed by calcium-controlled reorientation of macromolecular electric moment vectors. Here we sought to define the mechanism of C1q inhibition by low molecular weight disulphate compounds that bind to the globular (gC1q) domain, using experimental, computational docking and theoretical modelling approaches. Our experimental results demonstrate that betulin disulphate (B2S) and 9,9-bis(4'-hydroxyphenyl)fluorene disulphate (F2S) inhibit the interaction of C1q and its recombinant globular modules with target molecules IgG1, C-reactive protein (CRP) and long pentraxin 3 (PTX3). In most C1q-inhibitor docked complexes, there is a reduction of electric moment scalar values and similarly altered direction of electric/dipole moment vectors. This could explain the inhibitory effect by impaired electrostatic steering, lacking optimal target recognition and formation of functional complex. In the presence of the inhibitor, the tilt of gC1q domains is likely to be blocked by the altered direction of the electric moment vector. Thus, the transition from the inactive (closed) towards the active (open) conformation of C1q (i.e. the complement activation signal transmission) will be impaired and the cascade initiation disrupted. These results could serve as a starting point for the exploration of a new form of ‘electric moment inhibitors/effectors’. Copyright © 2007 John Wiley & Sons, Ltd.

Surfactant proteins SP-A and SP-D are pattern recognition innate immune molecules that belong to the C-type lectin family. In lungs, they play an important role in the clearance of pathogens and control of inflammation. SP-A and SP-D are also expressed in the female reproductive tract where they play an important role in pregnancy and parturition. However, the role of SP-A and SP-D expressed at the feto-maternal interface (decidua) remains unclear. Here, we have examined the expression of SP-A and SP-D in the murine decidua at 17.5 (pre-parturition) and 19.5dpc (near parturition) and their effect on lipopolysaccharide (LPS)-treated decidual macrophages. SP-A and SP-D were localized to stromal cells in the murine decidua at 17.5 and 19.5dpc in addition to cells lining the maternal spiral artery. Purified pre-parturition decidual cells were challenged with LPS with and without SP-A or SP-D, and expression of F4/80 and TNF-α were measured by flow cytometry. On their own, SP-A or SP-D did not affect the percentage of F4/80 positive cells while they suppressed the percentage of TNF-α positive cells. However, simultaneous addition of SP-A or SP-D, together with LPS, reduced TNF-α secreting F4/80 positive cells. It is likely that exogenous administration of SP-A and SP-D in decidua can potentially control infection and inflammation mediators during spontaneous term labor and infection-induced preterm labor. Thus, the presence of SP-A and SP-D in the murine decidua is likely to play a protective role against intrauterine infection during pregnancy.

C1q is the first subcomponent of the classical complement pathway that binds antigen-bound IgG or IgM and initiates complement activation via association of serine proteases C1r and C1s. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has some structural and functional autonomy. Although a number of studies have tried to identify IgG-binding sites on the gC1q domain, no such attempt has been made to localize IgM-binding site. On the basis of the information available via the gC1q crystal structure, molecular modeling, mutational studies, and bioinformatics, we have generated a series of substitution mutants of ghA, ghB, and ghC and examined their interactions with IgM. The comparative analysis of IgM- and IgG-binding abilities of the mutants suggests that the IgG- and IgM-binding sites within the gC1q domain are different but may overlap. Whereas Arg(B108), Arg (B109), and Tyr(B175) mainly constitute the IgM-binding site, the residues Arg(B114), Arg(B129), Arg(B163), and His(B117) that have been shown to be central to IgG binding are not important for the C1q-IgM interaction. Given the location of Arg(B108), Arg (B109), and Tyr(B175) in the gC1q crystal structure, it is likely that C1q interacts with IgM via the top of the gC1q domain.

C1q is the first subcomponent of the classical complement pathway that can interact with a range of biochemically and structurally diverse self and nonself ligands. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has specific and differential binding properties toward C1q ligands. It is largely considered that C1q-ligand interactions are ionic in nature; however, the complementary ligand-binding sites on C1q and the mechanisms of interactions are still unclear. To identify the residues on the gC1q domain that are likely to be involved in ligand recognition, we have generated a number of substitution mutants of ghA, ghB, and ghC modules and examined their interactions with three selected ligands: IgG1, C-reactive protein (CRP), and pentraxin 3 (PTX3). Our results suggest that charged residues belonging to the apex of the gC1q heterotrimer (with participation of all three chains) as well as the side of the ghB are crucial for C1q binding to these ligands, and their contribution to each interaction is different. It is likely that a set of charged residues from the gC1q surface participate via different ionic and hydrogen bonds with corresponding residues from the ligand, instead of forming separate binding sites. Thus, a recently proposed model suggesting the rotation of the gC1q domain upon ligand recognition may be extended to C1q interaction with CRP and PTX3 in addition to IgG1.

Decorin and biglycan are closely related abundant extracellular matrix proteoglycans that have been shown to bind to C1q. Given the overall structural similarities between C1q and mannose-binding lectin (MBL), the two key recognition molecules of the classical and the lectin complement pathways, respectively, we have examined functional consequences of the interaction of C1q and MBL with decorin and biglycan. Recombinant forms of human decorin and biglycan bound C1q via both collagen and globular domains and inhibited the classical pathway. Decorin also bound C1 without activating complement. Furthermore, decorin and biglycan bound efficiently to MBL, but only biglycan could inhibit activation of the lectin pathway. Other members of the collectin family, including human surfactant protein D, bovine collectin-43, and conglutinin also showed binding to decorin and biglycan. Decorin and biglycan strongly inhibited C1q binding to human endothelial cells and U937 cells, and biglycan suppressed C1q-induced MCP-1 and IL-8 production by human endothelial cells. In conclusion, decorin and biglycan act as inhibitors of activation of the complement cascade, cellular interactions, and proinflammatory cytokine production mediated by C1q. These two proteoglycans are likely to down-regulate proinflammatory effects mediated by C1q, and possibly also the collectins, at the tissue level.

As a charge pattern recognition molecule, human C1q can bind a range of immunoglobulin and non-immunoglobulin ligands via its carboxy-terminal globular domain and activate the classical complement pathway. Each globular domain has a heterotrimeric organization, composed of the carboxy-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Recently, we have found that the recombinant forms of individual ghA, ghB and ghC bind differentially to IgG, IgM, gp41 peptide 601-613 of human immunodeficiency virus-1 (HIV-1), gp21 peptide 400-429 of human T cell lymphotrophic virus-I (HTLV-I), beta-amyloid peptide, and apoptotic cells, suggesting a modular organization of the globular domain. This paper examines the interaction of ghA, ghB and ghC with two known C1q ligands: Klebsiella pneumoniae porin OmpK36 and salivary agglutinin. In addition, we have used a panel of recombinant single-chain antibodies (scFv) specific for ghA, ghB and ghC in order to map sites on the heterotrimeric globular domain which are likely to interact with IgG1, IgG3, IgM, OmpK36, salivary agglutinin and gp41 loop peptide. The combined use of recombinant ghA, ghB, ghC and single-chain antibodies has revealed at least three ligand-binding sites on the globular domain of C1q: one is IgG- and OmpK36-specific, the second (IgM-binding site) is most likely overlapping with IgG/OmpK36 binding site, and the third (the gp41-binding site) seems to be located at the junction between the collagen and globular domains.

Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. We reported a novel anti-cancer role of a recombinant fragment of human SP-D (rhSP-D) in leukemic and breast tumor cell lines. A recent study revealed correlation of SP-D expression in Prostate cancer tissues with increased Gleason score and tumor volume. In the present study, we elucidated the role of rhSP-D in prostate cancer using LNCaP (androgen dependent), PC3 (androgen independent) cell lines and primary prostate cancer cells. In accordance with our previous finding, rhSP-D induced apoptosis in LNCaP and PC3 cell lines in a time and dose dependent manner. Isolated primary prostate cancer epithelial cells from explant cultures of tissue biopsies of prostate cancer patients were characterised for the presence of Cytokeratin (epithelial cell), CD10 (negative) and CD164 (positive) markers at protein and transcript level. Anti-prostate tumor effect of rhSP-D was established in the isolated primary prostate cancer epithelial cells. Importantly, primary normal prostate epithelial cells treated with similar concentrations of rhSP-D showed no adverse effect on viability. rhSP-D upregulated phospho p53 and transcripts of Bax and reduced Bcl2 transcripts, suggesting p53 mediated apoptosis in LNCaP cells. rhSP-D induced apoptosis in PC3 cells by lowering phospho ERK1/2 levels and increased BAD transcripts, a distinct mechanism of programmed cell death. Increased release of cytochrome c upon rhSP-D confirmed the activation of mitochondrial intrinsic apoptotic pathway in both the cell types. rhSP-D treatment downregulated transcripts of Bcl2 while upregulated PUMA transcripts, suggesting p53 mediated apoptosis primary prostate cancer cells. Also, positive TUNEL assay confirmed induction of apoptosis by rhSP-D in cancer tissue biopsies. Collectively, our findings reveal an integral role of SP-D in immune surveillance against prostate cancer mediated by two distinct mitochondrial apoptotic mechanisms.

Properdin, an upregulator of the alternative complement pathway, is central to deposition of the activated complement fragment C3b on the surfaces of the pathogens, which it achieves by preventing the dissociation of the Bb catalytic subunit from the inherently labile C3bBb complexes. It is also known to bind sulphated glycoconjugates, such as sulphatides. Properdin has an unusual structure formed by oligomerisation of a rod-like monomer into cyclic dimers, trimers and tetramers. The monomer (approximately 53 kDa) contains an N-terminal region of no known homology, followed by six non-identical repeats of 60 amino acids (based on exon/intron boundaries), called 'thrombospondin type I repeats' or TSR modules. We have expressed and purified the N-terminal region and each of the individual TSR repeats in Escherichia coli. Although the individual recombinant TSRs, after a denaturation-renaturation cycle, appeared to be correctly folded modules, as judged by the one-dimensional (1D)- and 2D-nuclear magnetic resonance spectra of TSR3, they did not show binding to either C3b or sulphatide. Polyclonal antibodies were raised against each TSR and were found to be module-specific. The anti-TSR5 polyclonal antibody was found to inhibit binding of native human properdin to solid-phase C3b, or sulphatides. It could also block properdin-dependent haemolysis of rabbit erythrocytes. These results are consistent with the view that the TSR5 contains the major site in properdin which is involved in both C3b and sulphatide binding. It also suggests that a co-operative intramolecular interaction between TSRs, as found in the native molecule, is required for TSR5 to bind either C3b or sulphatides.

The first step in the activation of the classical pathway of complement cascade by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of immunoglobulin G (IgG) or immunoglobulin M (IgM). The globular heads of C1q are located C-terminal to the six triple-helical stalks present in the molecule, each head is considered to be composed of the C-terminal halves (3 x 135 residues) of one A-, one B- and one C-chain. It is not known if the C-terminal globular regions, present in each of the three types of chains, are independently folded modules (with each chain having distinct binding properties towards immunoglobulins) or whether the different binding functions of C1q are dependent upon a globular structure which relies on contributions from all three chains. Recent reports of recombinant production and characterisation of soluble globular head regions of all the three chains indicate that the globular regions of C1q may adopt a modular organization, i.e., each globular head of C1q may be composed of three, structurally and functionally, independent domains, thus retaining multivalency in the form of a heterotrimer. Modules of the same type as the C1q C-terminal module are also found in a variety of noncomplement proteins that include the C-terminal regions of the human type VIII and type X collagens, precerebellin, the chipmunk hibernation proteins, the human endothelial cell protein, multimerin, the serum protein, Acrp-30 which is secreted from mouse adipocytes, and the sunfish inner-ear specific structural protein. The C1q molecule is the only one of these proteins for which, to date, a function has been ascribed to the module. The existence of a shared structural region between C1q and certain collagens may suggest an evolutionarily common ancestral precursor. Various structural and biochemical data suggest that these modules may be responsible for multimerisation through patches of aromatic residues within them.