Human C1q Regulates Influenza A Virus Infection and Inflammatory Response via Its Globular Domain.

The Influenza A virus (IAV) is a severe respiratory pathogen. C1q is the first subcomponent of the complement system's classical pathway. C1q is composed of 18 polypeptide chains. Each of these chains contains a collagen-like region located at the N terminus, and a C-terminal globular head region organized as a heterotrimeric structure (ghA, ghB and ghC). This study was aimed at investigating the complement activation-independent modulation by C1q and its individual recombinant globular heads against IAV infection. The interaction of C1q and its recombinant globular heads with IAV and its purified glycoproteins was examined using direct ELISA and far-Western blotting analysis. The effect of the complement proteins on IAV replication kinetics and immune modulation was assessed by qPCR. The IAV entry inhibitory properties of C1q and its recombinant globular heads were confirmed using cell binding and luciferase reporter assays. C1q bound IAV virions via HA, NA and M1 IAV proteins, and suppressed replication in H1N1, while promoting replication in H3N2-infected A549 cells. C1q treatment further triggered an anti-inflammatory response in H1N1 and pro-inflammatory response in H3N2-infected cells as evident from differential expression of TNF-α, NF-κB, IFN-α, IFN-ß, IL-6, IL-12 and RANTES. Furthermore, C1q treatment was found to reduce luciferase reporter activity of MDCK cells transfected with H1N1 pseudotyped lentiviral particles, indicative of an entry inhibitory role of C1q against infectivity of IAV. These data appear to demonstrate the complement-independent subtype specific modulation of IAV infection by locally produced C1q.

Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells.

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 a potent innate immune molecule and offers protection against non-self and altered self, such as pathogens, allergens, and tumor. 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 characterized by varied expressions of estrogen (ER), progesterone (PR), and epidermal growth factor (EGF) receptors (HER2). The cell viability of HER2-overexpressing (SKBR3) and triple-positive (BT474) breast cancer cell lines [but not of a triple-negative cell line (BT20)] was reduced following rfhSP-D treatment at 24 h. 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 caspases 9 and 3 were detected in rfhSP-D-treated BT474 and SKBR3 cells, suggesting an involvement of the intrinsic apoptosis pathway. However, rfhSP-D-induced apoptosis was nullified in the presence of hyaluronic acid (HA) 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 HA showed decreased transcriptional levels of p53 when compared to cells treated with rfhSP-D only. Thus, HA appears to negate the anti-tumorigenic properties of rfhSP-D against HER2-overexpressing and triple-positive breast cancer cells.

Prognostic Value of Complement Properdin in Cancer.

The complement system is readily triggered by the presence of damage-associated molecular patterns on the surface of tumor cells. The complement alternative pathway provides rapid amplification of the molecular stress signal, leading to complement cascade activation to deal with pathogens or malignant cells. Properdin is the only known positive regulator of the alternative pathway. In addition, properdin promotes the phagocytic uptake of apoptotic T cells by macrophages and dendritic cells without activating the complement system, thus, establishing its ability to recognize "altered-self". Dysregulation of properdin has been implicated in substantial tissue damage in the host, and in some cases, chronic unresolved inflammation. A corollary of this may be the development of cancer. Hence, to establish a correlation between properdin presence/levels in normal and cancer tissues, we performed bioinformatics analysis, using Oncomine and UALCAN. Survival analyses were performed using UALCAN and PROGgeneV2 to assess if properdin can serve as a potential prognostic marker for human lung adenocarcinoma (LUAD), liver hepatocellular carcinoma (LIHC), cervical squamous cell carcinoma (CESC), and pancreatic adenocarcinoma (PAAD). We also analyzed levels of tumor-infiltrating immune cells using TIMER, a tool for characterizing immune cell composition in cancers. We found that in LUAD and LIHC, there was a lower expression of properdin in the tumors compared to normal tissues, while no significant difference was observed in CESC and PAAD. Survival analysis demonstrated a positive association between properdin mRNA expression and overall survival in all 4 types of cancers. TIMER analysis revealed that properdin expression correlated negatively with tumor purity and positively with levels of infiltrating B cells, cytotoxic CD8+ T cells, CD4+ helper T cells, macrophages, neutrophils and dendritic cells in LUAD, CESC and PAAD, and with levels of B cells, CD8+ T cells and dendritic cells in LIHC. Immunohistochemical analysis revealed that infiltrating immune cells were the most likely source of properdin in the tumor microenvironment. Thus, complement protein properdin shows promise as a prognostic marker in cancer and warrants further study.

Entry Inhibition and Modulation of Pro-Inflammatory Immune Response Against Influenza A Virus by a Recombinant Truncated Surfactant Protein D.

Surfactant protein D (SP-D) is expressed in the mucosal secretion of the lung and contributes to the innate host defense against a variety of pathogens, including influenza A virus (IAV). SP-D can inhibit hemagglutination and infectivity of IAV, in addition to reducing neuraminidase (NA) activity via its carbohydrate recognition domain (CRD) binding to carbohydrate patterns (N-linked mannosylated) on NA and hemagglutinin (HA) of 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 downregulates M1 expression considerably in A549 cells challenged with IAV of H1N1 and H3N2 subtypes at 2 h treatment. In addition, rfhSP-D downregulated mRNA levels of TNF-α, IFN-α, IFN-ß, IL-6, and RANTES, 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 HA binding. Furthermore, rfhSP-D was found to reduce luciferase reporter activity in MDCK cells transduced with H1+N1 pseudotyped lentiviral particles, where 50% of reduction was observed with 10 µg/ml rfhSP-D, suggestive of a critical role of rfhSP-D as an entry inhibitor against IAV infectivity. Multiplex cytokine array revealed that rfhSP-D treatment of IAV challenged A549 cells led to a dramatic suppression of key pro-inflammatory cytokines and chemokines. In the case of pH1N1, TNF-α, IFN-α, IL-10, IL-12 (p40), VEGF, GM-CSF, and eotaxin were considerably suppressed by rfhSP-D treatment at 24 h. However, these suppressive effects on IL-10, VEGF, eotaxin and IL-12 (p40) were not so evident in the case of H3N2 subtype, with the exception of TNF-α, IFN-α, 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 IAV subtype-specific manner. Thus, rfhSP-D treatment can downregulate pro-inflammatory milieu encouraged by IAV that otherwise causes aberrant inflammatory cell recruitment leading to cell death and lung damage.