Properdin Deficiency Impairs Phagocytosis and Enhances Injury at Kidney Repair Phase Post Ischemia-Reperfusion.

Properdin, a positive regulator of complement alternative pathway, participates in renal ischemia-reperfusion (IR) injury and also acts as a pattern-recognition molecule affecting apoptotic T-cell clearance. However, the role of properdin in tubular epithelial cells (TECs) at the repair phase post IR injury is not well defined. This study revealed that properdin knockout (PKO) mice exhibited greater injury in renal function and histology than wild-type (WT) mice post 72-h IR, with more apoptotic cells and macrophages in tubular lumina, increased active caspase-3 and HMGB1, but better histological structure at 24 h. Raised erythropoietin receptor by IR was furthered by PKO and positively correlated with injury and repair markers. Properdin in WT kidneys was also upregulated by IR, while H2O2-increased properdin in TECs was reduced by its small-interfering RNA (siRNA), with raised HMGB1 and apoptosis. Moreover, the phagocytic ability of WT TECs, analyzed by pHrodo Escherichia coli bioparticles, was promoted by H2O2 but inhibited by PKO. These results were confirmed by counting phagocytosed H2O2-induced apoptotic TECs by in situ end labeling fragmented DNAs but not affected by additional serum with/without properdin. Taken together, PKO results in impaired phagocytosis at the repair phase post renal IR injury. Properdin locally produced by TECs plays crucial roles in optimizing damaged cells and regulating phagocytic ability of TECs to effectively clear apoptotic cells and reduce inflammation.

Properdin Is a Modulator of Tumour Immunity in a Syngeneic Mouse Melanoma Model.

Background and Objectives: Tumours are often low immunogenic. The role of complement, an innate immune defence system, in tumour control has begun to be elucidated, but findings are conflicting. A role for properdin, an amplifier of complement activation, in tumour control has recently been implicated. Materials and Methods: Properdin-deficient and congenic wildtype mice were injected subcutaneously with B16F10 melanoma cells. Tumour mass and chemokine profile were assessed. The frequencies of CD45+CD11b+ Gr-1+ cells were determined from tumours and spleens, and CD206+ F4/80+ cells were evaluated in spleens. Sera were analysed for C5a, sC5b-9, and CCL2. Results: Whilst there was no difference in tumour growth at study endpoint, properdin-deficient mice had significantly fewer myeloid-derived suppressor cells (MDSCs) in their tumours and spleens. Splenic M2 type macrophages and serum levels of C5a, sC5b-9, and CCL2 were decreased in properdin-deficient compared to wildtype mice. Conclusions: The presence of intact complement amplification sustains an environment that lessens potential anti-tumour responses.

Dexamethasone acutely suppresses the anabolic SNAT2/SLC38A2 amino acid transporter protein in L6-G8C5 rat skeletal muscle cells.

Chronic metabolic acidosis plays a role in cachexia by enhancing total proteolysis in skeletal muscle. Glucocorticoid also triggers proteolysis and plays a permissive role in the effect of acidosis. The System A amino acid transporter SNAT2/SLC38A2 is ubiquitously expressed in mammalian cells including muscle, performing Na+-dependent active import of neutral amino acids, and is strongly inhibited by low pH. Exposure of rat skeletal muscle cell line L6-G8C5 to low pH rapidly inhibits SNAT2 transport activity and enhances total proteolysis rate. Pharmacological inhibition or silencing of SNAT2 also enhances proteolysis. This study tests the hypothesis that the glucocorticoid dexamethasone (DEX), like low pH, inhibits SNAT2 activity in L6-G8C5 myotubes, thus contributing to total proteolysis. Incubation with 500 nM DEX for 4 h reduced the System A amino acid transport rate to half the rate in control cultures. This inhibition depended on glucocorticoid receptor-mediated gene transcription, but SNAT2 mRNA levels were unaffected by DEX. In contrast, the SNAT2 protein assessed by immunoblotting was significantly depleted. The co-inhibitory effects of DEX and low pH on System A transport activity were additive in stimulating total proteolysis. In keeping with this mechanism, DEX's inhibitory effect on SNAT2 transport activity was significantly blunted by the proteasome inhibitor MG132. Proof of principle was achieved in similar experiments using recombinant expression of a GFP-tagged SNAT2 fusion protein in HEK293A cells. It is concluded that DEX acutely depletes the SNAT2 transporter protein, at least partly through proteasome-dependent degradation of this functionally important transporter.

Complement Properdin Determines Disease Activity in MRL/lpr Mice.

Background and objects: In systemic lupus erythematosus, circulating immune complexes activate complement and, when trapped in renal capillaries, cause glomerulonephritis. Mouse models have been used in the preclinical assessment of targeting complement activation pathways to manage chronic inflammation in lupus. Properdin is the only known positive regulator of complement activation, but its role in the severity of lupus nephritis has not been studied yet. Materials and Methods: Fully characterized properdin-deficient mice were crossed with lupus prone MRL/lpr mice on C57Bl/6 background. Results: Compared to MRL/lpr properdin wildtype mice, MRL/lpr properdin-deficient mice had significantly lower anti-DNA antibody titres, TNFα and BAFF levels in serum. The qualitative glomerulonephritic score was less severe and there was significantly less serum creatinine in MRL/lpr properdin-deficient mice compared to MRL/lpr properdin wildtype littermate mice. Conclusion: Properdin plays a significant role in the severity of lupus overall and specifically in the extent of glomerulonephritis observed in MRL/lpr mice. Because MRL/lpr properdin-deficient mice had lower levels of anti-DNA antibodies, inflammatory mediators and markers of renal impairment, the study implies that properdin could constitute a novel therapy target in lupus disease.

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.

In vitro Generation of Cytotoxic T Cells With Potential for Adoptive Tumor Immunotherapy of Multiple Myeloma.

Multiple myeloma is a life-threatening hematological malignancy, which is rarely curable by conventional therapies. Immunotherapy, using tumor antigen-specific, cytotoxic T-lymphocytes, may represent an alternative or additional treatment for multiple myeloma. In this study, we used hybrid cell lines, generated by fusion of an EBV B-lymphoblastoid cell line (B-LCL) and myeloma cells, to stimulate in vitro peripheral blood lymphocytes (PBLs) from patients with multiple myeloma. We investigated induction of antigen-specific, cytotoxic T-lymphocytes to the well-defined tumor associated antigens (TAAs) hTERT, MUC1, MAGE-C1 and CS1, which have been shown to be expressed in a high proportion of cases of multiple myeloma. HLA-A2-peptide pentamer staining, interferon-γ and perforin ELISpot assays, as well as cytotoxicity assays were used. Following several rounds of in vitro stimulation, the hybrid cell lines induced antigen-specific, cytotoxic T-lymphocytes to four candidate TAAs in PBLs from HLA-A2+ multiple myeloma patients, using known HLA-A2 restricted peptide epitopes of the TAAs. In contrast, the HLA-A2+ myeloma cell line U266 failed to induce antigen-specific, cytotoxic T-lymphocytes in vitro. Our data indicate that B-LCL/myeloma hybrid cell lines induce antigen-specific, cytotoxic T-lymphocytes in PBLs isolated from multiple myeloma patients in vitro and may represent a novel strategy for use in adoptive immunotherapy of multiple myeloma.

Human Properdin Opsonizes Nanoparticles and Triggers a Potent Pro-inflammatory Response by Macrophages without Involving Complement Activation.

Development of nanoparticles as tissue-specific drug delivery platforms can be considerably 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.

Experimentally-induced anti-myeloperoxidase vasculitis does not require properdin, MASP-2 or bone marrow-derived C5.

Anti-neutrophil cytoplasmic antibody vasculitis is a systemic autoimmune disease with glomerulonephritis and pulmonary haemorrhage as major clinical manifestations. The name reflects the presence of autoantibodies to myeloperoxidase and proteinase-3, which bind to both neutrophils and monocytes. Evidence of the pathogenicity of these autoantibodies is provided by the observation that injection of anti-myeloperoxidase antibodies into mice causes a pauci-immune focal segmental necrotizing glomerulonephritis which is histologically similar to the changes seen on renal biopsy in patients. Previous studies in this model have implicated the alternative pathway of complement activation and the anaphylatoxin C5a. Despite this progress, the factors that initiate complement activation have not been defined. In addition, the relative importance of bone marrow-derived and circulating C5 is not known. This is of interest given the recently identified roles for complement within leukocytes. We induced anti-myeloperoxidase vasculitis in mice and confirmed a role for complement activation by demonstrating protection in C3-deficient mice. We showed that neither MASP-2- nor properdin-deficient mice were protected, suggesting that alternative pathway activation does not require properdin or the lectin pathway. We induced disease in bone marrow chimaeric mice and found that circulating and not bone marrow-derived C5 was required for disease. We have therefore excluded properdin and the lectin pathway as initiators of complement activation and this means that future work should be directed at other potential factors within diseased tissue. In addition, in view of our finding that circulating and not bone marrow-derived C5 mediates disease, therapies that decrease hepatic C5 secretion may be considered as an alternative to those that target C5 and C5a. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Properdin provides protection from Citrobacter rodentium-induced intestinal inflammation in a C5a/IL-6-dependent manner.

Citrobacter rodentium is an attaching and effacing mouse pathogen that models enteropathogenic and enterohemorrhagic Escherichia coli in humans. The complement system is an important innate defense mechanism; however, only scant information is available about the role of complement proteins during enteric infections. In this study, we examined the impact of the lack of properdin, a positive regulator of complement, in C. rodentium-induced colitis. Following infection, properdin knockout (P(KO)) mice had increased diarrhea and exacerbated inflammation combined with defective epithelial cell-derived IL-6 and greater numbers of colonizing bacteria. The defect in the mucosal response was reversed by administering exogenous properdin to P(KO) mice. Then, using in vitro and in vivo approaches, we show that the mechanism behind the exacerbated inflammation of P(KO) mice is due to a failure to increase local C5a levels. We show that C5a directly stimulates IL-6 production from colonic epithelial cells and that inhibiting C5a in infected wild-type mice resulted in defective epithelial IL-6 production and exacerbated inflammation. These outcomes position properdin early in the response to an infectious challenge in the colon, leading to complement activation and C5a, which in turn provides protection through IL-6 expression by the epithelium. Our results unveil a previously unappreciated mechanism of intestinal homeostasis involving complement, C5a, and IL-6 during bacteria-triggered epithelial injury.

Exploring LPS-induced sepsis in rats and mice as a model to study potential protective effects of the nociceptin/orphanin FQ system.

The nociceptin receptor (NOP) and its ligand nociceptin/orphanin FQ (N/OFQ) have been shown to exert a modulatory effect on immune cells during sepsis. We evaluated the suitability of an experimental lipopolysaccharide (LPS)-induced sepsis model for studying changes in the nociceptin system. C57BL/6 mice BALB/c mice and Wistar rats were inoculated with different doses of LPS with or without a nociceptin receptor antagonist (UFP-101 or SB-612111). In C57BL/6 mice LPS 0.85 mg/kg injection produced no septic response, whereas 1.2mg/kg produced a profound response within 5h. In BALB/c mice, LPS 4 mg/kg produced no response, whereas 7 mg/kg resulted in a profound response within 24h. In Wistar rats LPS 15 mg/kg caused no septic response in 6/10 animals, whereas 25mg/kg resulted in marked lethargy before 24h. Splenic interleukin-1ß mRNA in BALB/c mice, and serum TNF-α concentrations in Wistar rats increased after LPS injection in a dose-dependent manner, but were undetectable in control animals, indicating that LPS had stimulated an inflammatory reaction. IL-1ß and TNF-α concentrations in LPS-treated animals were unaffected by administration of a NOP antagonist. Similarly NOP antagonists had no effect on survival or expression of mRNA for NOP or ppN/OFQ (the N/OFQ precursor) in a variety of tissues. In these animal models, the dose-response curve for LPS was too steep to allow use in survival studies and no changes in the N/OFQ system occurred within 24h. We conclude that LPS-inoculation in rodents is an unsuitable model for studying possible changes in the NOP-N/OFQ system in sepsis.

Properdin and factor h: opposing players on the alternative complement pathway "see-saw".

Properdin and factor H are two key regulatory proteins having opposite functions in the alternative complement pathway. Properdin up-regulates the alternative pathway by stabilizing the C3bBb complex, whereas factor H downregulates the pathway by promoting proteolytic degradation of C3b. While factor H is mainly produced in the liver, there are several extrahepatic sources. In addition to the liver, factor H is also synthesized in fetal tubuli, keratinocytes, skin fibroblasts, ocular tissue, adipose tissue, brain, lungs, heart, spleen, pancreas, kidney, muscle, and placenta. Neutrophils are the major source of properdin, and it is also produced by monocytes, T cells and bone marrow progenitor cell line. Properdin is released by neutrophils from intracellular stores following stimulation by N-formyl-methionine-leucine-phenylalanine (fMLP) and tumor necrosis factor alpha (TNF-α). The HEP G2 cells derived from human liver has been found to produce functional properdin. Endothelial cells also produce properdin when induced by shear stress, thus is a physiological source for plasma properdin. The diverse range of extrahepatic sites for synthesis of these two complement regulators suggests the importance and need for local availability of the proteins. Here, we discuss the significance of the local synthesis of properdin and factor H. This assumes greater importance in view of recently identified unexpected and novel roles of properdin and factor H that are potentially independent of their involvement in complement regulation.

Antibody directs properdin-dependent activation of the complement alternative pathway in a mouse model of abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a complex inflammatory vascular disease. There are currently limited treatment options for AAA when surgery is inapplicable. Therefore, insights into molecular mechanisms underlying AAA pathogenesis may reveal therapeutic targets that could be manipulated pharmacologically or biologically to halt disease progression. Using an elastase-induced AAA mouse model, we previously established that the complement alternative pathway (AP) plays a critical role in the development of AAA. However, the mechanism by which complement AP is initiated remains undefined. The complement protein properdin, traditionally viewed as a positive regulator of the AP, may also initiate complement activation by binding directly to target surfaces. In this study, we sought to determine whether properdin serves as a focal point for the initiation of the AP complement activation in AAA. Using a properdin loss of function mutation in mice and a mutant form of the complement factor B protein that produces a stable, properdin-free AP C3 convertase, we show that properdin is required for the development of elastase-induced AAA in its primary role as a convertase stabilizer. Unexpectedly, we find that, in AAA, natural IgG antibodies direct AP-mediated complement activation. The absence of IgG abrogates C3 deposition in elastase-perfused aortic wall and protects animals from AAA development. We also determine that blockade of properdin activity prevents aneurysm formation. These results indicate that an innate immune response to self-antigens activates the complement system and initiates the inflammatory cascade in AAA. Moreover, the study suggests that properdin-targeting strategies may halt aneurysmal growth.

Absence of the lectin activation pathway of complement does not increase susceptibility to Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa remains one of the major clinical pathogens that burden immuno-compromised patients and patients with cystic fibrosis. The present study aimed to define the role of the lectin pathway of complement in the immune-defence against P. aeruginosa in a mouse model of invasive pneumonia. Using in vitro assays specific for each of the three complement pathways, we demonstrate that some strains of P. aeruginosa bind lectin pathway recognition sub-components and initiate complement activation in a lectin pathway-specific mode. All of the tested strains activated complement via classical and alternative pathways. We assessed the importance of lectin pathway activation in fighting P. aeruginosa infections by testing a lectin pathway activating strain in a mouse model of intra-nasal infection. MASP-2 (mannan binding lectin associated serine protease-2) deficient mice, which have no lectin pathway activity, had no significant survival disadvantage compared to wild type littermates (72.7% and 81.8% survival, respectively, p=0.48). Likewise, no difference in opsonising activity was seen between MASP-2 sufficient and MASP-2 deficient mouse sera. Moreover, cytokine expression profiles in the lungs of WT mice and MASP-2-/- mice were similar throughout the course of P. aeruginosa infection. We conclude that the lectin pathway does not play an essential role in fighting P. aeruginosa infection in mice.

Direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin.

An intact complement system is crucial for limiting West Nile virus (WNV) dissemination. Herein, we define how complement directly restricts flavivirus infection in an antibody-independent fashion. Mannose-binding lectin (MBL) recognized N-linked glycans on the structural proteins of WNV and Dengue virus (DENV), resulting in neutralization through a C3- and C4-dependent mechanism that utilized both the canonical and bypass lectin activation pathways. For WNV, neutralization occurred with virus produced in insect cells, whereas for DENV, neutralization of insect and mammalian cell-derived virus was observed. Mechanism of action studies suggested that the MBL-dependent neutralization occurred, in part, by blocking viral fusion. Experiments in mice showed an MBL-dependent accelerated intravascular clearance of DENV or a WNV mutant with two N-linked glycans on its E protein, but not with wild-type WNV. Our studies show that MBL recognizes terminal mannose-containing carbohydrates on flaviviruses, resulting in neutralization and efficient clearance in vivo.

Properdin deficiency in murine models of nonseptic shock.

Hereditary properdin deficiency is linked to susceptibility to meningococcal disease (Neisseria meningitidis serotypes Y and W-135) with high mortality. Its relative contribution toward the outcome of nonseptic shock has not been investigated. Using properdin-deficient C57BL/6 mice and their littermates, this study examines their survival of zymosan-induced and LPS-induced shock. Properdin-deficient mice were more resistant to zymosan shock compared with wild-type mice, which showed greater impairment of end-organ function 24 h after zymosan injection, higher TNF-alpha production by alveolar and peritoneal macrophages, higher TNF-alpha, and, inversely, lower IL-10 levels in peritoneal lavage and circulation and higher plasma C5a levels. Properdin-deficient mice showed significantly higher mortality in LPS shock, elevated TNF-alpha, and, inversely, reduced IL-10 production by peritoneal macrophages as well as lower plasma C5a levels compared with wild-type littermates. NO production by peritoneal macrophages and plasma alpha1-antitrypsin levels at 24 h after the injection of LPS or zymosan were decreased in properdin-deficient mice in both models, and fewer histopathologic changes in liver were observed in properdin-deficient animals. This study provides evidence that properdin deficiency attenuates zymosan-induced shock and exacerbates LPS-induced shock.

Functional characterization of human mannose-binding lectin-associated serine protease (MASP)-1/3 and MASP-2 promoters, and comparison with the C1s promoter.

The 5'-flanking regions of the genes encoding human mannose-binding lectin-associated serine protease (MASP)-1/3 and MASP-2, key enzymes in the lectin complement pathway, were isolated and characterized. The features of their promoters were compared with those of the human gene for C1s, the effector component of the classical pathway. The sequences upstream from the transcription start sites of the three genes contained the elements essential for transcription and liver-specific expression. Transient expression of constructs of these genes fused to the luciferase reporter gene confirmed their liver-specific expression and showed that the MASP promoters were slightly up-regulated by the presence of IL-1beta. The stimulatory effects of IL-1beta on MASP1/3 and MASP2 gene expression were abolished by the simultaneous presence of IL-6. MASP-1/3 promoter activity was also down-regulated by IFN-gamma. In contrast, C1s promoter activity was strongly up-regulated by IL-6, IL-1beta and IFN-gamma. These results indicate that IL-6 and IFN-gamma affect the expression of the MASP genes in a different fashion from that of the C1s gene, implying differential regulatory effects of these cytokines on the biosynthesis of lectin pathway-specific serine proteases and classical pathway-specific serine proteases.