Complement Regulatory Proteins CD59 Research Articles

Navigating the Complement Pathway to Optimize PNH Treatment with Pegcetacoplan and Other Currently Approved Complement Inhibitors.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and potentially life-threatening hematologic disorder caused by a somatic mutation in a relevant portion of hematopoietic stem cells. Mutation of the phosphatidylinositol glycan biosynthesis class A (PIGA) gene prevents the expression of cell-surface proteins, including the complement regulatory proteins CD55 and CD59. With decreased or a lack of CD55 and CD59 expression on their membranes, PNH red blood cells become susceptible to complement-mediated hemolysis (symptoms of which include anemia, dysphagia, abdominal pain, and fatigue), leading to thrombosis. State-of-the-art PNH treatments act by inhibiting the dysregulated complement at distinct points in the activation pathway: late at the C5 level (C5 inhibitors, eculizumab, ravulizumab, and crovalimab), centrally at the C3 level (C3/C3b inhibitors and pegcetacoplan), and early at the initiation and amplification of the alternative pathway (factor B inhibitor, iptacopan; factor D inhibitor, danicopan). Through their differing mechanisms of action, these treatments elicit varying profiles of disease control and offer valuable insights into the molecular underpinnings of PNH. This narrative review provides an overview of the mechanisms of action of the six complement inhibitors currently approved for PNH, with a focus on the C3/C3b-targeted therapy, pegcetacoplan.

A Histological Analysis and Detection of Complement Regulatory Protein CD55 in SARS-CoV-2 Infected Lungs.

A complement imbalance in lung alveolar tissue can play a deteriorating role in COVID-19, leading to acute respiratory distress syndrome (ARDS). CD55 is a transmembrane glycoprotein that inhibits the activation of the complement system at the intermediate cascade level, blocking the activity of the C3 convertase. In our study, lung specimens from COVID-19 and ARDS-positive COVID+/ARDS+ patients were compared with COVID-19 and ARDS-negative COVID-/ARDS- as well as COVID-/ARDS+ patients. Histochemical staining and immunolabeling of CD55 protein were performed. The COVID-/ARDS- specimen showed higher expression and homogeneous distribution of glycosaminoglycans as well as compactly arranged elastic and collagen fibers of the alveolar walls in comparison to ARDS-affected lungs. In addition, COVID-/ARDS- lung tissues revealed stronger and homogenously distributed CD55 expression on the alveolar walls in comparison to the disrupted COVID-/ARDS+ lung tissues. Even though the collapse of the alveolar linings and the accumulation of cellular components in the alveolar spaces were characteristic of COVID+/ARDS+ lung tissues, evaluating CD55 expression could be relevant to understand its relation to the disease. Furthermore, targeting CD55 upregulation as a potential therapy could be an option for post-infectious complications of COVID-19 and other inflammatory lung diseases in the future.

Erythropoietin Effect on Complement Activation in Chronic Kidney Disease.

The complement system is an important part of innate immunity. Despite its known protective role, the complement system may contribute to increased inflammation and tissue injury in cases where its balanced activation is disrupted. The kidneys have been shown to be largely affected by complement dysregulation. The aim of the present study was to investigate the effect of erythropoietin administration, on the complement system, in chronic kidney disease patients. The study involved 20 patients with CKD who received erythropoietin and measurements of levels of complement factors C3a and C5a and complement regulatory proteins (CregPs) CD55, CD46, and CD59. An increase in serum C3a and C5a levels was observed in response to EPO therapy. The increase in C3a was statistically significant (p < 0.05) and concurrent with a statistically significant decrease in CD55 in CD4+ T cells (p < 0.05) and B cells (p < 0.05) and CD59 levels in CD4+ and CD8+ T cells (p < 0.05) at completion of EPO therapy compared with healthy controls. The above observations demonstrate that EPO induces complement activation in patients undergoing EPO therapy with a simultaneous restriction of CRegPs expression, thus possibly allowing the uncontrolled complement activation, which may contribute to tissue injury and disease progression.

Pozelimab, a human monoclonal immunoglobulin for the treatment of CHAPLE disease.

The complement is a crucial factor of the innate immune system. However, its activation can lead to various diseases, so it needs to be controlled. In mammals, surface-bound complement regulatory proteins safeguard cells from uncontrolled complement-mediated lysis. One of the human complement regulators is CD55, also known as the decay-accelerating factor (DAF), a single-chain, type I cell surface protein anchored to glycosylphosphatidylinositol (GPI). The genetic loss of the complement regulatory protein CD55 leads to a fatal illness known as CHAPLE disease. The complement and innate immunity become hyperactive in this disease, causing angiopathic thrombosis and protein-losing enteropathy. Patients with CHAPLE disease experience abdominal pain, nausea, vomiting, diarrhea, loss of appetite, weight loss, impaired growth, and swelling. This genetic condition has no known cure, and managing its symptoms can be challenging. Pozelimab, a human monoclonal immunoglobulin IgG4 antibody, is a drug that targets the terminal complement protein C5. The drug has a high affinity for both wild-type and variant human C5. Pozelimab has received designations such as fast track, orphan drug, and rare pediatric disease, making it a significant medical breakthrough. It is currently the only available treatment for this disease. In this review, we have summarized the preclinical and clinical data on pozelimab.

Abstract LB441: An intravenous compatible oncolytic vaccinia virus treated hepatic metastasis of colon cancer through extensive cancer cell killing and activation of cytotoxic effector differentiation

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and nearly 50% of CRC patients develop liver metastasis during the course of their disease. Treatment of colorectal cancer liver metastasis (CRLM) has been particularly challenging due to tolerogenic nature of liver immune microenvironment as well as an eventual development of therapy resistance to standard chemotherapy regimen. One promising area of cancer immunotherapy is oncolytic virotherapy that can selectively destroy tumor cells and stimulate anti-tumor immune response. In this study, we systemically administered mSJ-650, an intravenous compatible oncolytic Wyeth strain vaccinia virus that expresses human complement regulatory protein CD55 and murine GM-CSF in place of viral thymidine kinase gene and has a deletion of the viral K2L gene, and evaluated its anti-tumor efficacy in CRLM. Mouse CRLM was induced by intrasplenic injection of 5×105 MC38-OVA cancer cells. Following tumor nodules formation in the liver, low dose (1×106 pfu) or high dose (3×106 pfu) of mSJ-650 was administered multiple times via tail vein and liver was harvested for evaluation of anti-tumor efficacy. Flow cytometry analysis was performed to investigate changes in hepatic and tumor immune microenvironment upon mSJ-650 treatment. Liver and tumor tissue were stained for immunofluorescence imaging of viral replication within the tumor cells and subsequent oncolysis by the virus. Systemic administration of mSJ-650 showed potent anti-tumor efficacy and led to significant reduction in metastatic nodule formation in liver even at low dose. The anti-tumor efficacy was mediated by tumor cell-specific viral distribution and replication, followed by lytic egress of mSJ-650 from infected tumor cells. Anti-tumor efficacy of mSJ-650 also accompanied a dramatic shift in immunologically tolerant hepatic microenvironment to immune-stimulatory one by decreasing proportion of myeloid-derived suppressor cells, Kupffer cells, and monocyte-derived macrophages (MDMs) in the liver while increasing CD8+ T cell infiltration by 2~3 folds. Moreover, mSJ-650 treatment was associated with phenotypic change in hepatic immune cells, as evidenced by Kupffer cell and MDM polarization to M1 phenotype and elevated expression of activation markers and cytotoxic molecules in CD8+ T cells. Changes in hepatic immune microenvironment were also observed in tumor nodules, especially in those completely surrounded by adjacent liver tissue, suggesting that anti-tumor immune response in tumor bed is potentiated by reprogramming of tumor-adjacent hepatic immune microenvironment. Systemic administration of mSJ-650 demonstrated superior anti-tumor efficacy in CRLM which was mediated by direct tumor cell killing and tumor-adjacent hepatic immune microenvironment reprogramming by the virus. Citation Format: Eunhye Kim, Yeonsoo Yang, Solchan Won, Namhee Lee, Songyi Lee, Mi-Ju Park, Byung-Jin Jung, Yun-Kyoung Hong, Hang-Rae Kim, Dong-Sup Lee, Keunhee Oh. An intravenous compatible oncolytic vaccinia virus treated hepatic metastasis of colon cancer through extensive cancer cell killing and activation of cytotoxic effector differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB441.

Abstract LB354: A novel oncolytic vaccinia virus with intravenous compatibility evaded complement- and neutralizing antibody-mediated inhibition and provided superior anti-tumor activity to intra-tumoral administration in immunocompetent mice

Abstract Oncolytic viruses (OVs) are potent cancer therapeutics that can selectively kill tumor cells and promote anti-tumor immunity. mSJ-650 (Wyeth, K2L-, TK-, murine GM-CSF, human CD55) is the Wyeth strain of vaccinia virus with human complement regulatory protein CD55 incorporated on the intracellular mature virion membrane to evade complement-mediated attack during circulation. The virus also expresses mGM-CSF in replace of thymidine kinase to increase tumor selectivity and enhance immunogenicity. We previously reported that a single dose of mSJ-650 effectively evades complement and shows prominent anti-tumor efficacy in A549 and HCT116 xenograft model. Here, we further investigated the capability of mSJ-650 in promoting anti-tumor response after multiple systemic administration in immunocompetent breast cancer mouse model. In an orthotopic EMT6 breast cancer model, mSJ-650 or control mSJ-612 devoid of CD55 was intravenously injected at low dose (1x106 pfu) and high dose (3x106 pfu), twice weekly. mSJ-650 treatment dose-dependently suppressed tumor growth, showing superior antitumor efficacy compared to treatment of control mSJ-612. Moreover, multiple systemic administration of mSJ-650 was well-tolerated with no apparent signs of toxicity in serum tested via AST and ALT assay. To further elucidate whether mSJ-650 can evade neutralization by antibody and maintain its antitumor potency in vivo, we designed two experimental models. Firstly, endogenous vaccinia virus specific neutralizing antibody was generated by intravenous injection of mSJ-650 prior to tumor cell inoculation in tumor-bearing mouse, followed by systemic administration of mSJ-650 twice weekly. Second, serum containing vaccinia virus specific neutralizing antibody was intravenously injected 24 hours prior to mSJ-650 administration in tumor-bearing mouse. In both experimental models, mSJ-650 treated group significantly inhibited tumor growth despite the presence of neutralizing antibody, while control mSJ-612 treated group failed to suppress tumor growth. These results imply that mSJ-650 is capable of evading neutralization and maintaining its antitumor efficacy. Moreover, we performed multi-color flow cytometry analysis of tumor-infiltrating leukocytes to evaluate immunogenicity of mSJ-650. As a result, we observed a significant increase in recruitment of activated CD4+ and CD8+ T cells by 2-fold and high expression level of cytotoxic molecules (perforin, TNF-α, IFN-γ) in tumor-infiltrating T cells. In summary, multiple systemic administration of mSJ-650 markedly evaded neutralization and suppressed tumor growth by promoting anti-tumor response in immunocompetent breast cancer mouse model, thus implying that mSJ-650 is a promising candidate for cancer immunotherapy. Citation Format: Yeonsoo Yang, Solchan Won, Namhee Lee, Eunhye Kim, Songyi Lee, Mi-Ju Park, Byung-Jin Jung, Yun-Kyoung Hong, Hang-Rae Kim, Dong-Sup Lee, Keunhee Oh. A novel oncolytic vaccinia virus with intravenous compatibility evaded complement- and neutralizing antibody-mediated inhibition and provided superior anti-tumor activity to intra-tumoral administration in immunocompetent mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB354.

Metalloporphyrins Reduce Proteinuria in Podocyte Immune Injury: The Role of Metal and Porphyrin Moieties.

Depending on their central metal atom, metalloporphyrins (MPs) can attenuate or exacerbate the severity of immune-mediated kidney injury, and this has been attributed to the induction or inhibition of heme oxygenase (HO) activity, particularly the inducible isoform (HO-1) of this enzyme. The role of central metal or porphyrin moieties in determining the efficacy of MPs to attenuate injury, as well as mechanisms underlying this effect, have not been assessed. Using an antibody-mediated complement-dependent model of injury directed against rat visceral glomerular epithelial cells (podocytes) and two MPs (FePPIX, CoPPIX) that induce both HO-1 expression and HO enzymatic activity in vivo but differ in their chelated metal, we assessed their efficacy in reducing albuminuria. Podocyte injury was induced using rabbit immune serum raised against the rat podocyte antigen, Fx1A, and containing an anti-Fx1A antibody that activates complement at sites of binding. FePPIX or CoPPIX were injected intraperitoneally (5 mg/kg) 24 h before administration of the anti-Fx1A serum and on days 1, 3, 6, and 10 thereafter. Upon completion of urine collection on day 14, the kidney cortex was obtained for histopathology and isolation of glomeruli, from which total protein extracts were obtained. Target proteins were analyzed by capillary-based separation and immunodetection (Western blot analysis). Both MPs had comparable efficacy in reducing albuminuria in males, but the efficacy of CoPPIX was superior in female rats. The metal-free protoporphyrin, PPIX, had minimal or no effect on urine albumin excretion. CoPPIX was also the most potent MP in inducing glomerular HO-1, reducing complement deposition, and preserving the expression of the complement regulatory protein (CRP) CD55 but not that of CD59, the expression of which was reduced by both MPs. These observations demonstrate that the metal moiety of HO-1-inducing MPs plays an important role in reducing proteinuria via mechanisms involving reduced complement deposition and independently of an effect on CRPs.

Human papillomavirus-mediated expression of complement regulatory proteins in human cervical cancer cells

ObjectivesThis study aimed to evaluate the expression pattern of complement regulatory proteins (CRPs) CD46, CD59, and CD55 in HPV-positive (HPV+) & negative (HPV-) cervical cancer cell lines in search of a reliable differential biomarker. Study DesignWe analysed the expression of CRPs in HPV 16-positive SiHa cell line, HPV 18-positive HeLa cell line, and HPV-negative cell line C33a using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. ResultsWe observed a differential expression profile of CRPs in HPV+ and HPV- cervical cancer cell lines. The mRNA level of CD59 &CD55 showed a higher expression pattern in HPV+ cells when compared to HPV- cancer cells. However, flow cytometry-based experiments revealed that CD46 was preferentially expressed more in HPV 16-positive SiHa cells followed by HPV 18-positive HeLa cells when compared to HPV- C33a cells. Interestingly, confocal microscopy revealed a high level of CD59 expression in Hela cells and SiHa cells but low expression in HPV- C33a cells. In addition, HPV 18-positive HeLa cells expressed more CD55, which was lower in SiHa cells and very weak in C33a cells. ConclusionThe study demonstrates the differential expression of CRPs in both HPV+ and HPV- cervical cancer cells for the first time, and their potential to serve as an early diagnostic marker for cervical carcinogenesis.

Complement and complement regulatory proteins are upregulated in lungs of COVID-19 patients

We explored the pathological changes and the activation of local complement system in COVID-19 pneumonia. Lung paraffin sections of COVID-19 infected patients were analyzed by HE (hematoxylin-eosin) staining. The deposition of complement C3, the deposition of C3b/iC3b/C3d and C5b-9, and the expression of complement regulatory proteins, CD59, CD46 and CD55 were detected by immunohistochemistry. In COVID-19 patients’ lung tissues, fibrin exudation, mixed with erythrocyte, alveolar macrophage and shed pneumocyte are usually observed in the alveoli. The formation of an “alveolar emboli” structure may contribute to thrombosis and consolidation in lung tissue. In addition, we also found that compared to normal tissue, the lung tissues of COVID-19 patients displayed the hyper-activation of complement that is represented by extensive deposition of C3, C3b/iC3b/C3d and C5b-9, and the increased expression level of complement regulatory proteins CD55, and especially CD59 but not CD46. The thrombosis and consolidation in lung tissues may contribute to the pathogenesis of COVID-19. The increased expression of CD55 and CD59 may reflect a feedback of self-protection on the complement hyper-activation. Further, the increased C3 deposition and the strongly activated complement system in lung tissues may suggest the rationale of complement-targeted therapeutics in conquering COVID-19.

Relationship between the expression of complement inhibitory proteins and therapeutic efficacy of antibodies in breast cancer.

Complement regulatory proteins (mCRPs) CD55, CD46 and CD59 have been proposed as key elements in therapeutic resistance against cancer. mCRP-expressing tumor cells, in addition to hindering trastuzumab, pertuzumab and sacituzumab-govitecan therapeutic activity in breast cancer, can regulate biological processes that promote tumor progression. This review describes the structure of mCRPs and analyzes their expression using transcriptomic databases from breast cancer patients, in addition to collecting information on mCRPs interactions and signaling in tumor cells. Given that mCRPs are relevant targets, several strategies that have been explored for their inhibition and regulation in order to increase therapeutic efficacy and prevent cancer resistance and progression are described.

Lupus nephritis with corticosteroid responsiveness: molecular changes of CD46-mediated type 1 regulatory T cells.

The engagement of the complement regulatory proteins CD46 and CD3 in human CD4+ T cells induces the type 1 regulatory T cells (Tr1) and interleukin-10 (IL-10) secretion. This study aimed to elucidate the molecular changes of Tr1 cells through CD46 cytoplasmic Cyt1 tail in lupus nephritis (LN) respond to intravenous methylprednisolone (ivMP) therapy. We enrolled 40 pediatric patients with LN and 30 healthy controls. Clinical characteristics and peripheral blood mononuclear cells were collected before and 3 days after the administration of ivMP. Kidney specimens were taken from five LN and five minimal-change nephrotic syndrome patients. We found that defective CD46-mediated T-helper type 1 contraction (IL-10 switching) is present in active LN patients. The ivMP therapy enhanced LN remission, restored the production of IL-10, increased the CD46-Cyt1/Cyt2 ratio, AKT, and cAMP-responsive element-binding protein phosphorylation, and induced migration with the expression of chemokine receptor molecules CCR4, CCR6, and CCR7 of CD3/CD46-activated Tr1 cells. Pharmacologic interventions that alter the patterns of CD46-Cyt1/Cyt2 expression and the secretion of IL-10 by CD3/CD46-activated Tr1 cells can be used in patients with active LN. In patients with LN, ivMP was associated with increased IL-10 production and increased CD46-Cyt1/Cyt2 ratio and AKT phosphorylation by Tr1 cells, with enhanced potential to migration in response to CCL17. These results suggest that expression levels of CD46 isoforms Cyt1 and Cyt2 in CD4 + CD46 + Tr1 cells differ in patients with active LN but can be corrected by corticosteroid treatment. Enhancing the expression of functional CD4 + CD46 + Tr1 cells may be a useful therapeutic approach for LN.

Current Opinions on the Clinical Utility of Ravulizumab for the Treatment of Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder of hematopoietic stem cells genetically defined by the acquisition of somatic mutations in the X-linked phosphatidylinositol glycan anchor biosynthesis, class A (PIGA) gene. PIGA is essential for the synthesis of glycosyl phosphatidylinositol (GPI) anchor proteins and its mutations result in a deficiency of such molecules on the membrane of blood cells derived from the mutant clone. In particular, the lack of the GPI-linked complement regulatory proteins CD55 and CD59 is responsible for the increased sensitivity of PNH erythrocytes to complement-mediated destruction. Indeed, the classical clinical picture of PNH includes signs and symptoms of intravascular hemolysis along with variable degrees of cytopenia and a strong tendency to thrombosis, hallmarks of the disease. Before the introduction of anti-complement inhibitors, PNH was characterized by a high mortality primarily due to thrombotic events. The approval of the terminal anti-complement inhibitor eculizumab in 2007 introduced a paradigm shift in the treatment of the disease with improvement of the chronic hemolytic process and dramatic reduction of the thrombotic rate. However, eculizumab has a relatively short half-life when considering a life-long treatment, with obvious consequences as to the quality of life of treated patients necessitating relatively frequent drug administrations. Moreover, up to 30% of PNH patients undergoing eculizumab therapy show a suboptimal response, continuing to require red cell transfusions because of extravascular hemolysis or breakthrough hemolytic episodes. In 2019, the FDA approved the second-generation C5 inhibitor ravulizumab, a long-lasting agent with a better control of disease manifestations. Herein, we discuss the use of ravulizumab in PNH, its differences with first-generation C5 inhibitors, the research evidence supporting the safety and efficacy of this drug and its impact on costs for health systems and quality of life of PNH patients.

Anti-C2 Antibody ARGX-117 Inhibits Complement in a Disease Model for Multifocal Motor Neuropathy.

Background and ObjectivesTo determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN.MethodsiPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed.ResultsiPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs.DiscussionBinding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.

Interleukin-1\u03b2 and cathepsin D modulate formation of the terminal complement complex in cultured human disc tissue

PurposeFormation of terminal complement complex (TCC), a downstream complement system activation product inducing inflammatory processes and cell lysis, has been identified in degenerated discs. However, it remains unclear which molecular factors regulate complement activation during disc degeneration (DD). This study investigated a possible involvement of the pro-inflammatory cytokine interleukin-1β (IL-1β) and the lysosomal protease cathepsin D (CTSD).MethodsDisc biopsies were collected from patients suffering from DD (n = 43) and adolescent idiopathic scoliosis (AIS, n = 13). Standardized tissue punches and isolated cells from nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) were stimulated with 5% human serum (HS) alone or in combination with IL-1β, CTSD or zymosan. TCC formation and modulation by the complement regulatory proteins CD46, CD55 and CD59 were analysed.ResultsIn DD tissue cultures, IL-1β stimulation decreased the percentage of TCC + cells in AF and EP (P < 0.05), whereas CTSD stimulation significantly increased TCC deposition in NP (P < 0.01) and zymosan in EP (P < 0.05). Overall, the expression of CD46, CD55 and CD59 significantly increased in all isolated cells during culture (P < 0.05). Moreover, cellular TCC deposition was HS concentration dependent but unaffected by IL-1β, CTSD or zymosan.ConclusionThese results suggest a functional relevance of IL-1β and CTSD in modulating TCC formation in DD, with differences between tissue regions. Although strong TCC deposition may represent a degeneration-associated event, IL-1β may inhibit it. In contrast, TCC formation was shown to be triggered by CTSD, indicating a multifunctional involvement in disc pathophysiology.

Complement regulation in tenocytes under the influence of leukocytes in an indirect co-culture model.

The present in vitro study was undertaken to learn about the effects of leukocytes on tenocytes in respect to complement regulation simulating an inflammatory scenario of the traumatized tissue. Human hamstring tendon-derived tenocyte monolayers were co-cultured indirectly with human leukocytes (either Peripheral Blood Mononuclear Cells [PBMCs] or neutrophils) using a transwell system with/without (+ /wo) 10ng/ml tumor necrosis factor α (TNFα) for 4 and 24h. Tenocyte and leukocyte cell survival was assessed by live-dead assay. Tenocyte gene expression of TNFα, the anaphylatoxin receptor C5aR and the cytoprotective complement regulatory proteins (CRP) CD46, CD55 and CD59 was monitored using qPCR. TNFα was detected in the culture supernatants using ELISA. C5aR gene expression was significantly induced by TNFα after 4h, but impaired in the presence of leukocytes + TNFα after 24h. At 4h, PBMCs activated by TNFα induced the CRP CD46 gene expression. However, CD55 was significantly suppressed after 24h by neutrophils + /woTNFα. Leukocytes activated by TNFα decreased also significantly the gene expression of the more downstream acting CRP CD59 after 4h. TNFα gene expression and ELISA analysis revealed an amplified TNFα expression/release in tenocyte co-cultures with PBMC + /woTNFα, probably contributing to complement regulation. TNFα might represent a crucial soluble mediator exerting diverse time-dependent effects on tenocyte complement regulation.

Complement Regulation in Human Tenocytes under the Influence of Anaphylatoxin C5a.

A central part of the complement system, the anaphylatoxin C5a was investigated in this study to learn its effects on tenocytes in respect to understanding the potential expression of other crucial complement factors and pro-inflammatory mediators involved in tendinopathy. Human hamstring tendon-derived tenocytes were treated with recombinant C5a protein in concentrations of 25 ng/mL and 100 ng/mL for 0.5 h (early phase), 4 h (intermediate phase), and 24 h (late phase). Tenocytes survival was assessed after 24 h stimulation by live-dead assay. The gene expression of complement-related factors C5aR, the complement regulatory proteins (CRPs) CD46, CD55, CD59, and of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 was monitored using qPCR. Tenocytes were immunolabeled for C5aR and CD55 proteins. TNFα production was monitored by ELISA. Tenocyte survival was not impaired through C5a stimulation. Interestingly, the gene expression of C5aR and that of the CRPs CD46 and CD59 was significantly reduced in the intermediate and late phase, and that of TNFα only in an early phase, compared to the control group. ELISA analysis indicated a concomitant not significant trend of impaired TNFα protein synthesis at 4 h. However, there was also an early significant induction of CD55 and CD59 mediated by 25 ng/mL anaphylatoxin C5a. Hence, exposure of tenocytes to C5a obviously evokes a time and concentration-dependent response in their expression of complement and pro-inflammatory factors. C5a, released in damaged tendons, might directly contribute to tenocyte activation and thereby be involved in tendon healing and tendinopathy.

The expression of membrane-bound complement regulatory proteins CD46, CD55 and CD59 in oral lichen planus

ObjectiveTo investigate the expression levels of membrane-anchored complement regulatory proteins (mCRPs), CD46, CD55 and CD59 in oral lichen planus (OLP), and evaluate the activation status of complement. DesignThirty-seven cases of OLP patients (20 non-erosive OLP and 17 erosive OLP) and twenty healthy controls were recruited in this study. The proteins and mRNA expression levels of CD46, CD55 and CD59 in OLP tissues were detected by western blotting and RT-qPCR respectively, and the expression levels of complement C3 and sC5b-9 in OLP patients’ saliva were detected by ELISA to evaluate the activation status of complement. In addition, mucosa tissues of another 3 non-erosive OLP patients and another 3 healthy controls were collected, and the epithelial layer of two groups were separated to culture primary keratinocytes in vitro. Immunofluorescence was used to further detect the expression of mCRPs at the cellular level. ResultsThe levels of CD46, CD55 and CD59 in OLP tissues and cells were significantly decreased compared with those of the healthy control group, and the level of complement C3 in the patients’ saliva was significantly decreased, while the level of sC5b-9 was increased. ConclusionsThese results suggest that the reduced expression of mCRPs keeps the complement system in a continuously active state, which may be the reason of the persistent local immune inflammatory state in OLP. This study aimed to provide new insights for the etiology and therapy of OLP.

Broadly effective metabolic and immune recovery with C5 inhibition in CHAPLE disease.

Complement hyperactivation, angiopathic thrombosis, and protein-losing enteropathy (CHAPLE disease) is a lethal disease caused by genetic loss of the complement regulatory protein CD55 leading to overactivation of complement and innate immunity together with immunodeficiency due to immunoglobulin (Ig) wasting in the intestine. We report in vivo human data that we accumulated using the complement C5 inhibitor eculizumab for the medical treatment of CHAPLE patients and observed cessation of gastrointestinal pathology together with restoration of normal immunity and metabolism. We found that patients rapidly renormalized Ig concentrations and other serum proteins as revealed by aptamer profiling, re-established a healthy gut microbiome, discontinued Ig replacement and other treatments, and exhibited catch-up growth. Thus, we show blockade of C5 by eculizumab effectively re-establishes the regulation of the innate immune complement system to substantially reduce the pathophysiological manifestations of CD55 deficiency in humans.

Paroxysmal nocturnal hemoglobinuria: role of the complement system, pathogenesis, and pathophysiology.

The complement system is part of the innate immune response system, which comprises more than 50 distinct plasma and serum proteins that interact to opsonize pathogens (i.e., mark pathogens for destruction) and induce inflammatory responses to fight infection. The role of the complement system is 2-fold: immune surveillance and host defense. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, chronic, acquired, hematologic disease caused by somatic mutations in the gene PIGA in the hematopoietic stem cells. These stem cells produce abnormal clone blood cells that lack the complement regulatory proteins CD55 and CD59, causing the body to recognize these otherwise healthy red blood cells as damaged. The complement system destroys cells without these protective proteins, resulting in general hemolysis. PNH is characterized by fatigue; hemolytic anemia that can be severe and debilitating; increased lactic dehydrogenase level, reticulocyte count, and bilirubin level; propensity for thrombotic events; and renal dysfunction. Epidemiologic data, while sparse, suggest that an estimated 5,000-6,000 individuals in the United States are affected by PNH. If left untreated, PNH has a 10-year mortality rate of 29%, although the natural history of this disease has been recently altered by the introduction of complement inhibitors for the treatment of PNH. DISCLOSURES: This research was developed under a research contract between RTI Health Solutions and Apellis Pharmaceuticals and was funded by Apellis Pharmaceuticals. Bektas, Copley-Merriman, and Khan are employees of RTI Health Solutions. Sarda is an employee of Apellis Pharmaceuticals. Shammo consults for Apellis Pharmaceuticals.

IL-1/IL-1R signaling induced by all-trans-retinal contributes to complement alternative pathway activation in retinal pigment epithelium.

The underlying mechanisms of complement activation in Stargardt disease type 1 (STGD1) and age-related macular degeneration (AMD) are not fully understood. Overaccumulation of all-trans-retinal (atRAL) has been proposed as the pathogenic factor in both diseases. By incubating retinal pigment epithelium (RPE) cells with atRAL, we showed that C5b-9 membrane attack complexes (MACs) were generated mainly through complement alternative pathway. An increase in complement factor B (CFB) expression as well as downregulation of complement regulatory proteins CD46, CD55, CD59, and CFH were observed in RPE cells after atRAL treatment. Furthermore, interleukin-1β production was provoked in both atRAL-treated RPE cells and microglia/macrophages. Coincubation of RPE cells with interleukin-1 receptor antagonist (IL1Ra) and atRAL ameliorated complement activation and downregulated CFB expression by attenuating both p38 and c-Jun N-terminal kinase (JNK) signaling pathways. Our findings demonstrate that atRAL induces an autocrine/paracrine IL-1/IL-1R signaling to promote complement alternative pathway activation in RPE cells and provide a novel perspective on the pathomechanism of macular degeneration.