Formation Of Membrane Attack Complex Research Articles

Astrocyte- and Neuron-Derived Extracellular Vesicles from Alzheimer's Disease Patients Effect Complement-Mediated Neurotoxicity.

We have previously shown that blood astrocytic-origin extracellular vesicles (AEVs) from Alzheimer’s disease (AD) patients contain high complement levels. To test the hypothesis that circulating EVs from AD patients can induce complement-mediated neurotoxicity involving Membrane Attack Complex (MAC) formation, we assessed the effects of immunocaptured AEVs (using anti-GLAST antibody), in comparison with neuronal-origin (N)EVs (using anti-L1CAM antibody), and nonspecific CD81+ EVs (using anti-CD81 antibody), from the plasma of AD, frontotemporal lobar degeneration (FTLD), and control participants. AEVs (and, less effectively, NEVs) of AD participants induced Membrane Attack Complex (MAC) expression on recipient neurons (by immunohistochemistry), membrane disruption (by EthD-1 assay), reduced neurite density (by Tuj-1 immunohistochemistry), and decreased cell viability (by MTT assay) in rat cortical neurons and human iPSC-derived neurons. Demonstration of decreased cell viability was replicated in a separate cohort of autopsy-confirmed AD patients. These effects were not produced by CD81+ EVs from AD participants or AEVs/NEVs from FTLD or control participants, and were suppressed by the MAC inhibitor CD59 and other complement inhibitors. Our results support the stated hypothesis and should motivate future studies on the roles of neuronal MAC deposition and AEV/NEV uptake, as effectors of neurodegeneration in AD.

Thrombotic microangiopathy in rheumatology: a link between thrombosis and autoimmunity

Uncontrolled hypercoagulation and inflammation (thromboinflammation), which are both independent and closely related and amplifying each other pathological processes, form the basis for pathogenesis of a wide range of diseases and complications, including immuno-inflammatory (autoimmune) rheumatic diseases, with the development of potentially fatal injuries of internal organs. Thrombotic microangiopathy is one of the most prominent prototypes of thromboinflammatory pathological conditions. The close link between environmental factors, hemostasis genetic defects and the complement system, inflammation and autoimmunity as pathogenetic mechanisms of microthrombosis draws particular attention to studying thrombotic microangiopathy in immuno-inflammatory rheumatic diseases, primarily systemic lupus erythematosus, antiphospholipid syndrome and scleroderma renal crisis. In future, these studies may be important for expanding the idea of the role of autoimmune mechanisms in pathogenesis of critical hemostasis disorders in human diseases, and for developing new approaches to therapy. Recently, special attention has been paid to the treatment of systemic lupus erythematosus and antiphospholipid syndrome with eculizumab, which is humanized monoclonal IgG2/4k antibody that blocks the complement component C5a and the membrane attack complex (C5b-9) formation, and which is registered for the treatment of atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, as well as severe forms of myasthenia gravis and neuromyelitis optica. Further studies in this direction will create prerequisites for improving the prognosis not only in patients with orphan disorders, but also for widespread human diseases.

Vascular endothelial cells evade complement‐mediated membrane injury via Weibel‐Palade body mobilization

BackgroundDefective complement inhibition can lead to the formation of membrane attack complexes (MAC; C5b‐9) on the plasma membranes of vascular endothelial cells, resulting in injury that drives the progression of thrombotic microangiopathy (TMA), a key pathology in kidney disease. Objective/MethodsWe examined the response of human endothelial cells to complement‐mediated damage using blood outgrowth endothelial cells (BOECs) derived from healthy donors. BOECs were sensitized to complement factors present in normal human serum to induce the formation of C5b‐9 on their plasma membranes. ResultsThis triggered an expected abrupt rise in intracellular Ca2+ reflecting membrane leakage. Remarkably, while intracellular Ca2+ remained elevated, membrane leakage ceased within 30 minutes, and cells did not show significant death. Extensive mobilization of Weibel‐Palade bodies (WPBs) was observed along with secretion of von Willebrand factor (VWF). The potential role of WPBs and VWF in mitigating complement‐mediated damage was examined by comparing the effects of C5b‐9 on BOECs derived from von Willebrand disease (VWD) patients expressing reduced amounts of VWF, lacking expression of functional VWF, or lacking both VWF and WPBs. BOECs lacking WPBs were not resistant to complement‐mediated damage, but became resistant when transfected to express VWF (and thus WPBs). ConclusionWe conclude that BOECs exposed to C5b‐9 attack respond by mobilizing WPBs, which mitigate and repair damage by fusing with the plasma membrane. We propose that a similar cell‐specific response may protect the vascular endothelium from complement‐mediated damage in vivo.

Cloning, expression profile of the complement component C9 gene and influence of the recombinant C9 protein on peripheral mononuclear leukocytes transcriptome in half-smooth tongue sole (Cynoglossus semilaevis)

The ninth complement component (C9) is a terminal complement component (TCC) that is involved in creating the membrane attack complex (MAC) on the target cell surface. In this study, the CsC9 (C9 of Cynoglossus semilaevis) cDNA sequence was cloned and characterized. The full-length CsC9 cDNA measured 2,150 bp, containing an open reading frame (ORF) of 1,803 bp, a 5′-untranslated region (UTR) of 24 bp and a 3′-UTR of 323 bp. A domain search revealed that the CsC9 protein contains five domains, including two TSP1s, an LDLRA, an EGF, and a MACPF. Quantitative real-time PCR analysis showed that CsC9 at the mRNA level was expressed in all the tested tissues, with the highest expression being observed in the liver. CsC9 expression is significantly upregulated in the tested tissues after challenge with Vibrio anguillarum. To further characterize the role of CsC9, peripheral blood mononuclear cells of C. semilaevis were used for transcriptome analysis after incubation with recombinant CsC9 (rCsC9) protein. A total of 3,775 significant differentially expressed genes (DEGs) were identified between the control and the rCsC9-treated group, including 2,063 upregulated genes and 1,712 downregulated genes. KEGG analyses revealed that the DEGs were enriched in cell adhesion molecules, cytokine-cytokine receptor interactions, T cell receptor signaling pathways, B cell receptor signaling pathways and Toll-like receptor signaling pathways. The results of this study indicate that in addition to participating in MAC formation, CsC9 might play multiple roles in the innate and adaptive immunity of C. semilaevis.

Will Complement Inhibition Be the New Target in Treating COVID-19-Related Systemic Thrombosis?

Will Complement Inhibition Be the New Target in Treating COVID-19-Related Systemic Thrombosis?

Atypical Hemolytic Uremic Syndrome Successfully Treated with Eculizumab

Atypical hemolytic uremic syndrome (aHUS) is a rare syndrome characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal injury, which results from uncontrolled complement activation. Delayed diagnosis and treatment of aHUS may result in end-stage renal disease (ESRD) and an associated dependence on dialysis. In extreme cases, it may cause death due to multi-organ failure. Eculizumab, a humanized monoclonal antibody against C5, inhibits the formation of the terminal membrane attack complex and is used to treat aHUS. Here, we report a 46-year-old male patient who suffered from aHUS relapse, despite prior treatment with repeated plasma exchange and hemodialysis. Eculizumab therapy improved his hematologic findings without use of hemodialysis.

Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

SUMMARYRetinal degeneration is a form of neurodegenerative disease and is the leading cause of vision loss globally. The Toll-like receptors (TLRs) are primary components of the innate immune system involved in signal transduction. Here we show that TLR2 induces complement factors C3 and CFB, the common and rate-limiting factors of the alternative pathway in both retinal pigment epithelial (RPE) cells and mononuclear phagocytes. Neutralization of TLR2 reduces opsonizing fragments of C3 in the outer retina and protects photoreceptor neurons from oxidative stress-induced degeneration. TLR2 deficiency also preserves tight junction expression and promotes RPE resistance to fragmentation. Finally, oxidative stress-induced formation of the terminal complement membrane attack complex and Iba1+ cell infiltration are strikingly inhibited in the TLR2-deficient retina. Our data directly implicate TLR2 as a mediator of retinal degeneration in response to oxidative stress and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology.

Characterization and expression analysis of the C8α and C9 terminal complement components from pufferfish (Takifugu rubripes)

C8α and C9 mediate the membrane attack complex formation and bacterial lysis and are important components in the complement system. The cDNA sequences of the C8α and C9 genes were cloned from Takifugu rubripes. The full-length cDNA of Tr-C8α was 1893 bp and included a 5′-UTR of 69 bp and 3′-UTR of 83 bp. The full-length cDNA of Tr-C9 was 2083 bp and included a 5′-UTR of 72 bp and 3′-UTR of 250 bp. The expression of Tr-C8α and Tr-C9 was detected in newly fertilized eggs of T. rubripes. The expression of these two genes was at a higher level in the liver than in other tissues tested. After lipopolysaccharide (LPS) challenge, the gene expression of Tr-C8α and Tr-C9 increased more significantly in the liver. With these combined results, we further understood how Tr-C8α and Tr-C9 function in the innate immunity of pufferfish. Our findings could deepen the understanding of immune regulation in pufferfish.

The role of protein disulphide-isomerase A3 as autoantigen in the pathogenesis of autoimmune thyroiditis and related brain damage in adult mice

Autoimmune thyroiditis (AIT)-related brain damage is one of most severe extrathyroidal manifestations of AIT, but the mechanism remains unclear. In this study, we confirmed that protein disulfide-isomerase A3 (PDIA3) is expressed in both thyroid and brain tissues of mouse, and found the significantly increased serum levels of anti-PDIA3 antibody (PDIA3Ab) in classical mouse models of thyroiditis. In addition, we investigated the PDIA3-specific autoimmune reaction in thyroid and brain tissues in a mouse model with high–serum PDIA3Ab induced by immunization with recombinant PDIA3 protein. PDIA3-immunized mice had elevated serum thyrotropin and impaired learning and memory. PDIA3-expressing cells had IgG deposition, and IgG colocalized with C3 in the thyroid and brain tissues of PDIA3-immunized mice, resulting in membrane attack complex formation. Our results suggest that PDIA3 protein may be a common autoantigen shared by the thyroid and brain tissues and involve in the thyroidal and intracerebral damage through activating complement system.

Improved Outcomes of Transplant Associated Thrombotic Microangiopathy with Early Initiation of Eculizumab

Background Transplant-associated thrombotic microangiopathy (TA-TMA) is a rare, potentially lethal complication of allogeneic stem cell transplant. It is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal dysfunction. Endothelial injury and terminal complement activation contribute to the pathogenesis of TA-TMA. Several risk factors have been identified including certain conditioning regimens, acute graft versus host disease (GvHD) and/or calcineurin inhibitors (CNI), and viral infections. Eculizumab, a monoclonal antibody directed against C5 which prevents the formation of the C5b-9 membrane attack complex, is effective in treatment of TA-TMA, but with significant infection risk. Here, we review our institution's experience with TA-TMA treated with eculizumab. Methods A retrospective review was performed on all adult and pediatric patients with TA-TMA treated at our institution with eculizumab since 2015. Results Five patients with TA-TMA ages 4.5, 8.5, 11.4, 33, and 37 years (2 female) were treated with eculizumab. All patients received myeloablative conditioning, three TBI-based. Donor source was haploidentical (n=2), matched-unrelated (n=2), and cord (n=1). All patients were on a CNI at diagnosis. TA-TMA therapy required cessation of CNI in 3 of 5 patients. Median day of diagnosis from transplant was 73 days (range 17-251). All patients had renal impairment, three requiring dialysis. Median days from TMA diagnosis to treatment with eculizumab was 3 (range 1-5). Median doses received was 6 (range 4 - >25). Resolution of hemolysis with eculizumab was brisk, ranging from 25-59 days. Figure 1 illustrates a representative trend with regards to LDH, Haptoglobin, CH50, and eculizumab dosing. Four of five patients were alive with a median follow-up of 10.3 months (range 1 - 45 months). The one death was due to Stage IV GvHD and candidemia, present before diagnosis of TMA. Infections were common, including viral and fungal. There were no deaths due to TMA. Two patients continue on dialysis 2.5 and 34.5 months after diagnosis. Discussion Our experience confirms variable clinical presentations, as our patients differed in presence and severity of acute GvHD, confounding role of CNI or triggering of TA-TMA by viral infections. All patients were started on eculizumab promptly following diagnosis, with a high awareness for and prophylaxis of potential infectious complications, which has mitigated the reported high mortality risk of TA-TMA.

Molecular characterization and induced expression analysis of the terminal complement component C9 in rohu,Labeo rohita

The complement component 9 (C9) plays a significant role in the formation of membrane attack complex (MAC) on the targeted cell surface. The current study is dealt with molecular characterization of C9 gene from rohu, Labeo rohita, an important cultured carp species in India. An open reading frame (ORF) of 1998 bp was amplified by polymerase chain reaction (PCR) that encodes a polypeptide of 666 amino acids having a signal peptide of 19 amino acids and a mature peptide of 647 amino acids. The SMART domain architecture analysis revealed two thrombospondin type-1 domains (TSP1), a low-density lipoprotein receptor domain class A (LDLa), a membrane attack complex and perforin (MACPF) domain, and an epidermal growth factor (EGF)-like domain. Multiple sequence alignment and evolutionary analysis revealed a primitive C9 sequence of rohu with maximum similarity and clustering with common carp, grass carp and zebrafish. C9 was highly expressed in liver and constitutively expressed in wide array of tissues except in eye of rohu juveniles. It was expressed during early developmental days of rohu including in milt. A variable level of up-regulation in C9 expression was noticed upon poly I:C induction, Aeromonas hydrophila and Argulus siamensis infections in liver, spleen and gill tissues of rohu at different time points. A constitutive expression of C9 in different stages of rohu during the ontogeny and in response to pathogen exposures along with high degree of sequence homology with other fish species proved it as an important primitive immune molecule of the complement system lytic pathway.

Use of the terminal complement inhibitor eculizumab in paediatric heart transplant recipients.

Antibody-mediated rejection is a major clinical challenge that limits graft survival. Various modalities of treatment have been reported in small studies in paediatric heart recipients. A novel approach is to use complement-inhibiting agents, such as eculizumab, which inhibits cleavage of C5 to C5a thereby limiting the formation of membrane attack complex and terminal complement-mediated injury of tissue-bound antibodies. This medical modality of treatment has theoretical advantages but the collective experience in its use in the solid organ transplant community remains small. We add to this experience by combining 14 cases from 6 paediatric heart centres in this descriptive study.

A Phase 2 Open-Label Study of Danicopan (ACH-0144471) in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Who Have an Inadequate Response to Eculizumab Monotherapy

A Phase 2 Open-Label Study of Danicopan (ACH-0144471) in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Who Have an Inadequate Response to Eculizumab Monotherapy

Mechanistic Evaluation of Efficacy Using Biomarkers of the Oral, Small Molecule Factor D Inhibitor, Danicopan (ACH-4471), in Untreated Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Mechanistic Evaluation of Efficacy Using Biomarkers of the Oral, Small Molecule Factor D Inhibitor, Danicopan (ACH-4471), in Untreated Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

CFH exerts anti-oxidant effects on retinal pigment epithelial cells independently from protecting against membrane attack complex

Age Related Macular Degeneration (AMD) is the first cause of social blindness in people aged over 65 leading to atrophy of retinal pigment epithelial cells (RPE), photoreceptors and choroids, eventually associated with choroidal neovascularization. Accumulation of undigested cellular debris within RPE cells or under the RPE (Drusen), oxidative stress and inflammatory mediators contribute to the RPE cell death. The major risk to develop AMD is the Y402H polymorphism of complement factor H (CFH). CFH interacting with oxidized phospholipids on the RPE membrane modulates the functions of these cells, but the exact role of CFH in RPE cell death and survival remain poorly understood. The aim of this study was to analyze the potential protective mechanism of CFH on RPE cells submitted to oxidative stress. Upon exposure to oxidized lipids 4-HNE (4-hydroxy-2-nonenal) derived from photoreceptors, both the human RPE cell line ARPE-19 and RPE cells derived from human induced pluripotent stem cells were protected from death only in the presence of the full length human recombinant CFH in the culture medium. This protective effect was independent from the membrane attack complex (MAC) formation. CFH maintained RPE cells tight junctions’ structure and regulated the caspase dependent apoptosis process. These results demonstrated the CFH anti-oxidative stress functions independently of its capacity to inhibit MAC formation.

Sulfated Dendritic Polyglycerol Is a Potent Complement Inhibitor.

The complement system is a powerful mechanism of the innate immune defense system. Dysregulation may contribute to several diseases. Heparin is a known regulator of the complement system, but its application is limited due to its anticoagulative activity. A promising alternative is the synthetic analogue dendritic polyglycerol sulfate (dPGS). Although dPGS-mediated inhibition of the classical and alternative pathway has been roughly described previously, here we analyzed the effects of dPGS regarding the three pathways at different levels of the proteolytic cascades for the first time. Regarding the final outcome (membrane attack complex formation), IC50 values for dPGS varied between the alternative (900 nM), the classical (300 nM), and the lectin pathway (60 nM). In a backward approach, processing of proteins C5 and C3 via the respective convertase was analyzed by ELISA to narrow down dPGS targets. A dose-dependent reduction of C5a and C3a levels was detected. Further, the analysis via surface plasmon resonance revealed novel dPGS binding proteins; the pro-inflammatory anaphylatoxins C3a and C5a and the classical pathway activator C1q showed down to nanomolar binding affinities. The fully synthetic multivalent polymer dPGS seems to be a promising candidate for the further development to counteract excessive complement activation in disease.

Combination treatment with lipoteichoic acids isolated from Lactobacillus plantarum and Staphylococcus aureus alleviates atopic dermatitis via upregulation of CD55 and CD59

The innate immune complement system helps clear invading pathogens by forming membrane attack complexes (MACs) on their surface. Abnormal activation of the complement system may aggravate atopic dermatitis (AD) symptoms in AD patients. Here, we investigated the anti-AD effects of LTAs isolated from Lactobacillus plantarum (pLTA) and Staphylococcus aureus (aLTA) by examination of complement regulatory proteins (CRPs). Combination treatment with pLTA and aLTA increased CD55 and CD59 production in HaCaT cells. The regulation of CD55 and CD59 was mediated by p38 mitogen-activated protein kinase (p38) signaling pathways in pLTA- and aLTA-treated cells. Complement-dependent cytotoxicity (CDC) and bactericidal assays revealed that combination treatment with pLTA and aLTA down-regulated the complement system. In experiments using an irritant contact dermatitis (ICD)-induced mouse model, the levels of MAC and C3 convertase (C3C) were lower in serum collected from pLTA- and aLTA-injected mice than in serum from mice who were untreated or received pLTA or aLTA alone. Combination treatment also inhibited IgE and CCL2 levels in ICD mice. On the other hand, IFN-γ level was significantly increased, indicating that combination treatment switches the Th2 response to a Th1 response. Our results suggest that combination treatment with LTAs could be a good therapeutic approach to alleviate AD by reducing formation of MACs and inducing a Th1 response.

Reduced membrane attack complex formation in umbilical cord blood during Eculizumab treatment of the mother: a case report

BackgroundAtypical hemolytic uremic syndrome (aHUS) is a disorder of the microvasculature with hemolytic anemia, thrombocytopenia and acute kidney injury. Nowadays, aHUS is successfully treated with eculizumab, a humanized, chimeric IgG2/4 kappa antibody, which binds human complement C5 and blocks generation of C5a and membrane-attack-complex.Case presentationA 25-year-old woman with end stage renal disease due to relapsing atypical hemolytic uremic syndrome had a relapse of the disease during pregnancy. She was treated with eculizumab. We measured reduced formation of the membrane-attack complex in newborn’s umbilical cord vein blood using the sensitive and specific Palarasah-Nielsen-ELISA.ConclusionsEculizumab treatment of the mother with end stage renal disease may cause reduced innate immunity which could render newborns more susceptible to infections.

Glycine, serine and threonine metabolism confounds efficacy of complement-mediated killing

Serum resistance is a poorly understood but common trait of some difficult-to-treat pathogenic strains of bacteria. Here, we report that glycine, serine and threonine catabolic pathway is down-regulated in serum-resistant Escherichia coli, whereas exogenous glycine reverts the serum resistance and effectively potentiates serum to eliminate clinically-relevant bacterial pathogens in vitro and in vivo. We find that exogenous glycine increases the formation of membrane attack complex on bacterial membrane through two previously unrecognized regulations: 1) glycine negatively and positively regulates metabolic flux to purine biosynthesis and Krebs cycle, respectively. 2) α-Ketoglutarate inhibits adenosine triphosphate synthase, which in together promote the formation of cAMP/CRP regulon to increase the expression of complement-binding proteins HtrE, NfrA, and YhcD. The results could lead to effective strategies for managing the infection with serum-resistant bacteria, an especially valuable approach for treating individuals with weak acquired immunity but a normal complement system.

Staphylococcal phosphatidylinositol-specific phospholipase C potentiates lung injury via complement sensitisation.

Staphylococcus aureus is frequently isolated from patients with community-acquired pneumonia and acute respiratory distress syndrome (ARDS). ARDS is associated with staphylococcal phosphatidylinositol-specific phospholipase C (PI-PLC); however, the role of PI-PLC in the pathogenesis and progression of ARDS remains unknown. Here, we showed that recombinant staphylococcal PI-PLC possesses enzyme activity that causes shedding of glycosylphosphatidylinositol-anchored CD55 and CD59 from human umbilical vein endothelial cell surfaces and triggers cell lysis via complement activity. Intranasal infection with PI-PLC-positive S. aureus resulted in greater neutrophil infiltration and increased pulmonary oedema compared with a plc-isogenic mutant. Although indistinguishable proinflammatory genes were induced, the wild-type strain activated higher levels of C5a in lung tissue accompanied by elevated albumin instillation and increased lactate dehydrogenase release in bronchoalveolar lavage fluid compared with the plc- mutant. Following treatment with cobra venom factor to deplete complement, the wild-type strain with PI-PLC showed a reduced ability to trigger pulmonary permeability and tissue damage. PI-PLC-positive S. aureus induced the formation of membrane attack complex, mainly on type II pneumocytes, and reduced the level of CD55/CD59, indicating the importance of complement regulation in pulmonary injury. In conclusion, S. aureus PI-PLC sensitised tissue to complement activation leading to more severe tissue damage, increased pulmonary oedema, and ARDS progression.