Increased retinal drusen in IgA glomerulonephritis are further evidence for complement activation in disease pathogenesis.

Drusen are retinal deposits comprising cell debris, immune material and complement that are characteristic of macular degeneration but also found in glomerulonephritis. This was a pilot cross-sectional study to determine how often drusen occurred in IgA glomerulonephritis and their clinical significance. Study participants underwent non-mydriatic retinal photography, and their deidentified retinal images were examined for drusen by two trained graders, who compared central drusen counts, counts ≥ 10 and drusen size with those of matched controls. The cohort comprised 122 individuals with IgA glomerulonephritis including 89 males (73%), 49 individuals (40%) of East Asian or Southern European ancestry, with an overall median age of 54 years (34-64), and median disease duration of 9 years (4-17). Thirty-nine (33%) had an eGFR < 60 ml/min/1.73 m2 and 72 had previously reached kidney failure (61%). Overall mean drusen counts were higher in IgA glomerulonephritis (9 ± 27) than controls (2 ± 7, p < 0.001). Central counts ≥ 10 were also more common (OR = 3.31 (1.42-7.73, p = 0.006), and were associated with longer disease duration (p = 0.03) but not kidney failure (p = 0.31). Larger drusen were associated with more mesangial IgA staining (p = 0.004). Increased drusen counts were also present in IgA glomerulonephritis secondary to Crohn's disease but not with Henoch-Schonlein purpura. The finding of retinal drusen in IgA glomerulonephritis is consistent with complement activation and represents a model for better understanding glomerular immune deposition and a supporting argument for treatment with anti-complement therapies.

Complement C1s as a diagnostic marker and therapeutic target: Progress and propective.

The molecules of the complement system connect the effectors of innate and adaptive immunity and play critical roles in maintaining homeostasis. Among them, the C1 complex, composed of C1q, C1r, and C1s (C1qr2s2), is the initiator of the classical complement activation pathway. While deficiency of C1s is associated with early-onset systemic lupus erythematosus and increased susceptibility to bacteria infections, the gain-of- function variants of C1r and C1s may lead to periodontal Ehlers Danlos syndrome. As C1s is activated under various pathological conditions and associated with inflammation, autoimmunity, and cancer development, it is becoming an informative biomarker for the diagnosis and treatment of a variety of diseases. Thus, more sensitive and convenient methods for assessing the level as well as activity of C1s in clinic samples are highly desirable. Meanwhile, a number of small molecules, peptides, and monoclonal antibodies targeting C1s have been developed. Some of them are being evaluated in clinical trials and one of the antibodies has been approved by US FDA for the treatment of cold agglutinin disease, an autoimmune hemolytic anemia. In this review, we will summarize the biological properties of C1s, its association with development and diagnosis of diseases, and recent progress in developing drugs targeting C1s. These progress illustrate that the C1s molecule is an effective biomarker and promising drug target.

Ex Vivo Test for Measuring Complement Attack on Endothelial Cells: From Research to Bedside.

As part of the innate immune system, the complement system plays a key role in defense against pathogens and in host cell homeostasis. This enzymatic cascade is rapidly triggered in the presence of activating surfaces. Physiologically, it is tightly regulated on host cells to avoid uncontrolled activation and self-damage. In cases of abnormal complement dysregulation/overactivation, the endothelium is one of the primary targets. Complement has gained momentum as a research interest in the last decade because its dysregulation has been implicated in the pathophysiology of many human diseases. Thus, it appears to be a promising candidate for therapeutic intervention. However, detecting abnormal complement activation is challenging. In many pathological conditions, complement activation occurs locally in tissues. Standard routine exploration of the plasma concentration of the complement components shows values in the normal range. The available tests to demonstrate such dysregulation with diagnostic, prognostic, and therapeutic implications are limited. There is a real need to develop tools to demonstrate the implications of complement in diseases and to explore the complex interplay between complement activation and regulation on human cells. The analysis of complement deposits on cultured endothelial cells incubated with pathologic human serum holds promise as a reference assay. This ex vivo assay most closely resembles the physiological context. It has been used to explore complement activation from sera of patients with atypical hemolytic uremic syndrome, malignant hypertension, elevated liver enzymes low platelet syndrome, sickle cell disease, pre-eclampsia, and others. In some cases, it is used to adjust the therapeutic regimen with a complement-blocking drug. Nevertheless, an international standard is lacking, and the mechanism by which complement is activated in this assay is not fully understood. Moreover, primary cell culture remains difficult to perform, which probably explains why no standardized or commercialized assay has been proposed. Here, we review the diseases for which endothelial assays have been applied. We also compare this test with others currently available to explore complement overactivation. Finally, we discuss the unanswered questions and challenges to overcome for validating the assays as a tool in routine clinical practice.

Complement activation by RPE cells preexposed to TNFα and IFNγ.

Age-related macular degeneration (AMD) has been associated with both complement activation and increased levels of circulating cytokines. Here, we sougth to investigate if cytokine-preexposure of retinal pigment epithelial (RPE) leads to increased complement activation and deposition of membrane attack complex (MAC). Primary human RPE and the ARPE19 cell line cultured in serum-free conditions were preexposed to 100 ng/ml interferon-gamma (IFNγ) and 20 ng/ml tumor necrosis factor-alpha (TNFα) for 48 h followed by exposure to diluted serum from healthy donors or complement factor B deficient (CFBd) serum for 70 min. Deposition of membrane attack complexes (MAC) was examined by use of a MAC-ELISA kit and by immunofluorescence. Eculizumab (anti-C5) was examined for its ability to prevent deposition of MAC on RPE cells exposed to serum. Lactatdehydrogenase (LDH) and thiazolyl blue tetrazolium bromide (MTT) assays were used to assess cellular metabolism and survival. MAC was deposited only on RPE preexposed to both IFNγ and TNFα. Lack of complement factor B or inhibition of C5 abrogated the MAC-deposition on RPE cells, while reconstitution of CFBd serum with CFB resulted in MAC-deposition. MAC-deposition resulted in RPE-release of LDH, but unaltered mitochondrial activity estimated by MTT. We conclude that preexposure of primary RPE and ARPE19 with inflammatory cytokines promoted alternative pathway activation of complement and deposition of MAC. This implies that circulating inflammatory mediators may increase susceptibility to local complement activation and MAC-deposition, which may represent an early event in the pathogenesis leading to AMD development.

Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression.

Complement has emerged as a component of tumor promoting inflammation. We conducted a systematic assessment of the role of complement activation and effector pathways in sarcomas. C3-/-, MBL1/2-/- and C4-/- mice showed reduced susceptibility to 3-methylcholanthrene sarcomagenesis and transplanted sarcomas, whereas C1q and factor B deficiency had marginal effects. Complement 3a receptor (C3aR), but not C5aR1 and C5aR2, deficiency mirrored the phenotype of C3-/- mice. C3 and C3aR deficiency were associated with reduced accumulation and functional skewing of tumor-associated macrophages, increased T cell activation and response to anti-PD-1 therapy. Transcriptional profiling of sarcoma infiltrating macrophages and monocytes revealed the enrichment of MHC II-dependent antigen presentation pathway in C3-deficient cells. In patients, C3aR expression correlated with a macrophage population signature and C3 deficiency-associated signatures predicted better clinical outcome. These results suggest that the lectin pathway and C3a/C3aR axis are key components of complement and macrophage-mediated sarcoma promotion and immunosuppression.

The Benefits of Complement Measurements for the Clinical Practice.

The complement cascade is an evolutionary ancient innate immune defense system, playing a major role in the defense against infections. Its function in maintaining host homeostasis on activated cells has been emphasized by the crucial role of its overactivation in ever growing number of diseases, such as atypical hemolytic uremic syndrome (aHUS), autoimmune diseases as systemic lupus erythematosus (SLE), C3 glomerulopathies (C3GN), age-related macular degeneration (AMD), graft rejection, Alzheimer disease, and cancer, to name just a few. The last decade of research on complement has extended its implication in many pathological processes, offering new insights to potential therapeutic targets and asserting the necessity of reliable, sensitive, specific, accurate, and reproducible biomarkers to decipher complement role in pathology. We need to evaluate accurately which pathway or role should be targeted pharmacologically, and optimize treatment efficacy versus toxicity. This chapter is an introduction to the role of complement in human diseases and the use of complement-related biomarkers in the clinical practice. It is a part of a book intending to give reliable and standardized methods to evaluate complement according to nowadays needs and knowledge.

Complement-mediated release of fibroblast growth factor 2 from human RPE cells.

PURPOSE: Complement activation is associated with choroidal neovascularization (CNV) in age-related macular degeneration (AMD). Fibroblast growth factor 2 (FGF2) and membrane attack complex (MAC) are present in eyes of patients with CNV. Herein, we investigated the effect of complement activation on FGF2 release in human retinal pigment epithelial (RPE) cells. METHODS: Cultured human RPE cells were primed with an anti-RPE antibody and then treated with C1q-depleted human serum in the presence or absence of Tec kinases inhibitor (LFM-A13). 38 cytokines/chemokines levels were measured by Luminex technology. Secretion of FGF2 and interleukin (IL)-6 was assessed by ELISA. Tec protein was measured by Western blot. mRNA expression of FGF2, chemokine (C-X-C motif) ligand 1 (CXCL-1), and family members of Tec kinases was evaluated by qPCR. Cell viability and MAC deposition were determined by WST-1 assay and flow cytometry, respectively. RESULTS: Complement activation caused increased FGF2 and IL-6 release. FGF2 was released when C6-depleted human serum was reconstituted with C6. Anti-C5 antibody significantly attenuated complement-mediated FGF2 release, but not IL-6. FGF2 mRNA levels were not affected, while CXCL-1 mRNA levels were increased by complement activation. FGF2-containing extracellular vesicles were detected in response to complement challenge. Tec mRNA and protein were expressed in RPE cells. In the presence of LFM-A13, secretion of FGF2, but not IL-6, and MAC deposition were significantly decreased and cell viability was significantly increased in complement-treated cells when compared to controls. CONCLUSIONS: Complement plays an important role to release FGF2 from RPE cells. Tec kinase is involved in MAC formation and complement-mediated FGF2 release. This information suggests a role for complement activation to mediate neovascularization in conditions such as AMD, and may elucidate potential therapeutic targets.

The role of the lectin pathway of the complement system in SARS-CoV-2 lung injury.

Although some evidence showed the activation of complement systems in COVID-19 patients, proinflammatory status and lectin pathway remain unclear. Thus, the present study aimed to demonstrate the role of MBL and ficolin-3 in the complement system activation and compared to pandemic Influenza A virus H1N1 subtype infection (H1N1pdm09) and control patients. A total of 27 lungs formalin-fixed paraffin-embedded samples (10 from H1N1 group, 6 from the COVID-19 group, and 11 from the control group) were analyzed by immunohistochemistry using anti-IL-6, TNF-alfa, CD163, MBL e FCN3 antibodies. Genotyping of target polymorphisms in the MBL2 gene was performed by real-time PCR. Proinflammatory cytokines such as IL-6 and TNF-alpha presented higher tissue expression in the COVID-19 group compared to H1N1 and control groups. The same results were observed for ICAM-1 tissue expression. Increased expression of the FCN3 was observed in the COVID-19 group and H1N1 group compared to the control group. The MBL tissue expression was higher in the COVID-19 group compared to H1N1 and control groups. The genotypes AA for rs180040 (G/A), GG for rs1800451 (G/A) and CC for rs5030737 (T/C) showed a higher prevalence in the COVID-19 group. The intense activation of the lectin pathway, with particular emphasis on the MBL pathway, together with endothelial dysfunction and a massive proinflammatory cytokines production, possibly lead to a worse outcome in patients infected with SARS-Cov-2. Moreover, 3 SNPs of our study presented genotypes that might be correlated with high MBL tissue expression in the COVID-19 pulmonary samples.

Missing Self-Induced Activation of NK Cells Combines with Non-Complement-Fixing Donor-Specific Antibodies to Accelerate Kidney Transplant Loss in Chronic Antibody-Mediated Rejection.

BACKGROUND: Binding of donor-specific antibodies (DSAs) to kidney allograft endothelial cells that does not activate the classic complement cascade can trigger the recruitment of innate immune effectors, including NK cells. Activated NK cells contribute to microvascular inflammation leading to chronic antibody-mediated rejection (AMR). Recipient NK cells can also trigger antibody-independent microvascular inflammation by sensing the absence of self HLA class I molecules ("missing self") on allograft endothelial cells. This translational study investigated whether the condition of missing self amplifies DSA-dependent NK cell activation to worsen chronic AMR. METHODS AND RESULTS: Among 1682 kidney transplant recipients who underwent an allograft biopsy at Lyon University Hospital between 2004 and 2017, 135 fulfilled the diagnostic criteria for AMR and were enrolled in the study. Patients with complement-fixing DSAs identified by a positive C3d binding assay (n=73, 54%) had a higher risk of transplant failure (P=0.002). Among the remaining patients with complement-independent chronic AMR (n=62, 46%), those in whom missing self was identified through donor and recipient genotyping exhibited worse allograft survival (P=0.02). In multivariable analysis, only proteinuria (HR: 7.24; P=0.01) and the presence of missing self (HR: 3.57; P=0.04) were independent predictors for transplant failure following diagnosis of chronic AMR. Cocultures of human NK cells and endothelial cells confirmed that addition of missing self to DSA-induced NK cell activation increased endothelial damage. CONCLUSIONS: The assessment of missing self at the time of diagnosis of chronic AMR identifies patients at higher risk for kidney transplant failure.

Complement Activation Products and Cytokines in Pachychoroid Neovasculopathy and Neovascular Age-Related Macular Degeneration.

Purpose: To investigate the characteristics of complement activation products and angiogenic cytokines in the aqueous humor in eyes with pachychoroid neovasculopathy (PNV) and neovascular age-related macular degeneration (nAMD). Methods: This was a prospective, comparative, observational study. All patients with choroidal neovascularization were classified as PNV without polyps, PNV with polyps (polypoidal choroidal vasculopathy [PCV]), or drusen-associated nAMD according to the presence or absence of pachychoroid features and soft drusen. This study included a total of 105 eyes. Aqueous humor samples were collected from 25 eyes with PNV without polyps, 23 eyes with PCV, and 24 eyes with drusen-associated nAMD before intravitreal anti-vascular endothelial growth factor (VEGF) injection and cataract surgery in 33 control eyes. Clinical samples were measured for complement component 3a (C3a), C4a, C5a, VEGF, and macrophage chemoattractant protein 1 (MCP-1) using a bead-based immunoassay. Results: C3a and MCP-1 levels were significantly higher in PCV (P = 0.032 and P = 0.039, respectively) and drusen-associated nAMD (P = 0.01 for both comparisons) than in controls, and no difference was seen in C3a and MCP-1 levels between PNV and controls (P = 0.747 and P = 0.294, respectively). VEGF levels were significantly higher in PNV (P = 0.016), PCV (P = 0.009), and drusen-associated nAMD (P = 0.043) than in controls. In PNV, the VEGF levels elevated without elevated C3a and MCP-1. Conclusions: PNV, PCV, and drusen-associated nAMD had significantly distinct profiles of complement activation products and cytokines in the aqueous humor.

Modulation of complement activation by pentraxin-3 in prostate cancer.

Pentraxin 3 (PTX3) is an essential component of the innate immune system and a recognized modulator of Complement cascade. The role of Complement system in the pathogenesis of prostate cancer has been largely underestimated. The aim of our study was to investigate the role of PTX3 as possible modulator of Complement activation in the development of this neoplasia. We performed a single center cohort study; from January 2017 through December 2018, serum and prostate tissue samples were obtained from 620 patients undergoing prostate biopsy. A group of patients with benign prostatic hyperplasia (BPH) underwent a second biopsy within 12-36 months demonstrating the presence of a prostate cancer (Group A, n = 40) or confirming the diagnosis of BPH (Group B, N = 40). We measured tissue PTX3 protein expression together with complement activation by confocal microscopy in the first and second biopsy in group A and B patients. We confirmed that that PTX3 tissue expression in the first biopsy was increased in group A compared to group B patients. C1q deposits were extensively present in group A patients co-localizing and significantly correlating with PTX3 deposits; on the contrary, C1q/PTX3 deposits were negative in group B. Moreover, we found a significantly increased expression of C3a and C5a receptors within resident cells in group A patient. Interestingly, C1q/PTX3 deposits were not associated with activation of the terminal Complement complex C5b-9; moreover, we found a significant increase of Complement inhibitor CD59 in cancer tissue. Our data indicate that PTX3 might play a significant pathogenic role in the development of this neoplasia through recruitment of the early components of Complement cascade with hampered activation of terminal Complement pathway associated with the upregulation of CD59. This alteration might lead to the PTX3-mediated promotion of cellular proliferation, angiogenesis and insensitivity to apoptosis possible leading to cancer cell invasion and migration.

Biomarcadores y blancos moleculares del complemento en el diagnóstico de las microangiopatías trombóticas / Complement biomarkers and molecular targets in the diagnosis of thrombotic microangiopathies / Biomarcadores e alvos moleculares do complemento no diagnóstico das microangiopatias trombóticas

Resumen El sistema del complemento juega un papel central en la inmunidad innata, es una línea de defensa contra patógenos y participa en la homeostasis. La activación anormal del complemento contribuye al desarrollo de patologías de variable severidad, tanto inmunológicas y hematológicas como renales. Entre ellas, las microangiopatías trombóticas (MAT) representan un grupo de enfermedades raras con manifestaciones clínicas comunes caracterizadas por anemia hemolítica no inmune, trombocitopenia y daño de órgano(s) blanco. Si bien la clasificación de las MAT sigue siendo desafiante y no ha sido internacionalmente estandarizada, la descripción de entidades asociadas a anomalías del complemento fue comprobada con la eficiencia de la terapia anticomplemento en los pacientes. Las herramientas de diagnóstico desarrolladas en las últimas décadas son esenciales actualmente para diferenciar las MAT más características del grupo; esto es, la púrpura trombótica trombocitopénica (PTT) y el síndrome urémico hemolítico (SUH). En el presente trabajo se presenta una revisión del funcionamiento del sistema del complemento en condiciones fisiológicas, para poder explicar luego cuáles son las alteraciones del sistema implicadas en el desarrollo de las MAT y describir las herramientas disponibles para detectarlas en el laboratorio.

Abstract The complement system plays a crucial role in the innate immune response, being the first-line defense against pathogens and regulating homeostasis. Uncontrolled complement activation can cause immunologic, hematologic as well as renal syndromes of variable severity. Among them, thrombotic microangiopathies (TMA) represent a group of rare diseases characterised by similar clinical manifestations such as microangiopathic hemolytic anemia (MAHA), peripheral thrombocytopenia and organ injury. Although TMA classification is still challenging and no international consensus has been reached, complement-associated disorders have been described thanks to the efficiency of anti-complement therapy in patients. Diagnostic tools developed in the last decades are essential to differentiate the two most well characterized TMA: thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). This review will describe how the complement system works in physiological conditions in order to explain how complement abnormalities are involved in TMA, and finally how to detect those anomalies using laboratory tests.

Resumo O sistema do complemento desempenha um papel central na imunidade inata, sendo uma linha de defesa contra patógenos e participando da homeostase. A ativação anormal do complemento contribui para o desenvolvimento de patologias de gravidade variável, como imunológicas, hematológicas e renais. Entre elas, as microangiopatias trombóticas (MAT) representam um grupo de doenças raras com manifestações clínicas comuns caracterizadas por anemia hemolítica não imune, trombocitopenia e lesão de órgão(s) alvo. Embora a classificação das MAT continue sendo desafiadora e não tenha sido padronizada internacionalmente, a descrição de entidades associadas a anomalias do complemento foi comprovada com a eficiência da terapia anticomplemento nos pacientes. As ferramentas de diagnóstico desenvolvidas nas últimas décadas são atualmente essenciais para diferenciar as MAT mais características do grupo, que são a púrpura trombocitopênica trombótica (PTT) e a síndrome hemolítica urêmica atípica (SHU). Neste trabalho, é apresentada uma revisão do funcionamento do sistema de complemento em condições fisiológicas, a fim de explicar posteriormente quais são as alterações do sistema compreendidas no desenvolvimento das MAT, e descrever as ferramentas disponíveis para detectá-las em laboratório.

Activation of the alternative complement pathway predicts renal outcome in patients with lupus nephritis.

OBJECTIVES: The aims of this study were to clarify the activation of complement pathways in patients with lupus nephritis (LN), and to elucidate the association between these complement activation types and clinical outcomes. METHODS: We enrolled 115 patients with biopsy-proven LN from 2003 to 2016 from the lupus cohort at the Busan Paik Hospital and the Jeju National University Hospital in Korea. The patients were divided into two groups based on the patterns of glomerular complements deposits. The presence of C1q, C4 and/or C3 deposits in the glomerulus was considered evidence for the activation of the classical pathway with or without alternative pathway activation (group 1, N = 93), and glomerular C3 deposition without C1q and C4 deposits was considered as a marker for the alternative pathway limited activation (group 2, N = 22). The study end point was progression of kidney disease defined as a ≥50% reduction in estimated glomerular filtration rate from baseline values or advancement to end-stage renal disease. RESULTS: The mean estimated glomerular filtration rate and median urine protein-to-creatinine ratio of the patients were 85.7 ± 32.4 mL/min/1.73 m2 and 3.1 g/g, respectively, at the time of kidney biopsy. Forty-nine patients (43%) had nephrotic range of proteinuria. Compared to group 1 patients, those in group 2 were older, were more likely to be males and were more hypertensive. In addition, plasma C3 and C4 levels were significantly lower in group 1 patients compared to those in group 2. Moreover, anti-dsDNA concentration was significantly higher in group 1 patients compared to those in group 2. The mean follow-up time was 5.4 ± 3.4 years. The rates of response to one-year immunosuppressive treatment were poorer in group 2 patients compared to those in group 1. During the follow-up time, the progression of kidney disease was significantly higher in group 2 than in group 1 patients. CONCLUSION: This study showed that there was alternative complement pathway limited activation in the renal tissue in a small number of patients with LN, and these patients had worse renal outcomes compared to patients with glomerular classical complement pathway activation with or without alternative pathway activation.

Immunization Against Oxidized Elastin Exacerbates Structural and Functional Damage in Mouse Model of Smoke-Induced Ocular Injury.

Purpose: Age-related macular degeneration (AMD) is the leading cause of blindness in Western populations. While an overactive complement system has been linked to pathogenesis, mechanisms contributing to its activation are largely unknown. In aged and AMD eyes, loss of the elastin layer (EL) of Bruch's membrane (BrM) has been reported. Elastin antibodies are elevated in patients with AMD, the pathogenic significance of which is unclear. Here we assess the role of elastin antibodies using a mouse model of smoke-induced ocular pathology (SIOP), which similarly demonstrates EL loss. Methods: C57BL/6J mice were immunized with elastin or elastin peptide oxidatively modified by cigarette smoke (ox-elastin). Mice were then exposed to cigarette smoke or air for 6 months. Visual function was assessed by optokinetic response, retinal morphology by spectral-domain optical coherence tomography and electron microscopy, and complement activation and antibody deposition by Western blot. Results: Ox-elastin IgG and IgM antibodies were elevated in ox-elastin immunized mice following 6 months of smoke, whereas elastin immunization had a smaller effect. Ox-elastin immunization exacerbated smoke-induced vision loss, with thicker BrM and more damaged retinal pigment epithelium (RPE) mitochondria compared with mice immunized with elastin or nonimmunized controls. These changes were correlated with increased levels of IgM, IgG2, IgG3, and complement activation products in RPE/choroid. Conclusions: These data demonstrate that SIOP mice generate elastin-specific antibodies and that immunization with ox-elastin exacerbates ocular pathology. Elastin antibodies represented complement fixing isotypes that, together with the increased presence of complement activation seen in immunized mice, suggest that elastin antibodies exert pathogenic effects through mediating complement activation.

Anti-Factor B Antibodies and Acute Postinfectious GN in Children.

BACKGROUND: The pathophysiology of the leading cause of pediatric acute nephritis, acute postinfectious GN, including mechanisms of the pathognomonic transient complement activation, remains uncertain. It shares clinicopathologic features with C3 glomerulopathy, a complement-mediated glomerulopathy that, unlike acute postinfectious GN, has a poor prognosis. METHODS: This retrospective study investigated mechanisms of complement activation in 34 children with acute postinfectious GN and low C3 level at onset. We screened a panel of anticomplement protein autoantibodies, carried out related functional characterization, and compared results with those of 60 children from the National French Registry who had C3 glomerulopathy and persistent hypocomplementemia. RESULTS: All children with acute postinfectious GN had activation of the alternative pathway of the complement system. At onset, autoantibodies targeting factor B (a component of the alternative pathway C3 convertase) were found in a significantly higher proportion of children with the disorder versus children with hypocomplementemic C3 glomerulopathy (31 of 34 [91%] versus 4 of 28 [14%], respectively). In acute postinfectious GN, anti-factor B autoantibodies were transient and correlated with plasma C3 and soluble C5b-9 levels. We demonstrated that anti-factor B antibodies enhance alternative pathway convertase activity in vitro, confirming their pathogenic effect. We also identified crucial antibody binding sites on factor B, including one correlated to disease severity. CONCLUSIONS: These findings elucidate the pathophysiologic mechanisms underlying acute postinfectious GN by identifying anti-factor B autoantibodies as contributing factors in alternative complement pathway activation. At onset of a nephritic syndrome with low C3 level, screening for anti-factor B antibodies might help guide indications for kidney biopsy to avoid misdiagnosed chronic glomerulopathy, such as C3 glomerulopathy, and to help determine therapy.

Diagnostic and Risk Factors for Complement Defects in Hypertensive Emergency and Thrombotic Microangiopathy.

Hypertensive emergency can cause thrombotic microangiopathy (TMA) in the kidneys with high rates of end-stage renal disease (ESRD) and vice versa. The conundrum of hypertension as the cause of TMA or consequence of TMA on the background of defects in complement regulation remains difficult. Patients with hypertensive emergency and TMA on kidney biopsy were tested for ex vivo C5b9 formation on the endothelium and rare variants in complement genes to identify complement-mediated TMA. We identified factors associated with defects in complement regulation and poor renal outcomes. Massive ex vivo C5b9 formation was found on resting endothelial cells in 18 (69%) out of 26 cases at the presentation, including the 9 patients who carried at least one rare genetic variant. Thirteen (72%, N=18) and 3 (38%, N=8) patients with massive and normal ex vivo complement activation, respectively, progressed to ESRD (P=0.03). In contrast to BP control, inhibition of C5 activation prevented ESRD to occur in 5 (83%, N=6) patients with massive ex vivo complement activation. TMA-related graft failure occurred in 7 (47%, N=15) donor kidneys and was linked to genetic variants. The assessment of both ex vivo C5b9 formation and screening for rare variants in complement genes may categorize patients with hypertensive emergency and TMA into different groups with potential therapeutic and prognostic implications. We propose an algorithm to recognize patients at the highest risk for defects in complement regulation.

Complement and Coagulation: Cross Talk Through Time.

Two complex protein defense systems-complement and coagulation-are based on amplifying enzyme cascades triggered by specific local stimuli. Excess systemic activation of either system is pathologic and is normally prevented by a family of regulatory proteins. The 2 systems are ancient biological processes which share a common origin that predates vertebrate evolution. Recent research has uncovered multiple opportunities for cross talk between complement and coagulation including proteins traditionally viewed as coagulation factors that activate and regulate complement, and proteins traditionally seen as part of the complement system that participate in coagulation. Ten examples of cross talk between the 2 systems are described. The mutual engagement of both systems is increasingly recognized to occur in human diseases. Three conditions-paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and the antiphospholipid syndrome-provide examples of the importance of interactions between complement and coagulation in human biology. A better understanding of the mutual engagement of these 2 ancient defense systems is expected to result in improved diagnostics and new treatments for systemic diseases.

Exosomes Derived From Bone Marrow Mesenchymal Stem Cells Inhibit Complement Activation In Rats With Spinal Cord Injury.

PURPOSE: Spinal cord injury (SCI) is a relatively common, devastating traumatic condition resulting in permanent disability. In this study, the use of exosomes derived from bone mesenchymal stem cells (BMSCs-Exo) as a cell-free therapy for the treatment of SCI in rats was investigated to gain insights into their mechanisms of action. METHODS: Rats were randomly divided into three groups, Sham (treated with PBS), SCI (SCI injury + PBS) and SCI + Exo (SCI injury + BMSCs-Exo). Changes in the complement system between the three groups were assessed with the use of proteomics. The proteomic data were verified using reverse transcription-polymerase chain reaction (RT-PCR). In addition, the distributions of BMSCs-Exo in rats with SCI were detected by immunofluorescence. Moreover, SCI-activated NF-κB levels were determined using Western blot. RESULTS: SCI insult increased complement levels, including C4, C5, C6, C4 binding protein alpha and complement factor H. In contrast, the SCI + BMSCs-Exo group exhibited attenuated SCI-induced complement levels. Immunofluorescence assay results revealed that BMSCs-Exo mainly accumulated at the spinal cord injury site and were bound to microglia cells. Western blot analysis of tissue lysates showed that BMSCs-Exo treatment also inhibited SCI-activated nuclear factor kappa-B (NF-κB). CONCLUSION: BMSCs-Exo play a protective role in spinal cord injury by inhibiting complement mRNA synthesis and release and by inhibiting SCI-activated NF-κB by binding to microglia.

Complement activation sustains neuroinflammation and deteriorates adult neurogenesis and spatial memory impairment in rat hippocampus following sleep deprivation.

BACKGROUND: An association between neuroinflammation, reduced adult neurogenesis, and cognitive impairment has been established in sleep deprivation (SD). Complement receptors are expressed on neuronal and glial cells, thus, regulate the neuroinflammation, neurogenesis and learning/memory. However, understanding of the effect of SD on the brain-immune system interaction associated with cognitive dysfunction and its mechanisms is obscure. We hypothesized that complement activation induced changes in inflammatory and neurogenesis related proteins might be involved in the cognitive impairment during SD. METHODOLOGY: Adult male Sprague Dawley rats were used. Rats were sleep deprived for 48 h using a novel automated SD apparatus. Dosage of BrdU (50 mg/kg/day, i.p. in 0.07 N NaOH), complement C3a receptor antagonist (C3aRA; SB290157; 1 mg/kg/day, i.p.) in 1.16% v/v PBS and complement C5a receptor antagonist (C5aRA; W-54011; 1 mg/kg/day, i.p.) in normal saline were used. Rats were subjected to spatial memory evaluation following SD. Hippocampal tissue was collected for biochemical, molecular, and immunohistochemical studies. T-test and ANOVA were used for the statistical analysis. RESULTS: An up-regulation in the levels of complement components (C3, C5, C3a, C5a) and receptors (C3aR and C5aR) in hippocampus, displayed the complement activation during SD. Selective antagonism of C3aR/C5aR improved the spatial memory performance of sleep-deprived rats. C3aR antagonist (C3aRA) or C5aR antagonist (C5aRA) treatment inhibited the gliosis, maintained inflammatory cytokines balance in hippocampus during SD. Complement C3aR/C5aR antagonism improved hippocampal adult neurogenesis via up-regulating the BDNF level following SD. Administration of C3aRA and C5aRA significantly maintained synaptic homeostasis in hippocampus after SD. Gene expression analysis showed down-regulation in the mRNA levels of signal transduction pathways (Notch and Wnt), differentiation and axogenous proteins, which were found to be improved after C3aRA/C5aRA treatment. These findings were validated at protein and cellular level. Changes in the corticosterone level and ATP-adenosine-NO pathway were established as the key mechanisms underlying complement activation mediated consequences of SD. CONCLUSION: Our study suggests complement (C3a-C3aR and C5a-C5aR) activation as the novel mechanism underlying spatial memory impairment via promoting neuroinflammation and adult neurogenesis decline in hippocampus during SD, thereby, complement (C3aR/C5aR) antagonist may serve as the novel therapeutics to improve the SD mediated consequences.

Placental sFLT1 is associated with complement activation and syncytiotrophoblast damage in preeclampsia.

The immune complement system protects against pathogens; however, excess activation results in disease like hemolytic uremic syndrome, a clinical imitator of preeclampsia. Vascular endothelial factor (VEGF) protects against aberrant complement activation and is inhibited by soluble fms-like tyrosine kinase-1 (sFLT1) in other organs. We hypothesize that sFLT1 promotes complement-mediated placental damage through VEGF inhibition in preeclampsia. Objective: Quantify placental complement activity and sFLT1 expression in preeclampsia, and the subgroup of preeclampsia with hemolysis elevated liver enzymes low platelets (HELLP) syndrome. Methods: Placental complement activation marker C4d, membrane attack complex (MAC), and sFLT1 expression was quantified using immunofluores cence microscopy. Results: Placentas from 18 controls, 25 preeclampsia, including 6 cases of HELLP syndrome were identified. Placental C4d expression was greater in PE (median 6.4 [IQR: 5.1, 8.3]) compared to controls (4.4 [3.6, 5.5]; p = 0.003). MAC expression was also increased in preeclampsia compared to controls (6.5 [5.8, 8.7]; 5.4 [2.9, 5.9], p = 0.001). Placental sFLT1 expression was also higher in preeclampsia (p <0.0001). C4d and MAC were strongly correlated with sFLT1 levels in the placenta (R = 0.72; p < 0.0001 and R = 0.59; p = 0.01, respectively). Complement and sFLT1 expression was elevated in HELLP compared to preeclampsia without laboratory abnormalities, but this difference did not reach statistical significance. Conclusion: Increased placental complement activation and damage was seen in preeclampsia and correlates with sFLT1 expression. Our findings support the importance of the complement pathway in preeclampsia.