This study describes how the serum protein histidine-rich glycoprotein (HRG) affects the complement system. We show that HRG binds strongly to several complement proteins: C1q, factor H and C4b-binding protein and that it is found complexed with these proteins in human sera and synovial fluids of rheumatoid arthritis patients. HRG also binds C8 and to a lesser extent mannose-binding lectin, C4 and C3. However, HRG alone neither activates nor inhibits complement. Both HRG and C1q bind to necrotic cells and increase their phagocytosis. We found that C1q competes weakly with HRG for binding to necrotic cells whilst HRG does not compete with C1q. Furthermore, HRG enhances complement activation on necrotic cells measured as deposition of C3b. We show that HRG inhibits the formation of immune complexes of ovalbumin/anti-ovalbumin, whilst the reverse holds for C1q. Immune complexes formed in the presence of HRG show enhanced complement activation, whilst those formed in the presence of C1q show diminished complement activation. Taken together, HRG may assist in the maintenance of normal immune function by mediating the clearance of necrotic material, inhibiting the formation of insoluble immune complexes and enhancing their ability to activate complement, resulting in faster clearance.

Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. The complement system has been identified as one of the main AMD disease pathways. We performed a comprehensive expression analysis of 32 complement proteins in plasma samples of 255 AMD patients and 221 control individuals using mass spectrometry-based semi-quantitative multiplex profiling. We detected significant associations of complement protein levels with age, sex and body-mass index (BMI), and potential associations of C-reactive protein, factor H related-2 (FHR-2) and collectin-11 with AMD. In addition, we confirmed previously described associations and identified new associations of AMD variants with complement levels. New associations include increased C4 levels for rs181705462 at the C2/CFB locus, decreased vitronectin (VTN) levels for rs11080055 at the TMEM97/VTN locus and decreased factor I levels for rs10033900 at the CFI locus. Finally, we detected significant associations between AMD-associated metabolites and complement proteins in plasma. The most significant complement-metabolite associations included increased high density lipoprotein (HDL) subparticle levels with decreased C3, factor H (FH) and VTN levels. The results of our study indicate that demographic factors, genetic variants and circulating metabolites are associated with complement protein components. We suggest that these factors should be considered to design personalized treatment approaches and to increase the success of clinical trials targeting the complement system.

This study was aimed at examining the impact of common types of physical efforts used to determine the aerobic and anaerobic performance of the participants on the complement system in their peripheral blood. Fifty-one physically active young males aged 16 years old (range 15-21 years) were divided into two age groups (younger, 15-17 years old and older, 18-21 years old) and performed two types of intensive efforts: aerobic (endurance; 20-m shuttle run test; Beep) and anaerobic (speed; repeated speed ability test; RSA). Venous blood samples were collected before and after each exercise (5 and 60 min) to profile the complement system components, namely the levels of C2, C3, C3a, iC3b, and C4. The endurance effort caused a decrease in the post-test C3 (p < 0.001 for both age groups) and increase in post-test C3a (p < 0.001 and p < 0.01 for the younger and older group, respectively), recovery iC3b (p < 0.001 and p < 0.05 for younger and older group, respectively), recovery C2 (p < 0.01 for both age groups), and post-test C4 (p < 0.05 and p < 0.01 for the younger and older group, respectively) levels, while the speed effort caused a decrease only in the post-test C2 (p < 0.05 for younger participants) and post-test C4 levels (p < 0.001 and p < 0.01 for the younger and older group, respectively) and an increase in the recovery C3a level (p < 0.05). Our study provides evidence that different types of physical effort promote different immune responses in physically active young men. Aerobic exercise induced the activation of an alternative pathway of the complement system, whilst the anaerobic effort had little influence. A better understanding of the post-exercise immune response provides a framework to prescribe physical activity to achieve different health outcomes.

Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59 leading to loss of function have been described and, overall, 17/18 of patients with any mutation presented with recurrent GBS. Here we determine the localization and possible role of membrane-bound complement regulators, including CD59, in the peripheral nervous systems (PNS) of mice and humans.

Complement deficiencies are difficult to diagnose because of the variability of symptoms and the complexity of the diagnostic process. Here, we applied a novel 'complementomics' approach to study the impact of various complement deficiencies on circulating complement levels.

The complement system has been thought to originate exclusively in the deuterostomes. Here, we show that the central complement components already existed in the primitive protostome lineage. A functional homolog of vertebrate complement 3, CrC3, has been isolated from a 'living fossil', the horseshoe crab (Carcinoscorpius rotundicauda). CrC3 resembles human C3 and shows closest homology to C3 sequences of lower deuterostomes. CrC3 and plasma lectins bind a wide range of microbes, forming the frontline innate immune defense system. Additionally, we identified CrC2/Bf, a homolog of vertebrate C2 and Bf that participates in C3 activation, and a C3 receptor-like sequence. Furthermore, complement-mediated phagocytosis of bacteria by the hemocytes of horseshoe crab was also observed. Thus, a primitive yet complex opsonic complement defense system is revealed in the horseshoe crab, a protostome species. Our findings demonstrate an ancient origin of the critical complement components and the opsonic defense mechanism in the Precambrian ancestor of bilateral animals.

Innate immunity contributes effectively to the development of Alcohol-Associated liver disease (ALD). Particularly, human studies and murine models of ALD have shown that Complement activation plays an important role during the initial and later stages of ALD. The Complement System may contribute to the pathogenesis of this disease since it has been shown that ethanol-derived metabolic products activate the Complement cascade on liver membranes, leading to hepatocellular damage. However, studies evaluating the plasma levels of Complement proteins in ALD patients present contradictory results in some cases, and do not establish a well-marked role for each Complement component. The impairment of leukocyte chemoattractant activity observed in these patients may contribute to the susceptibility to bacterial infections in the latter stages of the disease. On the other hand, murine models of ALD have provided more detailed insights into the mechanisms that link the Complement System to the pathogenesis of the disease. It has been observed that Classical pathway can be activated via C1q binding to apoptotic cells in the liver and contributes to the development of hepatic inflammation. C3 contributes to the accumulation of triglycerides in the liver and in adipose tissue, while C5 seems to be involved with inflammation and liver injury after chronic ethanol consumption. In this review, we present a compendium of studies evaluating the role of Complement in human and murine models of ALD. We also discuss potential therapies to human ALD, highlighting the use of Complement inhibitors.

Several lines of evidence implicate immunological/inflammatory factors in development of schizophrenia. Complement is a key driver of inflammation, and complement dysregulation causes pathology in many diseases. Here we explored whether complement dysregulation occurred in first episode psychosis (FEP) and whether this provides a source of biomarkers. Eleven complement analytes (C1q, C3, C4, C5, factor B [FB], terminal complement complex [TCC], factor H [FH], FH-related proteins [FHR125], Properdin, C1 inhibitor [C1inh], soluble complement receptor 1 [CR1]) plus C-reactive protein (CRP) were measured in serum from 136 first episode psychosis (FEP) cases and 42 mentally healthy controls using established in-house or commercial ELISA. The relationship between caseness and variables (analytes measured, sex, age, ethnicity, tobacco/cannabis smoking) was tested by multivariate logistic regression. When measured individually, only TCC was significantly different between FEP and controls (p=0.01). Stepwise selection demonstrated interdependence between some variables and revealed other variables that significantly and independently contributed to distinguishing cases and controls. The final model included demographics (sex, ethnicity, age, tobacco smoking) and a subset of analytes (C3, C4, C5, TCC, C1inh, FHR125, CR1). A receiver operating curve analysis combining these variables yielded an area under the curve of 0.79 for differentiating FEP from controls. This model was confirmed by multiple replications using randomly selected sample subsets. The data suggest that complement dysregulation occurs in FEP, supporting an underlying immune/inflammatory component to the disorder. Classification of FEP cases according to biological variables rather than symptoms would help stratify cases to identify those that might most benefit from therapeutic modification of the inflammatory response.

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in Caucasian adults. Phospholipase A2 receptor (PLA2R)- and exostosin 1 (EXT1)/exostosin 2 (EXT2)-associated MN represent the most common primary and secondary forms of MN. The complement profile using a proteomics approach has not been studied in these 2 common forms of MN.

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin αMβ2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.

Poxviruses subvert the host immune response by producing immunomodulatory proteins, including a complement regulatory protein. Ectromelia virus provides a mouse model for smallpox where the virus and the host's immune response have co-evolved. Using this model, our study investigated the role of the complement system during a poxvirus infection. By multiple inoculation routes, ectromelia virus caused increased mortality by 7 to 10 days post-infection in C57BL/6 mice that lack C3, the central component of the complement cascade. In C3(-/-) mice, ectromelia virus disseminated earlier to target organs and generated higher peak titers compared to the congenic controls. Also, increased hepatic inflammation and necrosis correlated with these higher tissue titers and likely contributed to the morbidity in the C3(-/-) mice. In vitro, the complement system in naïve C57BL/6 mouse sera neutralized ectromelia virus, primarily through the recognition of the virion by natural antibody and activation of the classical and alternative pathways. Sera deficient in classical or alternative pathway components or antibody had reduced ability to neutralize viral particles, which likely contributed to increased viral dissemination and disease severity in vivo. The increased mortality of C4(-/-) or Factor B(-/-) mice also indicates that these two pathways of complement activation are required for survival. In summary, the complement system acts in the first few minutes, hours, and days to control this poxviral infection until the adaptive immune response can react, and loss of this system results in lethal infection.

The complement cascade and regulatory proteins are involved in the pathogenesis of the Sjögren's syndrome and other autoimmune diseases. The complement activation via the alternative pathway was recognized as a major pathogenic mechanism in autoimmune conditions. The aim of this study was to assess expression of complement cascade components and regulatory proteins in minor salivary glands in patients with primary Sjögren's syndrome (pSS).

The complement system plays critical roles in development, homeostasis, and regeneration in the central nervous system (CNS) throughout life; however, complement dysregulation in the CNS can lead to damage and disease. Complement proteins, regulators, and receptors are widely expressed throughout the CNS and, in many cases, are upregulated in disease. Genetic and epidemiological studies, cerebrospinal fluid (CSF) and plasma biomarker measurements and pathological analysis of post-mortem tissues have all implicated complement in multiple CNS diseases including multiple sclerosis (MS), neuromyelitis optica (NMO), neurotrauma, stroke, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Given this body of evidence implicating complement in diverse brain diseases, manipulating complement in the brain is an attractive prospect; however, the blood-brain barrier (BBB), critical to protect the brain from potentially harmful agents in the circulation, is also impermeable to current complement-targeting therapeutics, making drug design much more challenging. For example, antibody therapeutics administered systemically are essentially excluded from the brain. Recent protocols have utilized "Trojan horse" techniques to transport therapeutics across the BBB or used osmotic shock or ultrasound to temporarily disrupt the BBB. Most research to date exploring the impact of complement inhibition on CNS diseases has been in animal models, and some of these studies have generated convincing data; for example, in models of MS, NMO, and stroke. There have been a few recent clinical trials of available anti-complement drugs in CNS diseases associated with BBB impairment, for example the use of the anti-C5 monoclonal antibody (mAb) eculizumab in NMO, but for most CNS diseases there have been no human trials of anti-complement therapies. Here we will review the evidence implicating complement in diverse CNS disorders, from acute, such as traumatic brain or spine injury, to chronic, including demyelinating, neuroinflammatory, and neurodegenerative diseases. We will discuss the particular problems of drug access into the CNS and explore ways in which anti-complement therapies might be tailored for CNS disease.

Recent studies have revealed profound developmental consequences of mutations in genes encoding proteins of the lectin pathway of complement activation, a central component of the innate immune system. Apart from impairment of immunity against microorganisms, it is known that hereditary deficiencies of this system predispose one to autoimmune conditions. Polymorphisms in complement genes are linked to, for example, atypical hemolytic uremia and age-dependent macular degeneration. The complement system comprises three convergent pathways of activation: the classical, the alternative, and the lectin pathway. The recently discovered lectin pathway is less studied, but polymorphisms in the plasma pattern-recognition molecule mannan-binding lectin (MBL) are known to impact its level, and polymorphisms in the MBL-associated serine protease-2 (MASP-2) result in defects of complement activation. Recent studies have described roles outside complement and immunity of another MBL-associated serine protease, MASP-3, in the etiology of 3MC syndrome, an autosomal-recessive disorder involving a spectrum of developmental features, including characteristic facial dysmorphism. Syndrome-causing mutations were identified in MASP1, encoding MASP-3 and two additional proteins, MASP-1 and MAp44. Furthermore, an association was discovered between 3MC syndrome and mutations in COLEC11, encoding CL-K1, another molecule of the lectin pathway. The findings were confirmed in zebrafish, indicating that MASP-3 and CL-K1 underlie an evolutionarily conserved pathway of embryonic development. Along with the discovery of a role of C1q in pruning synapses in mice, these recent advances point toward a broader role of complement in development. Here, we compare the functional immunologic consequences of "conventional" complement deficiencies with these newly described developmental roles.

An emerging number of diseases and therapeutic approaches with defined involvement of the complement system justify a need for specific markers reflecting activation of particular effector arms of the complement cascade. Measurement of such soluble markers in circulation is a challenge since the specificity of antibodies must be limited to activated complement fragments but not predominant and ubiquitous parental molecules. Existing assays for the measurement of soluble, activated complement proteins are based on the detection of conformational neoepitopes. We tested an alternative approach based on detection of short linear neoepitopes exposed at the cleavage sites after activation of the actual complement component. Obtained antibodies reactive to C4d and C5b fragments enabled us to set up highly specific sandwich ELISAs, which ensured trustful measurements without false positive readouts characteristic for some of the widely used assays.

Entamoeba histolytica is the cause of amoebiasis. The trophozoite (amoeba) form of this parasite is capable of invading the intestine and can disseminate through the bloodstream to other organs. The mechanisms that allow amoebae to evade complement deposition during dissemination have not been well characterized. We previously discovered a novel complement-evasion mechanism employed by E. histolytica. E. histolytica ingests small bites of living human cells in a process termed trogocytosis. We demonstrated that amoebae were protected from lysis by human serum following trogocytosis of human cells and that amoebae acquired and displayed human membrane proteins from the cells they ingested. Here, we aimed to define how amoebae are protected from complement lysis after performing trogocytosis. We found that amoebae were protected from complement lysis after ingestion of both human Jurkat T cells and red blood cells and that the level of protection correlated with the amount of material ingested. Trogocytosis of human cells led to a reduction in deposition of C3b on the surface of amoebae. We asked whether display of human complement regulators is involved in amoebic protection, and found that CD59 was displayed by amoebae after trogocytosis. Deletion of a single complement-regulatory protein, CD59 or CD46, from Jurkat cells was not sufficient to alter amoebic protection from lysis, suggesting that multiple, redundant complement regulators mediate amoebic protection. However, exogeneous expression of CD46 or CD55 in amoebae was sufficient to confer protection from lysis. These studies shed light on a novel strategy for immune evasion by a pathogen. IMPORTANCE Entamoeba histolytica is the cause of amoebiasis, a diarrheal disease of global importance. While infection is often asymptomatic, the trophozoite (amoeba) form of this parasite is capable of invading and ulcerating the intestine and can disseminate through the bloodstream to other organs. Understanding how E. histolytica evades the complement system during dissemination is of great interest. Here, we demonstrate for the first time that amoebae that have performed trogocytosis (nibbling of human cells) resist deposition of the complement protein C3b. Amoebae that have performed trogocytosis display the complement-regulatory protein CD59. Overall, our studies suggest that acquisition and display of multiple, redundant complement regulators is involved in amoebic protection from complement lysis. These findings shed light on a novel strategy for immune evasion by a pathogen. Since other parasites use trogocytosis for cell killing, our findings may apply to the pathogenesis of other infections.

Selection pressure exerted by pathogens can influence patterns of genetic diversity in the host. In the immune system especially, numerous genes encode proteins involved in antagonistic interactions with pathogens, paving the way for coevolution that results in increased genetic diversity as a consequence of balancing selection. The complement system is a key component of innate immunity. Many complement proteins interact directly with pathogens, either by recognising pathogen molecules for complement activation, or by serving as targets of pathogen immune evasion mechanisms. Complement genes can therefore be expected to be important targets of pathogen-mediated balancing selection, but analyses of such selection on this part of the immune system have been limited.

SAPHO syndrome is an inflammatory disease invading the skin and bones, whose diagnosis has been difficult due to its low incidence and diversified manifestation. We investigated the serum proteomic profile of SAPHO patients to identify key proteins associated with SAPHO syndrome, trying to find clinical biomarkers or functional molecules for this rare disease. Blood samples from 8 SAPHO patients and 8 healthy controls were detected and analyzed using data independent acquisition (DIA) method to identify differentially expressed proteins (DEPs) specific to SAPHO. A total of 57 differentially expressed proteins were identified (p < 0.05, fold change >1.2), in which 27 proteins were upregulated and 30 downregulated. DEPs may participate in GO terms such as "lipid particle" and "Notch signaling pathway", as well as KEGG pathways including "complement and coagulation cascades" and "mTOR signaling pathway". The overexpression of inhibitors of the complement system (CFH and C4BP), were verified in a larger cohort (16 SAPHO patients, 8 AS patients and 24 healthy controls) with ELISA, and the combined diagnostic ability of CFH and C4BP was predicted by ROC curve with an AUC of 0.91, which may be molecular candidates for further study on diagnosis and pathology of this rare disease. SIGNIFICANCE: Our research provided the first insight into plasma proteomic profile for SAPHO patients，offering potential biomarkers for disease diagnosis. We found that inhibitors of complement system such as CFH and C4BP were up-regulated in SAPHO syndrome, which may play important roles in the pathogenesis of SAPHO syndrome.

Notothenioidei are typical Antarctic teleosts evolved to adapt to the very low temperatures of the Antarctic seas. Aim of the present paper is to investigate sequence and structure of C3, the third component of the complement system of the notothenioid Trematomus bernacchii and Chionodraco hamatus. We determined the complete nucleotide sequence of two C3 isoforms of T. bernacchii and a single C3 isoform of C. hamatus. These sequences were aligned against other homologous teleost sequences to check for the presence of diversifying selection. Evidence for positive selection was observed in the evolutionary lineage of Antarctic teleost C3 sequences, especially in that of C. hamatus, the most recently diverged species. Adaptive selection affected numerous amino acid positions including three residues located in the anaphylatoxin domain. In an attempt to evaluate the link between sequence variants and specific structural features, we constructed molecular models of Antarctic teleost C3s, of their proteolytic fragments C3b and C3a, and of the corresponding molecules of the phylogenetically related temperate species Epinephelus coioides, using human crystallographic structures as templates. Subsequently, we compared dynamic features of these models by molecular dynamics simulations and found that the Antarctic C3s models show higher flexibility, which likely allows for more pronounced movements of both the TED domain in C3b and the carboxyl-terminal region of C3a. As such dynamic features are associated to positively selected sites, it appears that Antarctic teleost C3 molecules positively evolved toward an increased flexibility, to cope with low kinetic energy levels of the Antarctic marine environment.

Several studies have investigated the interactions between C-reactive protein (CRP) and various complement proteins but none of them took into consideration the different structural forms of CRP. The aim of our study was to investigate whether the different antigenic forms of CRP are able to bind C1q, to trigger activation of the C1 complex and to study the ability of the various CRP forms to bind complement factor H (FH) and C4b-binding protein (C4BP). Interactions between various CRP forms and complement proteins were analysed in enzyme-linked immunosorbent assay and surface plasmon resonance tests and activation of the C1 complex was followed in a reconstituted system using purified C1q, C1r and C1s in the presence of C1-INH. Native, ligand-unbound CRP activated the classical pathway weakly. After binding to phosphocholine, native CRP bound C1q and significantly activated C1. Native CRP complexed to phosphocholine did not bind the complement regulatory proteins FH and C4BP. After disruption of the pentameric structure of CRP, as achieved by urea-treatment or by site-directed mutagenesis, C1q binding and C1 activation further increased and the ability of CRP to bind complement regulatory proteins was revealed. C1q binds to CRP through its globular head domain. The binding sites on CRP for FH and C4BP seemed to be different from that of C1q. In conclusion, in parallel with the increase in the C1-activating ability of different CRP structural variants, the affinity for complement regulatory proteins also increased, providing the biological basis for limitation of excess complement activation.