C3 is the central effector molecule of the complement system, mediating its multiple functions through different binding sites and their corresponding receptors. We will introduce the C3 forms (native C3, C3 [H2 O], and intracellular C3), the C3 fragments C3a, C3b, iC3b, and C3dg/C3d, and the C3 expression sites. To highlight the important role that C3 plays in human biological processes, we will give an overview of the diseases linked to C3 deficiency and to uncontrolled C3 activation. Next, we will present a structural description of C3 activation and of the C3 fragments generated by complement regulation. We will proceed by describing the C3a interaction with the anaphylatoxin receptor, followed by the interactions of opsonins (C3b, iC3b, and C3dg/C3d) with complement receptors, divided into two groups: receptors bearing complement regulatory functions and the effector receptors without complement regulatory activity. We outline the molecular architecture of the receptors, their binding sites on the C3 activation fragments, the cells expressing them, the diversity of their functions, and recent advances. With this review, we aim to give an up-to-date analysis of the processes triggered by C3 activation fragments on different cell types in health and disease contexts.

C3 glomerulopathy (C3G) is a severe kidney disease, which is caused by defective regulation of the alternative complement pathway. Disease pathogenesis is heterogeneous and is caused by both autoimmune and genetic factors. Here we characterized IgG autoantibodies derived from 33 patients with autoimmune C3 glomerulopathy. Serum antibodies from all 33 patients as well as purified IgGs bound to the in vitro assembled C3-convertase. Noteworthy, two groups of antibodies were identified: group 1 with strong (12 patients) and group 2 with weak binding C3-convertase autoantibodies (22 patients). C3Nef, as evaluated in a standard C3Nef assay, was identified in serum from 19 patients, which included patients from group 1 as well as group 2. The C3-convertase binding profile was independent of C3Nef. Group 1 antibodies, but not the group 2 antibodies stabilized the C3-convertase, and protected the enzyme from dissociation by Factor H. Also, only group 1 antibodies induced C3a release. However, both group 1 and group 2 autoantibodies bound to the C5-convertase and induced C5a generation, which was inhibited by monoclonal anti-C5 antibody Eculizumab in vitro. In summary, group 1 antibodies are composed of C3Nef and C5Nef antibodies and likely over-activate the complement system, as seen in hemolytic assays. Group 2 antibodies show predominantly C5Nef like activities and stabilize the C5 but not the C3-convertase. Altogether, these different profiles not only reveal a heterogeneity of the autoimmune forms of C3G (MPGN), they also show that in diagnosis of C3G not all autoimmune forms are identified and thus more vigorous autoantibody testing should be performed.

Podocytes are an important part of the glomerular filtration barrier and the key player in the development of proteinuria, which is an early feature of complement mediated renal diseases. Complement factors are mainly liver-born and present in circulation. Nevertheless, there is a growing body of evidence for additional sites of complement protein synthesis, including various cell types in the kidney. We hypothesized that podocytes are able to produce complement components and contribute to the local balance of complement activation and regulation. To investigate the relevant balance between inhibiting and activating sides, our studies focused on complement factor H (CFH), an important complement regulator, and on C3, the early key component for complement activation. We characterized human cultured podocytes for the expression and secretion of activating and regulating complement factors, and analyzed the secretion pathway and functional activity. We studied glomerular CFH and C3 expression in puromycin aminonucleoside (PAN) -treated rats, a model for proteinuria, and the physiological mRNA-expression of both factors in murine kidneys. We found, that C3 and CFH were expressed in cultured podocytes and expression levels differed from those in cultivated glomerular endothelial cells. The process of secretion in podocytes was stimulated with interferon gamma and located in the Golgi apparatus. Cultured podocytes could initiate the complement cascade by the splitting of C3, which can be shown by the generation of C3a, a functional C3 split product. C3 contributed to external complement activation. Podocyte-secreted CFH, in conjunction with factor I, was able to split C3b. Podocytes derived from a patient with a CFH mutation displayed impaired cell surface complement regulation. CFH and C3 were synthesized in podocytes of healthy C57Bl/6-mice and were upregulated in podocytes of PAN treated rats. These data show that podocytes produce functionally active complement components, and could therefore influence the local glomerular complement activation and regulation. This modulating effect should therefore be considered in all diseases where glomerular complement activation occurs. Furthermore, our data indicate a potential novel role of podocytes in the innate immune system.

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.

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3(-/-) and C5(-/-) as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3(-/-): -25%, p=0.02; C5(-/-): -20% p=0.052) and newly formed bone (C3(-/-): -38%, p=0.01; C5(-/-): -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3(-/-) mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.

The complement system, which is part of the innate immune response system, has been recently shown to participate in multiple key processes in the developing brain. Here we aimed to elucidate downstream signaling responses linking complement C3, a key molecule of the pathway, to small GTPases, known to affect the cytoskeleton. The expression pattern of the activated small GTPase Rac1 resembled that of complement C3. C3-deficient mice exhibited reduced Rac1 and elevated RhoA activity in comparison with control mice. The most pronounced reduction of Rac1 activity occurred at embryonic day 14. Rac1 has been implicated in neuronal migration as well as neuronal stem cell proliferation and differentiation. Consistent with the reduction in Rac1 activity, the expression of phospho-cofilin, decreased in migrating neurons. Reduced Rac1-GTP was also correlated with a decrease in the expression of progenitor markers (Nestin, Pax6 and Tbr2) and conversely the expression of neuronal markers (Dcx and NeuN) increased in C3 knockout (KO) cortices in comparison with wild-type (WT) cortices. More specifically, C3 deficiency resulted in a reduction in the number of the cells in S-phase and an elevation in the number of cells that precociously exited the cell cycle. Collectively, our findings suggest that C3 impacts the activity of small GTPases resulting in cell cycle defects and premature neuronal differentiation.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common metastatic skin cancers with increasing incidence. We examined the roles of complement component C3 and complement factor B (CFB) in the growth of cSCC. Analysis of cSCC cell lines (n = 8) and normal human epidermal keratinocytes (n = 11) with real-time quantitative PCR and Western blotting revealed up-regulation of C3 and CFB expression in cSCC cells. Immunohistochemical staining revealed stronger tumor cell-specific labeling for C3 and CFB in invasive cSCCs (n = 71) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n = 11) than in cSCC in situ (n = 69), actinic keratoses (n = 63), and normal skin (n = 5). Significant up-regulation of C3 and CFB mRNA expression was noted in chemically induced mouse cSCCs, compared to benign papillomas. Knockdown of C3 and CFB expression inhibited migration and proliferation of cSCC cells and resulted in potent inhibition of extracellular signal-regulated kinase 1/2 activation. Knockdown of C3 and CFB markedly inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the roles of C3 and CFB in the development of cSCC and identify them as biomarkers and potential therapeutic targets in this metastatic skin cancer.

Nasal polyposis often is characterized by a persistent inflammation of the sinonasal mucosa, disease recurrence after medical or surgical intervention, and asthma comorbidity. Dysregulated complement activation may contribute to immunologic alterations and disease. To date, there is only scattered knowledge on the source and regulation of the central complement factors in the pathogenesis of nasal polyps. Here, we aim to study complement signatures, especially the C3-C3aR axis, and focus on cellular sources and targets in nasal polyps. Expression of complement factors, including C3, C5, and the anaphylatoxin receptors, was analyzed in nasal polyp tissue samples, the corresponding inferior turbinates, and healthy controls using transcriptomic methods and protein measurements. Distinct patterns of complement expression were found in nasal polyps compared to controls, characterized by an increased C3 activation and an increase in C3aR-bearing cells. In contrast, no difference was shown for epithelial-dependent C3 production. Besides low intracellular C3-expression levels for lymphocytes in general, we could identify an enlarged B lymphocyte population in nasal polyps displaying high amounts of intracellular C3. Our data suggest a prominent role for the C3-C3aR-axis in nasal polyps and, for the first time, describe a B cell population containing high levels of intracellular C3, suggesting a new role of B cells in the maintenance of the inflammation by complement.

In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.

C3 glomerulopathy is a complement-mediated renal disease that is frequently associated with abnormalities in regulation of the complement alternative pathway. Mice with deficiency of factor H (Cfh(-/-)), a negative alternative pathway regulator, are an established experimental model of C3 glomerulopathy in which complement C3 fragments including iC3b accumulate along the glomerular basement membrane. Here we show that deficiency of complement receptor 3 (CR3), the main receptor for iC3b, enhances the severity of spontaneous renal disease in Cfh(-/-) mice. This effect was found to be dependent on CR3 expression on bone marrow-derived cells. CR3 also mediated renal protection outside the setting of factor H deficiency, as shown by the development of enhanced renal injury in CR3-deficient mice during accelerated nephrotoxic nephritis. The iC3b-CR3 interaction downregulated the proinflammatory cytokine response of both murine and human macrophages to lipopolysaccharide stimulation in vitro, suggesting that the protective effect of CR3 on glomerular injury was mediated via modulation of macrophage-derived proinflammatory cytokines. Thus, CR3 has a protective role in glomerulonephritis and suggests that pharmacologic potentiation of the macrophage CR3 interaction with iC3b could be therapeutically beneficial.

It has been found that the extensive use of anticancer drugs containing DNA-alkylating agents not only target cancer cells but also cause retinal inflammation through toxic intermediates. Complement C3 (C3) is a core component of the complement activation pathway, and dysregulation of the complement pathway is involved in several retinal degenerative diseases. However, whether C3 plays a critical role in alkylation-induced retinal degeneration is unclear.

C3 glomerulopathy (C3G) describes a pathologic pattern of injury diagnosed by renal biopsy. It is characterized by the dominant deposition of the third component of complement (C3) in the renal glomerulus as resolved by immunofluorescence microscopy. The underlying pathophysiology is driven by dysregulation of the alternative pathway of complement in the fluid-phase and in the glomerular microenvironment. Characterization of clinical features and a targeted evaluation for indices and drivers of complement dysregulation are necessary for optimal patient care. Autoantibodies to the C3 and C5 convertases of complement are the most commonly detected drivers of complement dysregulation, although genetic mutations in complement genes can also be found. Approximately half of patients progress to end-stage renal disease within 10 years of diagnosis, and, while transplantation is a viable option, there is high risk for disease recurrence and allograft failure. This poor outcome reflects the lack of disease-specific therapy for C3G, relegating patients to symptomatic treatment to minimize proteinuria and suppress renal inflammation. Fortunately, the future is bright as several anti-complement drugs are currently in clinical trials.

Complement C3 (C3) has been shown to be involved in the aging process. However, the role of C3 in kidney aging has not been fully elucidated. This study aimed to investigate the effect of C3 on senescence related kidney disorders in mice.

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.

Human T helper type 1 (Th1) responses are essential in defense. Although T cell receptor (TCR) and co-stimulator engagement are indispensable for T cell activation, stimulation of additional receptor pathways are also necessary for effector induction. For example, engagement of the complement regulator CD46 by its ligand C3b generated upon TCR activation is required for IFN-γ production as CD46-deficient patients lack Th1 responses. Utilizing T cells from two C3-deficient patients we demonstrate here that normal Th1 responses also depend on signals mediated by the anaphylatoxin C3a receptor (C3aR). Importantly, and like in CD46-deficient patients, whilst Th1 induction are impaired in C3-deficient patients in vitro, their Th2 responses are unaffected. Furthermore, C3-deficient CD4(+) T cells present with reduced expression of CD25 and CD122, further substantiating the growing notion that complement fragments regulate interleukin-2 receptor (IL-2R) assembly and that disturbance of complement-guided IL-2R assembly contributes to aberrant Th1 effector responses. Lastly, sustained intrinsic production of complement fragments may participate in the Th1 contraction phase as both C3a and CD46 engagement regulate IL-10 co-expression in Th1 cells. These data suggest that C3aR and CD46 activation via intrinsic generation of their respective ligands is an integral part of human Th1 (but not Th2) immunity.

Pancreatic cancer (PC) remains a devastating disease with a five-year survival rate of <5%. The difficulty in making an early diagnosis and the frequent occurrence of metastasis are important reasons for this poor prognosis. In China, the incidence of PC has been increasing steadily. Therefore, the present study aimed to identify effective markers in the early and advanced stages of PC. The expression levels of complement C3, complement C4b1 and apolipoprotein E (ApoE) in the various stages of PC were assessed by immunohistochemistry, RT-PCR and western blotting. Additionally, the statistical significance of the results was analyzed. The expression levels of complement C3, complement C4b1 and apoE were higher in PC compared with normal pancreatic tissues. No correlations were observed between complement C3 and tumor TNM staging or lymph node metastasis. However, complement C4b1 and apoE were markedly correlated with tumor TNM staging and lymph node metastasis. Complement C3 may be used as a marker for the diagnosis of early-stage PC, while complement C4b1 and apoE are closely correlated with tumor development, reflecting the biological behavior of PC, and thus may be used as diagnostic markers of advanced PC.

Abnormal regulation of the complement alternative pathway is associated with C3 glomerulopathy. Complement factor H is the main plasma regulator of the alternative pathway and consists of 20 short consensus repeat (SCR) domains. Although recombinant full-length factor H represents a logical treatment for C3 glomerulopathy, its production has proved challenging. We and others have designed recombinant mini-factor H proteins in which 'non-essential' SCR domains have been removed. Here, we report the in vitro and in vivo effects of a mini-complement factor H protein, FH1-5^18-20, using the unique factor H-deficient (Cfh-/-) mouse model of C3 glomerulopathy. FH1-5^18-20 is comprised of the key complement regulatory domains (SCRs 1-5) linked to the surface recognition domains (SCRs 18-20). Intraperitoneal injection of FH1-5^18-20 in Cfh-/- mice reduced abnormal glomerular C3 deposition, similar to full-length factor H. Systemic effects on plasma alternative pathway control were comparatively modest, in association with a short half-life. Thus, FH1-5^18-20 is a potential therapeutic agent for C3 glomerulopathy and other renal conditions with alternative pathway-mediated tissue injury.

The primary host response to Staphylococcus aureus infection occurs via complement. Complement is an elegant evolutionarily conserved system, playing essential roles in early defences by working in concert with immune cells to survey, label and destroy microbial intruders and coordinate inflammation. Currently the exact mechanisms employed by S. aureus to manipulate and evade complement is not clear and is hindered by the lack of accurate molecular tools that can report on complement deposition on the bacterial surface. Current gold-standard detection methods employ labelled complement-specific antibodies and flow cytometry to determine complement deposited on bacteria. These methods are restricted by virtue of the expression of the S. aureus immunoglobulin binding proteins, Protein A and Sbi. In this study we describe the use of a novel antibody-independent C3 probe derived from the staphylococcal Sbi protein, specifically Sbi-IV domain. Here we show that biotin-labelled Sbi-IV interacts specifically with deposited C3 products on the staphylococcal surface and thus can be used to measure complement fixation on wild-type cells expressing a full repertoire of immune evasion proteins. Lastly, our data indicates that genetically diverse S. aureus strains restrict complement to different degrees suggesting that complement evasion is a variable virulence trait among S. aureus isolates.

Preeclampsia (PE) is a common vascular disease of pregnancy with genetic predisposition. Dysregulation of the complement system has been implicated, but molecular mechanisms are incompletely understood. In this study, we determined the potential linkage of severe PE to the most central complement gene, C3. Three cohorts of Finnish patients and controls were recruited for a genetic case-control study. Participants were genotyped using Sequenom genotyping and Sanger sequencing. Initially, we studied 259 Finnish patients with severe PE and 426 controls from the Southern Finland PE and the Finnish population-based PE cohorts. We used a custom-made single nucleotide polymorphism (SNP) genotyping assay consisting of 98 SNPs in 18 genes that encode components of the complement system. Following the primary screening, C3 was selected as the candidate gene and consequently Sanger sequenced. Fourteen SNPs from C3 were also genotyped by a Sequenom panel in 960 patients with severe PE and 705 controls, including already sequenced individuals. Three of the 43 SNPs observed within C3 were associated with severe PE: rs2287845 (p = 0.038, OR = 1.158), rs366510 (p = 0.039, OR = 1.158), and rs2287848 (p = 0.041, OR = 1.155). We also discovered 16 SNP haplotypes with extreme linkage disequilibrium in the middle of the gene with a protective (p = 0.044, OR = 0.628) or a predisposing (p = 0.011, OR = 2.110) effect to severe PE depending on the allele combination. Genetic variants associated with PE are located in key domains of C3 and could thereby influence the function of C3. This is, as far as we are aware, the first candidate gene in the complement system with an association to a clinically relevant PE subphenotype, severe PE. The result highlights a potential role for the complement system in the pathogenesis of PE and may help in defining prognostic and therapeutic subgroups of preeclamptic women.

Prostate apoptosis response-4 (Par-4) is a tumor suppressor that induces apoptosis in cancer cells. However, the physiological function of Par-4 remains unknown. Here we show that conventional Par-4 knockout (Par-4-/-) mice and adipocyte-specific Par-4 knockout (AKO) mice, but not hepatocyte-specific Par-4 knockout mice, are obese with standard chow diet. Par-4-/- and AKO mice exhibit increased absorption and storage of fat in adipocytes. Mechanistically, Par-4 loss is associated with mdm2 downregulation and activation of p53. We identified complement factor c3 as a p53-regulated gene linked to fat storage in adipocytes. Par-4 re-expression in adipocytes or c3 deletion reversed the obese mouse phenotype. Moreover, obese human subjects showed lower expression of Par-4 relative to lean subjects, and in longitudinal studies, low baseline Par-4 levels denoted an increased risk of developing obesity later in life. These findings indicate that Par-4 suppresses p53 and its target c3 to regulate obesity.