C3-dependent effector functions of complement.

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.

Insights into the molecular basis and mechanism of heme-triggered TLR4 signalling: The role of heme-binding motifs in TLR4 and MD2.

Haemolytic disorders, such as sickle cell disease, are accompanied by the release of high amounts of labile heme into the intravascular compartment resulting in the induction of proinflammatory and prothrombotic complications in affected patients. In addition to the relevance of heme-regulated proteins from the complement and blood coagulation systems, activation of the TLR4 signalling pathway by heme was ascribed a crucial role in the progression of these pathological processes. Heme binding to the TLR4-MD2 complex has been proposed recently, however, essential mechanistic information of the processes at the molecular level, such as heme-binding kinetics, the heme-binding capacity and the respective heme-binding sites (HBMs) is still missing. We report the interaction of TLR4, MD2 and the TLR4-MD2 complex with heme and the consequences thereof by employing biochemical, spectroscopic, bioinformatic and physiologically relevant approaches. Heme binding occurs transiently through interaction with up to four HBMs in TLR4, two HBMs in MD2 and at least four HBMs in their complex. Functional studies highlight that mutations of individual HBMs in TLR4 preserve full receptor activation by heme, suggesting that heme interacts with TLR4 through different binding sites independently of MD2. Furthermore, we confirm and extend the major role of TLR4 for heme-mediated cytokine responses in human immune cells.

Basic Mechanisms of Hemolysis-Associated Thrombo-Inflammation and Immune Dysregulation.

Independent of etiology, hemolytic diseases are associated with thrombosis, inflammation and immune dysregulation, all together contributing to organ damage and poor outcome. Beyond anemia and the loss of the anti-inflammatory functions of red blood cells, hemolysis leads to the release of damage-associated molecular patterns including ADP, hemoglobin, and heme, which act through multiple receptors and signaling pathways fostering a hyperinflammatory and hypercoagulable state. Extracellular free heme is promiscuous alarmin capable of triggering oxido-inflammatory and thrombotic events by inducing the activation of platelets, endothelial and innate cells as well as the coagulation and complement cascades. In this review, we discuss the main mechanisms by which hemolysis and, in particular, heme, drive this thrombo-inflammatory milieu and discuss the consequences of hemolysis on the host response to secondary infections.

IgG Subclass-Dependent Pulmonary Antigen Retention during Acute IgG-Dependent Systemic Anaphylaxis in Mice.

Mouse models of active systemic anaphylaxis rely predominantly on IgG Abs forming IgG-allergen immune complexes that induce IgG receptor-expressing neutrophils and monocytes/macrophages to release potent mediators, leading to systemic effects. Whether anaphylaxis initiates locally or systemically remains unknown. In this study, we aimed at identifying the anatomical location of IgG-allergen immune complexes during anaphylaxis. Active systemic anaphylaxis was induced following immunization with BSA and i.v. challenge with fluorescently labeled BSA. Ag retention across different organs was examined using whole-body fluorescence imaging, comparing immunized and naive animals. Various mouse models and in vivo deletion strategies were employed to determine the contribution of IgG receptors, complement component C1q, myeloid cell types, and anaphylaxis mediators. We found that following challenge, Ag diffused systemically, but specifically accumulated in the lungs of mice sensitized to that Ag, where it formed large Ab-dependent aggregates in the vasculature. Ag retention in the lungs did not rely on IgG receptors, C1q, neutrophils, or macrophages. IgG2a-mediated, but neither IgG1- nor IgG2b-mediated, passive systemic anaphylaxis led to Ag retention in the lung. Neutrophils and monocytes significantly accumulated in the lungs after challenge and captured high amounts of Ag, which led to downmodulation of surface IgG receptors and triggered their activation. Thus, within minutes of systemic injection in sensitized mice, Ag formed aggregates in the lung and liver vasculature, but accumulated specifically and dose-dependently in the lung. Neutrophils and monocytes recruited to the lung captured Ag and became activated. However, Ag aggregation in the lung vasculature was not necessary for anaphylaxis induction.

Assessment of the breadth of binding promiscuity of heme towards human proteins.

Heme regulates important biological processes by transient interactions with many human proteins. The goal of the present study was to assess extends of protein binding promiscuity of heme. To this end we evaluated interaction of heme with >9000 human proteins. Heme manifested high binding promiscuity by binding to most of the proteins in the array. Nevertheless, some proteins have outstanding heme binding capacity. Bioinformatics analyses revealed that apart from typical haemoproteins, these proteins are frequently involved in metal binding or have the potential to recognize DNA. This study can contribute for understanding the regulatory functions of labile heme.

P-selectin drives complement attack on endothelium during intravascular hemolysis in TLR-4/heme-dependent manner.

Hemolytic diseases are frequently linked to multiorgan failure subsequent to vascular damage. Deciphering the mechanisms leading to organ injury upon hemolytic event could bring out therapeutic approaches. Complement system activation occurs in hemolytic disorders, such as sickle cell disease, but the pathological relevance and the acquisition of a complement-activating phenotype during hemolysis remain unclear. Here we found that intravascular hemolysis, induced by injection of phenylhydrazine, resulted in increased alanine aminotransferase plasma levels and NGAL expression. This liver damage was at least in part complement-dependent, since it was attenuated in complement C3-/- mice and by injection of C5-blocking antibody. We evidenced C3 activation fragments' deposits on liver endothelium in mice with intravascular hemolysis or injected with heme as well as on cultured human endothelial cells (EC) exposed to heme. This process was mediated by TLR4 signaling, as revealed by pharmacological blockade and TLR4 deficiency in mice. Mechanistically, TLR4-dependent surface expression of P-selectin triggered an unconventional mechanism of complement activation by noncovalent anchoring of C3 activation fragments, including the typical fluid-phase C3(H2O), measured by surface plasmon resonance and flow cytometry. P-selectin blockade by an antibody prevented complement deposits and attenuated the liver stress response, measured by NGAL expression, in the hemolytic mice. In conclusion, these results revealed the critical impact of the triad TLR4/P-selectin/complement in the liver damage and its relevance for hemolytic diseases. We anticipate that blockade of TLR4, P-selectin, or the complement system could prevent liver injury in hemolytic diseases like sickle cell disease.

Complement factor H: a guardian within?

The glomerular endothelium produces the key complement regulator factor H (FH), but its role in the endothelial cells protection and functional integrity is unclear. In this edition of Kidney International, Mahajan et al. demonstrate that the endothelial-intrinsic FH is important for the cytoskeletal architecture, monolayer integrity, proliferation control, metabolism, and inflammatory signaling regulation. These findings place the endothelium-derived FH in the center of the pathological process of diseases, characterized with FH genetic abnormalities.

Complement activation is a crucial driver of acute kidney injury in rhabdomyolysis.

Rhabdomyolysis is a life-threatening condition caused by skeletal muscle damage with acute kidney injury being the main complication dramatically worsening the prognosis. Specific treatment for rhabdomyolysis-induced acute kidney injury is lacking and the mechanisms of the injury are unclear. To clarify this, we studied intra-kidney complement activation (C3d and C5b-9 deposits) in tubules and vessels of patients and mice with rhabdomyolysis-induced acute kidney injury. The lectin complement pathway was found to be activated in the kidney, likely via an abnormal pattern of Fut2-dependent cell fucosylation, recognized by the pattern recognition molecule collectin-11 and this proceeded in a C4-independent, bypass manner. Concomitantly, myoglobin-derived heme activated the alternative pathway. Complement deposition and acute kidney injury were attenuated by pre-treatment with the heme scavenger hemopexin. This indicates that complement was activated in a unique double-trigger mechanism, via the alternative and lectin pathways. The direct pathological role of complement was demonstrated by the preservation of kidney function in C3 knockout mice after the induction of rhabdomyolysis. The transcriptomic signature for rhabdomyolysis-induced acute kidney injury included a strong inflammatory and apoptotic component, which were C3/complement-dependent, as they were normalized in C3 knockout mice. The intra-kidney macrophage population expressed a complement-sensitive phenotype, overexpressing CD11b and C5aR1. Thus, our results demonstrate a direct pathological role of heme and complement in rhabdomyolysis-induced acute kidney injury. Hence, heme scavenging and complement inhibition represent promising therapeutic strategies.

Heme Oxygenase 1: A Defensive Mediator in Kidney Diseases.

The incidence of kidney disease is rising, constituting a significant burden on the healthcare system and making identification of new therapeutic targets increasingly urgent. The heme oxygenase (HO) system performs an important function in the regulation of oxidative stress and inflammation and, via these mechanisms, is thought to play a role in the prevention of non-specific injuries following acute renal failure or resulting from chronic kidney disease. The expression of HO-1 is strongly inducible by a wide range of stimuli in the kidney, consequent to the kidney's filtration role which means HO-1 is exposed to a wide range of endogenous and exogenous molecules, and it has been shown to be protective in a variety of nephropathological animal models. Interestingly, the positive effect of HO-1 occurs in both hemolysis- and rhabdomyolysis-dominated diseases, where the kidney is extensively exposed to heme (a major HO-1 inducer), as well as in non-heme-dependent diseases such as hypertension, diabetic nephropathy or progression to end-stage renal disease. This highlights the complexity of HO-1's functions, which is also illustrated by the fact that, despite the abundance of preclinical data, no drug targeting HO-1 has so far been translated into clinical use. The objective of this review is to assess current knowledge relating HO-1's role in the kidney and its potential interest as a nephroprotection agent. The potential therapeutic openings will be presented, in particular through the identification of clinical trials targeting this enzyme or its products.

Impact of Autoantibodies to Complement Components on the Disease Activity in SLE.

INTRODUCTION: Systemic Lupus Erythematosus (SLE) is a chronic multi-system autoimmune disease with varied clinical presentations. Complement components are the major players in disease pathogenesis. This retrospective cross-sectional study was aimed at assessing the role of autoantibodies to these complement components and their association disease activity in newly diagnosed SLE patients from India. METHOD: Clinically diagnosed SLE patients (n=57) classified as per 2015 ACR/SLICC revised criteria were enrolled between November 2016 to April 2017. Patients' sera were tested for C3 and C4 by nephelometry, while serum levels of factor H, factor P (properdin) as well as autoantibodies to C3, C4, factor H and factor P were detected by ELISA. GraphPad Prism Version 6.01 was used for statistical analysis. Mean, SD, SEM were calculated. Mann Whittney U-test, ANOVA, Chi-square test, Odd's Ratio were calculated. Pearson's correlation was used to study relativeness of the study parameters. RESULTS: Among the 57 SLE patients, low C3 were seen in 51% patients, low C4 in 49%, low factor H in 19% and low factor P in 49% patients. Positivity for autoantibodies against complement components, anti-C3 were seen in 42% patients, anti-C4 in 7%, anti-factor H in 19% and anti-factor P in 28% patients. Serum levels of C3 (p=0.0009), C4 (p=0.0031) and anti-C3 autoantibodies (p=0.0029) were significantly associated with ACR/SLICC 2015 scores. CONCLUSION: Hypocomplementemia was found to be associated with higher disease damage score in newly diagnosed SLE patients. This study adds novel arguments for the importance of the anti-C3 autoantibodies as a marker of SLE.

Complement activation in sickle cell disease: Dependence on cell density, hemolysis and modulation by hydroxyurea therapy.

The complement system is an innate immune defense cascade that can cause tissue damage when inappropriately activated. Evidence for complement over activation has been reported in small cohorts of patients with sickle cell disease (SCD). However, the mechanism governing complement activation in SCD has not been elucidated. Here, we observe that the plasma concentration of sC5b-9, a reliable marker for terminal complement activation, is increased at steady state in 61% of untreated SCD patients. We show that greater complement activation in vitro is promoted by SCD erythrocytes compared to normal ones, although no significant differences were observed in the regulatory proteins CD35, CD55, and CD59 in whole blood. Complement activation is positively correlated with the percentage of dense sickle cells (DRBCs). The expression levels of CD35, CD55, and CD59 are reduced in DRBCs, suggesting inefficient regulation when cell density increases. Moreover, the surface expression of the complement regulator CD46 on granulocytes was inversely correlated with the plasma sC5b-9. We also show increased complement deposition in cultured human endothelial cells incubated with SCD serum, which is diminished by the addition of the heme scavenger hemopexin. Treatment of SCD patients with hydroxyurea produces substantial reductions in complement activation, measured by sC5b-9 concentration and upregulation of CD46, as well as decreased complement activation on RBCs in vitro. In conclusion, complement over activation is a common pathogenic event in SCD that is associated with formation of DRBCs and hemolysis. And, it affects red cells, leukocytes and endothelial cells. This complement over activation is partly alleviated by hydroxyurea therapy.

Endothelial cells: source, barrier, and target of defensive mediators.

Endothelium is strategically located at the interface between blood and interstitial tissues, placing thus endothelial cell as a key player in vascular homeostasis. Endothelial cells are in a dynamic equilibrium with their environment and constitute concomitantly a source, a barrier, and a target of defensive mediators. This review will discuss the recent advances in our understanding of the complex crosstalk between the endothelium, the complement system and the hemostasis in health and in disease. The first part will provide a general introduction on endothelial cells heterogeneity and on the physiologic role of the complement and hemostatic systems. The second part will analyze the interplay between complement, hemostasis and endothelial cells in physiological conditions and their alterations in diseases. Particular focus will be made on the prototypes of thrombotic microangiopathic disorders, resulting from complement or hemostasis dysregulation-mediated endothelial damage: atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Novel aspects of the pathophysiology of the thrombotic microangiopathies will be discussed.

Hemolytic uremic syndrome associated with Bordetella pertussis infection in a 2-month-old infant carrying a pathogenic variant in complement factor H.

BACKGROUND: Hemolytic uremic syndrome (HUS) has been associated with a number of infectious agents. We report here the case of an infant with severe Bordetella pertussis infection who developed HUS. CASE DIAGNOSIS/TREATMENT: A 2-month-old preterm male was admitted for severe Bordetella pertussis infection. Symptoms leading to a diagnosis of hemolytic uremic syndrome (HUS) rapidly appeared: hemolytic anemia, thrombocytopenia, and acute kidney injury. He was treated with 25 days of peritoneal dialysis and received complement-targeting therapy with eculizumab (five injections over 2 months), in addition to blood transfusions, antibiotics, and respiratory support. The outcome was favorable. The genetic workup found a complement factor H gene variant which has been associated with atypical HUS. This variant was located in the C3b-binding site and functional tests revealed that it perturbed the regulatory activity of factor H. CONCLUSION: This case suggests that pertussis is a strong trigger of HUS and that complement investigations are necessary to guide treatment and understand the pathophysiology.

Complement C3 is a novel modulator of the anti-factor VIII immune response.

Development of neutralizing antibodies against therapeutic Factor VIII (FVIII) is the most serious complication of the treatment of hemophilia A. There is growing evidence to show the multifactorial origin of the anti-FVIII immune response, combining both genetic and environmental factors. While a role for the complement system on innate as well as adaptive immunity has been documented, the implication of complement activation on the onset of the anti-FVIII immune response is unknown. Here, using in vitro assays for FVIII endocytosis by human monocyte-derived dendritic cells and presentation to T cells, as well as in vivo complement depletion in FVIII-deficient mice, we show a novel role for complement C3 in enhancing the immune response against therapeutic FVIII. In vitro, complement C3 and its cleavage product C3b enhanced FVIII endocytosis by dendritic cells and presentation to a FVIII-specific CD4+ T-cell hybridoma. The C1 domain of FVIII had previously been shown to play an important role in FVIII endocytosis, and alanine substitutions of the K2092, F2093 and R2090 C1 residues drastically reduce FVIII uptake in vitro Interestingly, complement activation rescued the endocytosis of the FVIII C1 domain triple mutant. In a mouse model of severe hemophilia A, transient complement C3 depletion by humanized cobra venom factor, which does not generate anaphylatoxin C5a, significantly reduced the primary anti-FVIII immune response, but did not affect anti-FVIII recall immune responses. Taken together, our results suggest an important adjuvant role for the complement cascade in the initiation of the immune response to therapeutic FVIII.

Von Willebrand Factor Interacts with Surface-Bound C1q and Induces Platelet Rolling.

Premature atherosclerosis and thrombotic complications are major causes of morbidity and mortality in patients with systemic lupus erythematosus (SLE). However, the high incidence of these complications cannot be explained by traditional risk factors alone, suggesting direct effects of an activated immune system on hemostasis. The unexpected nucleotide sequence homology between SLE patient-derived autoantibodies against complement C1q (Fab anti-C1q) and von Willebrand factor (VWF) led us to investigate a potential interaction between the complement and hemostatic systems on the level of initiating molecules. VWF was found to bind to surface-bound C1q under static conditions. The binding could specifically be inhibited by Fab anti-C1q and C1q-derived peptides. Under shear stress the C1q-VWF interaction was enhanced, resembling the binding of VWF to collagen I. Additionally, we could show that C1q-VWF complexes induced platelet rolling and firm adhesion. Furthermore, we observed VWF binding to C1q-positive apoptotic microparticles and cholesterol crystals, as well as increased VWF deposition in C1q-positive glomeruli of SLE patients compared with control nephropathy. We show, to our knowledge for the first time, binding of VWF to C1q and thus a direct interaction between starter molecules of hemostasis and the classical pathway of complement. This direct interaction might contribute to the pathogenic mechanisms in complement-mediated, inflammatory diseases.

Anti-Factor B and Anti-C3b Autoantibodies in C3 Glomerulopathy and Ig-Associated Membranoproliferative GN.

In C3 glomerulopathy (C3G), the alternative pathway of complement is frequently overactivated by autoantibodies that stabilize the C3 convertase C3bBb. Anti-C3b and anti-factor B (anti-FB) IgG have been reported in three patients with C3G. We screened a cohort of 141 patients with C3G and Ig-associated membranoproliferative GN (Ig-MPGN) for anti-FB and anti-C3b autoantibodies using ELISA. We identified seven patients with anti-FB IgG, three patients with anti-C3b IgG, and five patients with anti-FB and anti-C3b IgG. Of these 15 patients, ten were diagnosed with Ig-MPGN. Among those patients with available data, 92% had a nephrotic syndrome, 64% had AKI, and 67% had a documented infection. Patients negative for anti-C3b and anti-FB IgG had much lower rates of infection (17 [25%] patients with C3G and one [10%] patient with Ig-MPGN). After 48 months, four of 15 (26%) positive patients had developed ESRD or died. All 15 patients had high plasma Bb levels, six (40%) patients had low levels of C3, and nine (60%) patients had high levels of soluble C5b9. In vitro, IgG purified from patients with anti-FB Abs selectively enhanced C3 convertase activity; IgG from patients with anti-C3b/anti-FB Abs enhanced C3 and C5 cleavage. IgG from patients with anti-C3b Abs stabilized C3bBb and perturbed C3b binding to complement receptor 1 but did not perturb binding to factor H. In conclusion, the prevalence of anti-C3b/anti-FB Abs and alternative pathway activation is similar in Ig-MPGN and C3G, suggesting similar pathogenic mechanisms. Identification of the underlying defect in Ig-MPGN could lead to improved treatment.

C5 nephritic factors drive the biological phenotype of C3 glomerulopathies.

C3 Glomerulopathies, which include Dense Deposit Disease and C3 Glomerulonephritis, are associated with genetic and acquired dysregulation of the C3 convertase alternative pathway of complement. The potential role of the activation of the C5 convertase has not been studied extensively. Here we analyzed IgG samples from patients with C3 Glomerulopathies to identify circulating autoantibodies that stabilize the C3 alternative pathway (C3 Nephritic Factors) as well as C5 convertases (C5 Nephritic Factors), thus preventing decay of these enzyme complexes. Rare variants in alternative pathway genes were found in 28 of 120 tested patients. C3 and C5 Nephritic Factors were found in 76 of 101 (75%) and 29 of 59 (49%) of the patients, respectively. Therefore, we compared the results of the assays for the C3 and C5 nephritic factors functional activity: 29% were positive for C3 Nephritic Factors alone, 39% were positive for both C3 and C5 Nephritic Factors, and 10% were positive for C5 Nephritic Factors alone. We found that the addition of properdin-enhanced stabilization of C3 convertase in the presence of IgG doubly positive for both Nephritic Factors, while it did not modify the stabilization mediated by IgG solely positive for C3 Nephritic Factors. Both C3 and C5 Nephritic Factors correlated with C3 consumption, while only C5 Nephritic Factors correlated with sC5b9 levels. C5 Nephritic Factors-positive patients were more likely to have C3 Glomerulonephritis than Dense Deposit Disease. Thus, dysregulation of the C5 convertase contributes to C3 Glomerulopathies inter-disease differences and may have direct therapeutic implications.