Alternative pathway of complement activation has a beneficial role against Chandipura virus infection.

The complement system is a critical component of both innate and adaptive immune responses. It has both protective and pathogenic roles in viral infections. There are no studies regarding the role of complement system in Chandipura virus (CHPV) infection. The current study has investigated the role of complement pathways in the in vitro neutralization of CHPV in Vero E6 cells. Using normal human serum (NHS), heat-inactivated serum (HIS), human serum deficient of complement factor, respective reconstituted serum, assays like in vitro neutralization, real-time PCR, and flow cytometry-based tissue culture-based limited dose assay (TC-LDA) were carried out for assessing the activation of different complement pathways. NHS from 9/10 donors showed complement dependent neutralization, reduction in viral load and decrease in percentage of CHPV-positive cells compared to their HIS counterparts. EGTA or EDTA pretreatment experiments indicated that CHPV neutralization proceeds through the alternative pathway of the complement activation. Our data showed a strong dependence on C3 for the in vitro neutralization of CHPV. Disparity in CHPV neutralization levels between factor B-deficient and reconstituted sera could be attributed to amplification loop/"tick-over" mechanism. Assays using C3, C5, and C8 deficient sera indicated that complement-mediated CHPV neutralization and suppression of CHPV infectivity are primarily through C3 and C5, and not dependent on downstream complement factor C8. With no specific anti-viral treatment/vaccine against Chandipura, the current data, elucidating role of human complement system in the neutralization of CHPV, may help in designing effective therapeutics.

Impaired insulin signaling in the B10.D2-Hc0 H2d H2-T18c/oSnJ mouse model of complement factor 5 deficiency.

Given the chemoattractant potential of complement factor 5 (C5) and its increased expression in adipose tissue (AT) of obese mice, we determined whether this protein of the innate immune system impacts insulin action. C5 control (C5cont) and spontaneously C5-deficient (C5def, B10.D2-Hc0 H2d H2-T18c/oSnJ) mice were placed on low- and high-fat diets to investigate their inflammatory and metabolic phenotypes. Adenoviral delivery was used to evaluate the effects of exogenous C5 on systemic metabolism. C5def mice gained less weight than controls while fed a high-fat diet, accompanied by reduced AT inflammation, liver mass, and liver triglyceride content. Despite these beneficial metabolic effects, C5def mice demonstrated severe glucose intolerance and systemic insulin resistance, as well as impaired insulin signaling in liver and AT. C5def mice also exhibited decreased expression of insulin receptor (INSR) gene and protein, as well as improper processing of pro-INSR. These changes were not due to the C5 deficiency alone as other C5-deficient models did not recapitulate the INSR processing defect; rather, in addition to the mutation in the C5 gene, whole genome sequencing revealed an intronic 31-bp deletion in the Insr gene in the B10.D2-Hc0 H2d H2-T18c/oSnJ model. Irrespective of the genetic defect, adenoviral delivery of C5 improved insulin sensitivity in both C5cont and C5def mice, indicating an insulin-sensitizing function of C5.

Role of Murine Complement Component C5 in Acute in Vivo Infection by Pathogenic Leptospira interrogans.

Leptospirosis is considered one of the most important zoonosis worldwide. The activation of the Complement System is important to control dissemination of several pathogens in the host. Only a few studies have employed murine models to investigate leptospiral infection and our aim in this work was to investigate the role of murine C5 during in vivo infection, comparing wild type C57BL/6 (B6 C5+/+) and congenic C57BL/6 (B6 C5-/-, C5 deficient) mice during the first days of infection. All animals from both groups survived for at least 8 days post-infection with pathogenic Leptospira interrogans serovar Kennewicki strain Fromm (LPF). At the third day of infection, we observed greater numbers of LPF in the liver of B6 C5-/- mice when compared to B6 C5+/+ mice. Later, on the sixth day of infection, the LPF population fell to undetectable levels in the livers of both groups of mice. On the third day, the inflammatory score was higher in the liver of B6 C5+/+ mice than in B6 C5-/- mice, and returned to normal on the sixth day of infection in both groups. No significant histopathological differences were observed in the lung, kidney and spleen from both infected B6 C5+/+ than B6 C5-/- mice. Likewise, the total number of circulating leukocytes was not affected by the absence of C5. The liver levels of IL-10 on the sixth day of infection was lower in the absence of C5 when compared to wild type mice. No significant differences were observed in the levels of several inflammatory cytokines when B6 C5+/+ and B6 C5-/- were compared. In conclusion, C5 may contribute to the direct killing of LPF in the first days of infection in C57BL/6 mice. On the other hand, other effector immune mechanisms probably compensate Complement impairment since the mice survival was not affected by the absence of C5 and its activated fragments, at least in the early stage of this infection.

Development of Autologous C5 Vaccine Nanoparticles to Reduce Intravascular Hemolysis in Vivo.

The complement system is emerging as a new target for treating many diseases. For example, Eculizumab, a humanized monoclonal antibody against complement component 5 (C5), has been approved for paroxysmal nocturnal hemoglobinuria (PNH) in which patient erythrocytes are lysed by complement. In this study, we developed vaccines to elicit autologous anti-C5 antibody production in mice for complement inhibition. Immunization of mice with a conservative C5 xenoprotein raised high titers of IgG's against the xenogenous C5, but these antibodies did not reduce C5 activity in the blood. In contrast, an autologous mouse C5 vaccine containing multiple predicted epitopes together with a tolerance-breaking peptide was found to induce anti-C5 autoantibody production in vivo, resulting in decreased hemolytic activity in the blood. We further validated a peptide epitope within this C5 vaccine and created recombinant virus-like particles (VLPs) displaying this epitope fused with the tolerance breaking peptide. Immunizing mice with these novel nanoparticles elicited strong humoral responses against recombinant mouse C5, reduced hemolytic activity, and protected the mice from complement-mediated intravascular hemolysis in a model of PNH. This proof-of-concept study demonstrated that autologous C5-based vaccines could be an effective alternative or supplement for treating complement-mediated diseases such as PNH.

My contribution, my dream - a look at the future of APS.

We found, in 1994, that the epitope for anticardiolipin antibodies (aCL) developed when ß2-glycoprotein I (ß2GPI) was adsorbed on polyoxygenated polystyrene plates. We also found, in 2009, that the cleaved form of ß2GPI (nicked ß2GPI) was bound to angiostatin 4.5 and attenuated its antiangiogenic property. We described in 2000 that antiprothrombin antibodies were bound to prothrombin exposed to immobilized phosphatidylserine (aPS/PT) and more than 95% of antiphospholipid syndrome patients who were positive for aPS/PT had lupus anticoagulant. We demonstrated that in the cells stimulated by human monoclonal anti-ß2GPI antibodies, p38 mitogen-activated protein kinase phosphorylation was observed in the presence of ß2GPI. Furthermore, we found that complement activation was essential for thrombus formation in patients with the antiphospholipid syndrome in vivo.

Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase.

Lymphocyte depletion with antithymocyte globulin (ATG) can be complicated by systemic coagulation activation. We found that ATG activated tissue factor procoagulant activity (TF PCA) on monocytic cells more potently than other stimuli that decrypt TF, including cell disruption, TF pathway inhibitor inhibition, and calcium ionophore treatment. Induction of TF PCA by ATG was dependent on lipid raft integrity and complement activation. We showed that ATG-mediated TF activation required complement activation until assembly of the C5b-7 membrane insertion complex, but not lytic pore formation by the membrane attack complex C5b-9. Consistently, induction of TF PCA by ATG did not require maximal phosphatidylserine membrane exposure and was not correlated with the magnitude of complement-induced lytic cell injury. Blockade of free thiols, an inhibitory monoclonal antibody to protein disulfide isomerase (PDI), and the small-molecule PDI antagonist quercetin-3-rutinoside prevented ATG-mediated TF activation, and C5 complement activation resulted in oxidation of cell surface PDI. This rapid and potent mechanism of cellular TF activation represents a novel connection between the complement system and cell surface PDI-mediated thiol-disulfide exchange. Delineation of this clinically relevant mechanism of activation of the extrinsic coagulation pathway during immunosuppressive therapy with ATG may have broader implications for vascular thrombosis associated with inflammatory disorders.

Complement factors in newly diagnosed Nigerian schizoprenic patients and those on antipsychotic therapy.

The role of Complement factors in the pathogenesis of psychiatric disorders is enormous, but the data on levels and functions of complement factors in patients with schizophrenia are scanty and conflicting. To address this issue, levels of Complement regulators (C1 inhibitor and C3 activator) and complement factors (C1q, C3c, C4 and C5) were determined in the serum of newly diagnosed drug free schizophrenic patients, schizophrenic patients on medication and healthy subjects using immune-plates. C1q was significantly reduced in newly diagnosed schizophrenic patients or schizophrenic patients on medication compared with the controls. C3c was significantly reduced in newly diagnosed schizophrenic patients compared with controls or schizophrenic patients on medication. The levels of C3 activators, C1 inhibitors and C4 were similar in the two groups of schizophrenic patients compared with the controls. It may be concluded from this study that C1q is deficient in schizophrenic patients; and that C3c may differentiate newly diagnosed schizophrenia from schizophrenic patients on medication.

Structure, function and control of complement C5 and its proteolytic fragments.

As part of the innate immune system, the complement system recognises a wide range of non-self structures present on pathogens or altered self cells. Its activation elicits proteolytic cascades which eventually results in the cleavage of the C5 protein into two fragments, C5a and C5b. The small anaphylatoxin C5a induces a variety of biological responses upon binding to the 7TM receptors C5aR and the C5L2, while the large C5b fragment nucleates formation of the membrane attack complex capable of killing susceptible pathogens by the formation of a pore structure in association with complement components C6, C7, C8, and C9. A number of regulatory molecules help to control C5 mediated immune responses towards host cells, but in several major inflammatory conditions including sepsis and arthritis, C5a is believed to contribute significantly to disease etiology. Inhibition of membrane attack complex assembly is already approved for treatment of paroxysmal nocturnal haemoglobinuria and atypical hemolytic uremic syndrome. A number of recent crystal structures have provided a comprehensive insight into the architecture and properties of intact C5 and its fragments, and how pathogens interfere with their function. Here we review the functional and structural aspects of C5 and its fragments, the pathological conditions associated with them, and strategies employed by pathogens to interfere with the biological function of C5. Structural insight and elucidation of evasion strategies employed by pathogens present a unique opportunity for promoting the development of novel selective C5 inhibitors with therapeutic applications.

A novel role for the fifth component of complement (C5) in cardiac physiology.

We have previously demonstrated that C5-deficient A/J and recombinant congenic BcA17 mice suffer from cardiac dysfunction when infected with C. albicans blastospores intravenously. During these studies we had observed that, even in the control un-infected state, BcA17 hearts displayed alterations in gene expression that have been associated with pathological cardiac hypertrophy in comparison to parental C5-sufficient C57Bl/6J (B6) mice. Of note was an increase in the expression of Nppb, a member of the fetal gene program and a decrease in the expression of Rgs2, an inhibitor of the hypertrophic response. We now report that C5-deletion has also affected the expression of other elements of the fetal gene program. Moreover deleting the C5a receptor, C5aR, has essentially the same effect as deleting C5, indicating a key role for C5a-C5aR signaling in the phenotype. Having noted a pathological phenotype in the un-infected state, we investigated the role of C5 in the response to cardiac stress. In previous studies, comparison of the expression profiles of C. albicans-infected BcA17 and similarly infected B6 hearts had revealed a paucity of cardioprotective genes in the C5-deficient heart. To determine whether this was also directly linked to C5-deficiency, we tested the expression of 5 such genes in the C. albicans-infected C5aR(-/-) mice. We found again that deletion of C5aR recapitulated the alterations in stress response of BcA17. To determine whether our observations were relevant to other forms of cardiac injury, we tested the effect of C5-deficiency on the response to isoproterenol-induced hypertrophic stimulation. Consistent with our hypothesis, A/J, BcA17 and C5aR(-/-) mice responded with higher levels of Nppa expression than B6 and BALB/c mice. In conclusion, our results suggest that an absence of functional C5a renders the heart in a state of distress, conferring a predisposition to cardiac dysfunction in the face of additional injury.

Distinct roles for C3a and C5a in complement-induced tubulointerstitial injury.

To prevent injury to host tissues, complement activation is regulated by a number of plasma and membrane-associated proteins, most of which limit C3 and C5 activation. An influx of circulating C3 from a syngeneic host into donor kidneys deficient in Crry (a membrane protein that reduces C3 convertase activity) causes spontaneous complement activation, primarily in the tubulointerstitum, leading to renal failure. To determine the roles of the C3a and C5a anaphylatoxins in tubulointerstitial inflammation and fibrosis, kidneys from Crry-/-C3-/- mice were transplanted into hosts lacking the C3a and/or C5a receptor. While unrestricted complement activation in the tubules was not affected by receptor status in the transplant recipient, C3a receptor deficiency in the recipients led to significantly reduced renal leukocyte infiltration and the extent of tubulointerstitial inflammation and fibrosis, all of which led to preserved renal function. The absence of C5a receptors in recipients was not only inconsequential, but the protective effect of C3a receptor deficiency was also eliminated, suggesting distinct roles of C3a and C5a receptor signaling in this model. There was significant infiltration of the tubulointerstitum with 7/4+F4/80+CD11b+ myelomonocytic cells and Thy1.2+ T cells along injured tubules, and interstitial collagen I and III deposition, all of which were C3a receptor dependent. Thus, blockade of C3a receptor signaling is a possible treatment to reduce renal inflammation and preserve renal function associated with complement activation.

Experimental models of membranoproliferative glomerulonephritis, including dense deposit disease.

Membranoproliferative glomerulonephritis (MPGN) is characterised by mesangial expansion and hypercellularity and capillary wall thickening with capillary wall and mesangial deposits of immunoglobulin and/or complement. Two main forms are described in humans: MPGN type I with subendothelial and mesangial electron-dense deposits on electron microscopy, and MPGN type II, or dense deposit disease, with electron dense transformation of the glomerular capillary wall. Spontaneous MPGN type I has been described in dogs and sheep in association with C3 deficiency. Induced models of MPGN type I have been described in mice with cryoglobulinaemia. Glomerulonephritis resembling MPGN type II has occurred spontaneously in pigs that have a genetic deficiency of the complement control protein factor H. The animals develop capillary wall deposits of C3 before birth. Mice have been genetically engineered with a deficiency of factor H and similarly develop glomerular capillary wall C3 with MPGN. This model has been used to study both pathogenesis and therapeutic interventions. In particular, MPGN associated with factor H deficiency is absolutely dependent on both the ability to activate C3 and on the ability of factor I to cleave C3b. There is an important role for C5 activation in the development of glomerular inflammation in this model. Factor H dysfunction is associated with an increased susceptibility to complement-activating nephrotoxic insults and in these scenarios C5 activation appears to play a major role in mediating glomerular injury.

The development of atypical hemolytic uremic syndrome depends on complement C5.

Gene variants in the alternative pathway of the complement system strongly associate with atypical hemolytic uremic syndrome (aHUS), presumably by predisposing to increased complement activation within the kidney. Complement factor H (CFH) is the major regulator of complement activation through the alternative pathway. Factor H-deficient mice transgenically expressing a mutant CFH protein (Cfh(-/-).FHΔ16-20) that functionally mimics the CFH mutations reported in aHUS patients spontaneously develop thrombotic microangiopathy. To investigate the role of complement C5 activation in this aHUS model, we generated C5-deficient Cfh(-/-).FHΔ16-20 mice. Both C5-sufficient and C5-deficient Cfh(-/-).FHΔ16-20 mice had abnormal C3 deposition within the kidney, but spontaneous aHUS did not develop in any of the C5-deficient mice. Furthermore, although Cfh(-/-).FHΔ16-20 animals demonstrated marked hypersensitivity to experimentally triggered renal injury, animals with concomitant C5 deficiency did not. These data demonstrate a critical role for C5 activation in both spontaneous aHUS and experimentally triggered renal injury in animals with defective complement factor H function. This study provides a rationale to investigate therapeutic inhibition of C5 in human aHUS.

Novel insight into stem cell mobilization-plasma sphingosine-1-phosphate is a major chemoattractant that directs the egress of hematopoietic stem progenitor cells from the bone marrow and its level in peripheral blood increases during mobilization due to activation of complement cascade/membrane attack complex.

The complement cascade (CC) becomes activated and its cleavage fragments play a crucial role in the mobilization of hematopoietic stem/progenitor cells (HSPCs). Here, we sought to determine which major chemoattractant present in peripheral blood (PB) is responsible for the egress of HSPCs from the bone marrow (BM). We noticed that normal and mobilized plasma strongly chemoattracts HSPCs in a stromal-derived factor-1 (SDF-1)-independent manner because (i) plasma SDF-1 level does not correlate with mobilization efficiency; (ii) the chemotactic plasma gradient is not affected in the presence of AMD3100 and (iii) it is resistant to denaturation by heat. Surprisingly, the observed loss of plasma chemotactic activity after charcoal stripping suggested the involvement of bioactive lipids and we focused on sphingosine-1-phosphate (S1P), a known chemoattracant of HSPCs. We found that S1P (i) creates in plasma a continuously present gradient for BM-residing HSPCs; (ii) is at physiologically relevant concentrations a chemoattractant several magnitudes stronger than SDF-1 and (iii) its plasma level increases during mobilization due to CC activation and interaction of the membrane attack complex (MAC) with erythrocytes that are a major reservoir of S1P. We conclude and propose a new paradigm that S1P is a crucial chemoattractant for BM-residing HSPCs and that CC through MAC induces the release of S1P from erythrocytes for optimal egress/mobilization of HSPCs.

Mobilization studies in complement-deficient mice reveal that optimal AMD3100 mobilization of hematopoietic stem cells depends on complement cascade activation by AMD3100-stimulated granulocytes.

We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) induced by granulocyte colony-stimulating factor (G-CSF) and C5 cleavage has an important function in optimal egress of HSPCs. In this work, we explored whether CC is involved in mobilization of HSPCs induced by the CXCR4 antagonist, AMD3100. To address this question, we performed mobilization studies in mice that display a defect in the activation of the proximal steps of CC (Rag(-/-), severe combined immune deficient (SCID), C2.Cfb(-/-)) as well as in mice that do not activate the distal steps of CC (C5(-/-)). We noticed that proximal CC activation-deficient mice (above C5 level), in contrast to distal step CC activation-deficient C5(-/-) ones, mobilize normally in response to AMD3100 administration. We hypothesized that this discrepancy in mobilization could be explained by AMD3100-activating C5 in Rag(-/-), SCID, and C2.Cfb(-/-) animals in a non-canonical mechanism involving activated granulocytes. To support this, granulocytes (i) first egress from BM and (ii) secrete several proteases that cleave/activate C5 in response to AMD3100. We conclude that AMD3100-directed mobilization of HSPCs, similarly to G-CSF-induced mobilization, depends on activation of CC; however, in contrast to G-CSF, AMD3100 activates the distal steps of CC directly at the C5 level. Overall, these data support that C5 cleavage fragments and distal steps of CC activation are required for optimal mobilization of HSPCs.

[Eculizumab in paroxysmal nocturnal hemoglobinuria]. / Anticorps monoclonal anti-C5 (éculizumab) dans l'hémoglobinurie paroxystique nocturne.

Paroxysmal nocturnal hemoglobinuria is a rare acquired clonal of the hematopoietic stem cell due to acquired mutation of the PIG-A gene. This results in the lack of two GPI-anchored membrane proteins involved in the inhibition of complement attack, thus explaining red cells hemolysis. The development of an anti-C5 monoclonal antibody (eculizumab) had profoundly modified the treatment of the the hemolytic form of the disease.

Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria.

The complement system provides critical immunoprotective and immunoregulatory functions but uncontrolled complement activation can lead to severe pathology. In the rare hemolytic disease paroxysmal nocturnal hemoglobinuria (PNH), somatic mutations result in a deficiency of glycosylphosphatidylinositol-linked surface proteins, including the terminal complement inhibitor CD59, on hematopoietic stem cells. In a dysfunctional bone marrow background, these mutated progenitor blood cells expand and populate the periphery. Deficiency of CD59 on PNH red blood cells results in chronic complement-mediated intravascular hemolysis, a process central to the morbidity and mortality of PNH. A recently developed, humanized monoclonal antibody directed against complement component C5, eculizumab (Soliris; Alexion Pharmaceuticals Inc., Cheshire, CT, USA), blocks the proinflammatory and cytolytic effects of terminal complement activation. The recent approval of eculizumab as a first-in-class complement inhibitor for the treatment of PNH validates the concept of complement inhibition as an effective therapy and provides rationale for investigation of other indications in which complement plays a role.

C5-blocking antibody reduces fluid requirements and improves responsiveness to fluid infusion in hemorrhagic shock managed with hypotensive resuscitation.

Hypotensive resuscitation strategies and inhibition of complement may both be of benefit in hemorrhagic shock. We asked if C5-blocking antibody (anti-C5) could diminish the amount of fluid required and improve responsiveness to resuscitation from hemorrhage. Awake, male Sprague-Dawley rats underwent controlled hemorrhage followed by prolonged (3 h) hypotensive resuscitation with lactated Ringer's or Hextend, with or without anti-C5. Anti-C5 treatment led to an estimated 62.3 and 58.5% reduction in the volume of Hextend and lactated Ringer's, respectively. In the subgroup of animals with a positive mean arterial pressure (MAP) response to fluid infusion following prolonged hypotension, anti-C5 treatment led to an estimated 4.7- and 4.1-fold increase in mean arterial pressure response per unit Hextend and lactated Ringer's infused, respectively. We observed no significant postresuscitation metabolic differences between the anti-C5 groups and controls. Whether anti-C5 could serve as a volume-sparing adjunct that improves responsiveness to fluid administration in humans deserves further study.