Formation Of Terminal Complement Complex Research Articles

Dual inhibition of complement C5 and CD14 attenuates inflammation in a cord blood model.

Escherichia coli and Group B streptococci (GBS) are the main causes of neonatal early-onset sepsis (EOS). Despite antibiotic therapy, EOS is associated with high morbidity and mortality. Dual inhibition of complement C5 and the Toll-like receptor co-factor CD14 has in animal studies been a promising novel therapy for sepsis. Whole blood was collected from the umbilical cord after caesarean section (n = 30). Blood was anti-coagulated with lepirudin. C5 inhibitor (eculizumab) and anti-CD14 was added 8 min prior to, or 15 and 30 min after adding E. coli or GBS. Total bacterial incubation time was 120 min (n = 16) and 240 min (n = 14). Cytokines and the terminal complement complex (TCC) were measured using multiplex technology and ELISA. Dual inhibition significantly attenuated TCC formation by 25-79% when adding inhibitors with up to 30 min delay in both E. coli- and GBS-induced inflammation. TNF, IL-6 and IL-8 plasma concentration were significantly reduced by 28-87% in E. coli-induced inflammation when adding inhibitors with up to 30 min delay. The dual inhibition did not significantly reduce TNF, IL-6 and IL-8 plasma concentration in GBS-induced inflammation. Dual inhibition of C5 and CD14 holds promise as a potential future treatment for severe neonatal EOS. Neonatal sepsis can cause severe host inflammation with high morbidity and mortality, but there are still no effective adjunctive immunologic interventions available. Adding CD14 and complement C5 inhibitors up to 30 min after incubation of E. coli or Group B streptococci in a human umbilical cord blood model significantly reduced complement activation and cytokine release. Dual inhibition of C5 and CD14 is a potential future therapy to modulate systemic inflammation in severe cases of neonatal sepsis.

Interleukin-1\u03b2 and cathepsin D modulate formation of the terminal complement complex in cultured human disc tissue

PurposeFormation of terminal complement complex (TCC), a downstream complement system activation product inducing inflammatory processes and cell lysis, has been identified in degenerated discs. However, it remains unclear which molecular factors regulate complement activation during disc degeneration (DD). This study investigated a possible involvement of the pro-inflammatory cytokine interleukin-1β (IL-1β) and the lysosomal protease cathepsin D (CTSD).MethodsDisc biopsies were collected from patients suffering from DD (n = 43) and adolescent idiopathic scoliosis (AIS, n = 13). Standardized tissue punches and isolated cells from nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) were stimulated with 5% human serum (HS) alone or in combination with IL-1β, CTSD or zymosan. TCC formation and modulation by the complement regulatory proteins CD46, CD55 and CD59 were analysed.ResultsIn DD tissue cultures, IL-1β stimulation decreased the percentage of TCC + cells in AF and EP (P < 0.05), whereas CTSD stimulation significantly increased TCC deposition in NP (P < 0.01) and zymosan in EP (P < 0.05). Overall, the expression of CD46, CD55 and CD59 significantly increased in all isolated cells during culture (P < 0.05). Moreover, cellular TCC deposition was HS concentration dependent but unaffected by IL-1β, CTSD or zymosan.ConclusionThese results suggest a functional relevance of IL-1β and CTSD in modulating TCC formation in DD, with differences between tissue regions. Although strong TCC deposition may represent a degeneration-associated event, IL-1β may inhibit it. In contrast, TCC formation was shown to be triggered by CTSD, indicating a multifunctional involvement in disc pathophysiology.

Terminal complement complex formation is associated with intervertebral disc degeneration

PurposeThe complement system is a crucial part of innate immunity. Recent work demonstrated an unexpected contribution to tissue homeostasis and degeneration. This study investigated for the first time, in human disc tissues, the deposition profile of the complement activation product terminal complement complex (TCC), an inflammatory trigger and inducer of cell lysis, and its inhibitor CD59, and their correlation with the degree of disc degeneration (DD).MethodsDisc biopsies were collected from patients diagnosed with DD (n = 39, age 63 ± 12) and adolescent idiopathic scoliosis (AIS, n = 10, age 17 ± 4) and compared with discs from healthy Young (n = 11, age 7 ± 7) and Elder (n = 10, age 65 ± 15) donors. Immunohistochemical detection of TCC and CD59 in nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) was correlated with age, Pfirrmann grade and Modic changes.ResultsHigher percentage of TCC+ cells was detected in the NP and EP of DD compared to Elder (P < 0.05), and in the EP of Young versus Elder (P < 0.001). In DD, TCC deposition was positively correlated with Pfirrmann grade, but not with Modic changes, whereas for Young donors, a negative correlation was found with age, indicating TCC’s involvement not only in DD, but also in early stages of skeletal development. Higher CD59 positivity was found in AIS and DD groups compared to Young (P < 0.05), and it was negatively correlated with the age of the patients.ConclusionTCC deposition positively correlated with the degree of disc degeneration. A functional relevance of TCC may exist in DD, representing a potential target for new therapeutics.

Dihydro-stilbene gigantol relieves CCl4-induced hepatic oxidative stress and inflammation in mice via inhibiting C5b-9 formation in the liver.

In general, anti-inflammatory treatment is considered for multiple liver diseases despite the etiology. But current drugs for alleviating liver inflammation have defects, making it necessary to develop more potent and safer drugs for liver injury. In this study, we screened a series of (dihydro-)stilbene or (dihydro-)phenanthrene derivatives extracted from Pholidota chinensis for their potential biological activities. Among 31 compounds, the dihydro-stilbene gigantol exerted most potent protective effects on human hepatocytes against lithocholic acid toxicity, and exhibited solid antioxidative and anti-inflammatory effect in vitro. In mice with CCl4-induced acute liver injury, pre-administration of gigantol (10, 20, 40 mg· kg-1· d-1, po, for 7 days) dose-dependently decreased serum transaminase levels and improved pathological changes in liver tissues. The elevated lipid peroxidation and inflammatory responses in the livers were also significantly alleviated by gigantol. The pharmacokinetic studies showed that gigantol was highly concentrated in the mouse livers, which consisted with its efficacy in preventing liver injury. Using a label-free quantitative proteomic analysis we revealed that gigantol mainly regulated the immune system process in liver tissues of CCl4-treated mice, and the complement and coagulation cascades was the predominant pathway; gigantol markedly inhibited the expression of complement component C9, which was a key component for the formation of terminal complement complex (TCC) C5b-9. These results were validated by immunohistochemistry (IHC) or real time-PCR. Confocal microscopy analysis showed that gigantol significantly inhibited the vascular deposition of TCC in the liver. In conclusion, we demonstrate for the first time that oral administration of gigantol potently relieves liver oxidative stress and inflammation, possibly via a novel mechanism of inhibiting the C5b-9 formation in the liver.

Crucial role of the terminal complement complex in chondrocyte death and hypertrophy after cartilage trauma

Innate immune response and particularly terminal complement complex (TCC) deposition are thought to be involved in the pathogenesis of posttraumatic osteoarthritis. However, the possible role of TCC in regulated cell death as well as chondrocyte hypertrophy and senescence has not been unraveled so far and was first addressed using an ex vivo human cartilage trauma-model. Cartilage explants were subjected to blunt impact (0.59J) and exposed to human serum (HS) and cartilage homogenate (HG) with or without different potential therapeutics: RIPK1-inhibitor Necrostatin-1 (Nec), caspase-inhibitor zVAD, antioxidant N-acetyl cysteine (NAC) and TCC-inhibitors aurintricarboxylic acid (ATA) and clusterin (CLU). Cell death and hypertrophy/senescence-associated markers were evaluated on mRNA and protein level. Addition of HS resulted in significantly enhanced TCC deposition on chondrocytes and decrease of cell viability after trauma. This effect was potentiated by HG and was associated with expression of RIPK3, MLKL and CASP8. Cytotoxicity of HS could be prevented by heat-inactivation or specific inhibitors, whereby combination of Nec and zVAD as well as ATA exhibited highest cell protection. Moreover, HS+HG exposition enhanced the gene expression of CXCL1, IL-8, RUNX2 and VEGFA as well as secretion of IL-6 after cartilage trauma. Our findings imply crucial involvement of the complement system and primarily TCC in regulated cell death and phenotypic changes of chondrocytes after cartilage trauma. Inhibition of TCC formation or downstream signaling largely modified serum-induced pathophysiologic effects and might therefore represent a therapeutic target to maintain the survival and chondrogenic character of cartilage cells.

Combining MAP‐1:CD35 or MAP‐1:CD55 fusion proteins with pattern‐recognition molecules as novel targeted modulators of the complement cascade

Dysregulation of the complement system is involved in the pathogenesis of several diseases, and its inhibition has been shown to be a feasible therapeutic option. Therefore, there is an interest in the development of complement modulators to treat complement activation-related inflammatory pathologies. Mannose-binding lectin (MBL)/ficolin/collectin-associated protein-1 (MAP-1) is a regulatory molecule of the lectin pathway (LP), whereas complement receptor 1 (CD35) and decay-accelerating factor (CD55) are membrane-anchored regulators with effects on the central effector molecule C3. In this study, we developed 2 novel soluble chimeric inhibitors by fusing MAP-1 to the 3 first domains of CD35 (CD351-3) or the 4 domains of CD55 (CD551-4) to modulate the complement cascade at 2 different stages. The constructs showed biologic properties similar to those of the parent molecules. In functional complement activation assays, the constructs were very efficient in inhibiting LP activation at the level of C3 and in the formation of terminal complement complex. This activity was enhanced when coincubated with recombinant LP recognition molecules MBL and ficolin-3. Recombinant MAP-1 fusion proteins, combined with recombinant LP recognition molecules to target sites of inflammation, represent a novel and effective therapeutic approach involving the initiation and the central and terminal effector functions of the complement cascade.-Pérez-Alós, L., Bayarri-Olmos, R., Skjoedt, M.-O., Garred, P. Combining MAP-1:CD35 or MAP-1:CD55 fusion proteins with pattern-recognition molecules as novel targeted modulators of the complement cascade.

Reduced Terminal Complement Complex Formation in Mice Manifests in Low Bone Mass and Impaired Fracture Healing

The terminal complement complex (TCC) is formed on activation of the complement system, a crucial arm of innate immunity. TCC formation on cell membranes results in a transmembrane pore leading to cell lysis. In addition, sublytic TCC concentrations can modulate various cellular functions. TCC-induced effects may play a role in the pathomechanisms of inflammatory disorders of the bone, including rheumatoid arthritis and osteoarthritis. In this study, we investigated the effect of the TCC on bone turnover and repair. Mice deficient for complement component 6 (C6), an essential component for TCC assembly, and mice with a knockout of CD59, which is a negative regulator of TCC formation, were used in this study. The bone phenotype was analyzed invivo, and bone cell behavior was analyzed exvivo. In addition, the mice were subjected to a femur osteotomy. Under homeostatic conditions, C6-deficient mice displayed a reduced bone mass, mainly because of increased osteoclast activity. After femur fracture, the inflammatory response was altered and bone formation was disturbed, which negatively affected the healing outcome. By contrast, CD59-knockout mice only displayed minor skeletal alterations and uneventful bone healing, although the early inflammatory reaction to femur fracture was marginally enhanced. These results demonstrate that TCC-mediated effects regulate bone turnover and promote an adequate response to fracture, contributing to an uneventful healing outcome.

Urine Complement Proteins and the Risk of Kidney Disease Progression and Mortality in Type 2 Diabetes.

OBJECTIVEWe examined the association of urine complement proteins with progression to end-stage renal disease (ESRD) or death in people with type 2 diabetes and proteinuric diabetic kidney disease (DKD).RESEARCH DESIGN AND METHODSUsing targeted mass spectrometry, we quantified urinary abundance of 12 complement proteins in a predominantly Mexican American cohort with type 2 diabetes and proteinuric DKD (n = 141). The association of urine complement proteins with progression to ESRD or death was evaluated using time-to-event analyses.RESULTSAt baseline, median estimated glomerular filtration rate (eGFR) was 54 mL/min/1.73 m2 and urine protein-to-creatinine ratio 2.6 g/g. Sixty-seven participants developed ESRD or died, of whom 39 progressed to ESRD over a median of 3.1 years and 40 died over a median 3.6 years. Higher urine CD59, an inhibitor of terminal complement complex formation, was associated with a lower risk of ESRD (hazard ratio [HR] [95% CI per doubling] 0.50 [0.29–0.87]) and death (HR [95% CI] 0.56 [0.34–0.93]), after adjustment for demographic and clinical covariates, including baseline eGFR and proteinuria. Higher urine complement components 4 and 8 were associated with lower risk of death (HR [95% CI] 0.57 [0.41–0.79] and 0.66 [0.44–0.97], respectively); higher urine factor H–related protein 2, a positive regulator of the alternative complement pathway, was associated with greater risk of death (HR [95% CI] 1.61 [1.05–2.48]) in fully adjusted models.CONCLUSIONSIn a largely Mexican American cohort with type 2 diabetes and proteinuric DKD, urine abundance of several complement and complement regulatory proteins was strongly associated with progression to ESRD and death.

Calcineurin inhibitor-induced complement system activation via ERK1/2 signalling is inhibited by SOCS-3 in human renal tubule cells.

One factor that significantly contributes to renal allograft loss is chronic calcineurin inhibitor (CNI) nephrotoxicity (CIN). Among other factors, the complement (C-) system has been proposed to be involved CIN development. Hence, we investigated the impact of CNIs on intracellular signalling and the effects on the C-system in human renal tubule cells. In a qPCR array, CNI treatment upregulated C-factors and downregulated SOCS-3 and the complement inhibitors CD46 and CD55. Additionally, ERK1/-2 was required for these regulations. Following knock-down and overexpression of SOCS-3, we found that SOCS-3 inhibits ERK1/-2 signalling. Finally, we assessed terminal complement complex formation, cell viability and apoptosis. Terminal complement complex formation was induced by CNIs. Cell viability was significantly decreased, whereas apoptosis was increased. Both effects were reversed under complement component-depleted conditions. In vivo, increased ERK1/-2 phosphorylation and SOCS-3 downregulation were observed at the time of transplantation in renal allograft patients who developed a progressive decline of renal function in the follow-up compared to stable patients. The progressive cohort also had lower total C3 levels, suggesting higher complement activity at baseline. In conclusion, our data suggest that SOCS-3 inhibits CNI-induced ERK1/-2 signalling, thereby blunting the negative control of C-system activation.

Cyclodextrin Reduces Cholesterol Crystal-Induced Inflammation by Modulating Complement Activation.

Cholesterol crystals (CC) are abundant in atherosclerotic plaques and promote inflammatory responses via the complement system and inflammasome activation. Cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (BCD) is a compound that solubilizes lipophilic substances. Recently we have shown that BCD has an anti-inflammatory effect on CC via suppression of the inflammasome and liver X receptor activation. The putative effects of BCD on CC-induced complement activation remain unknown. In this study, we found that BCD bound to CC and reduced deposition of Igs, pattern recognition molecules, and complement factors on CC in human plasma. Furthermore, BCD decreased complement activation as measured by terminal complement complex and lowered the expression of complement receptors on monocytes in whole blood in response to CC exposure. In line with this, BCD also reduced reactive oxygen species formation caused by CC in whole blood. Furthermore, BCD attenuated the CC-induced proinflammatory cytokine responses (e.g., IL-1α, MIP-1α, TNF, IL-6, and IL-8) as well as regulated a range of CC-induced genes in human PBMC. BCD also regulated complement-related genes in human carotid plaques treated ex vivo. Formation of terminal complement complex on other complement-activating structures such as monosodium urate crystals and zymosan was not affected by BCD. These data demonstrate that BCD inhibits CC-induced inflammatory responses, which may be explained by BCD-mediated attenuation of complement activation. Thus, these findings support the potential for using BCD in treatment of atherosclerosis.

FHR-1 Binds to C-Reactive Protein and Enhances Rather than Inhibits Complement Activation.

Factor H-related protein (FHR) 1 is one of the five human FHRs that share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by competitive inhibition of FH binding to some surfaces and immune proteins. We have recently shown that FHR-1 binds to pentraxin 3. In this study, our aim was to investigate whether FHR-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction, and study the role of FHR-1 in complement activation and regulation. FHR-1 did not bind to native, pentameric CRP, but it bound strongly to monomeric CRP via its C-terminal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement deregulation also on this ligand. FHR-1 did not inhibit regulation of solid-phase C3 convertase by FH and did not inhibit terminal complement complex formation induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase formation and thereby enhanced complement activation. FHR-1/CRP interactions increased complement activation via the classical and alternative pathways on surfaces such as the extracellular matrix and necrotic cells. Altogether, these results identify CRP as a ligand for FHR-1 and suggest that FHR-1 enhances, rather than inhibits, complement activation, which may explain the protective effect of FHR-1 deficiency in age-related macular degeneration.

Three pentraxins C-reactive protein, serum amyloid p component and pentraxin 3 mediate complement activation using Collectin CL-P1

BackgroundPentraxins (PTXs) are a superfamily of multifunctional conserved proteins involved in acute-phase responses. Recently, we have shown that collectin placenta 1 (CL-P1) and C-reactive protein (CRP) mediated complement activation and failed to form terminal complement complex (TCC) in normal serum conditions because of complement factor H inhibition. MethodsWe used CL-P1 expressing CHO/ldlA7 cells to study the interaction with PTXs. Soluble type CL-P1 was used in an ELISA assay for the binding, C3 and TCC deposition experiments. Furthermore, we used our previously established CL-P1 expressing HEK293 cells for the C3 fragment and TCC deposition assay. ResultsWe demonstrated that CL-P1 also bound serum amyloid p component (SAP) and pentraxin 3 (PTX3) to activate the classical pathway and the alternative pathway using factor B. CRP and PTX3 further amplified complement deposition by properdin. We found that CRP and PTX3 recruit CFH, whereas SAP recruits C4 binding protein on CL-P1 expressing cell surfaces to prevent the formation of TCC in normal serum conditions. In addition, depletion of CFH, C4BP and complement factor I (CFI) failed to prevent TCC formation both in ELISA and cell experiments. Furthermore, soluble complement receptor 1, an inhibitor of all complement pathways prevents PTX induced TCC formation. ConclusionOur current study hypothesizes that the interaction of pentraxins with CL-P1 is involved in complement activation. General significanceCL-P1 might generally inhibit PTX induced complement activation and host damage to protect self-tissues.

Preformed mediators of defense-Gatekeepers enter the spotlight.

Preformed mediators of defense-Gatekeepers enter the spotlight.

NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways.

Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b–9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their “AP tool kit” to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases.

Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-β and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed complement activation (C3a and C5a) and soluble terminal complement complex formation (C5b-9) locally and active TGF-β1 systemically. C3aR/C5aR antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C5aR, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C5aR in lesions. The blockade of C3aR and C5aR arrested the progression of fibrosis by attenuating local complement activation and TGF-β/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with IPF.-Gu, H., Fisher, A. J., Mickler, E. A., Duerson, F., III, Cummings, O. W., Peters-Golden, M., Twigg, H. L., III, Woodruff, T. M., Wilkes, D. S., Vittal, R. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

The Plasmodium falciparum blood stages acquire factor H family proteins to evade destruction by human complement.

SummaryThe acquisition of regulatory proteins is a means of blood‐borne pathogens to avoid destruction by the human complement. We recently showed that the gametes of the human malaria parasite Plasmodium falciparum bind factor H (FH) from the blood meal of the mosquito vector to assure successful sexual reproduction, which takes places in the mosquito midgut. While these findings provided a first glimpse of a complex mechanism used by Plasmodium to control the host immune attack, it is hitherto not known, how the pathogenic blood stages of the malaria parasite evade destruction by the human complement. We now show that the human complement system represents a severe threat for the replicating blood stages, particularly for the reinvading merozoites, with complement factor C3b accumulating on the surfaces of the intraerythrocytic schizonts as well as of free merozoites. C3b accumulation initiates terminal complement complex formation, in consequence resulting in blood stage lysis. To inactivate C3b, the parasites bind FH as well as related proteins FHL‐1 and CFHR‐1 to their surface, and FH binding is trypsin‐resistant. Schizonts acquire FH via two contact sites, which involve CCP modules 5 and 20. Blockage of FH‐mediated protection via anti‐FH antibodies results in significantly impaired blood stage replication, pointing to the plasmodial complement evasion machinery as a promising malaria vaccine target.

Novel Small-Molecule Inhibitors Targeting Complement Factor D for Therapy of Paroxysmal Nocturnal Hemoglobinuria

INTRODUCTIONThe complement system is a pivotal player in multiple hematological conditions that include paroxysmal nocturnal hemoglobinuria (PNH). The current standard of care for PNH is intravenous infusion of eculizumab, a humanized monoclonal antibody that targets the terminal complement protein C5 and thereby efficiently impairs intravascular hemolysis. However, a significant fraction of PNH patients in clinical practice respond incompletely to eculizumab due to extravascular hemolysis by immune cells as eculizumab does not prevent erythrocyte opsonization. Additionally, a non-responsive sub-population has been identified with a rare genetic polymorphism in C5 that renders the variant incapable of binding eculizumab. Therefore, an unmet medical need remains for regimens that improve efficacy and that can be administered orally. To achieve this goal, we initiated a discovery program for small molecule inhibitors of complement factor D, a serine protease that is the rate limiting enzyme of the alternative complement pathway. We have obtained high resolution (1.5 Å) crystal structure of inhibitor-bound factor D. Through structure-guided optimization, we have discovered novel inhibitors of factor D that possess high potency and specificity as well as pharmacokinetic properties suitable for oral administration. Herein, we present a biochemical characterization of lead compounds and studies of their activities in various complement-mediated processes. METHODSThe binding affinity to factor D was determined by surface plasmon resonance (Biacore) analysis. The inhibitory effect on factor D protease was evaluated biochemically using both a nonspecific synthetic substrate and a natural substrate consisting of C3b and the complement factor B. The alternative pathway (AP) hemolytic assay was conducted with unsensitized rabbit erythrocytes, 8% human or non-human primate serum, and 10 mM MgEGTA. The classical pathway (CP) hemolytic assay was conducted with antibody-sensitized sheep erythrocytes and 0.5% human serum. To assess inhibition of CP activation through the AP-dependent amplification loop, inhibition of terminal complement complex (TCC) formation by 50% human serum was quantitated following activation by the CP-specific activator HAIGG. Inhibition of complement opsonization and lysis of PNH-like erythrocytes exposed to acidified serum (Ham test) was assessed ex vivo. Finally, selected lead compounds were administered to non-human primates to evaluate the efficacy of factor D inhibitors for blockade of alternative pathway-mediated events. RESULTSLead compounds obtained through optimization bound to factor D with Kd values below 1 nM. Tight binding to factor D of the compounds resulted in potent inhibition of factor D proteolytic activity assessed using a synthetic nonspecific substrate as well as a natural substrate. When evaluated in an AP hemolytic assay with human serum, the most potent compounds inhibited cell lysis with EC50 values below 10 nM. Similar inhibitory activity was observed when compounds were assessed in AP hemolytic assays with non-human primate serum, but not with rodent serum. The compounds showed no activity in a CP hemolytic assay using highly diluted human serum and antibody-coated erythrocytes, indicating specificity for factor D. When evaluated in the presence of 50% human serum and with TCC formation as an endpoint, however, the compounds blocked approximately 75% of TCC formation upon CP activation, a consequence of blockage of the AP-dependent amplification loop. Furthermore, the compounds prevented both opsonisation and lysis of PNH-like cells in the Ham test. Finally, when selected compounds were administered to non-human primates, potent inhibition of TCC formation following ex vivo AP activation was observed with EC50 values below 50 nM. CONCLUSIONSHighly potent and specific factor D inhibitors that block both the terminal pathway and opsonization were discovered using structure-guided design. These orally deliverable inhibitors hold promise as a novel therapeutic approach for treatment of complement-mediated hematological conditions including PNH. DisclosuresMorgan:Swedish Orphan Biovitrum: Consultancy, Research Funding; Glaxo Smith Kline: Consultancy; Achillion Pharmaceuticals: Consultancy, Research Funding. Thanassi:Achillion Pharmaceuticals: Employment. Podos:Achillion Pharmaceuticals : Employment. Phadke:Achillion Pharmaceuticals: Employment. Gadhachanda:Achillion Pharmaceuticals: Employment. Pais:Achillion Pharmaceuticals: Employment. Hashimoto:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Chen:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Agarwal:Achillion Pharmaceuticals: Employment. Deshpande:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Wiles:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment.

Role of Complement in Enterohemorrhagic Escherichia coli–Induced Hemolytic Uremic Syndrome

Hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in childhood, is mainly caused by infection with Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). Besides its cytotoxic activity, Stx has been shown to interact with the complement system. Complement breakdown products have been found in serum of HUS patients suggesting complement activation and in vitro studies have demonstrated that Stx2 directly activates complement leading to formation of terminal complement complex. Furthermore, Stx2 has been found to bind to factor H (FH) resulting in a reduced cofactor activity on the cell surface. Binding of Stx2 has also been shown for other members of the FH family, namely FH-like protein 1 and FH-related protein 1. Both proteins also compete with FH for Stx binding, so that in the presence of FHR-1 less FH is bound to Stx and therefore more is available for endothelial cell protection. In addition, Stx2 has been demonstrated to downregulate the membrane-bound regulator CD59 on the surface of glomerular endothelial and tubulus epithelial cells on protein and at the mRNA level. In conclusion, Stx modulates complement regulator proteins leading to an impaired control and thus to enhanced complement activation. Its implication in the pathogenesis of EHEC-induced HUS in vivo and whether complement blockage might be a therapeutic option still has to be elucidated.

TUNABLE COMPLEMENT ACTIVATION BY PARTICLES WITH VARIABLE SIZE AND Fc DENSITY

The complement system is an integral innate immune component that is made up of a cascade of enzymatic proteins that, once activated, results in lysis of invading pathogens, opsonization or recruitment of other innate and/or acquired immune responders, or some combination of the three. Due to the importance of the signal amplification and control points present in the cascade, complement is highly sensitive to subtle variations in initiation conditions, including nanoscale changes to molecular spacing. Using Fc-functionalized microparticles and nanoparticles, we find that activation requires a minimum threshold surface concentration of Fc of at least 20% surface coverage. This result indicates that a high surface density Fc is necessary for micro/nanoparticle complement activation through the classical pathway. In addition, the magnitude of the response was dependent on the size of the particle, with larger particles causing decreased activation. We hypothesize that a high density of Fc is needed to efficiently bind and closely appose molecular initiators of the complement cascade, from initiation to terminal complement complex formation. These fundamental studies of the interaction of microparticles and nanoparticles with the immune system suggest design rules for particle size and molecular density that impact immunostimulation through the complement system. Providing a therapeutic agent to modulate the complement response could aid a variety of treatment strategies. Engineered nanoparticles with controlled gaps between molecular activators could lead to new types of immunomodulatory agents.

A randomized, double‐blind, placebo‐controlled phase II study of eculizumab in patients with refractory generalized myasthenia gravis

Complement activation at the neuromuscular junction is a primary cause of acetylcholine receptor loss and failure of neuromuscular transmission in myasthenia gravis (MG). Eculizumab, a humanized monoclonal antibody, blocks the formation of terminal complement complex by specifically preventing the enzymatic cleavage of complement 5 (C5). This study was a randomized, double-blind, placebo-controlled, crossover trial involving 14 patients with severe, refractory generalized MG (gMG). Six of 7 patients treated with eculizumab for 16 weeks (86%) achieved the primary endpoint of a 3-point reduction in the quantitative myasthenia gravis (QMG) score. Examining both treatment periods, the overall change in mean QMG total score was significantly different between eculizumab and placebo (P = 0.0144). After assessing data obtained from all visits, the overall change in mean QMG total score from baseline was found to be significantly different between eculizumab and placebo (P < 0.0001). Eculizumab was well tolerated. The data suggest that eculizumab may have a role in treating severe, refractory MG.