Knocking 'em Dead: Pore-Forming Proteins in Immune Defense.

Immune cells use a variety of membrane-disrupting proteins [complement, perforin, perforin-2, granulysin, gasdermins, mixed lineage kinase domain-like pseudokinase (MLKL)] to induce different kinds of death of microbes and host cells, some of which cause inflammation. After activation by proteolytic cleavage or phosphorylation, these proteins oligomerize, bind to membrane lipids, and disrupt membrane integrity. These membrane disruptors play a critical role in both innate and adaptive immunity. Here we review our current knowledge of the functions, specificity, activation, and regulation of membrane-disrupting immune proteins and what is known about the mechanisms behind membrane damage, the structure of the pores they form, how the cells expressing these lethal proteins are protected, and how cells targeted for destruction can sometimes escape death by repairing membrane damage.

Genome-wide screen in human plasma identifies multifaceted complement evasion of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma. Here, using a gain-of-function transposon sequencing (Tn-seq) screen in plasma, we identified and validated previously unknown factors affecting bacterial persistence in plasma. Among them, we identified a small periplasmic protein, named SrgA, whose expression leads to up to a 100-fold increase in resistance to killing. Additionally, mutants in pur and bio genes displayed higher tolerance and persistence, respectively. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate (polyP) granules upon incubation in plasma of different size in purD and wild-type strains, implying the bacterial response to a stress signal. Indeed, inactivation of ppk genes encoding polyP-generating enzymes lead to significant elimination of persisting bacteria from plasma. Through this study, we shed light on a complex P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC-induced killing.

Neutrophils activated by membrane attack complexes increase the permeability of melanoma blood vessels.

The microenvironment of malignant melanomas defines the properties of tumor blood vessels and regulates infiltration and vascular dissemination of immune and cancer cells, respectively. Previous research in other cancer entities suggested the complement system as an essential part of the tumor microenvironment. Here, we confirm activation of the complement system in samples of melanoma patients and murine melanomas. We identified the tumor endothelium as the starting point of the complement cascade. Generation of complement-derived C5a promoted the recruitment of neutrophils. Upon contact with the vascular endothelium, neutrophils were further activated by complement membrane attack complexes (MACs). MAC-activated neutrophils release neutrophil extracellular traps (NETs). Close to the blood vessel wall, NETs opened the endothelial barrier as indicated by an enhanced vascular leakage. This facilitated the entrance of melanoma cells into the circulation and their systemic spread. Depletion of neutrophils or lack of MAC formation in complement component 6 (C6)-deficient animals protected the vascular endothelium and prevented vascular intravasation of melanoma cells. Our data suggest that inhibition of MAC-mediated neutrophil activation is a potent strategy to abolish hematogenous dissemination in melanoma.

Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies.

The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6 Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.

The activated complement pathway in the fibrous process of benign prostatic hyperplasia.

BACKGROUND: To elucidate the changes in activated complement pathway in the fibrous process of benign prostatic hyperplasia (BPH), we analyzed the correlation between complement component expression and histological types of fibrosis using human BPH tissue. METHODS: Fifty-six histological BPH patients who underwent prostate needle biopsy at our institution (mean age 68.6 ± 6.5 years), divided into two histological groups, fibromuscular and fibrous, were compared. Inflammatory cell infiltration in BPH tissue was evaluated by immunohistochemical staining using CD45, with complement expression analysis performed using C3, factor B, and C5b-9 antibody, and the occupancy ratio of the stained region was calculated. Further, correlation between the histological types of fibrous components in BPH tissue and lower urinary tract symptoms questionnaires was analyzed. RESULTS: Twenty-seven (48.2%) and 29 (51.8%) cases were classified in the fibromuscular and fibrous groups, respectively. The proportion of CD45-positive cells in BPH tissue was significantly higher in the fibromuscular group. In complement component analysis, factor B did not significantly differ between groups, while C3 (fibromuscular group; 10.7 ± 8.2%, fibrous group; 16.4 ± 12.7%) and C5b-9 (fibromuscular group; 15.9 ± 6.2%, fibrous group; 17.6 ± 9.2%) were significantly higher in the fibrous group (p = 0.04, p = 0.04, respectively). International Prostate Symptom Score Q5 subscore, indicating slow stream, was significantly higher in the fibrous group (p = 0.04). CONCLUSIONS: In fibrous BPH with abundant fibrosis, the late complement pathway in addition to alternative pathway was activated compared to fibromuscular BPH. These results suggested that the alternative and late complement pathways were involved in the histological fibrous process of BPH.

Differential contributions of the C5b-9 and C5a/C5aR pathways to microvascular and macrovascular thrombosis in complement-mediated thrombotic microangiopathy patients.

To clarify the role of the C5a/C5aR (C5a receptor) and C5b-9 pathways in macrovascular thrombosis (MAT) and renal microthrombosis (MIT), 73 renal biopsy-proven complement-mediated thrombotic microangiopathy (C-TMA) patients were enrolled; 9 patients with pure MAT and 13 patients with pure MIT were selected for further study. Twenty-five external C-TMA patients were selected as the validation cohort. Plasma C5a and sC5b-9 (soluble C5b-9) levels were significantly higher in patients with MAT than in those with MIT (P = 0.008, P = 0.041, respectively). The mean optical density of C5aR1 in the kidney was significantly higher in MAT patients than in those with MIT (P < 0.001). Both urinary sC5b-9 levels (MIT: P < 0.001, MAT: P = 0.004) and renal deposition of C5b-9 (MIT: P < 0.001, MAT: P = 0.001) were significantly higher in C-TMA patients compared to normal control, but were similar between MAT and MIT groups. In the correlation analysis within 22C-TMA patients, urinary sC5b-9 levels and renal deposition of C5b-9 were positively correlated to renal MIT formation (P = 0.009 and P = 0.031, respectively). Furthermore, the renal citrullinated histone H3 (CitH3)- and neutrophil elastase (NE)-positive area ratios were both significantly higher in the MAT group than in the MIT group (P = 0.006 and P = 0.020, respectively). Therefore, the local C5b-9 and C5a/C5aR1 pathways might have differential contributions to MIT and MAT formation in the disease.

Endothelial dysfunction and complement activation are independently associated with disease duration in patients with systemic vasculitis.

OBJECTIVES: Systemic vasculitis is a heterogenous group of autoimmune diseases characterized by enhanced cardiovascular mortality. Endothelial dysfunction is associated with accelerated vascular damage, representing a core pathophysiologic mechanism contributing to excess CV risk. Recent studies have also shown that complement activation holds significant role in the pathogenesis of Anti-Neutrophilic Cytoplasmic Autoantibody (ANCA) -associated vasculitis (AAV). Given the potential crosstalk between the endothelium and complement, we aimed to assess, for the first time simultaneously, easily accessible biomarkers of endothelial dysfunction and complement activation in SV. METHODS: We measured circulating endothelial microvesicles (EMVs) and soluble complement components representative of alternative, classical and terminal activation (C5b-9, C1q, Bb fragments, respectively) in a meticulously selected group of patients with systemic vasculitis, but without cardiovascular disease. Individuals free from systemic diseases, who were matched with patients for cardiovascular risk factors(hypertension, diabetes, smoking, dyslipidemia), comprised the control group. RESULTS: We studied 60 individuals (30 in each group). Patients with systemic vasculitis had elevated EMVs, higher levels of C5b-9 [536.4(463.4) vs 1200.94457.3), p = 0.003] and C1q [136.2(146.5 vs 204.2(232.9), p = 0.0129], compared to controls [232.0 (243.5) vs 139.3(52.1), p < 0.001]. In multivariate analysis both EMVs and C5b-9 were independently associated with disease duration (p = 0.005 and p = 0.004 respectively), yet not with disease activity. CONCLUSION: Patients with systemic vasculitis exhibit impaired endothelial function and complement activation, both assessed by easily accessible biomarkers, even in the absence of cardiovascular disease manifestations. EMVs and soluble complement components such as C5b-9 and C1q could be used as early biomarkers of endothelial dysfunction and complement activation, respectively, in clinical practice during the course of SV, yet their predictive value in terms of future cardiovascular disease warrants further verification in appropriately designed studies.

Klotho Stabilizes the Podocyte Actin Cytoskeleton in Idiopathic Membranous Nephropathy through Regulating the TRPC6/CatL Pathway.

INTRODUCTION: The aim of this study was to explore the renoprotective effects of Klotho on podocyte injury mediated by complement activation and autoantibodies in idiopathic membranous nephropathy (IMN). METHODS: Rat passive Heymann nephritis (PHN) was induced as an IMN model. Urine protein levels, serum biochemistry, kidney histology, and podocyte marker levels were assessed. In vitro, sublytic podocyte injury was induced by C5b-9. The expression of Klotho, transient receptor potential channel 6 (TRPC6), and cathepsin L (CatL); its substrate synaptopodin; and the intracellular Ca2+ concentration were detected via immunofluorescence. RhoA/ROCK pathway activity was measured by an activity quantitative detection kit, and the protein expression of phosphorylated-LIMK1 (p-LIMK1) and p-cofilin in podocytes was detected via Western blotting. Klotho knockdown and overexpression were performed to evaluate its role in regulating the TRPC6/CatL pathway. RESULTS: PHN rats exhibited proteinuria, podocyte foot process effacement, decreased Klotho and Synaptopodin levels, and increased TRPC6 and CatL expression. The RhoA/ROCK pathway was activated by the increased phosphorylation of LIMK1 and cofilin. Similar changes were observed in C5b-9-injured podocytes. Klotho knockdown exacerbated podocyte injury, while Klotho overexpression partially ameliorated podocyte injury. CONCLUSION: Klotho may protect against podocyte injury in IMN patients by inhibiting the TRPC6/CatL pathway. Klotho is a potential target for reducing proteinuria in IMN patients.

Voluntary wheel running prevents formation of membrane attack complexes and myelin degradation after peripheral nerve injury.

Regular aerobic activity is associated with a reduced risk of chronic pain in humans and rodents. Our previous studies in rodents have shown that prior voluntary wheel running can normalize redox signaling at the site of peripheral nerve injury, attenuating subsequent neuropathic pain. However, the full extent of neuroprotection offered by voluntary wheel running after peripheral nerve injury is unknown. Here, we show that six weeks of voluntary wheel running prior to chronic constriction injury (CCI) reduced the terminal complement membrane attack complex (MAC) at the sciatic nerve injury site. This was associated with increased expression of the MAC inhibitor CD59. The levels of upstream complement components (C3) and their inhibitors (CD55, CR1 and CFH) were altered by CCI, but not increased by voluntary wheel running. Since MAC can degrade myelin, which in turn contributes to neuropathic pain, we evaluated myelin integrity at the sciatic nerve injury site. We found that the loss of myelinated fibers and decreased myelin protein which occurs in sedentary rats following CCI was not observed in rats with prior running. Substitution of prior voluntary wheel running with exogenous CD59 also attenuated mechanical allodynia and reduced MAC deposition at the nerve injury site, pointing to CD59 as a critical effector of the neuroprotective and antinociceptive actions of prior voluntary wheel running. This study links attenuation of neuropathic pain by prior voluntary wheel running with inhibition of MAC and preservation of myelin integrity at the sciatic nerve injury site.

Synthetic Oligodeoxynucleotide CpG Motifs Activate Human Complement through Their Backbone Structure and Induce Complement-Dependent Cytokine Release.

Bacterial and mitochondrial DNA, sharing an evolutionary origin, act as danger-associated molecular patterns in infectious and sterile inflammation. They both contain immunomodulatory CpG motifs. Interactions between CpG motifs and the complement system are sparsely described, and mechanisms of complement activation by CpG remain unclear. Lepirudin-anticoagulated human whole blood and plasma were incubated with increasing concentrations of three classes of synthetic CpGs: CpG-A, -B, and -C oligodeoxynucleotides and their GpC sequence controls. Complement activation products were analyzed by immunoassays. Cytokine levels were determined via 27-plex beads-based immunoassay, and CpG interactions with individual complement proteins were evaluated using magnetic beads coated with CpG-B. In whole blood and plasma, CpG-B and CpG-C (p < 0.05 for both), but not CpG-A (p > 0.8 for all), led to time- and dose-dependent increase of soluble C5b-9, the alternative complement convertase C3bBbP, and the C3 cleavage product C3bc. GpC-A, -B, and -C changed soluble fluid-phase C5b-9, C3bBbP, and C3bc to the same extent as CpG-A, -B, and -C, indicating a DNA backbone-dependent effect. Dose-dependent CpG-B binding was found to C1q (r = 0.83; p = 0.006) and factor H (r = 0.93; p < 0.001). The stimulatory complement effect was partly preserved in C2-deficient plasma and completely preserved in MASP-2-deficient serum. CpG-B increased levels of IL-1ß, IL-2, IL-6, IL-8, MCP-1, and TNF in whole blood, which were completely abolished by inhibition of C5 and C5aR1 (p < 0.05 for all). In conclusion, synthetic analogs of bacterial and mitochondrial DNA activate the complement system via the DNA backbone. We suggest that CpG-B interacts directly with classical and alternative pathway components, resulting in complement-C5aR1-dependent cytokine release.

Temporal lobe epilepsy with GAD antibodies: neurons killed by T cells not by complement membrane attack complex.

Temporal lobe epilepsy (TLE) is one of the syndromes linked to antibodies against glutamic acid decarboxylase (GAD). It has been questioned whether 'limbic encephalitis with GAD antibodies' is a meaningful diagnostic entity. The immunopathogenesis of GAD-TLE has remained enigmatic. Improvement of immunological treatability is an urgent clinical concern. We retrospectively assessed the clinical, MRI and CSF course as well as brain tissue of 15 adult patients with GAD-TLE who underwent temporal lobe surgery. Brain tissue was studied by means of immunohistochemistry, multiplex fluorescent microscopy and transcriptomic analysis for inflammatory mediators and neuronal degeneration. In 10 patients, there was a period of mediotemporal swelling and T2 signal increase; in nine cases this occurred within the first 6 years after symptom onset. This resulted in unilateral or bilateral hippocampal sclerosis; three cases developed hippocampal sclerosis within the first 2 years. All CSF studies done within the first year (n = 6) revealed intrathecal synthesis of immunoglobulin G. Temporal lobe surgeries were done after a median disease duration of 9 years (range 3 weeks to 60 years). Only two patients became seizure-free. Brain parenchyma collected during surgery in the first 6 years revealed high numbers of plasma cells but no signs of antibody-mediated tissue damage. Even more dense was the infiltration by CD8+ cytotoxic T lymphocytes (CTLs) that were seen to locally proliferate. Further, a portion of these cells revealed an antigen-specific resident memory T cell phenotype. Finally, CTLs with cytotoxic granzyme B+ granules were also seen in microglial nodules and attached to neurons, suggesting a CTL-mediated destruction of these cells. With longer disease duration, the density of all lymphocytes decreased. Whole transcriptome analysis in early/active cases (but not in late/inactive stages) revealed 'T cell immunity' and 'Regulation of immune processes' as the largest overrepresented clusters. To a lesser extent, pathways associated with B cells and neuronal degeneration also showed increased representation. Surgically treated patients with GAD-TLE go through an early active inflammatory, 'encephalitic' stage (≤6 years) with CTL-mediated, antigen-driven neuronal loss and antibody-producing plasma cells but without signs of complement-mediated cell death. Subsequently, patients enter an apparently immunologically inactive or low-active stage with ongoing seizures, probably caused by the structural damage to the temporal lobe. 'Limbic encephalitis' with GAD antibodies should be subsumed under GAD-TLE. The early tissue damage explains why immunotherapy does not usually lead to freedom from seizures.

Soluble terminal complement complex blood levels are elevated in schizophrenia.

The role of the complement system in schizophrenia (Sz) is inconclusive due to heterogeneity of the disease and study designs. Here, we assessed the levels of complement activation products and functionality of the classical pathway in acutely ill unmedicated Sz patients at baseline and after 6 weeks of treatment versus matched controls. The study included analyses of the terminal complement complex (sTCC) and C5a in plasma from 96 patients and 96 controls by enzyme-linked immunosorbent assay. Sub-group analysis of serum was conducted for measurement of C4 component and activity of the classical pathway (28 and 24 cases per cohort, respectively). We found no differences in levels of C5a, C4 and classical pathway function in patients versus controls. Plasma sTCC was significantly higher in patients [486 (392-659) ng/mL, n = 96] compared to controls [389 (304-612) ng/mL, n = 96] (p = 0.027, δ = 0.185), but not associated with clinical symptom ratings or treatment. The differences in sTCC between Sz and controls were confirmed using an Aligned Rank Transformation model considering the covariates age and sex (p = 0.040). Additional analysis showed that sTCC was significantly associated with C-reactive protein (CRP; p = 0.006). These findings suggest that sTCC plays a role in Sz as a trait marker of non-specific chronic immune activation, as previously described for CRP. Future longitudinal analyses with more sampling time points from early recognition centres for psychoses may be helpful to better understand the temporal dynamics of innate immune system changes during psychosis development.

Negative Modulation of B Cell Activation by Melanocortin 1 Receptor Signaling Protects against Membranous Nephropathy.

SIGNIFICANCE STATEMENT: Emerging evidence suggests that melanocortin neuropeptides-specifically adrenocorticotropic hormone-offer a novel, steroidogenic-independent therapeutic modality for membranous nephropathy (MN). The molecular mechanism underlying this beneficial effect, however, remains largely elusive. To investigate whether melanocortins modulate humoral immunity, the authors induced passive Heymann nephritis, a model of human MN, in wild-type and melanocortin 1 receptor (MC1R) knockout rats and treated them with melanocortin agents. Additional rats received adoptive transfer of bone marrow-derived cells beforehand from wild-type or MC1R knockout rats. The findings indicate that MC1R signaling plays a key role in negative modulation of B-cell activation and thereby suppresses humoral immune responses in passive Heymann nephritis, and suggest that MC1R signaling might offer a novel B cell-targeted therapeutic strategy for MN. BACKGROUND: Emerging evidence suggests that the pituitary neuropeptide melanocortins-specifically, adrenocorticotropic hormone-offer a novel nonsteroidogenic therapeutic modality for membranous nephropathy (MN). However, the mechanism(s) of action remains elusive. METHODS: To investigate whether melanocortins modulate humoral immunity, we induced passive Heymann nephritis (PHN), a model of MN, in wild-type (WT) and melanocortin 1 receptor (MC1R) knockout (KO) rats. We treated the animals with melanocortin agents-repository corticotropin injection, the nonsteroidogenic pan-melanocortin receptor agonist [Nle 4 , DPhe 7 ]-α-melanocyte stimulating hormone, the selective MC1R agonist MS05, vehicle gel, or phosphate-buffered saline-and evaluated kidney function, histology, and molecular changes. Additional rats received adoptive transfer of syngeneic bone marrow-derived cells beforehand from WT or MC1R KO rats. RESULTS: KO of MC1R worsened PHN and this was associated with increased deposition of autologous immunoglobulin G (IgG) and complement C5b-9 in glomeruli and higher circulating levels of autologous IgG-evidence of a sensitized humoral immune response. Melanocortin therapy ameliorated PHN in WT rats, coinciding with reduced glomerular deposition of autologous IgG and C5b -9. The beneficial efficacy of melanocortins was blunted in KO rats but restored by adoptive transfer of syngeneic bone marrow-derived cells derived from WT rats. Mechanistically, MC1R was expressed in B lymphocytes and was negatively associated with B cell activation. MC1R agonism triggered the expression of microphthalmia-associated transcription factor in activated B cells in a cAMP-dependent mode and also repressed the expression of interferon regulatory factor 4 (a lymphoid transcription factor essential for B-cell development and maturation), resulting in suppressed plasma cell differentiation and IgG production. CONCLUSIONS: MC1R signaling negatively modulates B cell activation and suppresses humoral immune responses in PHN, suggesting that MC1R signaling might offer a novel therapeutic target for MN.

Efficacy and Safety of Eculizumab in Pediatric Patients Affected by Shiga Toxin-Related Hemolytic and Uremic Syndrome: A Randomized, Placebo-Controlled Trial.

SIGNIFICANCE STATEMENT: Shiga toxin-related hemolytic uremic syndrome (STEC-HUS) is a serious condition, characterized by multiorgan thrombotic microangiopathy, mainly affecting children. Renal involvement is severe, with approximately half of patients requiring dialysis. So far, no specific treatment has been proven efficient in STEC-HUS. The use of eculizumab, a monoclonal antibody inhibiting terminal complement complex, has demonstrated remarkable success in atypical hemolytic uremic syndrome, but its use in uncontrolled studies to treat STEC-HUS has yielded inconsistent results. In this Phase 3 randomized, placebo-controlled trial in 100 pediatric patients with STEC-HUS, the findings did not show efficacy of eculizumab during the acute phase of the disease. However, the results indicated a reduction of renal sequelae in eculizumab-treated patients at 1-year follow-up. Larger prospective studies would be needed to further explore eculizumab as a potential treatment. BACKGROUND: Shiga toxin-related hemolytic uremic syndrome (STEC-HUS) in children is a severe condition, resulting in approximately 50% of patients requiring RRT. Furthermore, at least 30% of survivors experience kidney sequelae. Recently, activation of the complement alternative pathway has been postulated as a factor in STEC-HUS pathophysiology, leading to compassionate use of eculizumab, a monoclonal antibody inhibiting the terminal complement complex, in affected patients. Given the lack of therapy for STEC-HUS, a controlled study of eculizumab efficacy in treating this condition is a priority. METHODS: We conducted a Phase 3 randomized trial of eculizumab in children with STEC-HUS. Patients were randomly assigned in a 1:1 ratio to receive either eculizumab or placebo during 4 weeks. Follow-up lasted for 1 year. The primary end point was RRT duration <48 hours after randomization. Secondary endpoints included hematologic and extrarenal involvement. RESULTS: Baseline characteristics were similar among the 100 patients who underwent randomization. The rate of RRT <48 hours did not differ significantly between the two groups (48% in the placebo versus 38% in the eculizumab group; P = 0.31) or in the course of ARF. The two groups also exhibited similar hematologic evolution and extrarenal manifestations of STEC-HUS. The proportion of patients experiencing renal sequelae at 1 year was lower in the eculizumab group than in the placebo group (43.48% and 64.44%, respectively, P = 0.04). No safety concern was reported. CONCLUSIONS: In pediatric patients with STEC-HUS, eculizumab treatment does not appear to be associated with improved renal outcome during acute phase of the disease but may reduce long-term kidney sequelae. CLINICAL TRIALS REGISTRATIONS: EUDRACT (2014-001169-28) ClinicalTrials.gov ( NCT02205541 ).

The rare C9 P167S risk variant for age-related macular degeneration increases polymerization of the terminal component of the complement cascade.

Age-related macular degeneration (AMD) is a complex neurodegenerative eye disease with behavioral and genetic etiology and is the leading cause of irreversible vision loss among elderly Caucasians. Functionally significant genetic variants in the alternative pathway of complement have been strongly linked to disease. More recently, a rare variant in the terminal pathway of complement has been associated with increased risk, Complement component 9 (C9) P167S. To assess the functional consequence of this variant, C9 levels were measured in two independent cohorts of AMD patients. In both cohorts, it was demonstrated that the P167S variant was associated with low C9 plasma levels. Further analysis showed that patients with advanced AMD had elevated sC5b-9 compared to those with non-advanced AMD, although this was not associated with the P167S polymorphism. Electron microscopy of membrane attack complexes (MACs) generated using recombinantly produced wild type or P167S C9 demonstrated identical MAC ring structures. In functional assays, the P167S variant displayed a higher propensity to polymerize and a small increase in its ability to induce hemolysis of sheep erythrocytes when added to C9-depleted serum. The demonstration that this C9 P167S AMD risk polymorphism displays increased polymerization and functional activity provides a rationale for the gene therapy trials of sCD59 to inhibit the terminal pathway of complement in AMD that are underway.

Bacterial killing by complement requires membrane attack complex formation via surface-bound C5 convertases.

The immune system kills bacteria by the formation of lytic membrane attack complexes (MACs), triggered when complement enzymes cleave C5. At present, it is not understood how the MAC perturbs the composite cell envelope of Gram-negative bacteria. Here, we show that the role of C5 convertase enzymes in MAC assembly extends beyond the cleavage of C5 into the MAC precursor C5b. Although purified MAC complexes generated from preassembled C5b6 perforate artificial lipid membranes and mammalian cells, these components lack bactericidal activity. In order to permeabilize both the bacterial outer and inner membrane and thus kill a bacterium, MACs need to be assembled locally by the C5 convertase enzymes. Our data indicate that C5b6 rapidly loses the capacity to form bactericidal pores; therefore, bacterial killing requires both in situ conversion of C5 and immediate insertion of C5b67 into the membrane. Using flow cytometry and atomic force microscopy, we show that local assembly of C5b6 at the bacterial surface is required for the efficient insertion of MAC pores into bacterial membranes. These studies provide basic molecular insights into MAC assembly and bacterial killing by the immune system.

Initial properdin binding contributes to alternative pathway activation at the surface of viable and necrotic cells.

Properdin, the only known positive regulator of the complement system, stabilizes the C3 convertase, thereby increasing its half-life. In contrast to most other complement factors, properdin is mainly produced extrahepatically by myeloid cells. Recent data suggest a role for properdin as a pattern recognition molecule. Here, we confirmed previous findings of properdin binding to different necrotic cells including Jurkat T cells. Binding can occur independent of C3, as demonstrated by HAP-1 C3 KO cells, excluding a role for endogenous C3. In view of the cellular source of properdin, interaction with myeloid cells was examined. Properdin bound to the surface of viable monocyte-derived pro- and anti-inflammatory macrophages, but not to DCs. Binding was demonstrated for purified properdin as well as fractionated P2, P3, and P4 properdin oligomers. Binding contributed to local complement activation as determined by C3 and C5b-9 deposition on the cell surfaces and seems a prerequisite for alternative pathway activation. Interaction of properdin with cell surfaces could be inhibited with the tick protein Salp20 and by different polysaccharides, depending on sulfation and chain length. These data identify properdin as a factor interacting with different cell surfaces, being either dead or alive, contributing to the local stimulation of complement activation.

Complement terminal pathway inhibition reduces peritoneal injuries in a rat peritonitis model.

Peritonitis and the resulting peritoneal injuries are common problems that prevent long-term peritoneal dialysis (PD) therapy in patients with end-stage kidney diseases. Previously, we have analyzed the relationship between the complement system and progression of peritoneal injuries associated with PD, particularly focusing on the early activation pathways and effects of the anaphylatoxins. We here utilized a novel mAb 2H2 that blocks assembly of the membrane attack complex (MAC) to investigate roles of the complement terminal pathway in PD-associated peritoneal injury. We intraperitoneally injected mAb 2H2 anti-C5b-7 (2.5 or 5 mg/rat) once or twice over the five-day course of the experiment to investigate the effects of inhibiting formation of MAC in a fungal rat peritonitis model caused by repeated intraperitoneal administration of zymosan after methylglyoxal pretreatment (Zy/MGO model). Rats were sacrificed on day 5 and macroscopic changes in both parietal and visceral peritoneum evaluated. Peritoneal thickness, the abundance of fibrinogen and complement C3 and MAC deposition in tissue and accumulation of inflammatory cells were pathologically assessed. The results showed that mAb 2H2, but not isotype control mAb, reduced peritoneal thickness and accumulation of inflammatory cells in a dose and frequency-dependent manner in the Zy/MGO model. These effects were accompanied by decreased C3, MAC, and fibrinogen deposition in peritoneum. In conclusion, in the rat Zy/MGO model, complement terminal pathway activation and MAC formation substantially contributed to development of peritoneal injuries, suggesting that MAC-targeted therapies might be effective in preventing development of peritoneal injuries in humans.

Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores.

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and 'locked' C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes.

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.