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Aegerolysin proteins are involved in various interactions by recognising a molecular receptor in the target organism. The formation of pores in combination with larger, non-aegerolysin-like protein partners (such as membrane attack complex/perforin proteins [MACPFs]) is one of the possible responses in the presumed competitive exclusion of other organisms from the ecological niche. Bicomponent pairs are already observed at the gene level. Fungi growing under extreme conditions can be divided into ubiquitous and extremotolerant generalists which can compete with mesophilic species and rare, isolated extremophilic and extremotolerant specialists with narrow ecological amplitude that cannot compete. Under extreme conditions, there are fewer competitors, so fungal specialists generally produce less diverse and complicated profiles of specialised molecules. Since extremotolerant and extremophilic fungi have evolved in numerous branches of the fungal tree of life and aegerolysins are unevenly distributed across fungal genomes, we investigated whether aegerolysins, together with their partner proteins, contribute to the extreme survival ecology of generalists and specialists. We compiled a list of 109 thermo-, psihro-, acido-, alkali-, halo-, metallo- and polyextremo-tolerant/-philic fungal species. Several challenges were identified that affected the outcome: renaming fungal species, defining extremotolerant/extremophilic traits, identifying extremotolerant/extremophilic traits as metadata in databases and linking fungal isolates to fungal genomes. The yield of genomes coding aegerolysins or MACPFs appears to be lower in extremotolerant/extremophilic fungi compared to all fungal genomes. No candidates for pore-forming gene pairs were identified in the genomes of extremophilic fungi. Aegerolysin and MACPFs partner pairs were identified in only two of 69 species with sequenced genomes, namely in the ubiquitous metallotolerant generalists Aspergillus niger and A. foetidus. These results support the hypothesised role of these pore-forming proteins in competitive exclusion., (© 2024 The Author(s). IUBMB Life published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Purpose: Polymorphisms in complement genes are risk-associated for age-related macular degeneration (AMD). Functional analysis revealed a common deficiency to control the alternative complement pathway by risk-associated gene polymorphisms. Thus, we investigated the levels of terminal complement complex (TCC) in the plasma of wet AMD patients with defined genotypes and the impact of the complement activation of their plasma on second-messenger signaling, gene expression, and cytokine/chemokine secretion in retinal pigment epithelium (RPE) cells., Design: Collection of plasma from patients with wet AMD (n = 87: 62% female and 38% male; median age 77 years) and controls (n = 86: 39% female and 61% male; median age 58 years), grouped for risk factor smoking and genetic risk alleles CFH 402HH and ARMS2 rs3750846, determination of TCC levels in the plasma, in vitro analysis on RPE function during exposure to patients' or control plasma as a complement source., Methods: Genotyping, measurement of TCC concentrations, ARPE-19 cell culture, Ca 2+ imaging, gene expression by qPCR, secretion by multiplex bead analysis of cell culture supernatants., Main Outcome Measures: TCC concentration in plasma, intracellular free Ca 2+ , relative mRNA levels, cytokine secretion., Results: TCC levels in the plasma of AMD patients were five times higher than in non-AMD controls but did not differ in plasma from carriers of the two risk alleles. Complement-evoked Ca 2+ elevations in RPE cells differed between patients and controls with a significant correlation between TCC levels and peak amplitudes. Comparing the Ca 2+ signals, only between the plasma of smokers and non-smokers, as well as heterozygous ( CFH 402YH ) and CFH 402HH patients, revealed differences in the late phase. Pre-stimulation with complement patients' plasma led to sensitization for complement reactions by RPE cells. Gene expression for surface molecules protective against TCC and pro-inflammatory cytokines increased after exposure to patients' plasma. Patients' plasma stimulated the secretion of pro-inflammatory cytokines in the RPE., Conclusion: TCC levels were higher in AMD patients but did not depend on genetic risk factors. The Ca 2+ responses to patients' plasma as second-messenger represent a shift of RPE cells to a pro-inflammatory phenotype and protection against TCC. We conclude a substantial role of high TCC plasma levels in AMD pathology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Busch, Rau, Sekulic, Perie, Huber, Gehrke, Joussen, Zipfel, Wildner, Skerka and Strauß.)

Introduction: Comprehensive genetic analysis is essential to clinical care of patients with atypical haemolytic uremic syndrome (aHUS) to reinforce diagnosis, and to guide treatment. However, the characterization of complement gene variants remains challenging owing to the complexity of functional studies with mutant proteins. This study was designed: 1) To identify a tool for rapid functional determination of complement gene variants; 2) To uncover inherited complement dysregulation in aHUS patients who do not carry identified gene variants., Methods: To address the above goals, we employed an ex-vivo assay of serum-induced C5b-9 formation on ADP-activated endothelial cells in 223 subjects from 60 aHUS pedigrees (66 patients and 157 unaffected relatives)., Results: Sera taken from all aHUS patients in remission induced more C5b-9 deposition than control sera, independently from the presence of complement gene abnormalities. To avoid the possible confounding effects of chronic complement dysregulation related to aHUS status, and considering the incomplete penetrance for all aHUS-associated genes, we used serum from unaffected relatives. In control studies, 92.7% of unaffected relatives with known pathogenic variants exhibited positive serum-induced C5b-9 formation test, documenting a high sensitivity of the assay to identify functional variants. The test was also specific, indeed it was negative in all non-carrier relatives and in relatives with variants non-segregating with aHUS. All but one variants in aHUS-associated genes predicted in-silico as likely pathogenic or of uncertain significance (VUS) or likely benign resulted as pathogenic in the C5b-9 assay. At variance, variants in putative candidate genes did not exhibit a functional effect, with the exception of a CFHR5 variant. The C5b-9 assay in relatives was helpful in defining the relative functional effect of rare variants in 6 pedigrees in which the proband carried more than one genetic abnormality. Finally, for 12 patients without identified rare variants, the C5b-9 test in parents unmasked a genetic liability inherited from an unaffected parent., Discussion: In conclusion, the serum-induced C5b-9 formation test in unaffected relatives of aHUS patients may be a tool for rapid functional evaluation of rare complement gene variants. When combined with exome sequencing the assay might be of help in variant selection, to identify new aHUS-associated genetic factors., Competing Interests: MN has received honoraria from Alexion Pharmaceuticals for giving lectures, and for participating in advisory boards, and she has received research grants from Omeros, Gemini, Novartis and BioCryst Pharmaceuticals. AB has received honoraria from Alexion Pharmaceuticals and BioCryst Pharmaceuticals. GR has consultancy agreements with AbbVie, Alexion Pharmaceuticals, Novartis Pharma and BioCryst Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gastoldi, Aiello, Galbusera, Breno, Alberti, Bresin, Mele, Piras, Liguori, Santarsiero, Benigni, Remuzzi and Noris.)

The cytolytic activity of the membrane attack complex (MAC) is pivotal in the complement-mediated elimination of pathogens. Terminal complement pathway (TCP) genes encode the proteins that form the MAC. Although the TCP genes are well conserved within most vertebrate species, the early evolution of the TCP genes is poorly understood. Based on the comparative genomic analysis of the early evolutionary history of the TCP homologs, we evaluated four possible scenarios that could have given rise to the vertebrate TCP. Currently available genomic data support a scheme of complex sequential protein domain gains that may be responsible for the birth of the vertebrate C6 gene. The subsequent duplication and divergence of this vertebrate C6 gene formed the C7, C8α, C8β, and C9 genes. Compared to the widespread conservation of TCP components within vertebrates, we discovered that C9 has disintegrated in the genomes of galliform birds. Publicly available genome and transcriptome sequencing datasets of chicken from Illumina short read, PacBio long read, and Optical mapping technologies support the validity of the genome assembly at the C9 locus. In this study, we have generated a > 120X coverage whole-genome Chromium 10x linked-read sequencing dataset for the chicken and used it to verify the loss of the C9 gene in the chicken. We find multiple CR1 (chicken repeat 1) element insertions within and near the remnant exons of C9 in several galliform bird genomes. The reconstructed chronology of events shows that the CR1 insertions occurred after C9 gene loss in an early galliform ancestor. Loss of C9 in galliform birds, in contrast to conservation in other vertebrates, may have implications for host-pathogen interactions. Our study of C6 gene birth in an early vertebrate ancestor and C9 gene death in galliform birds provides insights into the evolution of the TCP., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Extremely rare reactions characterized by thrombosis and thrombocytopenia have been described in subjects that received ChAdOx1 nCoV-19 vaccination 5-16 days earlier. Although patients with vaccine-induced thrombotic thrombocytopenia (VITT) have high levels of antibodies to platelet factor 4 (PF4)-polyanion complexes, the exact mechanism of the development of thrombosis is still unknown. Here we reported serum studies as well as proteomics and genomics analyses demonstrating a massive complement activation potentially linked to the presence of anti-PF4 antibodies in a patient with severe VITT. At admission, complement activity of the classical and lectin pathways were absent (0% for both) with normal levels of the alternative pathway (73%) in association with elevated levels of the complement activation marker sC5b-9 (630 ng/mL [n.v. 139-462 ng/mL]) and anti-PF4 IgG (1.918 OD [n.v. 0.136-0.300 OD]). The immunoblotting analysis of C2 showed the complete disappearance of its normal band at 110 kDa. Intravenous immunoglobulin treatment allowed to recover complement activity of the classical pathway (91%) and lectin pathway (115%), to reduce levels of sC5b-9 (135 ng/mL) and anti-PF4 IgG (0.681 OD) and to normalize the C2 pattern at immunoblotting. Proteomics and genomics analyses in addition to serum studies showed that the absence of complement activity during VITT was not linked to alterations of the C2 gene but rather to a strong complement activation leading to C2 consumption. Our data in a single patient suggest monitoring complement parameters in other VITT patients considering also the possibility to target complement activation with specific drugs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Rat Thy-1 nephritis (Thy-1N) is an animal model of human mesangioproliferative glomerulonephritis (MsPGN), accompanied by glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) deposition. Although sublytic C5b-9 formed on GMC membrane could induce cell proliferation, the mechanism is still unclear. In this study, we first demonstrated that the level of SRY related HMG-BOX gene 9 (SOX9), general control nonderepressible 5 (GCN5), fibroblast growth factor 1 (FGF1) and platelet-derived growth factor α (PDGFα) was all elevated both in the renal tissues of Thy-1N rats (in vivo) and in the GMCs (in vitro) with sublytic C5b-9 stimulation. Then, we not only discovered that sublytic C5b-9 caused GMC proliferation through increasing SOX9, GCN5, FGF1 and PDGFα expression, but also proved that SOX9 and GCN5 formed a complex and combined with FGF1 and PDGFα promoters, leading to FGF1 and PDGFα gene transcription. More importantly, GCN5 could mediate SOX9 acetylation at lysine 62 (K62) to enhance SOX9 binding to FGF1 or PDGFα promoter and promote FGF1 or PDGFα synthesis and GMC proliferation. Besides, the experiments in vivo also showed that FGF1 and PDGFα expression, GMC proliferation and urinary protein secretion in Thy-1N rats were greatly reduced by silencing renal SOX9, GCN5, FGF1 or PDGFα gene. Furthermore, the renal tissues of MsPGN patients also exhibited positive expression of these genes mentioned above. Collectively, our findings indicate that GCN5, SOX9 and FGF1/PDGFα can form an axis and play an essential role in sublytic C5b-9-triggered GMC proliferation, which might provide a novel insight into the pathogenesis of Thy-1N and MsPGN., (© 2021 Federation of American Societies for Experimental Biology.)

Aim: We aimed to explore the detailed molecular mechanism of immune-associated genes in membranous nephropathy (MN)., Methods: A microarray data set (GSE133288) was retrieved from the Gene Expression Omnibus database. Differentially expressed mRNAs (DEMs) in MN vs control groups were identified, and MN-related DEMs (MN-DEMs) were further verified and screened using the comparative toxicogenomics database (CTD) database. The publicly available database, InnateDB was used to investigate immune genes, and the overlapped genes between MN-DEMs and the immune genes were considered as MN-related immune genes (iDEMs). A protein-protein interaction network (PPI) was constructed based on these iDEMs, followed by function and pathway enrichment analysis. Finally, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) associated with iDEMs were predicted, followed by a lncRNA-miRNA-mRNA (competing endogenous RNAs, ceRNA) network construction., Results: A total of 327 DEMs and 48 iDEMs were revealed; a PPI network was constructed with 100 PPI pairs and 37 iDEMs. iDEMs including JUN and FOS were mainly enriched in pathways such as osteoclast differentiation and function including response to immobilization stress, respectively. Based on mRNA-associated miRNA and lncRNA prediction, 30 ceRNA interactions including KCNQ1OT1-miR-204-5p-SRY-Box Transcription Factor 4 (SOX4) were explored., Conclusion: mRNAs including FOS and JUN might participate in MN development via response to immobilization stress function and the osteoclast differentiation pathway. The mRNA SOX4 might contribute to MN progression via sponging KCNQ1OT1-miR-204-5p interaction., (© 2020 Asian Pacific Society of Nephrology.)

Historically, the membrane attack complex, composed of complement components C5b-9, has been connected to lytic cell death and implicated in secondary injury after a CNS insult. However, studies to date have utilized either non-littermate control rat models, or mouse models that lack significant C5b-9 activity. To investigate what role C5b-9 plays in spinal cord injury and recovery, we generated littermate PVG C6 wildtype and deficient rats and tested functional and histological recovery after moderate contusion injury using the Infinite Horizon Impactor. We compare the effect of C6 deficiency on recovery of locomotor function and histological injury parameters in PVG rats under two conditions: (1) animals maintained as separate C6 WT and C6-D homozygous colonies; and (2) establishment of a heterozygous colony to generate C6 WT and C6-D littermate controls. The results suggest that maintenance of separate homozygous colonies is inadequate for testing the effect of C6 deficiency on locomotor and histological recovery after SCI, and highlight the importance of using littermate controls in studies involving genetic manipulation of the complement cascade.

Membrane Attack Complex and Perforin (MACPF) proteins play crucial roles in plant development and plant responses to environmental stresses. To date, only four MACPF genes have been identified in Arabidopsis thaliana , and the functions of the MACPF gene family members in other plants, especially in important crop plants, such as the Poaceae family, remain largely unknown. In this study, we identified and analyzed 42 MACPF genes from six completely sequenced and well annotated species representing the major Poaceae clades. A phylogenetic analysis of MACPF genes resolved four groups, characterized by shared motif organizations and gene structures within each group. MACPF genes were unevenly distributed along the Poaceae chromosomes. Moreover, segmental duplications and dispersed duplication events may have played significant roles during MACPF gene family expansion and functional diversification in the Poaceae. In addition, phylogenomic synteny analysis revealed a high degree of conservation among the Poaceae MACPF genes. In particular, Group I, II, and III MACPF genes were exposed to strong purifying selection with different evolutionary rates. Temporal and spatial expression analyses suggested that Group III MACPF genes were highly expressed relative to the other groups. In addition, most MACPF genes were highly expressed in vegetative tissues and up-regulated by several biotic and abiotic stresses. Taken together, these findings provide valuable information for further functional characterization and phenotypic validation of the Poaceae MACPF gene family.

The serum complement system is a first line of defense against bacterial invaders. Resistance to killing by serum enhances the capacity of Klebsiella pneumoniae to cause infection, but it is an incompletely understood virulence trait. Identifying and characterizing the factors responsible for preventing activation of, and killing by, serum complement could inform new approaches to treatment of K. pneumoniae infections. Here, we used functional genomic profiling to define the genetic basis of complement resistance in four diverse serum-resistant K. pneumoniae strains (NTUH-K2044, B5055, ATCC 43816, and RH201207), and explored their recognition by key complement components. More than 90 genes contributed to resistance in one or more strains, but only three, rfaH , lpp , and arnD , were common to all four strains. Deletion of the antiterminator rfaH , which controls the expression of capsule and O side chains, resulted in dramatic complement resistance reductions in all strains. The murein lipoprotein gene lpp promoted capsule retention through a mechanism dependent on its C-terminal lysine residue; its deletion led to modest reductions in complement resistance. Binding experiments with the complement components C3b and C5b-9 showed that the underlying mechanism of evasion varied in the four strains: B5055 and NTUH-K2044 appeared to bypass recognition by complement entirely, while ATCC 43816 and RH201207 were able to resist killing despite being associated with substantial levels of C5b-9. All rfaH and lpp mutants bound C3b and C5b-9 in large quantities. Our findings show that, even among this small selection of isolates, K. pneumoniae adopts differing mechanisms and utilizes distinct gene sets to avoid complement attack., (Copyright © 2020 Short et al.)

Dynamic interactions that govern the balance between host and pathogen determine the outcome of infection and are shaped by evolutionary pressures. Eukaryotic hosts have evolved elaborate and formidable defense mechanisms that provide the basis for innate and adaptive immunity. Proteins containing a membrane attack complex/Perforin (MACPF) domain represent an important class of immune effectors. These pore-forming proteins induce cell killing by targeting microbial or host membranes. Intracellular bacteria can be shielded from MACPF-mediated killing, and Chlamydia spp. represent a successful paradigm of obligate intracellular parasitism. Ancestors of present-day Chlamydia likely originated at evolutionary times that correlated with or preceded many host defense pathways. We discuss the current knowledge regarding how chlamydiae interact with the MACPF proteins Complement C9, Perforin-1, and Perforin-2. Current evidence indicates a degree of resistance by Chlamydia to MACPF effector mechanisms. In fact, chlamydiae have acquired and adapted their own MACPF-domain protein to facilitate infection., (Copyright © 2020 Keb and Fields.)

Objective: 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., Design: Cartilage explants were subjected to blunt impact (0.59 J) 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., Results: 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., Conclusions: 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., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The complement system is now a therapeutic target for the management of serious and life-threatening conditions such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, glomerulonephritis and other diseases caused by complement deficiencies or genetic variants. As complement therapeutics expand into more clinical conditions, monitoring complement activation is increasingly important, as is the baseline levels of complement activation fragments in blood or other body fluid levels. Although baseline complement levels have been reported in the literature, the majority of these data were generated using non-standard assays and with variable sample handling, potentially skewing results. In this study, we examined the plasma and serum levels of the soluble membrane attack complex of complement (sMAC). sMAC is formed in the fluid phase when complement is activated through the terminal pathway. It binds the regulatory proteins vitronectin and/or clusterin and cannot insert into cell membranes, and can serve as a soluble diagnostic marker in infectious disease settings, as previously shown for intraventricular shunt infections. Here we show that in healthy adults, serum sMAC levels were significantly higher than those in plasma, that plasma sMAC levels were similar between in African Americans and Caucasians and that plasma sMAC levels increase with age. Plasma sMAC levels were significantly higher in virally suppressed people living with HIV (PLWH) compared to non-HIV infected healthy donors. More specifically, PLWH with CD4 + T cell counts below 200 had even greater sMAC levels, suggesting diagnostic value in monitoring sMAC levels in this group., (© 2019 British Society for Immunology.)

Proteins of the aegerolysin family have a high abundance in Fungi. Due to their specific binding to membrane lipids, and their membrane-permeabilization potential in concert with protein partner(s) belonging to a membrane-attack-complex/perforin (MACPF) superfamily, they were proposed as useful tools in different biotechnological and biomedical applications. In this work, we performed functional studies on expression of the genes encoding aegerolysin and MACPF-like proteins in Aspergillus niger. Our results suggest the sporulation process being crucial for strong induction of the expression of all these genes. However, deletion of either of the aegerolysin genes did not influence the growth, development, sporulation efficiency and phenotype of the mutants, indicating that aegerolysins are not key factors in the sporulation process. In all our expression studies we noticed a strong correlation in the expression of one aegerolysin and MACPF-like gene. Aegerolysins were confirmed to be secreted from the fungus. We also showed the specific interaction of a recombinant A. niger aegerolysin with an invertebrate-specific membrane sphingolipid. Moreover, using this protein labelled with mCherry we successfully stained insect cells membranes containing this particular sphingolipid. Our combined results suggest, that aegerolysins in this species, and probably also in other aspergilli, could be involved in defence against predators., (© 2019 John Wiley & Sons Ltd.)

Serum resistance is a poorly understood but common trait of some difficult-to-treat pathogenic strains of bacteria. Here, we report that glycine, serine and threonine catabolic pathway is down-regulated in serum-resistant Escherichia coli, whereas exogenous glycine reverts the serum resistance and effectively potentiates serum to eliminate clinically-relevant bacterial pathogens in vitro and in vivo. We find that exogenous glycine increases the formation of membrane attack complex on bacterial membrane through two previously unrecognized regulations: 1) glycine negatively and positively regulates metabolic flux to purine biosynthesis and Krebs cycle, respectively. 2) α-Ketoglutarate inhibits adenosine triphosphate synthase, which in together promote the formation of cAMP/CRP regulon to increase the expression of complement-binding proteins HtrE, NfrA, and YhcD. The results could lead to effective strategies for managing the infection with serum-resistant bacteria, an especially valuable approach for treating individuals with weak acquired immunity but a normal complement system.

Atypical hemolytic uremic syndrome (aHUS) is a form of thrombotic microangiopathy (TMA) caused by dysregulated complement activation. Clinically, aHUS is effectively treated by an anti-C5 monoclonal antibody (mAb) but whether the disease is mediated by the C5a receptor (C5aR) or C5b-9 pathway, or both, is unknown. Here we address this in a factor H mutant mouse (FH R/R ) which developed complement-mediated TMA as well as macrovascular thrombosis caused by an aHUS-related factor H point mutation (mouse W1206R, corresponding to human W1183R). C5 deficiency and anti-C5 mAb treatment blocked all disease manifestations in FH R/R mice. C5aR1 gene deficiency prevented macrovascular thrombosis in various organs but did not improve survival or reduce renal TMA. Conversely, C6 or C9 deficiency significantly improved survival and markedly diminished renal TMA but did not prevent macrovascular thrombosis. Interestingly, as they aged both FH R/R C6 -/- and FH R/R C9 -/- mice developed glomerular disease reminiscent of C3 glomerulonephritis. Thus, C5aR and C5b-9 pathways drove different aspects of disease in FH R/R mice with the C5aR pathway being responsible for macrovascular thrombosis and chronic inflammatory injury while the C5b-9 pathway caused renal TMA. Our data provide new understanding of the pathogenesis of complement-mediated TMA and macrovascular thrombosis in FH R/R mice and suggest that C5 blockade is more effective for the treatment of aHUS than selectively targeting the C5aR or C5b-9 pathway alone., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: It remains uncertain whether activation of the complement system, assessed by the soluble terminal C5b-9 complement complex (plasma TCC), is associated with future risk of incident venous thromboembolism (VTE)., Objectives: To investigate the association between plasma levels of TCC and future risk of incident VTE in a nested case-control study, and to explore genetic variants associated with TCC using protein quantitative trait loci analysis of exome sequencing data., Methods: We sampled 415 VTE cases and 848 age- and sex-matched controls from a population-based cohort, the Tromsø study. Logistic regression models were used to calculate odds ratios with 95% confidence intervals for VTE across quartiles of plasma levels of TCC. Whole exome sequencing was conducted using the Agilent SureSelect 50 Mb capture kit., Results: The risk of VTE increased across increasing quartiles of plasma TCC, particularly for unprovoked VTE. Participants with TCC in the highest quartile (>1.40 complement arbitrary units/mL) had an odds ratio for unprovoked VTE of 1.74 (95% confidence interval: 1.10-2.78) compared with those with TCC in the lowest quartile (≤0.80 complement arbitrary units/mL) in analyses adjusted for age, sex, and body mass index. A substantially higher risk for VTE was observed in samples taken shortly before VTE event. We found no association between genome-wide or complement-related gene variants and plasma levels of TCC., Conclusions: We found that high levels of plasma TCC were associated with VTE risk, and unprovoked events in particular. There was no genome-wide association between gene variants and plasma levels of TCC., (© 2019 International Society on Thrombosis and Haemostasis.)

Trichloroethylene (TCE) is a major occupational and environmental chemical compound which causes occupational dermatitis medicamentosa-like of TCE with severe liver damage. Our previous studies showed that complement activation was a newly recognized mechanism for TCE-induced liver damage. The objective of this study was to explore the role of the key complement regulatory protein, CD59a, in TCE-induced immune liver injury. We firstly evaluated the changes of CD59a expression in liver tissue and then investigated if the changes were associated with membrane attack complex (MAC) formation, nuclear factor kappa B (NF-κB) activation and liver damage in BALB/c mice model of TCE-induced skin sensitization in the absence or presence of soluble recombinant rat CD59-Cys. The results showed that low expression of CD59a accompanied by MAC deposition in the liver of TCE-sensitized BALB/c mice, which was consistent in time. In addition, activation of NF-κB pathway, upregulation of inflammatory cytokine and liver damage also occured. Additional experiment showed that recombinant rat sCD59-Cys alleviated inflammation and liver damage in TCE-sensitized BALB/c mice. Moreover, recombinant rat sCD59-Cys reduced MAC formation and inhibited NF-κB activation measured by P-IκBα and nuclear NF-κB p65 in the liver of TCE-sensitized BALB/c mice. In conclusion, recombinant rat sCD59-Cys plays a protective role in immune liver injury of TCE-sensitized BALB/c mice., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD., (© 2018 Wiley Periodicals, Inc.)

The crystal structure of the Gram-negative insecticidal protein, GNIP1Aa, has been solved at 2.5-Å resolution. The protein consists of two structurally distinct domains, a MACPF (membrane attack complex/PerForin) and a previously uncharacterized type of domain. GNIP1Aa is unique in being a prokaryotic MACPF member to have both its structure and function identified. It was isolated from a Chromobacterium piscinae strain and is specifically toxic to Diabrotica virgifera virgifera larvae upon feeding. In members of the MACPF family, the MACPF domain has been shown to be important for protein oligomerization and formation of transmembrane pores, while accompanying domains define the specificity of the target of the toxicity. In GNIP1Aa the accompanying C-terminal domain has a unique fold composed of three pseudosymmetric subdomains with shared sequence similarity, a feature not obvious from the initial sequence examination. Our analysis places this domain into a protein family, named here β-tripod. Using mutagenesis, we identified functionally important regions in the β-tripod domain, which may be involved in target recognition., Competing Interests: Conflict of interest statement: J.Z., D.V., and S.L.M. are current employees of BASF; J.F., J.D., C.K., and D.B. are current employees of Bayer; N.B.D. was formerly employed by Bayer. GNIP1Aa is protected under US Patent 8575425 and family members, and US Patent 9862965 and family members.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. The therapeutic approach to CLL includes chemotherapeutic regimens and immunotherapy. Complement-mediated cytotoxicity, which is one of the mechanisms activated by the therapeutic monoclonal antibodies, depends on the availability and activity of the complement (C) system. The aim was to study the structure of circulating C components and evaluate the importance of C5 structural integrity for C activity in CLL patients. Blood samples were collected from 40 naïve CLL patients and 15 normal controls (NC). The Western blot analysis showed abnormal C5 pattern in some CLL patients, while patterns of C3 and C4 were similar in all subjects. Levels of the C activation markers sC5b-9 and C5a were quantified before and after activation via the classical (CP) and alternative (AP) pathways. In patients with abnormal C5, basal levels of sC5b-9 and C5a were increased while activities of the CP and of the CP C5-convertase, the immediate C5-upstream complex, were decreased compared to NC and to patients with normal C5. The data indicate a link between CP activation and apparent C5 alterations in CLL. This provides a potential prognostic tool that may personalize therapy by identifying a sub-group of CLL patients who display an abnormal C5 pattern, high basal levels of sC5b-9 and C5a, and impaired CP activity, and are likely to be less responsive to immunotherapy due to compromised CP activity., Competing Interests: The authors have declared that no competing interests exist.

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 in vivo, and bone cell behavior was analyzed ex vivo. 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., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The redlip mullet (Liza haematocheila) is one of the most economically important fish in Korea and other East Asian countries; it is susceptible to infections by pathogens such as Lactococcus garvieae, Argulus spp., Trichodina spp., and Vibrio spp. Learning about the mechanisms of the complement system of the innate immunity of redlip mullet is important for efforts towards eradicating pathogens. Here, we report a comprehensive study of the terminal complement complex (TCC) components that form the membrane attack complex (MAC) through in-silico characterization and comparative spatial and temporal expression profiling. Five conserved domains (TSP1, LDLa, MACPF, CCP, and FIMAC) were detected in the TCC components, but the CCP and FIMAC domains were absent in MuC8β and MuC9. Expression analysis of four TCC genes from healthy redlip mullets showed the highest expression levels in the liver, whereas limited expression was observed in other tissues; immune-induced expression in the head kidney and spleen revealed significant responses against Lactococcus garvieae and poly I:C injection, suggesting their involvement in MAC formation in response to harmful pathogenic infections. Furthermore, the response to poly I:C may suggest the role of TCC components in the breakdown of the membrane of enveloped viruses. These findings may help to elucidate the mechanisms behind the complement system of the teleosts innate immunity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Thrombosis is the prognostic factor with the greatest effect on survival in patients with paroxysmal nocturnal hemoglobinuria (PNH), who lack dozens of membrane surface proteins. We recently described a primary homozygous Cys89Tyr congenital nonfunctioning CD59 in humans with clinical manifestation in infancy, associated with chronic hemolysis, recurrent strokes, and relapsing peripheral demyelinating neuropathy. Here we investigated hypercoagulability mechanisms characterizing the syndrome., Methods: Membrane attack complex (MAC) deposition (anti-SC5b-9) and free hemoglobin (colorimetric assay) were assessed. Platelet activation was identified (anti-CD61, anti-CD62P), and microparticles (MPs) of 0.5-0.9 μm, were characterized (Annexin V, anti-human GlyA, anti-CD15, anti-CD14, anti-CD61). Platelet-monocyte aggregation was assessed with FlowSight., Findings: 2/7 patients (29%) with homozygosity for Cys89Tyr and 6/12 (50%) with any of four described CD59 mutations had recurrent strokes. In plasma samples from four patients carrying identical mutations, MAC deposition was increased on RBCs (p < 0.0003), neutrophils (p < 0.009), and platelets (p < 0.0003). Free-plasma hemoglobin levels were abnormally high, up to 100 mg/dl. Patients with CD59 mutation had RBC-derived MP levels 9-fold higher than those in healthy controls (p < 0.01), and 2-2.5 fold higher than PNH patients (p < 0.09). Leukocyte-activated platelet aggregation was increased (p < 0.0062). Loss of CD59 was shown in the endothelium of these patients., Interpretation: Nonfunctioning CD59 is a major risk factor for stroke and hypercoagulability. Uncontrolled hemolysis causes massive MP release and endothelial heme damage. MAC attack on unprotected endothelium and platelet activation and aggregation with leukocytes mediate additional mechanisms leading to vascular occlusion. It is suggested that CD59 loss represents a major arterial prothrombotic factor in PNH and additional diseases., (Copyright © 2018. Published by Elsevier Ltd.)

The complement system participates in the pathogenesis of many diseases. Complement activation produces several active protein complexes and peptides, including the terminal C5b-9 complexes. It was reported that C5b-9 complexes insert into the plasma membrane and cause membrane perturbation, intracellular calcium surge, metabolic depletion, and osmotic lysis. Previously, we showed that complement-dependent cytotoxicity (CDC) is regulated by JNK and Bid. Here, we demonstrate that three mediators in TNFα-induced necroptosis (regulated necrosis), the receptor-interacting protein kinases, receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like protein (MLKL), are activated by complement and contribute to CDC. Cell treatment with necrostatin-1 (Nec-1), a RIPK1 inhibitor, GSK'872, a RIPK3 inhibitor, or necrosulfonamide and GW806742X, MLKL inhibitors, restrain CDC. These findings were confirmed by using specific siRNAs targeting the synthesis of these proteins. Mouse fibroblasts lacking RIPK3 or MLKL were found to be less sensitive to C5b-9 than were wild-type (WT) fibroblasts. Enhanced CDC was achieved by RIPK1 or RIPK3 overexpression but not by the overexpression of a RHIM-RIPK1 mutant nor by a kinase-dead RIPK3 mutant. Nec-1 reduces the CDC of WT but not of RIPK3-knockout fibroblasts. Cells treated with a sublytic dose of complement exhibit co-localization of RIPK3 with RIPK1 in the cytoplasm and co-localization of RIPK3 and MLKL with C5b-9 at the plasma membrane. Data supporting cooperation among the RIP kinases, MLKL, JNK, and Bid in CDC are presented. These results provide a deeper insight into the cell death process activated by complement and identify potential points of cross talk between complement and other inducers of inflammation and regulated necrosis.

The Membrane Attack Complex Perforin-like/Cholesterol-Dependent Cytolysin (MACPF) superfamily is an ancient and biologically diverse group of proteins that are best known for pore-forming roles in mammalian immunity and bacterial pathogenesis. Intriguingly, however, some eukaryotic proteins which contain the MACPF domain that defines this family do not act in attack or defence, and instead have distinct developmental functions. It remains unclear whether these proteins function via pore formation or have a different mechanism of action. Of these, by far the best characterised is Torso-like (Tsl), the only MACPF member that has been identified in the fruit fly, Drosophila melanogaster. While it has long been known to have a role in embryonic patterning, recent studies have shown that Tsl in fact has multiple roles in development. As such, it presents an excellent opportunity to investigate how the MACPF domain functions in a developmental context. Here, we review what is known about Tsl in Drosophila and other insects, and discuss the potential molecular mechanism by which Tsl and thus other developmental MACPF proteins may function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Astrotactins (ASTNs) and Bone morphogenetic protein/retinoic acid inducible neural-specific proteins (BRINPs) are two groups of Membrane Attack Complex/Perforin (MACPF) superfamily proteins that show overlapping expression in the developing and mature vertebrate nervous system. ASTN(1-2) and BRINP(1-3) genes are found at conserved loci in humans that have been implicated in neurodevelopmental disorders (NDDs). Here we review the tissue distribution and cellular localization of these proteins, and discuss recent studies that provide insight into their structure and interactions. We highlight the genetic relationships and co-expression of Brinps and Astns; and review recent knock-out mouse phenotypes that indicate a possible overlap in protein function between ASTNs and BRINPs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Complement C4 genes are linked to paediatric inflammatory bowel disease (PIBD), but the mechanisms have remained unclear. We examined the influence of C4B gene number on intestinal microbiota and in-vitro serum complement activation by intestinal microbes in PIBD patients. Complement C4A and C4B gene numbers were determined by genomic reverse transcription-polymerase chain reaction (RT-PCR) from 64 patients with PIBD (Crohn's disease or ulcerative colitis). The severity of the disease course was determined from faecal calprotectin levels. Intestinal microbiota was assessed using the HITChip microarray. Complement reactivity in patients was analysed by incubating their sera with Yersinia pseudotuberculosis and Akkermansia muciniphila and determining the levels of C3a and soluble terminal complement complex (SC5b-9) using enzyme immunoassays. The microbiota diversity was wider in patients with no C4B genes than in those with one or two C4B genes, irrespective of intestinal inflammation. C4B and total C4 gene numbers correlated positively with soluble terminal complement complex (TCC, SC5b-9) levels when patient serum samples were stimulated with bacteria. Our results suggest that the C4B gene number associates positively with inflammation in patients with PIBD. Multiple copies of the C4B gene may thus aggravate the IBD-associated dysbiosis through escalated complement reactivity towards the microbiota., (© 2017 British Society for Immunology.)

In patients with typical angina pectoris, inducible myocardial ischaemia and macroscopically normal coronaries (cardiac syndrome X (CSX)), a significantly elevated plasma level of terminal complement complex (TCC), the common end product of complement activation, has been observed without accompanying activation of the classical or the alternative pathways. Therefore, our aim was to clarify the role of the ficolin-lectin pathway in CSX. Eighteen patients with CSX, 37 stable angina patients with significant coronary stenosis (CHD) and 54 healthy volunteers (HC) were enrolled. Serum levels of ficolin-2 and ficolin-3, ficolin-3/MASP-2 complex and ficolin-3-mediated TCC deposition (FCN3-TCC) were determined. Plasma level of TCC was significantly higher in the CSX than in the HC or CHD group (5.45 versus 1.30 versus 2.04 AU/ml, P < 0.001). Serum levels of ficolin-2 and ficolin-3 were significantly lower in the CSX compared to the HC or CHD group (3.60 versus 5.80 or 5.20 μg/ml, P < 0.05; 17.80 versus 24.10 or 26.80 μg/ml, P < 0.05). The ficolin-3/MASP-2 complex was significantly lower in the CSX group compared to the HC group (92.90 versus 144.90 AU/ml, P = 0.006). FCN3-TCC deposition was significantly lower in the CSX group compared to the HC and CHD groups (67.8% versus 143.3% or 159.7%, P < 0.05). In the CSX group, a significant correlation was found between TCC and FCN3-TCC level (r = 0.507, P = 0.032) and between ficolin-3/MASP-2 complex level and FCN3-TCC deposition (r = 0.651, P = 0.003). In conclusion, in patients with typical angina and myocardial ischaemia despite macroscopically normal coronary arteries, low levels of several lectin pathway parameters were observed, indicating complement activation and consumption. Complement activation through the ficolin-lectin pathway might play a role in the complex pathomechanism of CSX., (© 2016 The Foundation for the Scandinavian Journal of Immunology.)

The complement system is a critical part of the immune response, acting in defense against viral infections, clearance of immune complexes, and maintenance of tissue homeostasis. Upregulated expression of the terminal complement complex, C5b-9, has been observed on various tumor cells, such as stomach carcinoma cells, and on cells in the necrotic regions of these tumors as well; however, whether and how C5b-9 is related to gastric cancer progression and severity remains unknown. In this study, human gastric adenocarcinoma (HGAC) tissues (n=47 cases) and patient-matched adjacent nontumoral parenchyma (n=20 cases) were evaluated by tissue microarray and immunohistochemistry. The HGAC tissues showed upregulated C5b-9 expression. Multinomial logistic regression and likelihood ratio testing showed that overexpression of C5b-9 in HGAC tissue was significantly correlated with clinical stage (P=0.007) and tumor stage (P=0.005), but not with tumor distant organ metastasis, lymphoid nodal status, sex, or age. Patients with late-stage gastric adenocarcinoma had a higher amount of tumor cells showing positive staining for C5b-9 than patients with early-stage disease. These results may help in diagnosis and assessment of disease severity of human gastric carcinoma.

Terminal complement membrane attack complex (MAC) formation is induced initially by C5b, followed by the sequential condensation of the C6, C7, C8. Polymerization of C9 to the C5b-8 complex forms the C5b-9 (or MAC). The C5b-9 forms lytic or non lytic pores in the cell membrane destroys membrane integrity. The biological functionalities of MAC has been previously investigated by using either the mice deficient in C5 and C6, or MAC's regulator CD59. However, there is no available C9 deficient mice (mC9(-/-)) for directly dissecting the role of C5b-9 in the pathogenesis of human diseases. Further, since C5b-7 and C5b-8 complexes form non lytic pore, it may also plays biological functionality. To better understand the role of terminal complement cascades, here we report a successful generation of mC9(-/-). We demonstrated that lack of C9 attenuates anti-erythrocyte antibody-mediated hemolysis or LPS-induced acute shock. Further, the rescuing effect on the acute shock correlates with the less release of IL-1β in mC9(-/-), which is associated with suppression of MAC-mediated inflammasome activation in mC9(-/-). Taken together, these results not only confirm the critical role of C5b-9 in complement-mediated hemolysis and but also highlight the critical role of C5b-9 in inflammasome activation.

Tumor development driven by inflammation is now an established phenomenon, but the role that complement plays remains uncertain. Recent evidence has suggested that various components of the complement (C) cascade may influence tumor development in disparate ways; however, little attention has been paid to that of the membrane attack complex (MAC). This is despite abundant evidence documenting the effects of this complex on cell behavior, including cell activation, protection from/induction of apoptosis, release of inflammatory cytokines, growth factors, and ECM components and regulators, and the triggering of the NLRP3 inflammasome. Here we present a novel approach to this issue by using global gene expression studies in conjunction with a systems biology analysis. Using network analysis of MAC-responsive expression changes, we demonstrate a cluster of co-regulated genes known to have impact in the extracellular space and on the supporting stroma and with well characterized tumor-promoting roles. Network analysis highlighted the central role for EGF receptor activation in mediating the observed responses to MAC exposure. Overall, the study sheds light on the mechanisms by which sublytic MAC causes tumor cell responses and exposes a gene expression signature that implicates MAC as a driver of tumor progression. These findings have implications for understanding of the roles of complement and the MAC in tumor development and progression, which in turn will inform future therapeutic strategies in cancer., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The MACPF domain-containing proteins potentially able to build a transmembrane pore structure are found in the transcriptome of a common periwinkle kidney loaded with trematode rediae. Two homologs of mammalian Macrophage expressed gene 1 (Mpeg1), LlMpeg1-1 and LIMpeg1-2, share similar domain structure with the only difference such as LIMpeg1-2 transcript lacks of a C-terminal transmembrane helix. Expression of membrane-anchored protein LlMpeg1-1 is similar in kidneys of naturally infected with trematode Himasthla elongata and uninfected snails. The expression of the second soluble LIMpeg1-2 protein is 4-fold upregulated under infection. The third MACPF protein found in Littorina littorea kidney is homologous with Perivitellin - 2 67 kDa subunit named LlPV2-67 and the expression of the transcript is 3-fold upregulated in the kidney of infected snails. The last two molecules are candidate effectors that may participate in the immune response of common periwinkles to trematode infestation. A single parasite-expressed MACPF-like protein was recorded from the transcriptome of Himasthla elongata., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (© FASEB.)

Complement activation has long been associated with inflammation, primarily due to the elaboration of the complement anaphylotoxins C5a and C3a. In this work, we demonstrate that the phagocytosis of complement-opsonized particles promotes host inflammatory responses by a new mechanism that depends on the terminal complement components (C5b-C9). We demonstrate that during the phagocytosis of complement-opsonized particles, the membrane attack complex (MAC) of complement can be transferred from the activating particle to the macrophage plasma membrane by a 'bystander' mechanism. This MAC-mediated bystander damage initiates NLRP3 inflammasome activation, resulting in caspase-1 activation and IL-1β and IL-18 secretion. Inflammasome activation is not induced when macrophages phagocytize unopsonized particles or particles opsonized with serum deficient in one of the terminal complement components. The secretion of IL-1β and IL-18 by macrophages depends on NLRP3, ASC (also known as PYCARD) and caspase-1, as macrophages deficient in any one of these components fail to secrete these cytokines following phagocytosis. The phagocytosis of complement-opsonized particles increases leukocyte recruitment and promotes T helper 17 cell (TH17) biasing. These findings reveal a new mechanism by which complement promotes inflammation and regulates innate and adaptive immunity., (© 2016. Published by The Company of Biologists Ltd.)

Properdin (P) stabilizes the alternative pathway (AP) convertases, being the only known positive regulator of the complement system. In addition, P is a pattern recognition molecule able to initiate directly the AP on non-self surfaces. Although P deficiencies have long been known to be associated with Neisseria infections and P is often found deposited at sites of AP activation and tissue injury, the potential role of P in the pathogenesis of complement dysregulation-associated disorders has not been studied extensively. Serum P levels were measured in 49 patients with histological and clinical evidence of C3 glomerulopathy (C3G). Patients were divided into two groups according to the presence or absence of C3 nephritic factor (C3NeF), an autoantibody that stabilizes the AP C3 convertase. The presence of this autoantibody results in a significant reduction in circulating C3 (P < 0·001) and C5 levels (P < 0·05), but does not alter factor B, P and sC5b-9 levels. Interestingly, in our cohort, serum P levels were low in 17 of the 32 C3NeF-negative patients. This group exhibited significant reduction of C3 (P < 0·001) and C5 (P < 0·001) and increase of sC5b-9 (P < 0·001) plasma levels compared to the control group. Also, P consumption was correlated significantly with C3 (r = 0·798, P = 0·0001), C5 (r = 0·806, P < 0·0001), sC5b-9 (r = -0·683, P = 0·043) and a higher degree of proteinuria (r = -0·862, P = 0·013). These results illustrate further the heterogeneity among C3G patients and suggest that P serum levels could be a reliable clinical biomarker to identify patients with underlying surface AP C5 convertase dysregulation., (© 2016 British Society for Immunology.)

The article presents diagnostic of night paroxysmal hemoglobinuria. The night paroxysmal hemoglobinuria is an orphan disease characterized by absence of GPI-anchor on blood cells as a result of mutation of PIG-A gene on the short arm of X-chromosome. The particular proteins bounded with GPI-anchor implement function of defense from activation of components of complement and development of membrane-attacking complex. The erythrocytes exposed to destruction in bloodstream are among the most impacted. Therefore, one of the main signs of night paroxysmal hemoglobinuria is complement-depending intravascular hemolysis which indicators for a long time played a key role in diagnostic of night paroxysmal hemoglobinuria. The actual technique of diagnostic of night paroxysmal hemoglobinuria is flow cytometry. The analysis of night paroxysmal hemoglobinuria clone is recommended to patients with hemolysis of unclear genesis, thrombosis of cerebral and abdominal veins, thrombocytopenia and macrocytosis and also patients with AA, myelodysplastic syndrome, myelofibrosis. The international protocol recommended by the International Society of Clinical Cytometry (2010) is implemented to diagnose night paroxysmal hemoglobinuria. The original technique of evaluation of reticulocytes was developed with purpose to detect night paroxysmal hemoglobinuria clone. The high correlation was substantiated between size of night paroxysmal hemoglobinuria clone measured among reticulocytes according to proposed mode and night paroxysmal hemoglobinuria clone measured among granulocytes and monocytes detected according international standardized approach.

Borrelia (B.) bavariensis exhibits a marked tropism for nervous tissues and frequently causes neurological manifestations in humans. The molecular mechanism by which B. bavariensis overcomes innate immunity, in particular, complement remains elusive. In contrast to other serum-resistant spirochetes, none of the B. bavariensis isolates investigated bound complement regulators of the alternative (AP) and classical pathway (CP) or proteolytically inactivated complement components. Focusing on outer surface proteins BGA66 and BGA71, we demonstrated that both molecules either inhibit AP, CP and terminal pathway (TP) activation, or block activation of the CP and TP respectively. Both molecules bind complement components C7, C8 and C9, and thereby prevent assembly of the terminal complement complex. This inhibitory activity was confirmed by the introduction of the BGA66 and BGA71 encoding genes into a serum-sensitive B. garinii strain. Transformed spirochetes producing either BGA66 or BGA71 overcome complement-mediated killing, thus indicating that both proteins independently facilitate serum resistance of B. bavariensis. The generation of C-terminally truncated proteins as well as a chimeric BGA71 protein lead to the localization of the complement-interacting binding site within the N-terminus. Collectively, our data reveal a novel immune evasion strategy of B. bavariensis that is directed against the activation of the TP., (© 2015 John Wiley & Sons Ltd.)

Background: Patients on haemodialysis (HD) have high rates of cardiovascular (CV) disease and activation of the complement system. Despite evidence in non-renal patients that these may be linked, this association has received little attention in HD patients to date. In the setting of a randomised controlled trial we evaluated the relationships between baseline complement levels and subsequent CV events and mortality, in addition to the effects of HD with a vitamin E (VE)-coated dialysis membrane on circulating complement levels., Methods: A total of 260 HD patients were randomised to dialysis with a VE-coated dialysis membrane or non-VE coated equivalent for 12 months. Blood samples were taken at baseline, 6 and 12 months for measurement of C3, factor D, factor H and SC5b-9 levels. Data were collected prospectively on deaths and CV events., Results: Higher C3 levels at baseline were associated with subsequent CV events (hazard ratio 1.20 (1.01-1.42) per 0.1 mg/ml). Patients with intermediate SC5b-9 levels had significantly lower CV event rates and mortality than those with either high or low levels (p < 0.01). There were no effects of the VE-membranes on the complement components measured nor the clinical endpoints considered., Conclusions: The levels of C3 and SC5b-9 may have prognostic utility for predicting future CV events and/or mortality in HD patients - a relationship that requires further investigation. Dialysing prevalent HD patients with VE-bonded polysulfone membranes for a period of 12 months did not alter the circulating levels of the alternative complement pathway components considered here., (© 2015 S. Karger AG, Basel.)

The opportunistic human pathogen Pseudomonas aeruginosa controls host innate immune and complement attack. Here we identify Dihydrolipoamide dehydrogenase (Lpd), a 57 kDa moonlighting protein, as the first P. aeruginosa protein that binds the two human terminal pathway inhibitors vitronectin and clusterin. Both human regulators when bound to the bacterium inhibited effector function of the terminal complement, blocked C5b-9 deposition and protected the bacterium from complement damage. P. aeruginosa when challenged with complement active human serum depleted from vitronectin was severely damaged and bacterial survival was reduced by over 50%. Similarly, when in human serum clusterin was blocked by a mAb, bacterial survival was reduced by 44%. Thus, demonstrating that Pseudomonas benefits from attachment of each human regulator and controls complement attack. The Lpd binding site in vitronectin was localized to the C-terminal region, i.e. to residues 354-363. Thus, Lpd of P. aeruginosa is a surface exposed moonlighting protein that binds two human terminal pathway inhibitors, vitronectin and clusterin and each human inhibitor when attached protected the bacterial pathogen from the action of the terminal complement pathway. Our results showed insights into the important function of Lpd as a complement regulator binding protein that might play an important role in virulence of P. aeruginosa.

Purpose: To determine the effects of chromosome 1 genotype and cigarette smoking on levels of complement activation and inflammation in the human macula., Methods: Donor macular tissue was stratified into three groups by diplotype at the AMD-associated CFH-to-F13B locus: homozygous "risk" (n = 9, 56-78 years), homozygous neutral (n = 2, 64-79 years), and homozygous "protective" (n = 6, 61-78 years) diplotype. Importantly, all donors were homozygous nonrisk at the ARMS2/HTRA1 locus, so that purely chromosome 1-directed pathways were examined. Immunohistochemistry was performed by using 14 antibodies, mostly against markers of complement and inflammation, followed by confocal microscopy and immunofluorescence quantification (all masked to donor status)., Results: Donors homozygous risk at CFH-to-F13B exhibited significantly higher levels of terminal complement complex (TCC) in macular Bruch's membrane (BM; P = 0.03), choriocapillaris (CC; P = 0.04), and choriocapillaris intercapillary septa (CC IS; P = 0.03), compared to homozygous protected donors. Smoking was associated with increased TCC in BM (P = 0.05), CC IS (P = 0.03), and choroidal stroma (CS; P = 0.01), and with substantially elevated C-reactive protein (CRP) levels in RPE (P = 0.04), BM (P = 0.01), CC (P = 0.05), and CS (P = 0.05). Smoking was associated with higher levels of oxidative stress in macular RPE (P = 0.04) and CS (P = 0.01)., Conclusions: Genetic risk at the CFH-to-F13B locus was associated with higher levels of complement activation at the human macular RPE-choroid interface, as was cigarette smoking. Levels of CRP were substantially elevated in risk donors with smoking history. Examination of human macular tissue from donors with "pure" diplotypes allows assessment of AMD-associated pathways driven solely by CFH-to-F13B. These findings have important implications for identifying chromosome 1-directed pathways and therapeutic targets.

Objective: The role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region., Methods: C5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method., Results: Even though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases., Conclusions: Our results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.

Age-related macular degeneration (AMD) is a common disease that can result in severe visual impairment. Abnormal regulation of the complement system has been implicated in its pathogenesis, and CFH polymorphisms contribute substantially to risk. How these polymorphisms exert their effects is poorly understood. We performed enzyme-linked immunosorbent assay (ELISA) analysis on young, aged, and AMD choroids to determine the abundance of the membrane attack complex (MAC) and performed immunofluorescence studies on eyes from 117 donors to evaluate the MAC in aging, early AMD, and advanced AMD. Morphometric studies were performed on eyes with high- or low-risk CFH genotypes. ELISA confirmed that MAC increases significantly with aging and with AMD. MAC was localized to Bruch's membrane and the choriocapillaris and was detectable at low levels as early as 5 years of age. Hard drusen were labeled with anti-MAC antibody, but large or confluent drusen and basal deposits were generally unlabeled. Labeling of retinal pigment epithelium was observed in some cases of advanced AMD, but not in early disease. Eyes homozygous for the high-risk CFH genotype had thinner choroids than low-risk homozygotes (P < 0.05). These findings suggest that increased complement activation in AMD and in high-risk genotypes can lead to loss of endothelial cells in early AMD. Treatments to protect the choriocapillaris in early AMD are needed.

Spinal cord pathology with inflammatory, demyelinating lesions spanning three or more vertebral segments is a characteristic feature of neuromyelitis optica (NMO). NMO pathogenesis is thought to involve binding of immunoglobulin G anti-aquaporin-4 autoantibodies (NMO-IgG) to astrocytes, causing complement-dependent cytotoxicity (CDC) and secondary inflammation, demyelination and neuron loss. We investigated the involvement of CD59, a glycophosphoinositol (GPI)-anchored membrane protein on astrocytes that inhibits formation of the terminal C5b-9 membrane attack complex. CD59 inhibition by a neutralizing monoclonal antibody greatly increased NMO-IgG-dependent CDC in murine astrocyte cultures and ex vivo spinal cord slice cultures. Greatly increased NMO pathology was also found in spinal cord slice cultures from CD59 knockout mice, and in vivo following intracerebral injection of NMO-IgG and human complement. Intrathecal injection (at L5-L6) of small amounts of NMO-IgG and human complement in CD59-deficient mice produced robust, longitudinally extensive white matter lesions in lumbar spinal cord. Pathology was most severe at day 2 after injection, showing loss of AQP4 and GFAP, C5b-9 deposition, microglial activation, granulocyte infiltration, and demyelination. Hind limb motor function was remarkably impaired as well. There was partial remyelination and recovery of motor function by day 5. Our results implicate CD59 as an important modulator of the immune response in NMO, and provide a novel animal model of NMO that closely recapitulates human NMO pathology. Up-regulation of CD59 on astrocytes may have therapeutic benefit in NMO., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Mannose binding lectin (MBL) has been linked to predisposition to systemic lupus erythematosus (SLE) and to disease activity. Some studies found deposits of MBL in glomerular tissue of patients with lupus nephritis. There is no research about the deposition of MBL in skin., Materials and Methods: Skin biopsies from lesional and non lesional skin of 4 discoid lupus erythematosus (DLE) and 10 SLE patients were submitted to immunofluorescence staining for IgG, IgA, IgM, C3, C4, C1q, C5b-9 and MBL. Charts were reviewed for demographic, clinical and serological data. Patients with SLE had disease activity measured by SLEDAI., Results: MBL was found only in SLE lesional skin and its presence showed an association trend towards higher disease activity. Deposition of C5b-9 occurred in vessels only in patients with SLE (70%) and in the two patients with kidney involvement., Conclusions: MBL deposition was found in the lesional skin of SLE patients but not in SLE non lesional skin nor in DLE patients, and it seems to be less frequent and less strong than observed in the kidneys biopsies, suggesting that the complement participation in the pathophysiology of SLE process may not be the same in these two clinical manifestations., (Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Visceral glomerular epithelial cells (GEC), also known as podocytes, are vital for the structural and functional integrity of the glomerulus. The actin cytoskeleton plays a central role in maintaining GEC morphology. In a rat model of experimental membranous nephropathy (passive Heymann nephritis (PHN)), complement C5b-9-induced proteinuria was associated with the activation of the actin regulator small GTPase, RhoA. The mechanisms of RhoA activation, however, remained unknown. In this study, we explored the role of the epithelial guanine nucleotide exchange factor, GEF-H1, in complement-induced RhoA activation. Using affinity precipitation to monitor GEF activity, we found that GEF-H1 was activated in glomeruli isolated from rats with PHN. Complement C5b-9 also induced parallel activation of GEF-H1 and RhoA in cultured GEC. In GEC in which GEF-H1 was knocked down, both basal and complement-induced RhoA activity was reduced. On the other hand, GEF-H1 knockdown augmented complement-mediated cytolysis, suggesting a role for GEF-H1 and RhoA in protecting GEC from cell death. The MEK1/2 inhibitor, U0126, and mutation of the ERK-dependent phosphorylation site (T678A) prevented complement-induced GEF-H1 activation, indicating a role for the ERK pathway. Further, complement induced GEF-H1 and microtubule accumulation in the perinuclear region. However, both the perinuclear accumulation and the activation of GEF-H1 were independent of microtubules and myosin-mediated contractility, as shown using drugs that interfere with microtubule dynamics and myosin II activity. In summary, we have identified complement-induced ERK-dependent GEF-H1 activation as the upstream mechanism of RhoA stimulation, and this pathway has a protective role against cell death.

Five different cholesterol-dependent cytolysins (CDCs) have now had their atomic structures solved. Here their structures are compared and shown to vary less in the C-terminal region than they do in their N-terminal MACPF/CDC homology region. The most variable region of the C-terminal domain is the undecapeptide, which is observed in two clusters of conformations, and comparison of this domain with the C2 domain of perforin shows that the two structures have a common ancestor. Structural studies of CDC pre-pore and pore oligomers by cryo-electron microscopy and atomic force microscopy have revealed much about their mechanism of action. Understanding the activity of CDCs has required a combination of structural, biophysical and functional assays but current models of pore formation still require development to account for variable functional pore size.

Membrane Attack Complex/Perforin (MACPF) and Cholesterol-Dependent Cytolysins (CDC) form the MACPF/CDC superfamily of important effector proteins widespread in nature. MACPFs and CDCs were discovered separately with no sequence similarity at that stage being apparent between the two protein families such that they were not, until recently, considered evolutionary related. The breakthrough showing they are came with recent structural work that also shed light on the molecular mechanism of action of various MACPF proteins. Similarity in structural properties and conserved functional features indicate that both protein families have the same evolutionary origin. We will describe the distribution of MACPF/CDC proteins in nature and discuss briefly their similarity and functional role in different biological processes.

Diabetic retinopathy is the leading cause of visual dysfunction in working adults and is attributed to retinal vascular and neural cell damage. Recent studies have described elevated levels of membrane attack complex (MAC) and reduced levels of membrane associated complement regulators including CD55 and CD59 in the retina of diabetic retinopathy patients as well as in animal models of this disease. We have previously described the development of a soluble membrane-independent form of CD59 (sCD59) that when delivered via a gene therapy approach using an adeno-associated virus vector (AAV2/8-sCD59) to the eyes of mice, can block MAC deposition and choroidal neovascularization. Here, we examine AAV2/8-sCD59 mediated attenuation of MAC deposition and ensuing complement mediated damage to the retina of mice following streptozotocin (STZ) induced diabetes. We observed a 60% reduction in leakage of retinal blood vessels in diabetic eyes pre-injected with AAV2/8-sCD59 relative to negative control virus injected diabetic eyes. AAV2/8-sCD59 injected eyes also exhibited protection from non-perfusion of retinal blood vessels. In addition, a 200% reduction in retinal ganglion cell apoptosis and a 40% reduction in MAC deposition were documented in diabetic eyes pre-injected with AAV2/8-sCD59 relative to diabetic eyes pre-injected with the control virus. This is the first study characterizing a viral gene therapy intervention that targets MAC in a model of diabetic retinopathy. Use of AAV2/8-sCD59 warrants further exploration as a potential therapy for advanced stages of diabetic retinopathy.

Complement-dependent cytotoxicity (CDC) is a potent promoter of tumor clearance during monoclonal antibody therapy. Complement activation on antibody-bearing tumor cells results in formation of the membrane attack complex (MAC), which activates cell death. The complement activation cascade that bridges between antibody binding and MAC formation is regulated by complement inhibitors that are over-expressed on tumor cells. In order to bypass those complement regulators, we have designed an immunoconjugate composed of a humanized single chain Fv of an anti-Tac (CD25) monoclonal antibody fused at its C terminus either to complement protein C9 (scFv-C9) or to complement C7 (scFv-C7) and tagged with six histidines at the C terminal end. Recombinant scFv-C9 and scFv-C7 were expressed in 293T cells and purified. Both are shown to efficiently bind to CD25-positive tumor cells. In addition, scFv-C9, but not scFv-C7, increases MAC deposition on the cells and enhances complement-mediated cell death of target CD25-positive cells. Thus, scFv-C9 fusion protein is potentially a novel reagent for application in cancer immunotherapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)