Research Highlights

Activation of the Complement System on Human Endothelial Cells by Urban Particulate Matter Triggers Inflammation-Related Protein Production

Exposure to particulate matter (PM) is becoming a major global health issue. The amount and time of exposure to PM are known to be closely associated with cardiovascular diseases. However, the mechanism through which PM affects the vascular system is still not clear. Endothelial cells line the interior surface of blood vessels and actively interact with plasma proteins, including the complement system. Unregulated complement activation caused by invaders, such as pollutants, may promote endothelial inflammation. In the present study, we sought to investigate whether urban PM (UPM) acts on the endothelial environment via the complement system. UPM-treated human endothelial cells with normal human serum showed the deposition of membrane attack complexes (MACs) on the cell surface via the alternative pathway of the complement system. Despite the formation of MACs, cell death was not observed, and cell proliferation was increased in UPM-mediated complement activation. Furthermore, complement activation on endothelial cells stimulated the production of inflammation-related proteins. Our results revealed that UPM could activate the complement system in human endothelial cells and that complement activation regulated inflammatory reaction in microenvironment. These findings provide clues with regard to the role of the complement system in pathophysiologic events of vascular disease elicited by air pollution.
Keywords: complement system; endothelial cell; inflammation; particulate matter.

Adipsin of the Alternative Complement Pathway Is a Potential Predictor for Preeclampsia in Early Pregnancy

Objective: The concentrations of complement proteins (adipsin, C3a, and C5a) and soluble endoglin (sENG) in the plasma were measured in this study, and their value as early-pregnancy predictors and potential diagnostic marker of preeclampsia was assessed, respectively.
Experimental design: Plasma samples were obtained from healthy and preeclampsia pregnant women before delivery for a cross-sectional study. Plasma samples were collected from healthy and preeclampsia pregnant women throughout pregnancy and postpartum for a follow-up study. Enzyme-linked immunosorbent assays were used to detect plasma levels of several complement proteins (adipsin, C3a, and C5a) and sENG.
Results: The plasma levels of adipsin, C5a, and sENG were significantly increased before delivery in pregnant women with preeclampsia. During pregnancy, the plasma adipsin, C5a, and sENG levels were increased from the third trimester in healthy pregnant women; plasma adipsin levels remained stable after delivery, while C3a levels increased in the second trimester and remained stable afterward. Furthermore, levels of adipsin, C5a, and sENG were higher in preeclampsia patients at different stages of pregnancy; the C3a level presents a similar change and no difference was found in the third trimester. In the first trimester, receiver-operating curve (ROC) curve analysis showed that adipsin (AUC, 0.83 ± 0.06, P=0.001) and sENG (AUC, 0.74 ± 0.09, P=0.021) presented high value as predictors of early pregnancy.
Conclusions: Adipsin is likely a novel plasma biomarker to monitor the increased risk of preeclampsia in early pregnancy. Moreover, the increased plasma levels of adipsin, C5a, and sENG before delivery may be associated with preeclampsia.
Keywords: adipsin; biomarker; complement system; preeclampsia; sENG.

Alternative C3 Complement System: Lipids and Atherosclerosis

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin αMβ2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.
Keywords: atherosclerosis; cardiovascular disease; complement system; mass spectrometry; proteomics.

Antimicrobial Peptide Arenicin-1 Derivative Ar-1-(C/A) as Complement System Modulator

Antimicrobial peptides (AMPs) are not only cytotoxic towards host pathogens or cancer cells but also are able to act as immunomodulators. It was shown that some human and non-human AMPs can interact with complement proteins and thereby modulate complement activity. Thus, AMPs could be considered as the base for complement-targeted therapeutics development. Arenicins from the sea polychaete Arenicola marina, the classical example of peptides with a β-hairpin structure stabilized by a disulfide bond, were shown earlier to be among the most prospective regulators. Here, we investigate the link between arenicins' structure and their antimicrobial, hemolytic and complement-modulating activities using the derivative Ar-1-(C/A) without a disulfide bond. Despite the absence of this bond, the peptide retains all important functional activities and also appears less hemolytic in comparison with the natural forms. These findings could help to investigate new complement drugs for regulation using arenicin derivatives.

ARGX-117, a therapeutic complement inhibiting antibody targeting C2

Background: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention.
Objective: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2.
Methods: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys.
Results: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks.
Conclusions: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.
Keywords: C2; Complement system; complement inhibitor; monoclonal antibody.

Autoantibodies Against the Complement Regulator Factor H in the Serum of Patients With Neuromyelitis Optica Spectrum Disorder

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory disease of the central nervous system (CNS), characterized by pathogenic, complement-activating autoantibodies against the main water channel in the CNS, aquaporin 4 (AQP4). NMOSD is frequently associated with additional autoantibodies and antibody-mediated diseases. Because the alternative pathway amplifies complement activation, our aim was to evaluate the presence of autoantibodies against the alternative pathway C3 convertase, its components C3b and factor B, and the complement regulator factor H (FH) in NMOSD. Four out of 45 AQP4-seropositive NMOSD patients (~9%) had FH autoantibodies in serum and none had antibodies to C3b, factor B and C3bBb. The FH autoantibody titers were low in three and high in one of the patients, and the avidity indexes were low. FH-IgG complexes were detected in the purified IgG fractions by Western blot. The autoantibodies bound to FH domains 19-20, and also recognized the homologous FH-related protein 1 (FHR-1), similar to FH autoantibodies associated with atypical hemolytic uremic syndrome (aHUS). However, in contrast to the majority of autoantibody-positive aHUS patients, these four NMOSD patients did not lack FHR-1. Analysis of autoantibody binding to FH19-20 mutants and linear synthetic peptides of the C-terminal FH and FHR-1 domains, as well as reduced FH, revealed differences in the exact binding sites of the autoantibodies. Importantly, all four autoantibodies inhibited C3b binding to FH. In conclusion, our results demonstrate that FH autoantibodies are not uncommon in NMOSD and suggest that generation of antibodies against complement regulating factors among other autoantibodies may contribute to the complement-mediated damage in NMOSD.
Keywords: aquaporin (AQP) 4; autoantibody; autoimmunity; central nervous system; complement; factor H; inflammation; neuromyelitis optica spectrum disorder.

C4b Binding Protein Acts as an Innate Immune Effector Against Influenza A Virus

C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation.
Keywords: C4BP; complement; inflammation; influenza A virus; pseudo-typed lentiviral particles.

Cerebrospinal fluid complement system biomarkers in demyelinating disease

Background: Multiple sclerosis (MS) can be difficult to differentiate from other demyelinating diseases, notably neuromyelitis optica spectrum disorder (NMOSD). We previously showed that NMOSD is distinguished from MS by plasma complement biomarkers.
Objective: Here, we measure cerebrospinal fluid (CSF) complement proteins in MS, NMOSD and clinically isolated syndrome (CIS), a neurological episode that may presage MS, to test whether these distinguish NMOSD from MS and CIS.
Materials and methods: CSF (53 MS, 17 CIS, 11 NMOSD, 35 controls) was obtained; complement proteins (C4, C3, C5, C9, C1, C1q, Factor B (FB)), regulators (Factor I (FI), Factor H (FH), FH-Related Proteins 1, 2 and 5 (FHR125), C1 Inhibitor (C1INH), Properdin) and activation products (terminal complement complex (TCC), iC3b) were quantified by ELISA and results expressed relative to CSF total protein (μg/mg).
Results: Compared to control CSF, (1) levels of C4, C1INH and Properdin were elevated in MS; (2) TCC, iC3b, FI and FHR125 were increased in CIS; and (3) all complement biomarkers except TCC, FHR125, Properdin and C5 were higher in NMOSD CSF. A statistical model comprising six analytes (C3, C9, FB, C1q, FI, Properdin) plus age/gender optimally differentiated MS from NMOSD.
Keywords: Multiple sclerosis; biomarkers; cerebrospinal fluid; complement; neuromyelitis optica.

Coagulation Complement Activation and Cerebral Hypoperfusion in Relapsing-Remitting Multiple Sclerosis

Introduction: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with an underlying immune-mediated and inflammatory pathogenesis. Innate immunity, in addition to the adaptive immune system, plays a relevant role in MS pathogenesis. It represents the immediate non-specific defense against infections through the intrinsic effector mechanism “immunothrombosis” linking inflammation and coagulation. Moreover, decreased cerebral blood volume (CBV), cerebral blood flow (CBF), and prolonged mean transit time (MTT) have been widely demonstrated by MRI in MS patients. We hypothesized that coagulation/complement and platelet activation during MS relapse, likely during viral infections, could be related to CBF decrease. Our specific aims are to evaluate whether there are differences in serum/plasma levels of coagulation/complement factors between relapsing-remitting (RR) MS patients (RRMS) in relapse and those in remission and healthy controls as well as to assess whether brain hemodynamic changes detected by MRI occur in relapse compared with remission. This will allow us to correlate coagulation status with perfusion and demographic/clinical features in MS patients.
Materials and Methods: This is a multi-center, prospective, controlled study. RRMS patients (1° group: 30 patients in relapse; 2° group: 30 patients in remission) and age/sex-matched controls (3° group: 30 subjects) will be enrolled in the study. Patients and controls will be tested for either coagulation/complement (C3, C4, C4a, C9, PT, aPTT, fibrinogen, factor II, VIII, and X, D-dimer, antithrombin, protein C, protein S, von-Willebrand factor), soluble markers of endothelial damage (thrombomodulin, Endothelial Protein C Receptor), antiphospholipid antibodies, lupus anticoagulant, complete blood count, viral serological assays, or microRNA microarray. Patients will undergo dynamic susceptibility contrast-enhanced MRI using a 3.0-T scanner to evaluate CBF, CBV, MTT, lesion number, and volume.
Statistical Analysis: ANOVA and unpaired t-tests will be used. The level of significance was set at p ≤ 0.05.
Discussion: Identifying a link between activation of coagulation/complement system and cerebral hypoperfusion could improve the identification of novel molecular and/or imaging biomarkers and targets, leading to the development of new effective therapeutic strategies in MS.
Clinical Trial Registration: Clinicaltrials.gov, identifier NCT04380220.
Keywords: multiple sclerosis, coagulation, complement, platelets, relapse, infection, cerebral hypoperfusion

Complement Activation in the Central Nervous System: A Biophysical Model for Immune Dysregulation in the Disease State

Complement, a feature of the innate immune system that targets pathogens for phagocytic clearance and promotes inflammation, is tightly regulated to prevent damage to host tissue. This regulation is paramount in the central nervous system (CNS) since complement proteins degrade neuronal synapses during development, homeostasis, and neurodegeneration. We propose that dysregulated complement, particularly C1 or C3b, may errantly target synapses for immune-mediated clearance, therefore highlighting regulatory failure as a major potential mediator of neurological disease. First, we explore the mechanics of molecular neuroimmune relationships for the regulatory proteins: Complement Receptor 1, C1-Inhibitor, Factor H, and the CUB-sushi multiple domain family. We propose that biophysical and chemical principles offer clues for understanding mechanisms of dysregulation. Second, we describe anticipated effects to CNS disease processes (particularly Alzheimer's Disease) and nest our ideas within existing basic science, clinical, and epidemiological findings. Finally, we illustrate how the concepts presented within this manuscript provoke new ways of approaching age-old neurodegenerative processes. Every component of this model is testable by straightforward experimentation and highlights the untapped potential of complement dysregulation as a driver of CNS disease. This includes a putative role for complement-based neurotherapeutic agents and companion biomarkers.
Keywords: Alzheimer's disease; C1 inhibitor; CR1; complement; factor H; neuroimmune; schizophrenia.

Complement Activation in the Disease Course of Coronavirus Disease 2019 and Its Effects on Clinical Outcomes

Background: Excessive activation of immune responses in coronavirus disease 2019 (COVID-19) is considered to be related to disease severity, complications, and mortality rate. The complement system is an important component of innate immunity and can stimulate inflammation, but its role in COVID-19 is unknown.
Methods: A prospective, longitudinal, single center study was performed in hospitalized patients with COVID-19. Plasma concentrations of complement factors C3a, C3c, and terminal complement complex (TCC) were assessed at baseline and during hospital admission. In parallel, routine laboratory and clinical parameters were collected from medical files and analyzed.
Results: Complement factors C3a, C3c, and TCC were significantly increased in plasma of patients with COVID-19 compared with healthy controls ( P < .05). These complement factors were especially elevated in intensive care unit patients during the entire disease course (P < .005 for C3a and TCC). More intense complement activation was observed in patients who died and in those with thromboembolic events.
Conclusions: Patients with COVID-19 demonstrate activation of the complement system, which is related to disease severity. This pathway may be involved in the dysregulated proinflammatory response associated with increased mortality rate and thromboembolic complications. Components of the complement system might have potential as prognostic markers for disease severity and as therapeutic targets in COVID-19.
Keywords: ARDS; COVID-19; Coagulation; Complement; Inflammation; SARS-CoV-2.

Complement Alternative and Mannose-Binding Lectin Pathway Activation Is Associated With COVID-19 Mortality

Background: The SARS-CoV-2 infection triggers excessive immune response resulting in increased levels of pro-inflammatory cytokines, endothelial injury, and intravascular coagulopathy. The complement system (CS) activation participates to this hyperinflammatory response. However, it is still unclear which activation pathways (classical, alternative, or lectin pathway) pilots the effector mechanisms that contribute to critical illness. To better understand the immune correlates of disease severity, we performed an analysis of CS activation pathways and components in samples collected from COVID-19 patients hospitalized in Grenoble Alpes University Hospital between 1 and 30 April 2020 and of their relationship with the clinical outcomes.
Methods: We conducted a retrospective, single-center study cohort in 74 hospitalized patients with RT-PCR-proven COVID-19. The functional activities of classical, alternative, and mannose-binding lectin (MBL) pathways and the antigenic levels of the individual components C1q, C4, C3, C5, Factor B, and MBL were measured in patients' samples during hospital admission. Hierarchical clustering with the Ward method was performed in order to identify clusters of patients with similar characteristics of complement markers. Age was included in the model. Then, the clusters were compared with the patient clinical features: rate of intensive care unit (ICU) admission, corticoid treatment, oxygen requirement, and mortality.
Results: Four clusters were identified according to complement parameters. Among them, two clusters revealed remarkable profiles: in one cluster (n = 15), patients exhibited activation of alternative and lectin pathways and low antigenic levels of MBL, C4, C3, Factor B, and C5 compared to all the other clusters; this cluster had the higher proportion of patients who died (27%) and required oxygen support (80%) or ICU care (53%). In contrast, the second cluster (n = 19) presented inflammatory profile with high classical pathway activity and antigenic levels of complement components; a low proportion of patients required ICU care (26%) and no patient died in this group.
Conclusion: These findings argue in favor of prominent activation of the alternative and MBL complement pathways in severe COVID-19, but the spectrum of complement involvement seems to be heterogeneous requiring larger studies.
Keywords: COVID-19; MBL; alternative pathway; complement; lectin pathway.

Complement as the enabler of carfilzomib-induced thrombotic microangiopathy

Carfilzomib has been associated with the development of thrombotic microangiopathy (TMA) in relapsed/refractory multiple myeloma patients, a severe disease with no currently available aetiological treatment. We evaluated the potential role of terminal complement pathway in four patients with carfilzomib-induced TMA. Membrane attack complex (C5b-9) deposition on endothelial cells in culture exposed to plasma from patients during the acute phase of the disease suggests complement overactivation as a mechanism of potential endothelial damage in three out of four patients. If confirmed in larger cohorts, C5b-9 evaluation will allow early identification of patients who could benefit from complement blockade and treatment monitoring.
Keywords: C5b-9 deposits; carfilzomib; drug-induced thrombotic microangiopathy; eculizumab; endothelial cells.

Complement C4 Is Reduced in iPSC-Derived Astrocytes of Autism Spectrum Disorder Subjects

In recent years, accumulating evidence has shown that the innate immune complement system is involved in several aspects of normal brain development and in neurodevelopmental disorders, including autism spectrum disorder (ASD). Although abnormal expression of complement components was observed in post-mortem brain samples from individuals with ASD, little is known about the expression patterns of complement molecules in distinct cell types in the developing autistic brain. In the present study, we characterized the mRNA and protein expression profiles of a wide range of complement system components, receptors and regulators in induced pluripotent stem cell (iPSC)-derived neural progenitor cells, neurons and astrocytes of individuals with ASD and neurotypical controls, which constitute in vitro cellular models that recapitulate certain features of both human brain development and ASD pathophysiology. We observed that all the analyzed cell lines constitutively express several key complement molecules. Interestingly, using different quantification strategies, we found that complement C4 mRNA and protein are expressed in significantly lower levels by astrocytes derived from ASD individuals compared to control astrocytes. As astrocytes participate in synapse elimination, and diminished C4 levels have been linked to defective synaptic pruning, our findings may contribute to an increased understanding of the atypically enhanced brain connectivity in ASD.
Keywords: autism spectrum disorder; complement C4; complement system; iPSC-derived astrocytes; synaptic pruning.

Complement component C4 levels in the cerebrospinal fluid and plasma of patients with schizophrenia

Abnormalities in the complement system have been described in patients with schizophrenia, with those individuals having greater frequency of complement component 4A (C4A) alleles and higher C4A transcript levels in postmortem brain tissue. Importantly, abnormalities in C4A and other complement molecules have been associated with synaptic pruning abnormalities that occur during neurodevelopment. A few studies have investigated C4 levels in living patients with schizophrenia, but all of them did so using peripheral blood samples. No studies have examined C4 levels in cerebrospinal fluid (CSF), presumably a better biofluid choice given its intimate contact with the brain. Therefore, we report for the first time on C4 levels in CSF and plasma of patients with schizophrenia. In this study, we obtained CSF in 32 patients with schizophrenia spectrum disorders and 32 healthy volunteers and peripheral blood samples in 33 SSD and 31 healthy volunteers. C4 levels were measured using Abcam ELISA assays. Univariate analysis did not show a statistically significant difference in CSF C4 values between groups. However, a multivariable analysis showed a statistically significant increase in CSF C4 levels between groups after adjusting for sex and age. We also observed a high correlation between CSF C4 levels and age. By contrast, plasma C4 levels were not significantly different between groups. CSF and plasma C4 levels were not significantly correlated. Therefore, the use of CSF samples is critical and should be complementary to the use of peripheral blood samples to allow for a comprehensive understanding of complement C4 abnormalities in schizophrenia.

Complement Decay-Accelerating Factor is a modulator of influenza A virus lung immunopathology

Clearance of viral infections, such as SARS-CoV-2 and influenza A virus (IAV), must be fine-tuned to eliminate the pathogen without causing immunopathology. As such, an aggressive initial innate immune response favors the host in contrast to a detrimental prolonged inflammation. The complement pathway bridges innate and adaptive immune system and contributes to the response by directly clearing pathogens or infected cells, as well as recruiting proinflammatory immune cells and regulating inflammation. However, the impact of modulating complement activation in viral infections is still unclear. In this work, we targeted the complement decay-accelerating factor (DAF/CD55), a surface protein that protects cells from non-specific complement attack, and analyzed its role in IAV infections. We found that DAF modulates IAV infection in vivo, via an interplay with the antigenic viral proteins hemagglutinin (HA) and neuraminidase (NA), in a strain specific manner. Our results reveal that, contrary to what could be expected, DAF potentiates complement activation, increasing the recruitment of neutrophils, monocytes and T cells. We also show that viral NA acts on the heavily sialylated DAF and propose that the NA-dependent DAF removal of sialic acids exacerbates complement activation, leading to lung immunopathology. Remarkably, this mechanism has no impact on viral loads, but rather on the host resilience to infection, and may have direct implications in zoonotic influenza transmissions.

Complement Regulation in Human Tenocytes under the Influence of Anaphylatoxin C5a

A central part of the complement system, the anaphylatoxin C5a was investigated in this study to learn its effects on tenocytes in respect to understanding the potential expression of other crucial complement factors and pro-inflammatory mediators involved in tendinopathy. Human hamstring tendon-derived tenocytes were treated with recombinant C5a protein in concentrations of 25 ng/mL and 100 ng/mL for 0.5 h (early phase), 4 h (intermediate phase), and 24 h (late phase). Tenocytes survival was assessed after 24 h stimulation by live-dead assay. The gene expression of complement-related factors C5aR, the complement regulatory proteins (CRPs) CD46, CD55, CD59, and of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 was monitored using qPCR. Tenocytes were immunolabeled for C5aR and CD55 proteins. TNFα production was monitored by ELISA. Tenocyte survival was not impaired through C5a stimulation. Interestingly, the gene expression of C5aR and that of the CRPs CD46 and CD59 was significantly reduced in the intermediate and late phase, and that of TNFα only in an early phase, compared to the control group. ELISA analysis indicated a concomitant not significant trend of impaired TNFα protein synthesis at 4 h. However, there was also an early significant induction of CD55 and CD59 mediated by 25 ng/mL anaphylatoxin C5a. Hence, exposure of tenocytes to C5a obviously evokes a time and concentration-dependent response in their expression of complement and pro-inflammatory factors. C5a, released in damaged tendons, might directly contribute to tenocyte activation and thereby be involved in tendon healing and tendinopathy.
Keywords: C5a; anaphylatoxin; complement regulation; complement system; tendinopathy; tenocytes.

Danicopan: an oral complement factor D inhibitor for paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is characterised by complement-mediated intravascular hemolysis (IVH) due to absence of complement regulators CD55 and CD59 on affected erythrocytes. Danicopan is a first-in-class oral proximal, complement alternative pathway factor D (FD) inhibitor. Therapeutic FD inhibition was designed to control IVH and prevent C3-mediated extravascular hemolysis (EVH). In this open-label, phase 2, dose-finding trial, 10 untreated hemolytic PNH patients received danicopan monotherapy (100-200 mg thrice daily). Endpoints included change in lactate dehydrogenase (LDH) at day 28 (primary) and day 84 and hemoglobin. Safety, pharmacokinetics/pharmacodynamics, and patient-reported outcomes were measured. Ten patients reached the primary endpoint; two later discontinued: one for a serious adverse event (elevated aspartate aminotransferase/alanine aminotransferase coincident with breakthrough hemolysis, resolving without sequelae) and one for personal reasons unrelated to safety. Eight patients completed treatment. IVH was inhibited, demonstrated by mean decreased LDH (5.7 times upper limit of normal [ULN] at baseline vs 1.8 times ULN [day 28] and 2.2 times ULN [day 84]; both p<0.001). Mean baseline hemoglobin, 9.8 g/dL, increased by 1.1 (day 28) and 1.7 (day 84) g/dL (both p<0.005). No significant C3 fragment deposition occurred on GPI-deficient erythrocytes. Mean baseline Functional Assessment of Chronic Illness Therapy–Fatigue score, 34, increased by 9 (day 28) and 13 (day 84) points. Most common adverse events were headache and upper respiratory tract infection. This phase 2, monotherapy data shows proximal inhibition with danicopan provides clinically meaningful IVH inhibition and hemoglobin improvement in untreated PNH patients, without evidence of C3- mediated EVH. Registered at www.clinicaltrials.gov (#NCT03053102).

Dendrimer end-terminal motif-dependent evasion of human complement and complement activation through IgM hitchhiking

Complement is an enzymatic humoral pattern-recognition defence system of the body. Non-specific deposition of blood biomolecules on nanomedicines triggers complement activation through the alternative pathway, but complement-triggering mechanisms of nanomaterials with dimensions comparable to or smaller than many globular blood proteins are unknown. Here we study this using a library of <6 nm poly(amido amine) dendrimers bearing different end-terminal functional groups. Dendrimers are not sensed by C1q and mannan-binding lectin, and hence do not trigger complement activation through these pattern-recognition molecules. While, pyrrolidone- and carboxylic acid-terminated dendrimers fully evade complement response, and independent of factor H modulation, binding of amine-terminated dendrimers to a subset of natural IgM glycoforms triggers complement activation through lectin pathway-IgM axis. These findings contribute to mechanistic understanding of complement surveillance of dendrimeric materials, and provide opportunities for dendrimer-driven engineering of complement-safe nanomedicines and medical devices.

Endothelial C3a receptor mediates vascular inflammation and blood-brain barrier permeability during aging

Dysfunction of immune and vascular systems has been implicated in aging and Alzheimer disease; however, their interrelatedness remains poorly understood. The complement pathway is a well-established regulator of innate immunity in the brain. Here, we report robust age-dependent increases in vascular inflammation, peripheral lymphocyte infiltration, and blood-brain barrier (BBB) permeability. These phenotypes were subdued by global inactivation and by endothelial cell-specific ablation of C3ar1. Using an in vitro model of the BBB, we identified intracellular Ca2+ as a downstream effector of C3a/C3aR signaling and a functional mediator of vascular endothelial cadherin junction and barrier integrity. Endothelial C3ar1 inactivation also dampened microglia reactivity and improved hippocampal and cortical volumes in the aging brain, demonstrating a crosstalk between brain vasculature dysfunction and immune cell activation and neurodegeneration. Further, prominent C3aR-dependent vascular inflammation was also observed in a tau-transgenic mouse model. Our studies suggest that heightened C3a/C3aR signaling through endothelial cells promotes vascular inflammation and BBB dysfunction and contributes to overall neuroinflammation in aging and neurodegenerative disease.
Keywords: Aging; Alzheimer's disease.

Gamma subunit of complement component 8 is a neuroinflammation inhibitor

The complement system is part of the innate immune system that comprises several small proteins activated by sequential cleavages. The majority of these complement components, such as components 3a (C3a) and C5a, are chemotactic and pro-inflammatory. However, in this study, we revealed an inhibitory role of complement component 8 gamma (C8G) in neuroinflammation. In patients with Alzheimer's disease, who exhibit strong neuroinflammation, we found higher C8G levels in brain tissue, CSF, and plasma. Our novel findings also showed that the expression level of C8G increases in the inflamed mouse brain, and that C8G is mainly localized to brain astrocytes. Experiments using recombinant C8G protein and shRNA-mediated knockdown showed that C8G inhibits glial hyperactivation, neuroinflammation, and cognitive decline in acute and chronic animal models of Alzheimer's disease. Additionally, we identified sphingosine-1-phosphate receptor 2 (S1PR2) as a novel interaction protein of C8G and demonstrated that astrocyte-derived C8G interacts with S1PR2 to antagonize the pro-inflammatory action of S1P in microglia. Taken together, our results reveal the previously unrecognized role of C8G as a neuroinflammation inhibitor. Our findings pave the way towards therapeutic containment of neuroinflammation in Alzheimer's disease and related neurological diseases.
Keywords: astrocytes; complement component 8 gamma; microglia; neuroinflammation; sphingosine-1-phosphate receptor 2.

Genetic and molecular evidence for complement dysregulation in patients with HELLP syndrome

Background: HELLP (Hemolysis, elevated liver enzymes and low platelets) syndrome is a severe and acute pregnancy-related disorder that occurs in approximately 2.5 per 1000 deliveries and represents a major cause of maternal and perinatal morbidity and mortality. This syndrome has been suggested to be a microangiopathy and delivery is the only effective treatment.
Objectives: The aim of this study was to investigate the pathophysiology of HELLP syndrome by simultaneously exploring complement, haemostasis, autoimmunity and inflammation in relation to the clinical outcome.
Methods: We investigated 19 HELLP patients at the time of diagnosis and 3 months after delivery, for complement function, haemostasis and inflammation with immunoenzymatic methods. Complement-related gene variants were also analyzed by next generation sequencing and multiplex ligation-dependent probe amplification. Nineteen age-matched healthy pregnant women served as controls.
Results: At diagnosis, HELLP patients, compared to controls, showed significantly higher plasma levels of SC5b-9 (median 710 ng/ml [range 216-1499] vs 253 ng/ml [19-371], P < 0.0001) and of C5a (20.8 ng/ml [5.6-27.5] vs 12.7 ng/ml [3.2-24.6]; P=0.004), which decreased three months after delivery (SC5b9: 190 ng/ml [83-446] vs 160 ng/ml [107-219]; C5a: 9.28 ng/ml [2.3-21.6] vs 10.7 ng/ml [2.5-21.2]). A significantly higher frequency of genetic variants involving complement regulatory genes was also observed (52.6% vs 15.8%; P=0.016). Moreover, at HELLP diagnosis, patients showed increased coagulation markers (fragment F1 + 2 and D-dimer; P=0.0001) while both patients and controls had high thrombin-generation potential that decreased after delivery.
Conclusions: In the pathophysiology of HELLP syndrome, complement dysregulation, in addition to coagulation activation, is involved and may represent a potential target for treatment with the aim of delaying delivery.
Keywords: Complement system proteins; Genetic variants; HELLP syndrome; Haemostasis; Pregnancy.

Inhibition of complement C5a receptor protects lung cells and tissues against lipopolysaccharide-induced injury via blocking pyroptosis

Acute lung injury (ALI) is the injury of alveolar epithelial cells and capillary endothelial cells caused by various factors. Complement system and pyroptosis have been proved to be involved in ALI, and inhibition of C5a/C5a receptor (C5aR) could alleviate ALI. This study aimed to investigate whether C5a/C5aR inhibition could protect against LPS-induced ALI via mediating pyroptosis. Rats were assigned into four groups: Control, LPS, LPS+W-54011 1mg/kg, and LPS+W-54011 5mg/kg. Beas-2B cells pretreated with or without C5a and W-54011, alone and in combination, were challenged with LPS+ATP. Results unveiled that LPS caused lung tissue injury and inflammatory response, increased pyroptotic and apoptotic factors, along with elevated C5a concentration and C5aR expressions. However, W-54011 pretreatment alleviated lung damage and pulmonary edema, reduced inflammation and prevented cell pyroptosis. In vitro studies confirmed that LPS+ATP reduced cell viability, promoted cell death, generated inflammatory factors and promoted expressions of pyroptosis-related proteins, which could be prevented by W-54011 pretreatment while intensified by C5a pretreatment. The co-treatment of C5a and W-54011 could blunt the effects of C5a on LPS+ATP-induced cytotoxicity. In conclusion, inhibition of C5a/C5aR developed protective effects against LPS-induced ALI and the cytotoxicity of Beas-2B cells, and these effects may depend on blocking pyroptosis.
Keywords: C5a; acute lung injury; complement; inflammation; pyroptosis.

Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

The complement system has developed different strategies to clear infections by several effector mechanisms, such as opsonization, which supports phagocytosis, attracting immune cells by C3 and C5 cleavage products, or direct killing of pathogens by the formation of the membrane attack complex (MAC). As the Zika virus (ZIKV) activates the classical complement pathway and thus has to avoid clearance by the complement system, we analyzed putative viral escape mechanisms, which limit virolysis. We identified binding of the recombinant viral envelope E protein to components of the terminal pathway complement (C5b6, C7, C8, and C9) by ELISA. Western blot analyses revealed that ZIKV E protein interfered with the polymerization of C9, induced on cellular surfaces, either by purified terminal complement proteins or by normal human serum (NHS) as a source of the complement. Further, the hemolytic activity of NHS was significantly reduced in the presence of the recombinant E protein or entire viral particles. This data indicates that ZIKV reduces MAC formation and complement-mediated lysis by binding terminal complement proteins to the viral E protein.
Keywords: complement, lysis, membrane attack complex, Zika virus, envelope protein, terminal complement pathway

Lectin Pathway Mediates Complement Activation by SARS-CoV-2 Proteins

Early and persistent activation of complement is considered to play a key role in the pathogenesis of COVID-19. Complement activation products orchestrate a proinflammatory environment that might be critical for the induction and maintenance of a severe inflammatory response to SARS-CoV-2 by recruiting cells of the cellular immune system to the sites of infection and shifting their state of activation towards an inflammatory phenotype. It precedes pathophysiological milestone events like the cytokine storm, progressive endothelial injury triggering microangiopathy, and further complement activation, and causes an acute respiratory distress syndrome (ARDS). To date, the application of antiviral drugs and corticosteroids have shown efficacy in the early stages of SARS-CoV-2 infection, but failed to ameliorate disease severity in patients who progressed to severe COVID-19 pathology. This report demonstrates that lectin pathway (LP) recognition molecules of the complement system, such as MBL, FCN-2 and CL-11, bind to SARS-CoV-2 S- and N-proteins, with subsequent activation of LP-mediated C3b and C4b deposition. In addition, our results confirm and underline that the N-protein of SARS-CoV-2 binds directly to the LP- effector enzyme MASP-2 and activates complement. Inhibition of the LP using an inhibitory monoclonal antibody against MASP-2 effectively blocks LP-mediated complement activation. FACS analyses using transfected HEK-293 cells expressing SARS-CoV-2 S protein confirm a robust LP-dependent C3b deposition on the cell surface which is inhibited by the MASP-2 inhibitory antibody. In light of our present results, and the encouraging performance of our clinical candidate MASP-2 inhibitor Narsoplimab in recently published clinical trials, we suggest that the targeting of MASP-2 provides an unsurpassed window of therapeutic efficacy for the treatment of severe COVID-19.
Keywords: COVID-19; SARS-CoV-2; complement system; innate immunity; lectin pathway.

Microglial trogocytosis and the complement system regulate axonal pruning in vivo

Partial phagocytosis-called trogocytosis-of axons by microglia has been documented in ex vivo preparations but has not been directly observed in vivo. The mechanisms that modulate microglial trogocytosis of axons and its function in neural circuit development remain poorly understood. Here, we directly observe axon trogocytosis by microglia in vivo in the developing Xenopus laevis retinotectal circuit. We show that microglia regulate pruning of retinal ganglion cell axons and are important for proper behavioral response to dark and bright looming stimuli. Using bioinformatics, we identify amphibian regulator of complement activation 3, a homolog of human CD46, as a neuronally expressed synapse-associated complement inhibitory molecule that inhibits trogocytosis and axonal pruning. Using a membrane-bound complement C3 fusion protein, we demonstrate that enhancing complement activity enhances axonal pruning. Our results support the model that microglia remodel axons via trogocytosis and that neurons can control this process through expression of complement inhibitory proteins.
Keywords: axon; behavior; immunology; inflammation; microglia; morphometry; neuroscience; pruning; retinotectal; xenopus.

Modulation of complement activation by pentraxin-3 in prostate cancer

Pentraxin 3 (PTX3) is an essential component of the innate immune system and a recognized modulator of Complement cascade. The role of Complement system in the pathogenesis of prostate cancer has been largely underestimated. The aim of our study was to investigate the role of PTX3 as possible modulator of Complement activation in the development of this neoplasia. We performed a single center cohort study; from January 2017 through December 2018, serum and prostate tissue samples were obtained from 620 patients undergoing prostate biopsy. A group of patients with benign prostatic hyperplasia (BPH) underwent a second biopsy within 12-36 months demonstrating the presence of a prostate cancer (Group A, n = 40) or confirming the diagnosis of BPH (Group B, N = 40). We measured tissue PTX3 protein expression together with complement activation by confocal microscopy in the first and second biopsy in group A and B patients. We confirmed that that PTX3 tissue expression in the first biopsy was increased in group A compared to group B patients. C1q deposits were extensively present in group A patients co-localizing and significantly correlating with PTX3 deposits; on the contrary, C1q/PTX3 deposits were negative in group B. Moreover, we found a significantly increased expression of C3a and C5a receptors within resident cells in group A patient. Interestingly, C1q/PTX3 deposits were not associated with activation of the terminal Complement complex C5b-9; moreover, we found a significant increase of Complement inhibitor CD59 in cancer tissue. Our data indicate that PTX3 might play a significant pathogenic role in the development of this neoplasia through recruitment of the early components of Complement cascade with hampered activation of terminal Complement pathway associated with the upregulation of CD59. This alteration might lead to the PTX3-mediated promotion of cellular proliferation, angiogenesis and insensitivity to apoptosis possible leading to cancer cell invasion and migration.

Monitoring of the Complement System Status in Patients With B-Cell Malignancies Treated With Rituximab

Rituximab is a pioneering anti-CD20 monoclonal antibody that became the first-line drug used in immunotherapy of B-cell malignancies over the last twenty years. Rituximab activates the complement system in vitro, but there is an ongoing debate on the exact role of this effector mechanism in therapeutic effect. Results of both in vitro and in vivo studies are model-dependent and preclude clear clinical conclusions. Additional confounding factors like complement inhibition by tumor cells, loss of target antigen and complement depletion due to excessively applied immunotherapeutics, intrapersonal variability in the concentration of main complement components and differences in tumor burden all suggest that a personalized approach is the best strategy for optimization of rituximab dosage and therapeutic schedule. Herein we critically review the existing knowledge in support of such concept and present original data on markers of complement activation, complement consumption, and rituximab accumulation in plasma of patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphomas (NHL). The increase of markers such as C4d and terminal complement complex (TCC) suggest the strongest complement activation after the first administration of rituximab, but not indicative of clinical outcome in patients receiving rituximab in combination with chemotherapy. Both ELISA and complement-dependent cytotoxicity (CDC) functional assay showed that a substantial number of patients accumulate rituximab to the extent that consecutive infusions do not improve the cytotoxic capacity of their sera. Our data suggest that individual assessment of CDC activity and rituximab concentration in plasma may support clinicians' decisions on further drug infusions, or instead prescribing a therapy with anti-CD20 antibodies like obinutuzumab that more efficiently activate effector mechanisms other than complement.

Monoclonal Antibodies Capable of Inhibiting Complement Downstream of C5 in Multiple Species

Better understanding of roles of complement in pathology has fuelled an explosion of interest in complement-targeted therapeutics. The C5-blocking monoclonal antibody (mAb) eculizumab, the first of the new wave of complement blocking drugs, was FDA approved for treatment of Paroxysmal Nocturnal Hemoglobinuria in 2007; its expansion into other diseases has been slow and remains restricted to rare and ultra-rare diseases such as atypical hemolytic uremic syndrome. The success of eculizumab has provoked other Pharma to follow this well-trodden track and made C5 blockade the busiest area of complement drug development. C5 blockade inhibits generation of C5a and C5b, the former an anaphylatoxin, the latter the nidus for formation of the pro-inflammatory membrane attack complex. In order to use anti-complement drugs in common complement-driven diseases, more affordable and equally effective therapeutics are needed. To address this, we explored complement inhibition downstream of C5. Novel blocking mAbs targeting C7 and/or the C5b-7 complex were generated, identified using high throughput functional assays and specificity confirmed by immunochemical assays and surface plasmon resonance (SPR). Selected mAbs were tested in rodents to characterize pharmacokinetics, and therapeutic capacity. Administration of a mouse C7-selective mAb to wildtype mice, or a human C7 specific mAb to C7-deficient mice reconstituted with human C7, completely inhibited serum lytic activity for >48 h. The C5b-7 complex selective mAb 2H2, most active in rat serum, efficiently inhibited serum lytic activity in vivo for over a week from a single low dose (10 mg/kg); this mAb effectively blocked disease and protected muscle endplates from destruction in a rat myasthenia model. Targeting C7 and C7-containing terminal pathway intermediates is an innovative therapeutic approach, allowing lower drug dose and lower product cost, that will facilitate the expansion of complement therapeutics to common diseases.
Keywords: C5b-7; C7; complement; human; monoclonal antibody; mouse; rat; therapeutics.

Overexpression of schizophrenia susceptibility factor human complement C4A promotes excessive synaptic loss and behavioral changes in mice

The complement component 4 (C4) gene is linked to schizophrenia and synaptic refinement. In humans, greater expression of C4A in the brain is associated with an increased risk of schizophrenia. To investigate this genetic finding and address how C4A shapes brain circuits in vivo, here, we generated a mouse model with primate-lineage-specific isoforms of C4, human C4A and/or C4B. Human C4A bound synapses more efficiently than C4B. C4A (but not C4B) rescued the visual system synaptic refinement deficits of C4 knockout mice. Intriguingly, mice without C4 had normal numbers of cortical synapses, which suggests that complement is not required for normal developmental synaptic pruning. However, overexpressing C4A in mice reduced cortical synapse density, increased microglial engulfment of synapses and altered mouse behavior. These results suggest that increased C4A-mediated synaptic elimination results in abnormal brain

Quantitative multiplex profiling of the complement system to diagnose complement-mediated diseases

Objectives: Complement deficiencies are difficult to diagnose because of the variability of symptoms and the complexity of the diagnostic process. Here, we applied a novel 'complementomics' approach to study the impact of various complement deficiencies on circulating complement levels.
Methods: Using a quantitative multiplex mass spectrometry assay, we analysed 44 peptides to profile 34 complement proteins simultaneously in 40 healthy controls and 83 individuals with a diagnosed deficiency or a potential pathogenic variant in 14 different complement proteins.
Results: Apart from confirming near or total absence of the respective protein in plasma of complement-deficient patients, this mass spectrometry-based profiling method led to the identification of additional deficiencies. In many cases, partial depletion of the pathway up- and/or downstream of the absent protein was measured. This was especially found in patients deficient for complement inhibitors, such as angioedema patients with a C1-inhibitor deficiency. The added value of complementomics was shown in three patients with poorly defined complement deficiencies.
Conclusion: Our study shows the potential clinical utility of profiling circulating complement proteins as a comprehensive read-out of various complement deficiencies. Particularly, our approach provides insight into the intricate interplay between complement proteins due to functional coupling, which contributes to the better understanding of the various disease phenotypes and improvement of care for patients with complement-mediated diseases.
Keywords: complement deficiencies; complement system; complementomics; complement‐mediated diseases; multiplex targeted mass spectrometry; pathway analysis.

REV-ERBα mediates complement expression and diurnal regulation of microglial synaptic phagocytosis

The circadian clock regulates various aspects of brain health including microglial and astrocyte activation. Here, we report that deletion of the master clock protein BMAL1 in mice robustly increases expression of complement genes, including C4b and C3 , in the hippocampus. BMAL1 regulates expression of the transcriptional repressor REV-ERBα, and deletion of REV-ERBα causes increased expression of C4b transcript in neurons and astrocytes as well as C3 protein primarily in astrocytes. REV-ERBα deletion increased microglial phagocytosis of synapses and synapse loss in the CA3 region of the hippocampus. Finally, we observed diurnal variation in the degree of microglial synaptic phagocytosis which was antiphase to REV-ERBα expression. This daily variation in microglial synaptic phagocytosis was abrogated by global REV-ERBα deletion, which caused persistently elevated synaptic phagocytosis. This work uncovers the BMAL1-REV-ERBα axis as a regulator of complement expression and synaptic phagocytosis in the brain, linking circadian proteins to synaptic regulation.
Keywords: circadian rhythm; complement; mouse; neuron-glia interactions; neuroscience; synaptic terminals.

SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation

Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

Specific Evolution and Gene Family Expansion of Complement 3 and Regulatory Factor H in Fish

The complement system comprises a large family of plasma proteins that play a central role in innate and adaptive immunity. To better understand the evolution of the complement system in vertebrates and the contribution of complement to fish immunity comprehensive in silico and expression analysis of the gene repertoire was made. Particular attention was given to C3 and the evolutionary related proteins C4 and C5 and to one of the main regulatory factors of C3b, factor H (Cfh). Phylogenetic and gene linkage analysis confirmed the standing hypothesis that the ancestral c3/c4/c5 gene duplicated early. The duplication of C3 (C3.1 and C3.2) and C4 (C4.1 and C4.2) was likely a consequence of the (1R and 2R) genome tetraploidization events at the origin of the vertebrates. In fish, gene number was not conserved and multiple c3 and cfh sequence related genes were encountered, and phylogenetic analysis of each gene generated two main clusters. Duplication of c3 and cfh genes occurred across the teleosts in a species-specific manner. In common, with other immune gene families the c3 gene expansion in fish emerged through a process of tandem gene duplication. Gilthead sea bream (Sparus aurata), had nine c3 gene transcripts highly expressed in liver although as reported in other fish, extra-hepatic expression also occurs. Differences in the sequence and protein domains of the nine deduced C3 proteins in the gilthead sea bream and the presence of specific cysteine and N-glycosylation residues within each isoform was indicative of functional diversity associated with structure. The diversity of C3 and other complement proteins as well as Cfh in teleosts suggests they may have an enhanced capacity to activate complement through direct interaction of C3 isoforms with pathogenic agents.
Keywords: complement system; environment; evolution; fish; innate immunity; liver; skin.

Syk facilitates phagosome-lysosome fusion by regulating actin-remodeling in complement-mediated phagocytosis

Effective phagocytosis is crucial for host defense against pathogens. Macrophages entrap pathogens into a phagosome and subsequently acidic lysosomes fuse to the phagosome. Previous studies showed the pivotal role of actin-remodeling mediated by phosphoinositide-related signaling in phagosome formation, but the mechanisms of phagosome-lysosome fusion remain unexplored. Here we show that in complement-mediated phagocytosis, phagosome-lysosome fusion requires the disappearance of F-actin structure surrounding the phagosome and a tyrosine kinase Syk plays a key role in this process. Using macrophage-like differentiated HL60 and Syk-knockout (Syk-KO) HL60 cells, we found that Syk-KO cells showed insufficient phagosome acidification caused by impaired fusion with lysosomes and permitted the survival of Candida albicans in complement-mediated phagocytosis. Phagosome tracking analysis showed that during phagosome internalization process, F-actin surrounding phagosomes disappeared in both parental and Syk-KO cells but this structure was reconstructed immediately only in Syk-KO cells. In addition, F-actin-stabilizing agent induced a similar impairment of phagosome-lysosome fusion. Collectively, Syk-derived signaling facilitates phagosome-lysosome fusion by regulating actin-remodeling.

Synaptic and complement markers in extracellular vesicles in multiple sclerosis

Background: Synaptic loss is a feature of multiple sclerosis pathology that can be seen even in normal-appearing gray matter. Opsonization of synapses with complement components may underlie pathologic synapse loss.
Objective: We sought to determine whether circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (NEVs and AEVs) provide biomarkers reflecting complement-mediated synaptic loss in multiple sclerosis.
Methods: From plasma of 61 people with multiple sclerosis (46 relapsing-remitting multiple sclerosis (RRMS) and 15 progressive MS) and 31 healthy controls, we immunocaptured L1CAM + NEVs and GLAST + AEVs. We measured pre- and post-synaptic proteins synaptopodin and synaptophysin in NEVs and complement components (C1q, C3, C3b/iC3b, C4, C5, C5a, C9, Factor B, and Factor H) in AEVs, total circulating EVs, and neat plasma.
Results: We found lower levels of NEV synaptopodin and synaptophysin in MS compared to controls (p < 0.0001 for both). In AEVs, we found higher levels of multiple complement cascade components in people with MS compared to controls; these differences were not noted in total EVs or neat plasma. Strikingly, there were strong inverse correlations between NEV synaptic proteins and multiple AEV complement components in MS, but not in controls.
Conclusion: Circulating EVs could identify synaptic loss in MS and suggest a link between astrocytic complement production and synaptic loss.
Keywords: Extracellular vesicles; astrocytes; biomarkers; complement; multiple sclerosis; synaptic dysfunction.

The allosteric modulation of complement C5 by knob domain peptides

Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining regions that harbour cysteine-rich knob domains. To produce high-affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors, and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.
Keywords: bovine immunoglobulin; complement C5; human; immunology; inflammation; knob-domain peptides; molecular biophysics; structural biology.

The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.
Keywords: arthritis; cell metabolism; cellular senescence; complement system; inflammasome; inflammation; mechanistic target of rapamycin; synovial fibroblasts; tissue priming; trained immunity.

The solution structure of the complement deregulator FHR5 reveals a compact dimer and provides new insights into CFHR5 nephropathy

The human complement Factor H-related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (R_g) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26-29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy.
Keywords: FHR5; Monte Carlo simulations; analytical ultracentrifugation; atomistic modeling; complement; molecular dynamics; molecular modeling; small-angle X-ray scattering (SAXS); small-angle X-ray scattering analytical ultracentrifugation.

Two human antibodies to a meningococcal serogroup B vaccine antigen enhance binding of complement Factor H by stabilizing the Factor H binding site

Microbial pathogens bind host complement regulatory proteins to evade the immune system. The bacterial pathogen Neisseria meningitidis, or meningococcus, binds several complement regulators, including human Factor H (FH). FH binding protein (FHbp) is a component of two licensed meningococcal vaccines and in mice FHbp elicits antibodies that inhibit binding of FH to FHbp, which defeat the bacterial evasion mechanism. However, humans vaccinated with FHbp develop antibodies that enhance binding of FH to the bacteria, which could limit the effectiveness of the vaccines. In the present study, we show that two vaccine-elicited antibody fragments (Fabs) isolated from different human subjects increase binding of complement FH to meningococcal FHbp by ELISA. The two Fabs have different effects on the kinetics of FH binding to immobilized FHbp as measured by surface plasmon resonance. The 1.7- and 2.0-Å resolution X-ray crystal structures of the Fabs in complexes with FHbp illustrate that the two Fabs bind to similar epitopes on the amino-terminal domain of FHbp, adjacent to the FH binding site. Superposition models of ternary complexes of each Fab with FHbp and FH show that there is likely minimal contact between the Fabs and FH. Collectively, the structures reveal that the Fabs enhance binding of FH to FHbp by altering the conformations and mobilities of two loops adjacent to the FH binding site of FHbp. In addition, the 1.5 Å-resolution structure of one of the isolated Fabs defines the structural rearrangements associated with binding to FHbp. The FH-enhancing human Fabs, which are mirrored in the human polyclonal antibody responses, have important implications for tuning the effectiveness of FHbp-based vaccines.