Complement-mediated Activation Research Articles

Soluble collectin-12 mediates C3-independent docking of properdin that activates the alternative pathway of complement.

Properdin stabilizes the alternative C3 convertase (C3bBb), whereas its role as pattern-recognition molecule mediating complement activation is disputed for decades. Previously, we have found that soluble collectin-12 (sCL-12) synergizes complement alternative pathway (AP) activation. However, whether this observation is C3 dependent is unknown. By application of the C3-inhibitor Cp40, we found that properdin in normal human serum bound to Aspergillus fumigatus solely in a C3b-dependent manner. Cp40 also prevented properdin binding when properdin-depleted serum reconstituted with purified properdin was applied, in analogy with the findings achieved by C3-depleted serum. However, when opsonized with sCL-12, properdin bound in a C3-independent manner exclusively via its tetrameric structure and directed in situ C3bBb assembly. In conclusion, a prerequisite for properdin binding and in situ C3bBb assembly was the initial docking of sCL-12. This implies a new important function of properdin in host defense bridging pattern recognition and specific AP activation.

P0392AHUS ASSOCIATED WITH IGA NEPHROPATHY

Abstract Background and Aims IgAN is characterized by the presence of intense deposits of C3 accompanying IgA. As documented in several studies this is due to the activation of the complement system by pathological IgA; the mechanism of IgA–mediated complement activation remains poorly understood but is thought to act with different mechanisms such as the stabilization of the C3 convertase, the activation of the lectin pathway through polymeric IgA and eventually inherited partial deficiencies of complement regulatory genes [Maillard N, Wyatt RJ, Julian BA, Kiryluk K, Gharavi A, Fremeaux-Bacchi V, Novak J.J Current Understanding of the Role of Complement in IgA Nephropathy.Am Soc Nephrol 26:, 2015] . Dysregulation of the C3 convertase is a recognized cause of thrombotic microangiopathy (TMA) in atypical hemolytic uremic syndrome (aHUS). The association between aHUS and IgAN has been sporadically described . In this study we report a series of 6 patients, observed in our department, with biopsy proven IgAN, who developed aHUS during the follow-up. We report a series of aHUS associated IgA nephropathy. Method This report describes 6 patients with aHUS IgAN who were identified among all patients with biopsy proven IgAN observed at the Department of Nephrology, University of Parma, from March 2010 to March 2015. The diagnosis of aHUS was suspected in the presence of renal biopsy with documented typical signs of TMA (glomerular or arteriolar thrombi, onion skin lesions) associated with non-immune microangiopathic haemolytic anemia, increased reticulocyte count, negative Coomb test, thrombocytopenia (platelets <150000/mm3). Results Data collected on the 6 patients with IgA nephropathy associated with aHUS are described in Figure 1. All the cases of aHUS-IgAN were characterized by worsening of nephrotic proteinuria and reduced serum C3. Pathogenetic variant of CFH, predisposing for aHUS were identified in 2/6 cases; in the remaining patients a predisposing risk aplotype for aHUS was documented. All patients were treatment resistant for IgA nephropathy, had persistent proteinuria and progressed to ESRD. Conclusion It is known that most cases of aHUS are associated with the presence of genetic or acquired alterations of the regulation of the complement AP. However overlapping of other triggering conditions was documented in 70% of aHUS cases [Noris M, Caprioli J, Bresin E, et al. Relative Role of Genetic Complement Abnormalities in Sporadic and Familial aHUS and Their Impact on Clinical Phenotype. Clin J Am Soc Nephrol. 2010;5: 1844–1859]. Our case series shows that the presence of genetic factors predisposing to uncontrolled activation of the C3 convertase associated to continuous activation of the same enzyme from pathological IgA, could lead to sC3 consumption, development of aHUS and consequent rapid progression towards ESRD. In conclusion during the natural history of IgAN, appearance of hemolysis, thrombocytopenia, serum C3 reduction and increasing proteinuria would suggest to search for aHUS, allowing to early treatment with plasmapheresis and/or new available complement targeted inhibitors to prevent renal failure.

Suboptimal treatment response to anti-IL-5 monoclonal antibodies in severe eosinophilic asthmatics with airway autoimmune phenomena.

In clinical trials, the two anti-interleukin (IL)-5 monoclonal antibodies (mAbs: mepolizumab and reslizumab) approved to treat severe eosinophilic asthma reduce exacerbations by ∼50-60%. To observe response to anti-IL-5 mAbs in a real-life clinical setting, and to evaluate predictors of suboptimal response. In four Canadian academic centres, predefined clinical end-points in 250 carefully characterised moderate-to-severe asthmatic patients were collected prospectively to assess response to the two anti-IL-5 mAbs. Suboptimal response was determined based on failure to reduce maintenance corticosteroid (MCS) or asthma symptoms scores (Asthma Control Questionnaire (ACQ)) or exacerbations, in addition to persistence of sputum/blood eosinophils. Worsening in suboptimal responders was assessed based on reduced lung function by 25% or increase in MCS/ACQ. A representative subset of 39 patients was evaluated for inflammatory mediators, autoantibodies and complement activation in sputum (by ELISA) and for immune-complex deposition by immunostaining formalin-fixed paraffin-embedded sputum plugs. Suboptimal responses were observed in 42.8% (107 out of 250) patients treated with either mepolizumab or reslizumab. Daily prednisone requirement, sinus disease and late-onset asthma diagnoses were the strongest predictors of suboptimal response. Asthma worsened in 13.6% (34 out of 250) of these patients. The majority (79%) of them were prednisone-dependent. Presence of sputum anti-eosinophil peroxidase immunoglobulin (Ig)G was a predictor of suboptimal response to an anti-IL-5 mAb. An increase in sputum C3c (marker of complement activation) and deposition of C1q-bound/IL-5-bound IgG were observed in the sputa of those patients who worsened on therapy, suggesting an underlying autoimmune-mediated pathology. A significant number of patients who meet currently approved indications for anti-IL5 mAbs show suboptimal response to them in real-life clinical practice, particularly if they are on high doses of prednisone. Monitoring blood eosinophil count is not helpful to identify these patients. The concern of worsening of symptoms associated with immune-complex mediated complement activation in a small proportion of these patients highlights the relevance of recognising airway autoimmune phenomena and this requires further evaluation.

A clinical trial comparing aliskiren with enalapril for the treatment of C3 glomerulopathy

Abstract Introduction To date there is no established, effective treatment for C3 glomerulopathy, encompassing both Dense Deposit Disease and C3 glomerulonephritis. We have previously reported that renin mediates complement activation by cleaving C3 and found that the direct renin inhibitor aliskiren blocked complement activation by renin and inhibited complement activation systemically and in the kidneys in three patients with Dense Deposit Disease. Theoretically enalapril should increase renin without blocking its effect and therefore we will conduct a clinical trial to compare these two treatments. Material and methods We will include patients to be treated with aliskiren or enalapril (15 in each arm). Recruitment will take place in Swedish hospitals. Children 6 years or older with a glomerular filtration rate equal to or above 50 ml/min/1.73 m2 and adults equal to or above 30 ml/min/1.73 m2 will be included. Patients will be randomized for treatment with aliskiren or enalapril, as monotherapy or in combination with immune suppressive medications. After 6 months patients will switch to the other treatment and continue on the second treatment for an additional 2.5 years. Patients will be followed regularly regarding renal function, proteinuria, blood pressure, activation of the renin-angiotensin system and complement activation assays. After 1–3 years, when medically indicated or at the end of the study, a repeat renal biopsy will be performed to validate the effect of treatment on renal morphology and complement deposition. Conclusions The results of this study may provide more scientific evidence for, or against, the use of aliskiren in patients with C3 glomerulopathy.

Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses.

Following fifteen years of research, neutrophil extracellular traps (NETs) are widely reported in a large range of inflammatory infectious and non-infectious diseases. Cumulating evidences from in vitro, in vivo and clinical diagnostics suggest that NETs may play a crucial role in inflammation and autoimmunity in a variety of autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Most likely, NETs contribute to breaking self-tolerance in autoimmune diseases in several ways. During this review, we discuss the current knowledge on how NETs could drive autoimmune responses. NETs can break self-tolerance by being a source of autoantigens for autoantibodies found in autoimmune diseases, such as anti-citrullinated protein antibodies (ACPAs) in RA, anti-dsDNA in SLE and anti-myeloperoxidase and anti-protein 3 in AAV. Moreover, NET components could accelerate the inflammatory response by mediating complement activation, acting as danger-associated molecular patterns (DAMPs) and inflammasome activators, for example. NETs also can activate other immune cells, such as B cells, antigen-presenting cells and T cells. Additionally, impaired clearance of NETs in autoimmune diseases prolongs the presence of active NETs and their components and, in this way, accelerate immune responses. NETs have not only been implicated as drivers of inflammation, but also are linked to resolution of inflammation. Therefore, NETs may be central regulators of inflammation and autoimmunity, serve as biomarkers, as well as promising targets for future therapeutics of inflammatory autoimmune diseases.

Immunization Against Oxidized Elastin Exacerbates Structural and Functional Damage in Mouse Model of Smoke-Induced Ocular Injury.

PurposeAge-related macular degeneration (AMD) is the leading cause of blindness in Western populations. While an overactive complement system has been linked to pathogenesis, mechanisms contributing to its activation are largely unknown. In aged and AMD eyes, loss of the elastin layer (EL) of Bruch's membrane (BrM) has been reported. Elastin antibodies are elevated in patients with AMD, the pathogenic significance of which is unclear. Here we assess the role of elastin antibodies using a mouse model of smoke-induced ocular pathology (SIOP), which similarly demonstrates EL loss.MethodsC57BL/6J mice were immunized with elastin or elastin peptide oxidatively modified by cigarette smoke (ox-elastin). Mice were then exposed to cigarette smoke or air for 6 months. Visual function was assessed by optokinetic response, retinal morphology by spectral-domain optical coherence tomography and electron microscopy, and complement activation and antibody deposition by Western blot.ResultsOx-elastin IgG and IgM antibodies were elevated in ox-elastin immunized mice following 6 months of smoke, whereas elastin immunization had a smaller effect. Ox-elastin immunization exacerbated smoke-induced vision loss, with thicker BrM and more damaged retinal pigment epithelium (RPE) mitochondria compared with mice immunized with elastin or nonimmunized controls. These changes were correlated with increased levels of IgM, IgG2, IgG3, and complement activation products in RPE/choroid.ConclusionsThese data demonstrate that SIOP mice generate elastin-specific antibodies and that immunization with ox-elastin exacerbates ocular pathology. Elastin antibodies represented complement fixing isotypes that, together with the increased presence of complement activation seen in immunized mice, suggest that elastin antibodies exert pathogenic effects through mediating complement activation.

Labile Heme Aggravates Renal Inflammation and Complement Activation After Ischemia Reperfusion Injury.

Background: Ischemia reperfusion injury (IRI) plays a major role in solid organ transplantation. The length of warm ischemia time is critical for the extent of tissue damage in renal IRI. In this experimental study we hypothesized that local release of labile heme in renal tissue is triggered by the duration of warm ischemia (15 vs. 45 min IRI) and mediates complement activation, cytokine release, and inflammation.Methods: To induce IRI, renal pedicle clamping was performed in male C57BL/6 mice for short (15 min) or prolonged (45 min) time periods. Two and 24 h after experimental ischemia tissue injury labile heme levels in the kidney were determined with an apo-horseradish peroxidase assay. Moreover, renal injury, cytokines, and C5a and C3a receptor (C5aR, C3aR) expression were determined by histology, immunohistochemistry and qPCR, respectively. In addition, in vitro studies stimulating bone marrow-derived macrophages with LPS and the combination of LPS and heme were performed and cytokine expression was measured.Results: Inflammation and local tissue injury correlated with the duration of warm ischemia time. Labile heme concentrations in renal tissue were significantly higher after prolonged (45 min) as compared to short (15 min) IRI. Notably, expression of the inducible heme-degrading enzyme heme oxygenase-1 (HO-1) was up-regulated in kidneys after prolonged, but not after short IRI. C5aR, the pro-inflammatory cytokines IL-6 and TNF-α as well as pERK were up-regulated after prolonged, but not after short ischemia times. Consecutively, neutrophil infiltration and up-regulation of pro-fibrotic cytokines such as CTGF and PAI were more pronounced in prolonged IRI in comparison to short IRI. In vitro stimulation of macrophages with LPS revealed that IL-6 expression was enhanced in the presence of heme. Finally, administration of the heme scavenger human serum albumin (HSA) reduced the expression of pro-inflammatory cytokines, C3a receptor and improved tubular function indicated by enhanced alpha 1 microglobulin (A1M) absorption after IRI.Conclusions: Our data show that prolonged duration of warm ischemia time increased labile heme levels in the kidney, which correlates with IRI-dependent inflammation and up-regulation of anaphylatoxin receptor expression.

Prevention and Management of Daratumumab Mediated Infusion Related Hypersensitivity Reactions Pre-and Post-Implementation of Rapid Infusion Protocol

Background: Multiple Myeloma (MM) is defined as a clonal proliferation of malignant bone marrow plasma cells with high and uniform expression of CD 38 (Kumar et al, J Natl Compr Canc Netw. 2019; 3 and Lin et al, Am J Clin Pathol. 2004; 121:482-488). Daratumumab, a human IgGk monoclonal antibody, targets and binds to CD38, induces antibody dependent cell mediated cytotoxicity, complement activation, and antibody mediated phagocytosis (Prescribing information. Daratumumab; Janssen Biotech, Inc 2016). Daratumumab is associated with infusion related reactions (IRRs), which present with symptoms of rhinitis, cough, dyspnea, bronchospasm, chills and nausea. In two phase 3 trials, CASTOR (Palumbo et al, N Engl J Med. 2016; 375:754-66) and POLLUX (Dimopolous et al, N Engl J Med. 2016; 375(14):1319-31), IRRs occurred in 45% and 48% of patients, respectively. Of these, 98% and 96%, respectively, occurred during the first infusion. Grade 3 IRRs occurred in 5.3% and 8.6% of patients in CASTOR and POLLUX, respectively. No grade 4 IRRs were observed in either trial. Standard of care includes extended infusion times for the first, second, and subsequent infusions with a three-drug pre-infusion prophylaxis strategy including diphenhydramine, dexamethasone or methylprednisolone, and acetaminophen with slow prolonged infusion times is recommended(Bhatnagar et al, Oncologist. 2017; 22:1347-53). A single-arm safety study of accelerated infusion rate indicate that administering daratumumab using a time-saving 90-minute infusion protocol can be safe. Of 28 patients treated with accelerated daratumumab infusion during their third and subsequent infusions, no IRRs were observed, and there was only one mild reaction with no further reactions during subsequent infusions at the 90-minute rate (Barr et al, Leukemia. Mar. 2018). Subsequently, a similar protocol for all daratumumab infusions was implemented at The Johns Hopkins Health System including five premedications and three medications (5+3) during the first two infusions. Premedication can be eliminated for lack of tolerance. The purpose of this study is to evaluate the safety and success of rapid daratumumab administration in clinical practice based on infusion times, IRRs prophylaxis medication administration, IRRs frequency, and management of IRRs in the ambulatory setting of the Johns Hopkins Health System, pre- and post-implementation of the 90-minute infusion protocol using a more robust premedication regimen (5+3) than previously published. Study Design and Methods: This study is approved by the institutional review board at The Johns Hopkins Hospital (IRB 00195519). This is a retrospective, chart review of daratumumab infusions prior to implementing the accelerated infusion rate. The inclusion criterion was adult patients who received daratumumab infusion in the ambulatory infusion clinic July 1, 2016 to May 25, 2018 for pre-implementation and July 1 2018 to June 30 2019 for the post-implementation period. Patients who only received inpatient administration of daratumumab were excluded due to limited infusion related documentation. The primary endpoint is the proportion of patients who experienced IRRs after daratumumab infusion. Secondary endpoints include proportion of patients with grade 1/2 or grade 3/4 IRRs, infusion duration (hours), and the number of pre and post infusion prophylaxis medications given. Other data collected will include but not limited to demographics, prior history of anaphylaxis, eczema, asthma, or other drug allergies to identify risk factors not previously defined. Results: Data collection and analysis are ongoing. The sample size for pre-implementation of rapid infusion is ninety-three patients. Documented IRRs occurred in 18 (19%) patients. The mean infusion time was 7.4 hours for first infusions (n=70), 8 hours for patients with IRRs in the first infusion (n=18) and 7.1 hours for patients without IRRs in the first infusion (n=52). The mean number of IRR prophylaxis medications given was 7.8 (range 2-9) for first or second infusions. All IRRs documented were grade 1 or 2. Additional data analysis will include descriptive statistics as well as comparison of infusion duration, and will use paired t-test within samples and student t-test across different samples. Final results will report safety of daratumumab rapid infusion in largest single center patient population to date. Disclosures Ali: Celgene: Research Funding; Poseida: Research Funding.

KCNH2-3.1 mediates aberrant complement activation and impaired hippocampal-medial prefrontal circuitry associated with working memory deficits.

Increased expression of the 3.1 isoform of the KCNH2 potassium channel has been associated with cognitive dysfunction and with schizophrenia, yet little is known about the underlying pathophysiological mechanisms. Here, by using in vivo wireless local field potential recordings during working memory processing, in vitro brain slice whole-cell patching recordings and in vivo stereotaxic hippocampal injection of AAV-encoded expression, we identified specific and delayed disruption of hippocampal-mPFC synaptic transmission and functional connectivity associated with reductions of SERPING1, CFH, and CD74 in the KCNH2-3.1 overexpression transgenic mice. The differentially expressed genes in mice are enriched in neurons and microglia, and reduced expression of these genes dysregulates the complement cascade, which has been previously linked to synaptic plasticity. We find that knockdown of these genes in primary neuronal-microglial cocultures from KCNH2-3.1 mice impairs synapse formation, and replenishing reduced CFH gene expression rescues KCNH2-3.1-induced impaired synaptogenesis. Translating to humans, we find analogous dysfunctional interactions between hippocampus and prefrontal cortex in coupling of the fMRI blood oxygen level-dependent (BOLD) signal during working memory in healthy subjects carrying alleles associated with increased KCNH2-3.1 expression in brain. Our data uncover a previously unrecognized role of the truncated KCNH2-3.1 potassium channel in mediating complement activation, which may explain its association with altered hippocampal-prefrontal connectivity and synaptic function. These results provide a potential molecular link between increased KCNH2-3.1 expression, synapse alterations, and hippocampal-prefrontal circuit abnormalities implicated in schizophrenia.

Decoy nanoparticles bearing native C5a receptors as a new approach to inhibit complement-mediated neutrophil activation

Complement-activated neutrophils are integrally involved in many pathological conditions as well as in dampening the efficacy of cell-based therapies. Mesenchymal stem cells (MSCs) hold promise for regenerative medicine and inflammatory disease therapy, but current MSC-based therapies still require further improvements to ensure success. We recently reported that immediately upon delivery to the bloodstream, MSCs activate complement to produce C5a, which binds to its receptor, C5aR, on neutrophils and thus activates these cells to damage MSCs. Thus, blocking this C5a-C5aR interaction should yield improvements in MSC survival and treatment efficacy. In this project, we developed decoy nanoparticles with surface displaying native C5aR by coating membrane vesicles derived from macrophages expressing high levels of C5aR onto poly(lactic-co-glycolic acid) (PLGA) cores. These C5aR-displaying decoy nanoparticles effectively inhibited neutrophil activation and thus reduced sequential injury to MSCs upon exposure to blood both in vitro and in vivo. Consequently, survival and treatment potency of the MSCs were significantly improved by these decoy nanoparticles. This finding suggests that the C5aR-displaying decoy nanoparticles represent a unique approach toward improving current MSC-based therapies. Additionally, these decoy nanoparticles can be useful as a new reagent for the treatment of other pathological conditions that involve C5a-C5aR signaling. Statement of SignificanceComplement C5aR has been implied in the pathogenesis of many disorders and is emerging as a new target for the development of therapeutics. So far all the inhibitors of C5aR are either biologicals or small compounds with various shortcomings. Since C5aR is a G-protein coupled receptor that features a multi-loop binding interface with its ligand, C5a, soluble forms of C5aR as decoys for cell surface C5aR are unlikely. We believe this is the first evidence suggesting that C5aR decoy nanoparticles can be developed to treat various C5aR-mediated pathological conditions.

C1s Inhibition by BIVV009 (Sutimlimab) Prevents Complement-Enhanced Activation of Autoimmune Human B Cells In Vitro.

The classical pathway of complement (CP) can mediate C3 opsonization of Ags responsible for the costimulation and activation of cognate B lymphocytes. In this manner, the complement system acts as a bridge between the innate and adaptive immune systems critical for establishing a humoral response. However, aberrant complement activation is often observed in autoimmune diseases in which C3 deposition on self-antigens may serve to activate self-reactive B cell clones. In this study, we use BIVV009 (Sutimlimab), a clinical stage, humanized mAb that specifically inhibits the CP-specific serine protease C1s to evaluate the impact of upstream CP antagonism on activation and proliferation of normal and autoimmune human B cells. We report that BIVV009 significantly inhibited complement-mediated activation and proliferation of primary human B cells. Strikingly, CP antagonism suppressed human Ig-induced activation of B cells derived from patients with rheumatoid arthritis. These results suggest that clinical use of CP inhibitors in autoimmune patients may not only block complement-mediated tissue damage, but may also prevent the long-term activation of autoimmune B cells and the production of autoantibodies that contribute to the underlying pathologic condition of these diseases.

Decoy nanoparticles bearing native C5a receptor as a new approach to inhibit complement-mediated neutrophil activation

Decoy nanoparticles bearing native C5a receptor as a new approach to inhibit complement-mediated neutrophil activation

Aberrant early endosome biogenesis mediates complement activation in the retinal pigment epithelium in models of macular degeneration

Abnormally enlarged early endosomes (EEs) are pathological features of neurodegenerative diseases, yet insight into the mechanisms and consequences of EE expansion remains elusive. Here, we report swollen apical EEs in the retinal pigment epithelium (RPE) of aged human donors and in the pigmented Abca4-/- mouse model of Stargardt early-onset macular degeneration. Using high-resolution live-cell imaging, we show that age-related and pathological accumulation of lipofuscin bisretinoids increases ceramide at the apical surface of the RPE, which promotes inward budding and homotypic fusion of EEs. These enlarged endosomes internalize the complement protein C3 into the RPE, resulting in the intracellular generation of C3a fragments. Increased C3a in turn activates the mechanistic target of rapamycin (mTOR), a regulator of critical metabolic processes such as autophagy. The antidepressant desipramine, which decreases ceramide levels by inhibiting acid sphingomyelinase, corrects EE defects in the RPE of Abca4-/- mice. This prevents C3 internalization and limits the formation of C3a fragments within the RPE. Although uncontrolled complement activation is associated with macular degenerations, how complement contributes to pathology in a progressive disease is not well understood. Our studies link expansion of the EE compartment with intracellular complement generation and aberrant mTOR activation, which could set the stage for chronic metabolic reprogramming in the RPE as a prelude to disease. The pivotal role of ceramide in driving EE biogenesis and fusion in the Abca4-/- mice RPE suggests that therapeutic targeting of ceramide could be effective in Stargardt disease and other macular degenerations.

High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation.

High-mobility group box 1 (HMGB1), a well-known danger-associated molecular pattern molecule, acts as a pro-inflammatory molecule when secreted by activated immune cells or released after necrotic cell damage. HMGB1 binds to immunogenic bacterial components and augments septic inflammation. In this study, we show how HMGB1 mediates complement activation, promoting sterile inflammation. We show that HMGB1 activates the classical pathway of complement system in an antibody-independent manner after binding to C1q. The C3a complement activation product in human plasma and C5b-9 membrane attack complexes on cell membrane surface are detected after the addition of HMGB1. In an acetaminophen (APAP)-induced hepatotoxicity model, APAP injection reduced HMGB1 levels and elevated C3 levels in C1q-deficient mouse serum samples, compared to that in wild-type (WT) mice. APAP-induced C3 consumption was inhibited by sRAGE treatment in WT mice. Moreover, in a mouse model of brain ischemia–reperfusion injury based on middle cerebral arterial occlusion, C5b-9 complexes were deposited on vessels where HMGB1 was accumulated, an effect that was suppressed upon HMGB1 neutralization. We propose that the HMGB1 released after cell necrosis and in ischemic condition can trigger the classical pathway of complement activation to exacerbate sterile inflammation.

Complement-Mediated Activation of the NLRP3 Inflammasome and Its Inhibition by AAV-Mediated Delivery of CD59 in a Model of Uveitis.

Uveitis is an inflammatory disorder of the eye responsible for approximately 10%-15% of blindness in the US. In this study, we examined the role of the complement membrane attack complex (MAC) and the NLRP3 inflammasome in the pathogenesis of experimental autoimmune uveitis (EAU) in normal and C9-/- mice that are incapable of assembling the MAC. We discovered that the MAC and the NLRP3 inflammasome and associated production of IL-1β are elevated in EAU mice and that MAC may be involved in regulation of Th1 and Th17 cell differentiation. In contrast, MAC and the NLRP3 inflammasome were not elevated in C9-/- mice. However, EAU-associated pathophysiology including retinal structure and function were not rescued in C9-/- mice. Unexpectedly, AAV-mediated delivery of sCD59, an inhibitor of C9 incorporation into the MAC, successfully attenuated activation of the NLRP3 inflammasome and EAU pathology as well as MAC. Our studies provide an improved understanding of the role of the MAC and the NLRP3 inflammasome in EAU as well as suggest a novel approach for the treatment of uveitis.

Serum mannan-binding lectin in patients with pulmonary tuberculosis: Its lack of a relationship to the disease and response to treatment.

Background: Lectin pathway mediates complement activation, which is activated by many microorganisms. This study aimed at determining the serum levels of mannose-binding lectin (MBL) in patients with pulmonary tuberculosis, assessing its relationship to antiuberculosis treatment response, and comparing them with a control group. Methods: This cross-sectional study was conducted on patients with pulmonary tuberculosis during 2012 and 2013 in South West of Iran. PPD-ST-negative individuals were selected as controls from healthy relatives of patients. Serum MBL levels were measured using ELISA kit (Human MBL HK323, Hycultbiotech Company, Netherlands). All patients were followed- up for response to treatment. We applied Mann-Whitney and Fisher’s exact tests and used SPSS Version 17 software for statistical analysis. Results: The study included 62 patients as the case group and 63 noninfected TB patients as the control group. The MBL (ng/mL) in patients with pulmonary tuberculosis (median = 1012) was significantly (p= 0.037) higher than that of the control group (median= 296.2). No significant difference was found in the MBL level (ng/mL) between patients with response to antituberculosis treatment (median= 1012) and patients with treatment failure (median= 798.9) (p= 0.84). Conclusion: MBL may be involved in the pathogenesis of tuberculosis and in the low values that are protective against tuberculosis, and it seems that it has no effect on the antituberculosis treatment response.

Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) constitutes life-threatening autoimmune diseases affecting every organ, including the kidneys, where they cause necrotizing crescentic glomerulonephritis. ANCA activates neutrophils and activated neutrophils damage the endothelium, leading to vascular inflammation and necrosis. Better understanding of neutrophil-mediated AAV disease mechanisms may reveal novel treatment strategies. Here we report that ANCA induces neutrophil extracellular traps (NETs) via receptor-interacting protein kinase (RIPK) 1/3- and mixed-lineage kinase domain-like (MLKL)-dependent necroptosis. NETs from ANCA-stimulated neutrophils caused endothelial cell (EC) damage in vitro. This effect was prevented by (i) pharmacologic inhibition of RIPK1 or (ii) enzymatic NET degradation. The alternative complement pathway (AP) was recently implicated in AAV, and C5a inhibition is currently being tested in clinical studies. We observed that NETs provided a scaffold for AP activation that in turn contributed to EC damage. We further established the in vivo relevance of NETs and the requirement of RIPK1/3/MLKL-dependent necroptosis, specifically in the bone marrow-derived compartment, for disease induction using murine AAV models and in human kidney biopsies. In summary, we identified a mechanistic link between ANCA-induced neutrophil activation, necroptosis, NETs, the AP, and endothelial damage. RIPK1 inhibitors are currently being evaluated in clinical trials and exhibit a novel therapeutic strategy in AAV.

Impact of carbamylation on IgG mediated complement activation

To improve the wear resistance of a C45E4 steel substrate, a Mo coating was deposited by plasma transferred arc (PTA) process. The phase and microstructure of the coating were characterized by X-ray diffraction (XRD), optical microscope (OM), scanning electron microscopy equipped with an energy dispersive spectrometer (SEM-EDS) and transmission electron microscopy (TEM), respectively. The hardness and wear resistance were investigated by Vickers hardness and pin-on-disk wear testers. It was found that the metastable Fe63Mo37, Fe solid solution, Fe7Mo6, and a certain amount of amorphous phases co-existed in the as-deposited Mo coating. The peritectic coupled growth in Fe-Mo alloys was observed. Based on the undercooling and eutectic growth theory, the possibility of the Fe63Mo37/α-Fess peritectic coupled growth has been discussed. The microhardness was much higher than that of the C45E4 steel substrate. The results showed that the coating exhibited higher wear resistance ascribed to the high hardness phases and low-friction MoO3 film on the worn surface of the coating under dry sliding wear test against ZrO2 disc at room temperature.

NLRP3 is Required for Complement-Mediated Caspase-1 and IL-1beta Activation in ICH.

Complement-mediated inflammation plays a vital role in intracerebral hemorrhage (ICH), implicating pro-inflammatory factor interleukin-1beta (IL-1β) secretion. Brain samples and contralateral hemiencephalon were all collected and detected by Western blot. NLRP3 expression was located by dual immunofluorescence staining at 1, 3, and 5days post-ICH. Brain water content was examined post-ICH. The neural deficit scores were evaluated by observers blindly. ILs were detected by ELISA. SiRNAs targeting NLRP3 (siNLRP3), siASC, and siControl were injected to inhibit NLRP3 function. To test the complement activation via Nod-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), normal rabbit complement (NRC) was injected with lipopolysaccharide (LPS) to facilitate the complement function. As a result, complement 3a (C3a) and complement 5a (C5a) were upregulated during the ICH-induced neuroinflammation, and ablation of C3 attenuates ICH-induced IL-1β release. Though the LPS rescues the neuroinflammation in the ICH model, C3 deficiency attenuates the LPS-induced inflammatory effect. The NLRP3 inflammasome was activated after ICH and was located in the microglial cell of the mouse brain, which exhibits a time-dependent manner. However, the number of NLRP3/Iba-1 dual-labeled cells in the C3-/- group is less than that in the WT group in each time course, respectively. IL-1β and IL-18 released in perihematoma tissue, caspase-1-p20, brain water content, and behavioral outcomes were attenuated in the siNLRP3 and siASC groups than in the siControl and ICH groups. We also found that 5% of complement supplement enhances ICH-induced IL-1β release, while NLRP3 and ASC inhibition attenuates it. In conclusion, complement-induced ICH neuroinflammation depended on NLRP3 activation, which facilities LPS- and ICH-induced neuroinflammation, and NLRP3 is required for ICH-induced inflammation.

TNT009 Prevents Erythrocyte C3 Fragment Opsonization and Rescues Reticulocytes from Destruction in Patients with Cold Agglutinin Disease

▪Autoantibody mediated classical complement pathway (CP) activation has been hypothesized to drive hemolytic anemia in cold agglutinin disease (CAD) patients. Red blood cell (RBC) destruction is believed to occur as a result of C3 opsonin mediated extravascular hemolysis in the liver. We recently reported that TNT009, a humanized monoclonal antibody targeting the CP specific serine protease C1s, rapidly restores hemoglobin levels in severely anemic CAD patients. Here we describe the pharmacodynamic changes in the complement profile of patients to provide a mechanistic understanding of the hematological responses and therapeutic benefit observed following TNT009 treatment.In a Phase 1b trial, we enrolled 5 female CAD patients with severe anemia, one of whom had a lymphoplasmacytic lymphoma with >70% bone marrow infiltration and no measurable CP activity prior to dosing. This patient did not respond to TNT009 while on study and will be omitted from subsequent analyses. Patients were given an initial 10 mg/kg test dose of TNT009 on Day 1, followed by four weekly doses of 60 mg/kg on Day 2 or Day 5. Patients were followed for 4 weeks following the last dose (washout). Plasma and serum samples were collected throughout the study to measure TNT009 concentrations and to monitor serological markers of anemia and hemolysis. Additionally, futhan-containing plasma samples were collected to assess the levels of CP specific components including C1s, C1s-C1INH, and C1q by ELISA. RBCs were collected to monitor cell surface complement deposition (C3 fragments) via flow cytometry. Finally, C4 levels and an ELISA-based readout of CP activity were examined as measures of the pharmacodynamic effect of TNT009.Baseline levels of circulating C4 were either low or undetectable in CAD patients. Accordingly, serum CP activity was reduced compared to normal human serum samples. Following the first 60 mg/kg TNT009 dose, CP activity was immediately and completely inhibited within 15 minutes of dosing in all patients and remained inhibited for 3 weeks after the last dose. During this period of inhibition, C4 levels rose from a median circulating concentration of <90 mcg/mL (range: <70 - 145) to 251 mcg/mL (range: 238 - 353; p < .001). Plasma C1s levels, on the other hand, decreased from a median plasma concentration of 53.3 mcg/mL (range: 49.4 - 60.3) to a nadir of <3.13 mcg/mL, the lower limit of quantification (LLOQ), (p < .001). Similarly, C1s-C1INH decreased from a median value of 4.5 mcg/mL (range: 4.2 - 5.3) to a nadir of <0.16 mcg/mL (LLOQ; p < .001). Notably, circulating plasma C1q levels were unaffected.Classical pathway inhibition led to a significant increase in reticulocytes in all patients by, on average, 69% within 24 hours of dosing (p < .05). Interestingly, within 1 week after the first TNT009 dose, reticulocyte counts returned to pre-treatment levels and continued to decrease throughout the study, as expected when hemoglobin normalizes. Similarly, within 24 hours of the first dose of TNT009, bilirubin levels dropped from a median value of 2.1 mg/dL (range 1.6 - 3.8) to 0.7 mg/dL (range 0.6 - 1.2), resulting in an average reduction of 66% from baseline levels (p < .05) and returning to pre-treatment levels following washout. Finally, we monitored in vivo complement activation by staining for C3 fragment deposition on RBCs. In general, we observed a gradual reduction in the percentage of C3 fragment positive RBCs over the course of the study from a median value of 49% (range: 37 - 81) to 29% (range: 20 - 38) before washout of TNT009. The decrease in opsonized RBCs was concomitant with the rise in hemoglobin (example shown in Figure 1).Figure 1: Elevation of hemoglobin is associated with a decrease in C3 fragment coated erythrocytesHere we report that TNT009 administration depletes circulating C1s and immediately halts in vivo CP activity, normalizing plasma C4 levels in CAD patients. The abrupt increase in reticulocyte count within 24 hours of dosing suggests that cold agglutinin mediated complement activation affects reticulocyte survival, preventing their maturation into erythrocytes. The observed reduction in C3 opsonized RBCs suggests that TNT009 ameliorates anemia by preventing complement mediated hepatic RBC sequestration, supported by the immediate normalization of circulating bilirubin levels. These results provide a mechanistic interpretation of the therapeutic effects of TNT009 in CAD patients. [Display omitted] DisclosuresPanicker:True North Therapeutics, Inc.: Employment, Equity Ownership. Hussain:True North Therapeutics, Inc.: Employment, Equity Ownership. Parry:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Gilbert:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Jaeger:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.