A guide to complement biology, pathology and therapeutic opportunity.

Complement has long been considered a key innate immune effector system that mediates host defence and tissue homeostasis. Yet, growing evidence has illuminated a broader involvement of complement in fundamental biological processes extending far beyond its traditional realm in innate immunity. Complement engages in intricate crosstalk with multiple pattern-recognition and signalling pathways both in the extracellular and intracellular space. Besides modulating host-pathogen interactions, this crosstalk guides early developmental processes and distinct cell trajectories, shaping tissue immunometabolic and regenerative programmes in different physiological systems. This Review provides a guide to the system-wide functions of complement. It highlights illustrative paradigm shifts that have reshaped our understanding of complement pathobiology, drawing examples from evolution, development of the central nervous system, tissue regeneration and cancer immunity. Despite its tight spatiotemporal regulation, complement activation can be derailed, fuelling inflammatory tissue pathology. The pervasive contribution of complement to disease pathophysiology has inspired a resurgence of complement therapeutics with major clinical developments, some of which have challenged long-held dogmas. We thus highlight major therapeutic concepts and milestones in clinical complement intervention.

A High-Performance Antibacterial Nanostructured ZnO Microfluidic Device for Controlled Bacterial Lysis and DNA Release.

In this work, the antibacterial properties of nanostructured zinc oxide (ZnO) surfaces are explored by incorporating them as walls in a simple-to-fabricate microchannel device. Bacterial cell lysis is demonstrated and quantified in such a device, which functions due to the action of its nanostructured ZnO surfaces in contact with the working fluid. To shed light on the mechanism responsible for lysis, E. coli bacteria were incubated in zinc and nanostructured ZnO substrates, as well as the here-investigated ZnO-based microfluidic devices. The unprecedented killing efficiency of E. coli in nanostructured ZnO microchannels, effective after a 15 min incubation, paves the way for the implementation of such microfluidic chips in the disinfection of bacteria-containing solutions. In addition, the DNA release was confirmed by off-chip PCR and UV absorption measurements. The results indicate that the present nanostructured ZnO-based microfluidic chip can, under light, achieve partial inactivation of the released bacterial DNA via reactive oxygen species-mediated oxidative damage. The present device concept can find broader applications in cases where the presence of DNA in a sample is not desirable. Furthermore, the present microchannel device enables, in the dark, efficient release of bacterial DNA for downstream genomic DNA analysis. The demonstrated potential of this antibacterial device for tailored dual functionality in light/dark conditions is the main novel contribution of the present work.

The Complement-Targeted Inhibitor Mini-FH Protects against Experimental Periodontitis via Both C3-Dependent and C3-Independent Mechanisms.

A minimized version of complement factor H (FH), designated mini-FH, was previously engineered combining the N-terminal regulatory domains (short consensus repeat [SCR]1-4) and C-terminal host-surface recognition domains (SCR19-20) of the parent molecule. Mini-FH conferred enhanced protection, as compared with FH, in an ex vivo model of paroxysmal nocturnal hemoglobinuria driven by alternative pathway dysregulation. In the current study, we tested whether and how mini-FH could block another complement-mediated disease, namely periodontitis. In a mouse model of ligature-induced periodontitis (LIP), mini-FH inhibited periodontal inflammation and bone loss in wild-type mice. Although LIP-subjected C3-deficient mice are protected relative to wild-type littermates and exhibit only modest bone loss, mini-FH strikingly inhibited bone loss even in C3-deficient mice. However, mini-FH failed to inhibit ligature-induced bone loss in mice doubly deficient in C3 and CD11b. These findings indicate that mini-FH can inhibit experimental periodontitis even in a manner that is independent of its complement regulatory activity and is mediated by complement receptor 3 (CD11b/CD18). Consistent with this notion, a complement receptor 3-interacting recombinant FH segment that lacks complement regulatory activity (specifically encompassing SCRs 19 and 20; FH19-20) was also able to suppress bone loss in LIP-subjected C3-deficient mice. In conclusion, mini-FH appears to be a promising candidate therapeutic for periodontitis by virtue of its ability to suppress bone loss via mechanisms that both include and go beyond its complement regulatory activity.

Temporal Pattern of Neuroinflammation Associated with a Low Glycemic Index Diet in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is associated with brain amyloid-ß (Aß) peptide accumulation and neuroinflammation. Currants, a low glycemic index dried fruit, and their components display pleiotropic neuroprotective effects in AD. We examined how diet containing 5% Corinthian currant paste (CurD) administered in 1-month-old 5xFAD mice for 1, 3, and 6 months affects Aß levels and neuroinflammation in comparison to control diet (ConD) or sugar-matched diet containing 3.5% glucose/fructose (GFD). No change in serum glucose or insulin levels was observed among the three groups. CurD administered for 3 months reduced brain Aß42 levels in male mice as compared to ConD and GFD, but after 6 months, Aß42 levels were increased in mice both on CurD and GFD compared to ConD. CurD for 3 months also reduced TNFα and IL-1ß levels in male and female mouse cortex homogenates compared to ConD and GFD. However, after 6 months, TNFα levels were increased in cortex homogenates of mice both on CurD and GFD as compared to ConD. A similar pattern was observed for TNFα-expressing cells, mostly co-expressing the microglial marker CD11b, in mouse hippocampus. IL-1ß levels were similarly increased in the brain of all groups after 6 months. Furthermore, a time dependent decrease of secreted TNFα levels was found in BV2 microglial cells treated with currant phenolic extract as compared to glucose/fructose solution. Overall, our findings suggest that a short-term currant consumption reduces neuroinflammation in 5xFAD mice as compared to sugar-matched or control diet, but longer-term intake of currant or sugar-matched diet enhances neuroinflammation.

Complement C3 inhibition in severe COVID-19 using compstatin AMY-101.

Complement C3 activation contributes to COVID-19 pathology, and C3 targeting has emerged as a promising therapeutic strategy. We provide interim data from ITHACA, the first randomized trial evaluating a C3 inhibitor, AMY-101, in severe COVID-19 (PaO2/FiO2 ≤ 300 mmHg). Patients received AMY-101 (n = 16) or placebo (n = 15) in addition to standard of care. AMY-101 was safe and well tolerated. Compared to placebo (8 of 15, 53.3%), a higher, albeit nonsignificant, proportion of AMY-101-treated patients (13 of 16, 81.3%) were free of supplemental oxygen at day 14. Three nonresponders and two placebo-treated patients succumbed to disease-related complications. AMY-101 significantly reduced CRP and ferritin and restrained thrombin and NET generation. Complete and sustained C3 inhibition was observed in all responders. Residual C3 activity in the three nonresponders suggested the presence of a convertase-independent C3 activation pathway overriding the drug's inhibitory activity. These findings support the design of larger trials exploring the potential of C3-based inhibition in COVID-19 or other complement-mediated diseases.

Complement Is Required for Microbe-Driven Induction of Th17 and Periodontitis.

In both mice and humans, complement and Th17 cells have been implicated in periodontitis, an oral microbiota-driven inflammatory disease associated with systemic disorders. A recent clinical trial showed that a complement C3 inhibitor (AMY-101) causes sustainable resolution of periodontal inflammation, the main effector of tissue destruction in this oral disease. Although both complement and Th17 are required for periodontitis, it is uncertain how these immune components cooperate in disease development. In this study, we dissected the complement-Th17 relationship in the setting of ligature-induced periodontitis (LIP), a model that previously established that microbial dysbiosis drives Th17 cell expansion and periodontal bone loss. Complement was readily activated in the periodontal tissue of LIP-subjected mice but not when the mice were placed on broad-spectrum antibiotics. Microbiota-induced complement activation generated critical cytokines, IL-6 and IL-23, which are required for Th17 cell expansion. These cytokines as well as Th17 accumulation and IL-17 expression were significantly suppressed in LIP-subjected C3-deficient mice relative to wild-type controls. As IL-23 has been extensively studied in periodontitis, we focused on IL-6 and showed that LIP-induced IL-17 and bone loss required intact IL-6 receptor signaling in the periodontium. LIP-induced IL-6 was predominantly produced by gingival epithelial cells that upregulated C3a receptor upon LIP challenge. Experiments in human gingival epithelial cells showed that C3a upregulated IL-6 production in cooperation with microbial stimuli that upregulated C3a receptor expression in ERK1/2- and JNK-dependent manner. In conclusion, complement links the periodontal microbiota challenge to Th17 cell accumulation and thus integrates complement- and Th17-driven immunopathology in periodontitis.

Emerging opportunities for C3 inhibition in the eye.

The eye presents a unique opportunity for complement component 3 (C3) therapeutics. Drugs can be delivered directly to specific parts of the eye, and growing evidence has established a pivotal role for C3 in age-related macular degeneration (AMD). Emerging data show that C3 may be important to the pathophysiology of other eye diseases as well. This article will discuss the location of C3 expression in the eye as well as the preclinical and clinical data regarding C3's functions in AMD. We will provide a comprehensive review of developing C3 inhibitors for the eye, including the Phase 2 and 3 data for the C3 inhibitor pegcetacoplan as a treatment for the geographic atrophy of AMD. Developing evidence also points toward C3 as a therapeutic target for stages of AMD preceding geographic atrophy. We will also discuss data illuminating C3's relationship to other eye diseases, such as Stargardt disease, diabetic retinopathy, and glaucoma. In addition to being a converging point and centerpiece of the complement cascade, C3 has broad effects as a multifaceted controller of opsonophagocytosis, microglia/macrophage recruitment, and downstream terminal pathway activity. C3 is a crucial player in the pathophysiology of AMD but also seems to have importance in other diseases that are major causes of blindness. Directions for further investigation will be highlighted, as culminating evidence suggests that we may be approaching an era of C3 therapeutics for the eye.

Complement C3 activation in the ICU: Disease and therapy as Bonnie and Clyde.

Patients in the intensive care unit (ICU) often straddle the divide between life and death. Understanding the complex underlying pathomechanisms relevant to such situations may help intensivists select broadly acting treatment options that can improve the outcome for these patients. As one of the most important defense mechanisms of the innate immune system, the complement system plays a crucial role in a diverse spectrum of diseases that can necessitate ICU admission. Among others, myocardial infarction, acute lung injury/acute respiratory distress syndrome (ARDS), organ failure, and sepsis are characterized by an inadequate complement response, which can potentially be addressed via promising intervention options. Often, ICU monitoring and existing treatment options rely on massive intervention strategies to maintain the function of vital organs, and these approaches can further contribute to an unbalanced complement response. Artificial surfaces of extracorporeal organ support devices, transfusion of blood products, and the application of anticoagulants can all trigger or amplify undesired complement activation. It is, therefore, worth pursuing the evaluation of complement inhibition strategies in the setting of ICU treatment. Recently, clinical studies in COVID-19-related ARDS have shown promising effects of central inhibition at the level of C3 and paved the way for prospective investigation of this approach. In this review, we highlight the fundamental and often neglected role of complement in the ICU, with a special focus on targeted complement inhibition. We will also consider complement substitution therapies to temporarily counteract a disease/treatment-related complement consumption.

C3-targeted host-modulation approaches to oral inflammatory conditions.

Periodontitis is an inflammatory disease caused by biofilm accumulation and dysbiosis in subgingival areas surrounding the teeth. If not properly treated, this oral disease may result in tooth loss and consequently poor esthetics, deteriorated masticatory function and compromised quality of life. Epidemiological and clinical intervention studies indicate that periodontitis can potentially aggravate systemic diseases, such as, cardiovascular disease, type 2 diabetes mellitus, rheumatoid arthritis, and Alzheimer disease. Therefore, improvements in the treatment of periodontal disease may benefit not only oral health but also systemic health. The complement system is an ancient host defense system that plays pivotal roles in immunosurveillance and tissue homeostasis. However, complement has unwanted consequences if not controlled appropriately or excessively activated. Complement overactivation has been observed in patients with periodontitis and in animal models of periodontitis and drives periodontal inflammation and tissue destruction. This review places emphasis on a promising periodontal host-modulation therapy targeting the complement system, namely the complement C3-targeting drug, AMY-101. AMY-101 has shown safety and efficacy in reducing gingival inflammation in a recent Phase 2a clinical study. We also discuss the potential of AMY-101 to treat peri-implant inflammatory conditions, where complement also seems to be involved and there is an urgent unmet need for effective treatment.

Compstatins: the dawn of clinical C3-targeted complement inhibition.

Despite the growing recognition of the complement system as a major contributor to a variety of clinical conditions, the therapeutic arsenal has remained scarce. The introduction of an anti-C5 antibody in 2007 raised confidence in complement-targeted therapy. However, it became apparent that inhibition of late-stage effector generation might not be sufficient in multifactorial complement disorders. Upstream intervention at the level of C3 activation has therefore been considered promising. The approval of pegcetacoplan, a C3 inhibitor of the compstatin family, in 2021 served as critical validation of C3-targeted treatment. This review delineates the evolution of the compstatin family from its academic origins to the clinic and highlights current and potential future applications of this promising drug class in complement diseases.

From discovery to approval: A brief history of the compstatin family of complement C3 inhibitors.

The FDA approval of pegcetacoplan (Empaveli), a PEGylated compstatin-based C3 therapeutic, as a new treatment for paroxysmal nocturnal hemoglobinuria (PNH) marks a milestone in the history of complement drug discovery. Almost 15 years after the approval of the first complement-specific drug for PNH, the anti-C5 antibody eculizumab, a novel class of complement inhibitors with a distinct mechanism of action finally enters the clinic. This landmark decision broadens the spectrum of available complement therapeutics, offering patients with unmet clinical needs or insufficient responses to anti-C5 therapy an alternative treatment option with a broad activity profile. Here we present a brief historical account of this newly approved complement drug, consolidating its approval within the long research record of the compstatin family of peptidic C3 inhibitors.

Isothermal Recombinase Polymerase Amplification (RPA) of E. coli gDNA in Commercially Fabricated PCB-Based Microfluidic Platforms.

Printed circuit board (PCB) technology has been recently proposed as a convenient platform for seamlessly integrating electronics and microfluidics in the same substrate, thus facilitating the introduction of integrated and low-cost microfluidic devices to the market, thanks to the inherent upscaling potential of the PCB industry. Herein, a microfluidic chip, encompassing on PCB both a meandering microchannel and microheaters to accommodate recombinase polymerase amplification (RPA), is designed and commercially fabricated for the first time on PCB. The developed microchip is validated for RPA-based amplification of two E. coli target genes compared to a conventional thermocycler. The RPA performance of the PCB microchip was found to be well-comparable to that of a thermocycler yet with a remarkably lower power consumption (0.6 W). This microchip is intended for seamless integration with biosensors in the same PCB substrate for the development of a point-of-care (POC) molecular diagnostics platform.

Phase IIa clinical trial of complement C3 inhibitor AMY-101 in adults with periodontal inflammation.

BackgroundGingivitis and periodontitis are prevalent inflammatory diseases of the periodontal tissues. Current treatments are often ineffective or do not prevent disease recurrence. Uncontrolled complement activation and the resulting chronic gingival inflammation are hallmarks of periodontal diseases. We determined the efficacy and safety of a complement 3-targeted therapeutic, AMY-101, which was locally administered to adult patients with periodontal inflammation.MethodsThirty-two patients with gingival inflammation were enrolled in a randomized, placebo-controlled, double-blind, split-mouth phase IIa trial that followed a dose escalation study to select a safe and effective dose in an additional 8 patients. Half of the patient's mouth was randomly assigned to AMY-101 (0.1 mg/site) or placebo injections at sites of inflammation, administered on days 0, 7, and 14, and then evaluated for safety and efficacy outcomes on days 28, 60, and 90. The primary efficacy outcome was a change in gingival inflammation, measured by a modified gingival index (MGI), and secondary outcomes included changes in bleeding on probing (BOP), the amount of plaque, pocket depth, clinical attachment level, and gingival crevicular fluid levels of matrix metalloproteinases (MMPs) over 90 days.ResultsA once-weekly intragingival injection of AMY-101 for 3 weeks was safe and well tolerated in all participants and resulted in significant (P < 0.001) reductions in clinical indices measuring gingival inflammation (MGI and BOP). AMY-101 significantly (P < 0.05) reduced MMP-8 and MMP-9 levels, indicators of inflammatory tissue destruction. These therapeutic effects persisted for at least 3 months after treatment.ConclusionAMY-101 treatment resulted in a significant and sustainable reduction in gingival inflammation without adverse events and, we believe, merits further investigation for the treatment of periodontitis and other oral or peri-implant inflammatory conditions.Trial registrationClinicalTrials.gov identifier NCT03694444.FundingAmyndas Pharmaceuticals.

Targeting complement components C3 and C5 for the retina: Key concepts and lingering questions.

Age-related macular degeneration (AMD) remains a major cause of legal blindness, and treatment for the geographic atrophy form of AMD is a significant unmet need. Dysregulation of the complement cascade is thought to be instrumental for AMD pathophysiology. In particular, C3 and C5 are pivotal components of the complement cascade and have become leading therapeutic targets for AMD. In this article, we discuss C3 and C5 in detail, including their roles in AMD, biochemical and structural aspects, locations of expression, and the functions of C3 and C5 fragments. Further, the article critically reviews developing therapeutics aimed at C3 and C5, underscoring the potential effects of broad inhibition of complement at the level of C3 versus more specific inhibition at C5. The relationships of complement biology to the inflammasome and microglia/macrophage activity are highlighted. Concepts of C3 and C5 biology will be emphasized, while we point out questions that need to be settled and directions for future investigations.

Complement C3 vs C5 inhibition in severe COVID-19: Early clinical findings reveal differential biological efficacy.

Growing clinical evidence has implicated complement as a pivotal driver of COVID-19 immunopathology. Deregulated complement activation may fuel cytokine-driven hyper-inflammation, thrombotic microangiopathy and NET-driven immunothrombosis, thereby leading to multi-organ failure. Complement therapeutics have gained traction as candidate drugs for countering the detrimental consequences of SARS-CoV-2 infection. Whether blockade of terminal complement effectors (C5, C5a, or C5aR1) may elicit similar outcomes to upstream intervention at the level of C3 remains debated. Here we compare the efficacy of the C5-targeting monoclonal antibody eculizumab with that of the compstatin-based C3-targeted drug candidate AMY-101 in small independent cohorts of severe COVID-19 patients. Our exploratory study indicates that therapeutic complement inhibition abrogates COVID-19 hyper-inflammation. Both C3 and C5 inhibitors elicit a robust anti-inflammatory response, reflected by a steep decline in C-reactive protein and IL-6 levels, marked lung function improvement, and resolution of SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). C3 inhibition afforded broader therapeutic control in COVID-19 patients by attenuating both C3a and sC5b-9 generation and preventing FB consumption. This broader inhibitory profile was associated with a more robust decline of neutrophil counts, attenuated neutrophil extracellular trap (NET) release, faster serum LDH decline, and more prominent lymphocyte recovery. These early clinical results offer important insights into the differential mechanistic basis and underlying biology of C3 and C5 inhibition in COVID-19 and point to a broader pathogenic involvement of C3-mediated pathways in thromboinflammation. They also support the evaluation of these complement-targeting agents as COVID-19 therapeutics in large prospective trials.

Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis.

Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyperinflammation and thrombotic microangiopathy, thereby increasing coronavirus 2019 (COVID-19) mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies, and NET/human aortic endothelial cell (HAEC) cocultures. Increased plasma levels of NETs, tissue factor (TF) activity, and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAECs. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against severe acute respiratory syndrome coronavirus 2 that exploit complement or NETosis inhibition.