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Complement component 3a (C3a) plays a crucial role in the immune response and host defense, but it is also involved in pro-inflammatory responses, causing many inflammatory disorders. Blockade of C3a has been regarded as a potent therapeutic strategy for inflammatory diseases. Here, we present the development of a human C3a (hC3a)-specific protein binder, which effectively inhibits pro-inflammatory responses. The protein binder, which is composed of leucine-rich repeat modules, was selected against hC3a through phage display, and its binding affinity was matured up to 600 pM by further expanding the binding interface in a module-by-module manner. The developed protein binder was shown to have more than 10-fold higher specificity to hC3a compared with human C5a, exhibiting a remarkable suppression effect on pro-inflammatory response in monocyte, by blocking the interaction between hC3a and its receptor. The hC3a-specific protein binder is likely to have a therapeutic potential for C3a-mediated inflammatory diseases., (© 2020 Wiley Periodicals, Inc.)

Background and Objective: Complement activation as an early and important inflammatory process contributes to multiple organ dysfunction after trauma. We have recently shown that complement inhibition by decay-accelerating factor (DAF) protects brain from blast-overpressure (BOP)-induced damage. This study was conducted to determine the effect of DAF on acute lung injury induced by BOP exposure and to elucidate its possible mechanisms of action., Methods: Anesthetized adult male Sprague-Daley rats were exposed to BOP (120 kPa) from a compressed air-driven shock tube. Rats were randomly assigned to three experimental groups: 1) Control (no BOP and no DAF treatment), 2) BOP (120 kPa BOP exposure), and 3) BOP followed by treatment with rhDAF (500μg/kg, i.v) at 30 minutes after blast. After a recovery period of 3, 24, or 48 hours, animals were euthanized followed by the collection of blood and tissues at each time point. Samples were subjected to the assessment of cytokines and histopathology as well as for the interaction of high-mobility-group box 1 (HMGB1) protein, NF-κB, receptor for advanced glycation end products (RAGE), C3a, and C3aR., Results: BOP exposure significantly increased in the production of systemic pro- and anti-inflammatory cytokines, and obvious pathological changes as characterized by pulmonary edema, inflammation, endothelial damage and hemorrhage in the lungs. These alterations were ameliorated by early administration of rhDAF. The rhDAF treatment not only significantly reduced the expression levels of HMGB1, RAGE, NF-κB, C3a, and C3aR, but also reversed the interaction of C3a-C3aR and nuclear translocation of HMGB1 in the lungs., Conclusions: Our findings indicate that early administration of DAF efficiently inhibits systemic and local inflammation, and mitigates blast-induced lung injury. The underlying mechanism might be attributed to its potential modulation of C3a-C3aR-HMGB1-transcriptional factor axis. Therefore, complement and/or HMGB1 may be potential therapeutic targets in amelioration of acute lung injury after blast injury., Competing Interests: The authors have declared that no competing interests exist.

Regeneration of skeletal muscle following injury is accompanied by transient inflammation. Here we show that complement is activated in skeletal muscle injury and plays a key role during regeneration. Genetic ablation of complement C3 or its inactivation with Cobra Venom Factor (CVF) result in impaired muscle regeneration following cardiotoxin-induced injury in mice. The effect of complement in muscle regeneration is mediated by the alternative pathway and C3a receptor (C3aR) signaling, as deletion of Cfb, a key alternative pathway component, or C3aR leads to impaired regeneration and reduced monocyte/macrophage infiltration. Monocytes from C3aR-deficient mice express a reduced level of adhesion molecules, cytokines and genes associated with antigen processing and presentation. Exogenous administration of recombinant CCL5 to C3aR-deficient mice rescues the defects in inflammatory cell recruitment and regeneration. These findings reveal an important role of complement C3a in skeletal muscle regeneration, and suggest that manipulating complement system may produce therapeutic benefit in muscle injury and regeneration.

Larvae of the blowfly Lucilia sericata facilitate wound healing by removing dead tissue and biofilms from non-healing and necrotic wounds. Another beneficial action of larvae and their excretions/secretions (ES) is down-regulation of excessive inflammation. As prolonged complement activation is key to excessive inflammation, the aim of this study was to elucidate the mechanisms underlying the anti-complement activities of ES. Results revealed that heat sensitive serine proteases in ES degrade multiple complement proteins in all steps of the three complement activation pathways. Importantly, C3a and C5a-major activators of inflammation-were also degraded by ES and pretreatment of these factors with ES completely blocked their ability to induce activation of human neutrophils. Pre-exposure of the neutrophils to ES did not affect their responsiveness to C3a/C5a and fMLP, indicating that the receptors for these activators on neutrophils were not affected by ES. Surprisingly, heat and serine protease inhibitor pretreatment did not affect the ability of ES to inhibit C5b-9 complex formation despite degrading complement proteins, indicating a second complement-inhibiting molecule in ES. Heated ES was as effective as intact ES in inhibiting C3 deposition upon activation of the alternative pathway, but was significantly less effective in wells with a classical or lectin pathway-specific coating. Unfortunately, the molecules affecting the complement system could not be identified due to an insufficient database for L. sericata. Together, larval ES inhibit complement activation by two different mechanisms and down-regulate the C3a/C5a-mediated neutrophil activation. This attenuates the inflammatory process, which may facilitate wound healing., (© 2016 by the Wound Healing Society.)

Background and Purpose: The restoration of blood-flow following cerebral ischemia incites a series of deleterious cascades that exacerbate neuronal injury. Pharmacologic inhibition of the C3a-receptor ameliorates cerebral injury by attenuating post-ischemic inflammation. Recent reports also implicate C3a in the modulation of tissue repair, suggesting that complement may influence both injury and recovery at later post-ischemic time-points., Methods: To evaluate the effect of C3a-receptor antagonism on post-ischemic neurogenesis and neurological outcome in the subacute period of stroke, transient focal cerebral ischemia was induced in adult male C57BL/6 mice treated with multiple regimens of a C3a receptor antagonist (C3aRA)., Results: Low-dose C3aRA administration during the acute phase of stroke promotes neuroblast proliferation in the subventricular zone at 7 days. Additionally, the C3a receptor is expressed on T-lymphocytes within the ischemic territory at 7 days, and this cellular infiltrate is abrogated by C3aRA administration. Finally, C3aRA treatment confers robust histologic and functional neuroprotection at this delayed time-point., Conclusions: Targeted complement inhibition through low-dose antagonism of the C3a receptor promotes post-ischemic neuroblast proliferation in the SVZ. Furthermore, C3aRA administration suppresses T-lymphocyte infiltration and improves delayed functional and histologic outcome following reperfused stroke. Post-ischemic complement activation may be pharmacologically manipulated to yield an effective therapy for stroke.