Efficient complement-mediated clearance of immunosuppressed T cells by macrophages.

Cancer is one of the leading causes of death worldwide. Treatment outcome is largely dictated by the tumor type, disease stage, and treatment success rates, but also by the variation among patients in endogenous anti-tumor responses. Studies indicate that the presence of neutrophils in the tumor microenvironment is associated with a worse patient outcome due to their ability to suppress local anti-tumor T cell activity. Our previous studies investigated the mechanisms by which neutrophils suppress and damage T cells to become smaller in size (small T cells), debilitating their effector activities. Several studies indicate a role for tumor-associated macrophages in scavenging damaged or dead cells. We hypothesized that the observed lack of small T cells in the TME by confocal microscopy is due to immediate uptake by macrophages. In this study, we confirmed that indeed only the smaller, damaged T cells are taken up by macrophages, once serum-opsonized. Damaged T cells opsonized with complement factor C3 fragments were phagocytosed by macrophages, resulting in almost instantaneous and highly efficient uptake of these small T cells. Inhibition of the complement receptors CR1, CR3 and CR4 expressed by macrophages completely blocked phagocytosis. By contrast, actively proliferating T cells (large T cells) were neither impaired in neutrophil-MDSC activity nor opsonized for phagocytosis by macrophages. Rapid removal of damaged T cells suggests a role of complement and macrophages within the tumor microenvironment to clear suppressed T cells in cancer patients.

Targeting Complement C3a Receptor to Improve Outcome After Ischemic Brain Injury.

Ischemic stroke is a major cause of disability. No efficient therapy is currently available, except for the removal of the occluding blood clot during the first hours after symptom onset. Loss of function after stroke is due to cell death in the infarcted tissue, cell dysfunction in the peri-infarct region, as well as dysfunction and neurodegeneration in remote brain areas. Plasticity responses in spared brain regions are a major contributor to functional recovery, while secondary neurodegeneration in remote regions is associated with depression and impedes the long-term outcome after stroke. Hypoxic-ischemic encephalopathy due to birth asphyxia is the leading cause of neurological disability resulting from birth complications. Despite major progress in neonatal care, approximately 50% of survivors develop complications such as mental retardation, cerebral palsy or epilepsy. The C3a receptor (C3aR) is expressed by many cell types including neurons and glia. While there is a body of evidence for its deleterious effects in the acute phase after ischemic injury to the adult brain, C3aR signaling contributes to better outcome in the post-acute and chronic phase after ischemic stroke in adults and in the ischemic immature brain. Here we discuss recent insights into the novel roles of C3aR signaling in the ischemic brain with focus on the therapeutic opportunities of modulating C3aR activity to improve the outcome after ischemic stroke and birth asphyxia.

C3a Receptor Signaling Inhibits Neurodegeneration Induced by Neonatal Hypoxic-Ischemic Brain Injury.

Hypoxic-ischemic neonatal encephalopathy due to perinatal asphyxia is the leading cause of brain injury in newborns. Clinical data suggest that brain inflammation induced by perinatal insults can persist for years. We previously showed that signaling through the receptor for complement peptide C3a (C3aR) protects against cognitive impairment induced by experimental perinatal asphyxia. To investigate the long-term neuropathological effects of hypoxic-ischemic injury to the developing brain and the role of C3aR signaling therein, we subjected wildtype mice, C3aR deficient mice, and mice expressing biologically active C3a in the CNS to mild hypoxic-ischemic brain injury on postnatal day 9. We found that such injury triggers neurodegeneration and pronounced reactive gliosis in the ipsilesional hippocampus both of which persist long into adulthood. Transgenic expression of C3a in reactive astrocytes reduced hippocampal neurodegeneration and reactive gliosis. In contrast, neurodegeneration and microglial cell density increased in mice lacking C3aR. Intranasal administration of C3a for 3 days starting 1 h after induction of hypoxia-ischemia reduced neurodegeneration and reactive gliosis in the hippocampus of wildtype mice. We conclude that neonatal hypoxic-ischemic brain injury leads to long-lasting neurodegeneration. This neurodegeneration is substantially reduced by treatment with C3aR agonists, conceivably through modulation of reactive gliosis.

The Role of Macrophages in Staphylococcus aureus Infection.

Staphylococcus aureus is a member of the human commensal microflora that exists, apparently benignly, at multiple sites on the host. However, as an opportunist pathogen it can also cause a range of serious diseases. This requires an ability to circumvent the innate immune system to establish an infection. Professional phagocytes, primarily macrophages and neutrophils, are key innate immune cells which interact with S. aureus, acting as gatekeepers to contain and resolve infection. Recent studies have highlighted the important roles of macrophages during S. aureus infections, using a wide array of killing mechanisms. In defense, S. aureus has evolved multiple strategies to survive within, manipulate and escape from macrophages, allowing them to not only subvert but also exploit this key element of our immune system. Macrophage-S. aureus interactions are multifaceted and have direct roles in infection outcome. In depth understanding of these host-pathogen interactions may be useful for future therapeutic developments. This review examines macrophage interactions with S. aureus throughout all stages of infection, with special emphasis on mechanisms that determine infection outcome.

Deficiency of C3a receptor attenuates the development of diabetic nephropathy.

Objective: Diabetic nephropathy (DN) is the leading cause of chronic kidney disease and end-stage renal disease. Emerging evidence suggests that complement activation is involved in the pathogenesis of DN. The aim of this study was to investigate the pathogenic role of C3a and C3a receptor (C3aR) in DN. Research design and methods: The expression of C3aR was examined in the renal specimen of patients with DN. Using a C3aR gene knockout mice (C3aR-/-), we evaluated kidney injury in diabetic mice. The mouse gene expression microarray was performed to further explore the pathogenic role of C3aR. Then the underlying mechanism was investigated in vitro with macrophage treated with C3a. Results: Compared with normal controls, the renal expression of C3aR was significantly increased in patients with DN. C3aR-/- diabetic mice developed less severe diabetic renal damage compared with wild-type (WT) diabetic mice, exhibiting significantly lower level of albuminuria and milder renal pathological injury. Microarray profiling uncovered significantly suppressed inflammatory responses and T-cell adaptive immunity in C3aR-/- diabetic mice compared with WT diabetic mice, and this result was further verified by immunohistochemical staining of renal CD4+, CD8+ T cells and macrophage infiltration. In vitro study demonstrated C3a can enhance macrophage-secreted cytokines which could induce inflammatory responses and differentiation of T-cell lineage. Conclusions: C3aR deficiency could attenuate diabetic renal damage through suppressing inflammatory responses and T-cell adaptive immunity, possibly by influencing macrophage-secreted cytokines. Thus, C3aR may be a promising therapeutic target for DN.

CRIg plays an essential role in intravascular clearance of bloodborne parasites by interacting with complement.

Although CRIg was originally identified as a macrophage receptor for binding complement C3b/iC3b in vitro, recent studies reveal that CRIg functions as a pattern recognition receptor in vivo for Kupffer cells (KCs) to directly bind bacterial pathogens in a complement-independent manner. This raises the critical question of whether CRIg captures circulating pathogens through interactions with complement in vivo under flow conditions. Furthermore, the role of CRIg during parasitic infection is unknown. Taking advantage of intravital microscopy and using African trypanosomes as a model, we studied the role of CRIg in intravascular clearance of bloodborne parasites. Complement C3 is required for intravascular clearance of African trypanosomes by KCs, preventing the early mortality of infected mice. Moreover, antibodies are essential for complement-mediated capture of circulating parasites by KCs. Interestingly, reduced antibody production was observed in the absence of complement C3 during infection. We further demonstrate that CRIg but not CR3 is critically involved in KC-mediated capture of circulating parasites, accounting for parasitemia control and host survival. Of note, CRIg cannot directly catch circulating parasites and antibody-induced complement activation is indispensable for CRIg-mediated parasite capture. Thus, we provide evidence that CRIg, by interacting with complement in vivo, plays an essential role in intravascular clearance of bloodborne parasites. Targeting CRIg may be considered as a therapeutic strategy.

The Role of Complement in Organ Transplantation.

The current immunosuppressive protocols used in transplant recipients have improved short-term outcomes, but long-term allograft failure remains an important clinical problem. Greater understanding of the immunologic mechanisms that cause allograft failure are needed, as well as new treatment strategies for protecting transplanted organs. The complement cascade is an important part of the innate immune system. Studies have shown that complement activation contributes to allograft injury in several clinical settings, including ischemia/reperfusion injury and antibody mediated rejection. Furthermore, the complement system plays critical roles in modulating the responses of T cells and B cells to antigens. Therapeutic complement inhibitors, therefore, may be effective for protecting transplanted organs from several causes of inflammatory injury. Although several anti-complement drugs have shown promise in selected patients, the role of these drugs in transplantation medicine requires further study.

New insights into the immune functions of complement.

The recognition of microbial or danger-associated molecular patterns by complement proteins initiates a cascade of events that culminates in the activation of surface complement receptors on immune cells. Such signalling pathways converge with those activated downstream of pattern recognition receptors to determine the type and magnitude of the immune response. Intensive investigation in the field has uncovered novel pathways that link complement-mediated signalling with homeostatic and pathological T cell responses. More recently, the observation that complement proteins also act in the intracellular space to shape T cell fates has added a new layer of complexity. Here, we consider fundamental mechanisms and novel concepts at the interface of complement biology and immunity and discuss how these affect the maintenance of homeostasis and the development of human pathology.

The Role of Complement C3a Receptor in Stroke.

The complement system is a key regulator of the innate immune response against diseased tissue that functions across multiple organ systems. Dysregulation of complement contributes to the pathogenesis of a number of neurological diseases including stroke. The C3a anaphylatoxin, via its cognate C3a receptor (C3aR), mediates inflammation by promoting breakdown of the blood-brain barrier and the massive infiltration of leukocytes into ischemic brain in experimental stroke models. Studies utilizing complement deficient mice as well as pharmacologic C3aR antagonists have shown a reduction in tissue injury and mortality in murine stroke models. The development of tissue-specific C3aR knockout mice and more specific C3aR antagonists is warranted to facilitate our understanding of the role of the C3aR in brain ischemia with the ultimate goal of clinical translation of therapies targeting C3aR in stroke patients.

[Molecular Mechanisms for Learning and Memory: What Happens at the Synapses?]

The synapse is a structure connecting neurons in the brain, which is crucial for learning and memory. Accumulating evidence suggests that synapses continuously change in function and structure in response to learning and memory. Especially, in the cerebellum, which underlies motor learning and memory, synapses are highly dynamic throughout life. Recently, various types of molecules involving synapse integrity, learning and memory, such as δ-type glutamate receptors (GluD receptors) and C1q-family proteins, have been identified.

Complement component 3a receptor deficiency attenuates chronic stress-induced monocyte infiltration and depressive-like behavior.

Major depressive disorder (MDD) is one of the most common and debilitating neuropsychiatric illnesses. Accumulating evidence suggests a potential role of the immune system in the pathophysiology of MDD. The complement system represents one of the major effector mechanisms of the innate immune system, and plays a critical role in inflammation. However, the role of complement components in MDD is not well understood. Here, we found significant increase in component 3 (C3) expression in the prefrontal cortex (PFC) of depressed suicide subjects. We tested the role of altered C3 expression in mouse model of depression and found that increased C3 expression in PFC as a result of chronic stress causes depressive-like behavior. Conversely, mice lacking C3 were resilient to stress-induced depressive-like behavior. Moreover, selective overexpression of C3 in PFC was sufficient to cause depressive-like behavior in mice. We found that C3a (activated product of C3) receptor, C3aR+ monocytes were infiltrated into PFC following chronic stress. However, C3aR knockout mice displayed significantly reduced monocyte recruitment into PFC and reduced levels of the proinflammatory cytokine IL-1ß in PFC after chronic stress. In addition, C3aR knockout mice did not exhibit chronic stress-induced behavior despair. Similarly, chronic stress-induced increases in C3aR+ monocytes and IL-1ß in PFC, and depressive-like behavior were attenuated by myeloid cell depletion. These postmortem and preclinical studies identify C3aR signaling as a key factor in MDD pathophysiology.

The C3aR promotes macrophage infiltration and regulates ANCA production but does not affect glomerular injury in experimental anti-myeloperoxidase glomerulonephritis.

The anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides are autoimmune diseases associated with significant morbidity and mortality. They often affect the kidney causing rapidly progressive glomerulonephritis. While signalling by complement anaphylatoxin C5a though the C5a receptor is important in this disease, the role of the anaphylatoxin C3a signalling via the C3a receptor (C3aR) is not known. Using two different murine models of anti-myeloperoxidase (MPO) glomerulonephritis, one mediated by passive transfer of anti-MPO antibodies, the other by cell-mediated immunity, we found that the C3aR did not alter histological disease severity. However, it promoted macrophage recruitment to the inflamed glomerulus and inhibited the generation of MPO-ANCA whilst not influencing T cell autoimmunity. Thus, whilst the C3aR modulates some elements of disease pathogenesis, overall it is not critical in effector responses and glomerular injury caused by autoimmunity to MPO.

Role of complement anaphylatoxin receptors in a mouse model of acute burn-induced pain.

The complement system is an essential component of the innate immune response. The anaphylatoxins C3a and C5a are key drivers of the complement system, acting through the receptors C3aR, C5aR1 and C5aR2 to regulate inflammation. While a role for C5a activation of C5aR1 in inflammatory and neuropathic pain has been established, the role of the complement system in burn-induced pain has not been investigated. To address this gap, we assessed the role of complement receptors C3aR, C5aR1 and C5aR2 in a mouse model of acute burn-induced pain. Superficial burn injury was induced in C57BL/6 mice by firm application of left hind paw plantar surface to a hot plate set at 52.5 °C for 25 s. Development of burn-induced mechanical allodynia, thermal allodynia, weight bearing changes and edema was assessed in C3aR-/-, C5aR1-/- and C5aR2-/- mice and compared to their wild type controls over three days. Burn-induced mechanical allodynia, thermal allodynia and weight bearing changes developed normally C3aR-/-, C5aR1-/- and C5aR2-/- mice. However, burn-induced edema was significantly reduced in C5aR2-/- male mice, but not C5aR2-/- female mice. These results suggest that the complement system has a limited role in the development of acute burn-induced pain.

Complement C3a signaling facilitates skeletal muscle regeneration by regulating monocyte function and trafficking.

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.

Peripheral complement interactions with amyloid ß peptide: Erythrocyte clearance mechanisms.

INTRODUCTION: Although amyloid ß peptide (Aß) is cleared from the brain to cerebrospinal fluid and the peripheral circulation, mechanisms for its removal from blood remain unresolved. Primates have uniquely evolved a highly effective peripheral clearance mechanism for pathogens, immune adherence, in which erythrocyte complement receptor 1 (CR1) plays a major role. METHODS: Multidisciplinary methods were used to demonstrate immune adherence capture of Aß by erythrocytes and its deficiency in Alzheimer's disease (AD). RESULTS: Aß was shown to be subject to immune adherence at every step in the pathway. Aß dose-dependently activated serum complement. Complement-opsonized Aß was captured by erythrocytes via CR1. Erythrocytes, Aß, and hepatic Kupffer cells were colocalized in the human liver. Significant deficits in erythrocyte Aß levels were found in AD and mild cognitive impairment patients. DISCUSSION: CR1 polymorphisms elevate AD risk, and >80% of human CR1 is vested in erythrocytes to subserve immune adherence. The present results suggest that this pathway is pathophysiologically relevant in AD.

Basics of the medical use of ayahuasca: physiology of dimethyltryptamine.

Ayahuasca is a brew made of two admixture plants containing dimethyltryptamine (DMT) and b-carbolines (harmine and tetrahydroharmine). The indigenous groups of the Amazonas basin have been using it for centuries as an ethnomedical substance in healing and spiritual-religious rituals. During the last two decades the brew has raised increased scientific and public interest worldwide about its healing effects. Present paper addresses the therapeutic potentials of ayahuasca use and outlines the cellular mechanisms behind - in focus of the Q-1 receptor mediated action of DMT. The scientific investigation of ayahuasca is complicated by methodical problems, legal issues, and sociocultural pre-conceptions.

Complement anaphylatoxin receptors C3aR and C5aR are required in the pathogenesis of experimental autoimmune uveitis.

Recent studies have suggested that reagents inhibiting complement activation could be effective in treating T cell mediated autoimmune diseases such as autoimmune uveitis. However, the precise role of the complement anaphylatoxin receptors (C3a and C5a receptors) in the pathogenesis of autoimmune uveitis remains elusive and controversial. We induced experimental autoimmune uveitis in mice deficient or sufficient in both C3a and C5a receptors and rigorously compared their retinal phenotype using various imaging techniques, including indirect ophthalmoscopy, confocal scanning laser ophthalmoscopy, spectral domain optical coherence tomography, topical endoscopic fundus imaging, and histopathological analysis. We also assessed retinal function using electroretinography. Moreover, we performed Ag-specific T cell recall assays and T cell adoptive transfer experiments to compare pathogenic T cell activity between wild-type and knockout mice with experimental autoimmune uveitis. These experiments showed that C3a receptor/C5a receptor-deficient mice developed much less severe uveitis than did control mice using all retinal examination methods and that these mice had reduced pathogenic T cell responses. Our data demonstrate that both complement anaphylatoxin receptors are important for the development of experimental autoimmune uveitis, suggesting that targeting these receptors could be a valid approach for treating patients with autoimmune uveitis.

Tumor necrosis-initiated complement activation stimulates proliferation of medulloblastoma cells.

OBJECTIVE AND DESIGN: We sought to determine the effect of necrosis-induced activation of the complement protein C3 in medulloblastoma. MATERIALS/METHODS: Twelve medulloblastoma surgical specimens were evaluated for complement activation using immunohistochemistry, with H&E stains performed on adjacent tissue sections to determine the relationship of complement activation to necrotic tissue. Flow cytometry and Western blot were performed on three established medulloblastoma lines and one surgically-procured cell culture to determine expression of C3a receptor (C3aR) in medulloblastoma. In vitro proliferation of siRNA C3aR knockdown cells was compared to that of control siRNA cells with cell line Daoy. RESULTS: Three surgical specimens were found to have necrosis on H&E sections. In each case, iC3b staining was identified on adjacent sections, limited to the necrotic region. In no case did necrosis occur without iC3b staining on adjacent sections. C3aR protein was demonstrated on both the three established cell lines and on the surgical culture. Proliferation assays of Daoy cells with siRNA knockdown vs. control siRNA revealed significantly reduced proliferation at 72 h (p = 0.001). CONCLUSIONS: Necrosis is associated with complement activation in medulloblastoma. Medulloblastoma cells express C3aR, and siRNA-mediated knockdown of C3aR inhibits proliferation of these cells in vitro.

Optimization of a flow cytometric assay to evaluate the human neutrophil ability to phagocytose immune complexes via Fcγ and complement receptors.

INTRODUCTION: This study aims to optimize some experimental conditions of a flow cytometric assay to examine the human neutrophil ability to phagocytose immune complexes (ICs) via Fcγ and complement receptors (FcγR and CR, respectively). The parameters assessed were: number of cells, concentration of ICs, reaction time, pH and concentration of the Trypan Blue quenching solution. METHODS: Neutrophils were isolated from peripheral blood of healthy volunteers. Precipitated ICs composed of IgG and fluorescein isothiocyanate (FITC)-labeled ovalbumin, opsonized or not with serum complement, were used to trigger the neutrophil phagocytosis via FcγR, CR, and FcγR+CR. Fluorescence of the internalized ICs was measured by flow cytometry, after quenching the extracellular fluorescence with Trypan Blue. RESULTS: The optimal experimental conditions established for the phagocytosis assay were: 1 × 10(6) cells mL(-1) and 40 µg mL(-1) FITC-labeled ICs, incubated for 30 min, at 37°C, in 0.5 mL of reaction volume. Trypan Blue solution at 1.25 mg mL(-1) pH4.4 was the best fluorescence quencher of FITC-labeled ICs attached to the outer surface of neutrophils. DISCUSSION: The selected experimental conditions were viable to evaluate IC phagocytosis by neutrophils; they are also suitable to compare the efficiency of IC phagocytosis mediated by FcγR and CR classes of membrane receptors, alone or in combination. This method finds application in studies of (i) the receptor-specific phagocytic function of normal and pathogenic neutrophils as well as (ii) the impact of drugs and therapies on this essential effector function of neutrophils.

The staphylococcal toxins γ-haemolysin AB and CB differentially target phagocytes by employing specific chemokine receptors.

Evasion of the host phagocyte response by Staphylococcus aureus is crucial to successful infection with the pathogen. γ-haemolysin AB and CB (HlgAB, HlgCB) are bicomponent pore-forming toxins present in almost all human S. aureus isolates. Cellular tropism and contribution of the toxins to S. aureus pathophysiology are poorly understood. Here we identify the chemokine receptors CXCR1, CXCR2 and CCR2 as targets for HlgAB, and the complement receptors C5aR and C5L2 as targets for HlgCB. The receptor expression patterns allow the toxins to efficiently and differentially target phagocytic cells. Murine neutrophils are resistant to HlgAB and HlgCB. CCR2 is the sole murine receptor orthologue compatible with γ-haemolysin. In a murine peritonitis model, HlgAB contributes to S. aureus bacteremia in a CCR2-dependent manner. HlgAB-mediated targeting of CCR2(+) cells highlights the involvement of inflammatory macrophages during S. aureus infection. Functional quantification identifies HlgAB and HlgCB as major secreted staphylococcal leukocidins.