Complement C3a treatment accelerates recovery after stroke via modulation of astrocyte reactivity and cortical connectivity.

Despite advances in acute care, ischemic stroke remains a major cause of long-term disability. Approaches targeting both neuronal and glial responses are needed to enhance recovery and improve long-term outcome. The complement C3a receptor (C3aR) is a regulator of inflammation with roles in neurodevelopment, neural plasticity, and neurodegeneration. Using mice lacking C3aR (C3aR-/-) and mice overexpressing C3a in the brain, we uncovered 2 opposing effects of C3aR signaling on functional recovery after ischemic stroke: inhibition in the acute phase and facilitation in the later phase. Peri-infarct astrocyte reactivity was increased and density of microglia reduced in C3aR-/- mice; C3a overexpression led to the opposite effects. Pharmacological treatment of wild-type mice with intranasal C3a starting 7 days after stroke accelerated recovery of motor function and attenuated astrocyte reactivity without enhancing microgliosis. C3a treatment stimulated global white matter reorganization, increased peri-infarct structural connectivity, and upregulated Igf1 and Thbs4 in the peri-infarct cortex. Thus, C3a treatment from day 7 after stroke exerts positive effects on astrocytes and neuronal connectivity while avoiding the deleterious consequences of C3aR signaling during the acute phase. Intranasal administration of C3aR agonists within a convenient time window holds translational promise to improve outcome after ischemic stroke.

Macrophages promote heat stress nephropathy in mice via the C3a-C3aR-TNF pathway.

Heat-stress nephropathy (HSN) is associated with recurrent dehydration. However, the mechanisms underlying HSN remain largely unknown. In this study, we evaluated the role of dehydration in HSN and kidney injury in mice. Firstly, we found that complement was strongly activated in the mice that were exposed to dehydration; and among complement components, the interaction between C3a and its receptor, C3aR, was more closely associated with kidney injury. Then two-month-old mice were intraperitoneally injected with 2% dimethyl sulfoxide (DMSO) or the C3aR inhibitor SB290157 during dehydration. DMSO-treated mice exhibited excessive macrophage infiltration, renal cell apoptosis, and kidney fibrosis. In contrast, SB290157-treated mice had no apparent kidney injury. By fluorescence-activated cell sorting (FACS), we found that SB290157 treatment in mice remarkably inhibited macrophage infiltration and suppressed CCR2 expression in macrophages. In addition, C3a binding to C3aR promoted macrophage polarization toward the M1 phenotype and increased the production of TNF-α, which induced renal tubular epithelial cell (RTEC) apoptosis in vivo and in vitro. Interestingly, C3a treatment failed to directly induce TNF-α production and apoptosis in RTECs. However, TNF-α production in response to C3a treatment was significantly elevated when RTECs were cocultured with macrophages, suggesting that macrophages rather than RTECs are the target of C3a-C3aR interaction. At last, we proved that infusion of macrophages which highly expressed TNF-α would significantly deteriorate HSN in TNF-KO mice when they were exposed to recurrent dehydration. This study uncovers a novel mechanism underlying the pathogenesis of HSN, and a potential pathway to prevent kidney injury during dehydration.

Aristolochic acid I promotes the invasion and migration of hepatocellular carcinoma cells by activating the C3a/C3aR complement system.

Aristolochic acid is an established human carcinogen. Previous reports have demonstrated a link between aristolochic acid exposure and liver cancer prevalence in Asia. The C3a/C3AR axis plays an essential role in regulating cancer cell migration and invasion. Here, we focused on the relationship between AA I-induced migration, invasion and epithelial-mesenchymal transition in HCC cells, as well as the possible role of the C3a/C3AR axis in these effects. HCC cells were exposed to different concentrations of AA I for 24 h. Cell migration and invasion abilities were evaluated with wound healing assays and Transwell invasion assays. The protein and mRNA expression levels were detected by western blot, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR) assays. Furthermore, the level of complement component C3a in the cell supernatant was determined by enzyme-linked immunosorbent assay. C3aRA, a C3a receptor antagonist, was used to block the C3a-C3aR axis. The results showed that aristolochic acid I promoted HCC cell invasion and migration. AAI exposure also induced EMT in HCC cells through E-cadherin downregulation and Snail, N-cadherin, and vimentin upregulation. AAI exposure increased the levels of secreted C3a and the expression of C3aR protein and mRNA in HCC cells. We further found that AA I-induced C3a/C3AR activation was involved in these effects. AA I-induced epithelial-to-mesenchymal transition (EMT), cell migration, and invasion were decreased by C3aR inhibition. Overall, our results suggest that AA I induces HCC cell migration and invasion through the EMT process, which is regulated by C3a/C3aR axis activation.

Elevated Expression Levels of Lung Complement Anaphylatoxin, Neutrophil Chemoattractant Chemokine IL-8, and RANTES in MERS-CoV-Infected Patients: Predictive Biomarkers for Disease Severity and Mortality.

The complement system, a network of highly-regulated proteins, represents a vital part of the innate immune response. Over-activation of the complement system plays an important role in inflammation, tissue damage, and infectious disease severity. The prevalence of MERS-CoV in Saudi Arabia remains significant and cases are still being reported. The role of complement in Middle East Respiratory Syndrome coronavirus (MERS-CoV) pathogenesis and complement-modulating treatment strategies has received limited attention, and studies involving MERS-CoV-infected patients have not been reported. This study offers the first insight into the pulmonary expression profile including seven complement proteins, complement regulatory factors, IL-8, and RANTES in MERS-CoV infected patients without underlying chronic medical conditions. Our results significantly indicate high expression levels of complement anaphylatoxins (C3a and C5a), IL-8, and RANTES in the lungs of MERS-CoV-infected patients. The upregulation of lung complement anaphylatoxins, C5a, and C3a was positively correlated with IL-8, RANTES, and the fatality rate. Our results also showed upregulation of the positive regulatory complement factor P, suggesting positive regulation of the complement during MERS-CoV infection. High levels of lung C5a, C3a, factor P, IL-8, and RANTES may contribute to the immunopathology, disease severity, ARDS development, and a higher fatality rate in MERS-CoV-infected patients. These findings highlight the potential prognostic utility of C5a, C3a, IL-8, and RANTES as biomarkers for MERS-CoV disease severity and mortality. To further explore the prediction of functional partners (proteins) of highly expressed proteins (C5a, C3a, factor P, IL-8, and RANTES), the computational protein-protein interaction (PPI) network was constructed, and six proteins (hub nodes) were identified.

Complement and Chlamydia psittaci: Early Complement-Dependent Events Are Important for DC Migration and Protection During Mouse Lung Infection.

The zoonotic intracellular bacterium Chlamydia psittaci causes life-threatening pneumonia in humans. During mouse lung infection, complement factor C3 and the anaphylatoxin C3a augment protection against C. psittaci by a so far unknown mechanism. To clarify how complement contributes to the early, innate and the late, specific immune response and resulting protection, this study addresses the amount of C3, the timing when its presence is required as well as the anaphylatoxin receptor(s) mediating its effects and the complement-dependent migration of dendritic cells. Challenge experiments with C. psittaci on various complement KO mice were combined with transient decomplementation by pharmacological treatment, as well as the analysis of in vivo dendritic cells migration. Our findings reveal that a plasma concentration of C3 close to wildtype levels was required to achieve full protection. The diminished levels of C3 of heterozygote C3+/- mice permitted already relative effective protection and improved survival as compared to C3-/- mice, but overall recovery of these animals was delayed. Complement was in particular required during the first days of infection. However, additionally, it seems to support protection at later stages. Migration of CD103+ dendritic cells from the infected lung to the draining lymph node-as prerequisite of antigen presentation-depended on C3 and C3aR and/or C5aR. Our results provide unique mechanistic insight in various aspects of complement-dependent immune responses under almost identical, rather physiological experimental conditions. Our study contributes to an improved understanding of the role of complement, and C3a in particular, in infections by intracellular bacteria.

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

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

Astrocyte-microglia interaction drives evolving neuromyelitis optica lesion.

Neuromyelitis optica (NMO) is a severe inflammatory autoimmune CNS disorder triggered by binding of an IgG autoantibody to the aquaporin 4 (AQP4) water channel on astrocytes. Activation of cytolytic complement has been implicated as the major effector of tissue destruction that secondarily involves myelin. We investigated early precytolytic events in the evolving pathophysiology of NMO in mice by continuously infusing IgG (NMO patient serum-derived or AQP4-specific mouse monoclonal), without exogenous complement, into the spinal subarachnoid space. Motor impairment and sublytic NMO-compatible immunopathology were IgG dose dependent, AQP4 dependent, and, unexpectedly, microglia dependent. In vivo spinal cord imaging revealed a striking physical interaction between microglia and astrocytes that required signaling from astrocytes by the C3a fragment of their upregulated complement C3 protein. Astrocytes remained viable but lost AQP4. Previously unappreciated crosstalk between astrocytes and microglia involving early-activated CNS-intrinsic complement components and microglial C3a receptor signaling appears to be a critical driver of the precytolytic phase in the evolving NMO lesion, including initial motor impairment. Our results indicate that microglia merit consideration as a potential target for NMO therapeutic intervention.

The Potential Role of Complement System in the Progression of Ovarian Clear Cell Carcinoma Inferred from the Gene Ontology-Based Immunofunctionome Analysis.

Ovarian clear cell carcinoma (OCCC) is the second most common epithelial ovarian carcinoma (EOC). It is refractory to chemotherapy with a worse prognosis after the preliminary optimal debulking operation, such that the treatment of OCCC remains a challenge. OCCC is believed to evolve from endometriosis, a chronic immune/inflammation-related disease, so that immunotherapy may be a potential alternative treatment. Here, gene set-based analysis was used to investigate the immunofunctionomes of OCCC in early and advanced stages. Quantified biological functions defined by 5917 Gene Ontology (GO) terms downloaded from the Gene Expression Omnibus (GEO) database were used. DNA microarray gene expression profiles were used to convert 85 OCCCs and 136 normal controls into to the functionome. Relevant offspring were as extracted and the immunofunctionomes were rebuilt at different stages by machine learning. Several dysregulated pathogenic functions were found to coexist in the immunopathogenesis of early and advanced OCCC, wherein the complement-activation-alternative-pathway may be the headmost dysfunctional immunological pathway in duality for carcinogenesis at all OCCC stages. Several immunological genes involved in the complement system had dual influences on patients' survival, and immunohistochemistrical analysis implied the higher expression of C3a receptor (C3aR) and C5a receptor (C5aR) levels in OCCC than in controls.

Adipsin preserves beta cells in diabetic mice and associates with protection from type 2 diabetes in humans.

Type 2 diabetes is characterized by insulin resistance and a gradual loss of pancreatic beta cell mass and function1,2. Currently, there are no therapies proven to prevent beta cell loss and some, namely insulin secretagogues, have been linked to accelerated beta cell failure, thereby limiting their use in type 2 diabetes3,4. The adipokine adipsin/complement factor D controls the alternative complement pathway and generation of complement component C3a, which acts to augment beta cell insulin secretion5. In contrast to other insulin secretagogues, we show that chronic replenishment of adipsin in diabetic db/db mice ameliorates hyperglycemia and increases insulin levels while preserving beta cells by blocking dedifferentiation and death. Mechanistically, we find that adipsin/C3a decreases the phosphatase Dusp26; forced expression of Dusp26 in beta cells decreases expression of core beta cell identity genes and sensitizes to cell death. In contrast, pharmacological inhibition of DUSP26 improves hyperglycemia in diabetic mice and protects human islet cells from cell death. Pertaining to human health, we show that higher concentrations of circulating adipsin are associated with a significantly lower risk of developing future diabetes among middle-aged adults after adjusting for body mass index (BMI). Collectively, these data suggest that adipsin/C3a and DUSP26-directed therapies may represent a novel approach to achieve beta cell health to treat and prevent type 2 diabetes.

Aristolochic acid I aggravates renal injury by activating the C3a/C3aR complement system.

Previous studies have reported that the complement system is unconventionally activated in many kinds of glomerulonephritis. Multiple complement components participate in the pathogenic process by triggering immune response or other intracellular signaling pathways. Here, we have investigated the role of C3a and its receptor C3aR in aristolochic acid nephropathy (AAN), which, is featured with progressive interstitial fibrosis. Over release of C3a and increased expression of C3aR parallels to the up-regulation of α-SMA and TGF-ß1 in AAN, which appeared to promote epithelial-mesenchymal-transition (EMT). To identify the role of complement activation in AAN, we used an inhibitor of C3aR (C3aRA) to block the coupling of C3a to its receptor. Our results confirmed from decreased EMT, the protective effect of C3aRA in cell apoptosis and inflammatory response induced by aristolochic acid I. These results showed that C3a and its receptor C3aR played pathogenic roles in AAN, and renal tubular epithelial cells were potentially pivotal targets of complement activation that could cause pro-fibrotic effects.

Association of age-related macular degeneration with complement activation products, smoking, and single nucleotide polymorphisms in South Carolinians of European and African descent.

Purpose: Smoking and the incidence of age-related macular degeneration (AMD) have been linked to an overactive complement system. Here, we examined in a retrospective cohort study whether AMD-associated single nucleotide polymorphisms (SNPs), smoking, ethnicity, and disease status are correlated with blood complement levels. Methods: Population: The study involved 91 AMD patients and 133 controls, which included 73% Americans of European descent (EUR) and 27% Americans of African descent (AFR) in South Carolina. Readouts: Participants were genotyped for 10 SNPs and systemic levels of complement factor H (CFH) activity, and the complement activation products C3a, C5a, and Bb were assessed. Main Outcome Measures: Univariate and multivariable logistic regression models were used to examine associations between AMD status and distinct readouts. Results: AMD affects EUR individuals more than AFRs. EUR but not AFR AMD subjects revealed higher levels of Factors C3a and Bb. In all subjects, a 10-unit increase in C3a levels was associated with an approximately 10% increase in the odds of being AMD-positive, and C3a and Bb were associated with smoking. While CFH activity levels were not correlated with AMD, a significant interaction was evident between patient age and CFH activity. Finally, EURs had lower odds of AMD with enhanced copies of rs1536304 (VEGFA) and higher odds with more copy numbers of rs3766404 (CFH). Conclusions: Our results support previous studies of systemic complement components being potential biomarkers for AMD, but they suggest that smoking and disease do not synergistically affect complement levels. We also suggest a novel susceptibility and protective haplotypes in the South Carolinian AMD population. Our studies indicate that augmented complement activation associated with advanced AMD could be attributed to a decrease in CFH activity in younger patients.

Complement C3a promotes proliferation, migration and stemness in cutaneous squamous cell carcinoma.

BACKGROUND: Complement C3 has been shown to be highly expressed in cutaneous squamous cell carcinoma (cSCC) tumour tissues and is correlated with tumour cell growth. This study aimed to investigate the mechanism of C3 in cSCC malignant transformation. METHODS: C3 expression was analysed in cSCC cell lines A431, Tca8113, SCC13, HSC-5 and HSC-1 and in immortalized HaCaT keratinocytes. Proliferation and migration of cSCC were determined after C3a exposure. Expression of cyclin D1, cyclin E, vascular endothelial growth factor (VEGF), pro-matrix metalloproteinase 1 (pro-MMP1), pro-matrix metalloproteinase 2 (pro-MMP2), stemness factors, GSK-3ß, and ß-catenin were analyzed. Tumour growth was examined in a murine xenograft model. RESULTS: C3 expression was much more highly expressed in all cSCC cell lines than in HaCaT cells. C3a treatment significantly promoted cSCC cell proliferation and migration and upregulated cyclin D1, cyclin E, VEGF, pro-MMP1 and pro-MMP2 expression, which were impeded by the C3aR antagonist. Moreover, the expression of stemness factors Sox-2, Nanog, Oct-4, c-Myc and CD-44 was stimulated by C3a and slowed by C3aR disruption. Knockdown of Sox-2 by siRNA transfection suppressed cell proliferation and migration, constrained VEGF secretion and inhibited pro-MMP1 and pro-MMP2 expression. C3a also activated the Wnt and ß-catenin pathway in cSCC cells. Disruption of C3aR expression dampened tumour growth and the expression of Wnt-1, ß-catenin and Sox-2 in the xenograft model. CONCLUSIONS: C3a enhanced cell proliferation, migration and stemness in cSCC, and this activity was correlated with activation of the Wnt and ß-catenin pathway.

Upregulated long noncoding RNA LOC105375913 induces tubulointerstitial fibrosis in focal segmental glomerulosclerosis.

Tubulointerstitial fibrosis impacts renal prognosis of focal segmental glomerulosclerosis (FSGS). Based on transcriptomic analysis, we found that the level of LOC105375913 was increased in tubular cells of FSGS patients. C3a induced the expression of LOC105375913, which promoted the expression of fibronectin and collagen I in tubular cells. Silence of snail reversed the level of fibronectin and collagen I in cells overexpressing LOC105375913. MiR-27b was predicted and confirmed to regulate the expression of snail in tubular cells, and LOC105375913 contained the response element of miR-27b. The competitive binding between LOC105375913 and miR-27b increased the level of snail and promoted fibrogenesis in tubular cells. Upstream, p38 and XBP-1s regulated the expression of LOC105375913. Inhibition of p38 or silence of XBP-1s decreased the level of LOC105375913, and suppressed the expression of snail, fibronectin and collagen I in tubular cells treated with C3a. Overexpression of LOC105375913 decreased the level of miR-27b, increased the level of snail and caused tubulointerstitial fibrosis in mice. In conclusion, the activation of C3a/p38/XBP-1s pathway induces the expression of LOC105375913 in tubular cells, and LOC105375913 increases the level of snail and induces tubulointerstitial fibrosis through competitive binding of miR-27b in tubular cells of FSGS patients.

Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) enhance islet function both in vitro and in vivo, at least in part by secreting ligands that activate islet G-protein coupled receptors (GPCRs). We assessed whether pre-treatment with a defined "cocktail" of MSC-secreted GPCR ligands enhances islet functional survival in vitro and improves the outcomes of islet transplantation in an experimental model of diabetes. METHODS: Isolated islets were cultured for 48 h with ANXA1, SDF-1 or C3a, alone or in combination. Glucose-stimulated insulin secretion (GSIS) and cytokine-induced apoptosis were measured immediately after the 48 h culture period and at 24 h or 72 h following removal of the ligands from the culture media. Islets were syngeneically transplanted underneath the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice and blood glucose levels monitored for 28 days. RESULTS: Pre-culturing islets with a cocktail of ANXA1/SDF-1/C3a potentiated GSIS and protected islet cells from cytokine-induced apoptosis in vitro. These effects were maintained for up to 72 h after the removal of the factors from the culture medium, suggesting a sustained protection of islet graft functional survival during the immediate post-transplantation period. Islets pre-treated with the cocktail of MSC secretory factors were more effective in reducing blood glucose in diabetic mice, consistent with their improved functional survival in vivo. DISCUSSION: Pre-culturing islets with a cocktail of MSC secretory products offers a well-defined, cell-free approach to improve clinical islet transplantation outcomes while avoiding many of the safety, regulatory and logistical hurdles of incorporating MSCs into transplantation protocols.

Complement C3aR Inactivation Attenuates Tau Pathology and Reverses an Immune Network Deregulated in Tauopathy Models and Alzheimer's Disease.

Strong evidence implicates the complement pathway as an important contributor to amyloid pathology in Alzheimer's disease (AD); however, the role of complement in tau modulation remains unclear. Here we show that the expression of C3 and C3a receptor (C3aR1) are positively correlated with cognitive decline and Braak staging in human AD brains. Deletion of C3ar1 in PS19 mice results in the rescue of tau pathology and attenuation of neuroinflammation, synaptic deficits, and neurodegeneration. Through RNA sequencing and cell-type-specific transcriptomic analysis, we identify a C3aR-dependent transcription factor network that regulates a reactive glial switch whose inactivation ameliorates disease-associated microglia and neurotoxic astrocyte signatures. Strikingly, this C3aR network includes multiple genes linked to late-onset AD. Mechanistically, we identify STAT3 as a direct target of C3-C3aR signaling that functionally mediates tau pathogenesis. All together our findings demonstrate a crucial role for activation of the C3-C3aR network in mediating neuroinflammation and tau pathology.

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.

Astrocytes in primary cultures express serine racemase, synthesize d-serine and acquire A1 reactive astrocyte features.

d-Serine is a co-agonist at forebrain N-methyl-d-aspartate receptors (NMDAR) and is synthesized by serine racemase (SR). Although d-serine and SR were originally reported to be localized to glia, recent studies have provided compelling evidence that under healthy physiologic conditions both are localized primarily in neurons. However, in pathologic conditions, reactive astrocytes can also express SR and synthesize d-serine. Since cultured astrocytes exhibit features of reactive astrocytes, we have characterized d-serine synthesis and the expression of enzymes involved in its disposition in primary glial cultures. The levels of SR were quite low early in culture and increased markedly in all astrocytes with the duration in vitro. The concentration of d-serine in the culture medium increased in parallel with SR expression in the astrocytes. Microglia, identified by robust expression of Iba1, did not express SR. While the levels of glial fibrillary acidic protein (GFAP), glycine decarboxylase (GLDC) and phosphoglycerate dehydrogenase (PHGDH), the initial enzyme in the pathway converting glycine to l-serine, remained constant in culture, the expression of lipocalin-2, a marker for pan-reactive astrocytes, increased several-fold. The cultured astrocytes also expressed Complement-3a, a marker for a subpopulation of reactive astrocytes (A1). Astrocytes grown from mice with a copy number variant associated with psychosis, which have four copies of the GLDC gene, showed a more rapid production of d-serine and a reduction in glycine in the culture medium. These results substantiate the conclusion that A1 reactive astrocytes express SR and release d-serine under pathologic conditions, which may contribute to their neurotoxic effects by activating extra-synaptic NMDARs.

Reduced order modeling and analysis of the human complement system.

Complement is an important pathway in innate immunity, inflammation, and many disease processes. However, despite its importance, there are few validated mathematical models of complement activation. In this study, we developed an ensemble of experimentally validated reduced order complement models. We combined ordinary differential equations with logical rules to produce a compact yet predictive model of complement activation. The model, which described the lectin and alternative pathways, was an order of magnitude smaller than comparable models in the literature. We estimated an ensemble of model parameters from in vitro dynamic measurements of the C3a and C5a complement proteins. Subsequently, we validated the model on unseen C3a and C5a measurements not used for model training. Despite its small size, the model was surprisingly predictive. Global sensitivity and robustness analysis suggested complement was robust to any single therapeutic intervention. Only the simultaneous knockdown of both C3 and C5 consistently reduced C3a and C5a formation from all pathways. Taken together, we developed a validated mathematical model of complement activation that was computationally inexpensive, and could easily be incorporated into pre-existing or new pharmacokinetic models of immune system function. The model described experimental data, and predicted the need for multiple points of therapeutic intervention to fully disrupt complement activation.

Exploiting a novel conformational switch to control innate immunity mediated by complement protein C3a.

Complement C3a is an important protein in innate and adaptive immunity, but its specific roles in vivo remain uncertain because C3a degrades rapidly to form the C3a-desArg protein, which does not bind to the C3a receptor and is indistinguishable from C3a using antibodies. Here we develop the most potent, stable and highly selective small molecule modulators of C3a receptor, using a heterocyclic hinge to switch between agonist and antagonist ligand conformations. This enables characterization of C3 areceptor-selective pro- vs. anti-inflammatory actions in human mast cells and macrophages, and in rats. A C3a receptor-selective agonist induces acute rat paw inflammation by first degranulating mast cells before activating macrophages and neutrophils. An orally administered C3a receptor-selective antagonist inhibits mast cell degranulation, thereby blocking recruitment and activation of macrophages and neutrophils, expression of inflammatory mediators and inflammation in a rat paw edema model. These novel tools reveal the mechanism of C3a-induced inflammation and provide new insights to complement-based medicines.Complement C3a is an important protein in innate and adaptive immunity, but its roles in vivo are unclear. Here the authors develop novel chemical agonists and antagonists for the C3a receptor, and show that they modulate mast cell degranulation and inflammation in a rat paw edema model.

Neutrophils Induce Astroglial Differentiation and Migration of Human Neural Stem Cells via C1q and C3a Synthesis.

Inflammatory processes play a key role in pathophysiology of many neurologic diseases/trauma, but the effect of immune cells and factors on neurotransplantation strategies remains unclear. We hypothesized that cellular and humoral components of innate immunity alter fate and migration of human neural stem cells (hNSC). In these experiments, conditioned media collected from polymorphonuclear leukocytes (PMN) selectively increased hNSC astrogliogenesis and promoted cell migration in vitro. PMN were shown to generate C1q and C3a; exposure of hNSC to PMN-synthesized concentrations of these complement proteins promoted astrogliogenesis and cell migration. Furthermore, in vitro, Abs directed against C1q and C3a reversed the fate and migration effects observed. In a proof-of-concept in vivo experiment, blockade of C1q and C3a transiently altered hNSC migration and reversed astroglial fate after spinal cord injury. Collectively, these data suggest that modulation of the innate/humoral inflammatory microenvironment may impact the potential of cell-based therapies for recovery and repair following CNS pathology.