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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.

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.

Type 2 diabetes is characterized by insulin resistance and a gradual loss of pancreatic beta cell mass and function 1 , 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 diabetes 3 , 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 secretion 5 . 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. Targeting the adipokine adipsin and its downstream pathway may provide an approach for preservation of beta cell loss in type 2 diabetes.

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.

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

Greater production of interleukin 17A is associated with severe asthma. Wills-Karp and colleagues show that the complement anaphylatoxins C3a and C5a have opposing roles in enhancing or suppressing IL-17a in allergic asthma. Severe asthma is associated with the production of interleukin 17A (IL-17A). The exact role of IL-17A in severe asthma and the factors that drive its production are unknown. Here we demonstrate that IL-17A mediated severe airway hyperresponsiveness (AHR) in susceptible strains of mice by enhancing IL-13-driven responses. Mechanistically, we demonstrate that IL-17A and AHR were regulated by allergen-driven production of anaphylatoxins, as mouse strains deficient in complement factor 5 (C5) or the complement receptor C5aR mounted robust IL-17A responses, whereas mice deficient in C3aR had fewer IL-17-producing helper T cells (T H 17 cells) and less AHR after allergen challenge. The opposing effects of C3a and C5a were mediated through their reciprocal regulation of IL-23 production. These data demonstrate a critical role for complement-mediated regulation of the IL-23–T H 17 axis in severe asthma.

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 −/− mousemodel 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.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30-50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD.

Francisella tularensis ( Ft ), a Gram-negative bacterium that causes tularemia, possesses a non-inflammatory atypical LPS (LPS Ft ) that is highly immunogenic through unknown mechanism. We previously showed that immunization with LPS Ft , a type 2 T-independent (TI) antigen, elicits protective LPS Ft -specific IgM (IgM Ft ) and IgG3 Ft by B1 cells in a mechanism dependent on the IL-5 produced by innate lymphoid cells type 2 (ILC2). Here, we examined the role of complement in the B1 cells’ response against LPS Ft . C3 -/- , C1q -/- and C4 -/- mice immunized with LPS Ft failed to produce IgM Ft and IgG3 Ft . In contrast, the response of Cfb -/- and Mbl1/Mbl2 -/- mice was comparable to that of WT mice. Thus, activation of the classical complement cascade, but not the alternative or the Mannose Binding Lectin pathway, is required for activation of B1 cells and production of LPS Ft -specific antibodies. Complement activation generates the C3d fragment, which opsonizes antigens for recognition by complement receptor-2 (CR2), and the C3a and C5a anaphylatoxins. Our results show that C3d opsonized LPS Ft and that the response to immunization was dependent on CR2 expression by B1 cells. Importantly, the response to LPS Ft immunization was also drastically decreased in C3ar1 -/- , but not in C5ar1 -/- mice. C3a induced IL-5 in ILC2, which supported B1 cells activation. Decreased antibody production in C3ar1 -/- and Cr2 -/- mice correlated with increased susceptibility to tularemia. Together, these results demonstrate that the high immunogenicity of LPS Ft depends on two effector mechanisms triggered by activation of the classical complement pathway: 1) tagging of LPS Ft with C3d fragment, leading to its interaction with CR2 expressed by B1 cells; 2) production of the anaphylatoxin C3a that stimulated IL-5 secretion by ILC2. Our study increases our understanding of the B1 cells’ response to TI-2 antigens and identifies two complement effector mechanisms that can be harnessed for therapeutic interventions.

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.