Intracellular C3 prevents hepatic steatosis by promoting autophagy and very-low-density lipoprotein secretion.

Complement component C3, mainly synthesized by hepatocytes, acts as the convergence point of three different pathways in activating the complement cascade. Besides its well-established roles in the extracellular milieu, C3 performs various intracellular functions such as immunomodulation and pathogen recognition. Although C3 is present at extremely high concentrations in hepatocytes, little is known about its intrahepatic function. In this study, we found that C3 knockout (C3-/- ) mice displayed accelerated hepatic triglyceride (TG) accumulation compared with C57BL/6J wild type mice. Mechanistically, C3 deficiency impaired lipophagy in hepatocytes, owing to the disrupted interaction between C3 and autophagy-related 16 like 1, which is essential for autolysosome assembly. Furthermore, lipophagy deficiency affected the function of the endoplasmic reticulum in C3-/- mice, subsequently affecting the expression of protein disulfide isomerase and activity of microsomal TG transfer protein, and ultimately impairing the production of hepatic very-low-density lipoproteins (VLDLs). Rapamycin and thapsigargin treatment accelerated VLDL secretion and alleviated hepatic lipid accumulation in C3-/- mice. Our study demonstrates that C3 promotes lipophagy to facilitate VLDL secretion in hepatocytes, thus playing an essential role in balancing TG levels in the liver.

Complement Component C3 Loss leads to Locomotor Deficits and Altered Cerebellar Internal Granule Cell In Vitro Synaptic Protein Expression in C57BL/6 Mice.

Complement component 3 (C3) expression is increased in the cerebellum of aging mice that demonstrate locomotor impairments and increased excitatory synapse density. However, C3 regulation of locomotion, as well as C3 roles in excitatory synapse function, remains poorly understood. Here, we demonstrate that constitutive loss of C3 function in mice evokes a locomotor phenotype characterized by decreased speed, increased active state locomotor probability, and gait ataxia. C3 loss does not alter metabolism or body mass composition. No evidence of significant muscle weakness or degenerative arthritis was found in C3 knockout mice to explain decreased gait speeds. In an enriched primary cerebellar granule cell culture model, loss of C3 protein results in increased excitatory synaptic density and increased response to KCl depolarization. Our analysis of excitatory synaptic density in the cerebellar internal granule cell and molecular layers did not demonstrate increased synaptic density in vivo, suggesting the presence of compensatory mechanisms regulating synaptic development. Functional deficits in C3 knockout mice are therefore more likely to result from altered synaptic function and/or connectivity than gross synaptic deficits. Our data demonstrate a novel role for complement proteins in cerebellar regulation of locomotor output and control.

Inherited Kidney Complement Diseases.

In the past 20 years, we have witnessed tremendous advances in our ability to diagnose and treat genetic diseases of the kidney caused by complement dysregulation. Staggering progress was realized toward a better understanding of the genetic underpinnings and pathophysiology of many forms of atypical hemolytic uremic syndrome (aHUS) and C3-dominant glomerulopathies that are driven by complement system abnormalities. Many of these seminal discoveries paved the way for the design and characterization of several innovative therapies, some of which have already radically improved patients' outcomes. This review offers a broad overview of the exciting developments that have occurred in the recent past, with a particular focus on single-gene (or Mendelian), complement-driven aHUS and C3-dominant glomerulopathies that should be of interest to both nephrologists and kidney researchers. The discussion is restricted to genes with robust associations with both aHUS and C3-dominant glomerulopathies (complement factor H, complement component 3, complement factor H-related proteins) or only aHUS (complement factor B, complement factor I, and membrane cofactor protein). Key questions and challenges are highlighted, along with potential avenues for future directions.

Complement C3 participates in the development of goose fatty liver potentially by regulating the expression of FASN and ETNK1.

Non-alcoholic fatty liver disease (NAFLD) occurs in humans, domestic animals and poultry. Different from upregulation of complement C3 in human NAFLD, C3 expression is inhibited in goose fatty liver (GFL), implying a specific role of C3 in GFL. This study was mainly focused on uncovering the uniqueness of goose liver cells in the regulation of C3 expression and identifying the downstream genes of C3 to improve understanding on the specific role of C3 in GFL. The results showed that C3 expression was inhibited in the liver, muscle and fat tissues of the overfed versus control (normally fed) geese. Oleate and insulin could inhibit C3 expression in goose primary hepatocytes but induce it in mouse primary hepatocytes. A total of 1,123 differentially expressed genes (DEGs) were affected by C3 overexpression and were mainly enriched in immune response/inflammation and catabolism-related KEGG pathways. Additionally, the representative downstream genes (FASN and ETNK1) of C3 could mediate the role of C3 in the development of GFL. In conclusion, the suppression of C3 in GFL is at least partially attributed to hyperinsulinemia, hyperlipidemia and uniqueness of goose liver cells. Complement C3 does not only affect hepatic steatosis but also affect inflammation/immune response in GFL.

Complement controls the immune synapse and tumors control complement.

The synapses between immune cells and their targets are 150 Å wide. They regulate immune cell responses (IRs) to cognate antigens. Here, I outline a potential mechanism for self-nonself discrimination based on the C3d and iC3b proteolytic fragments of complement protein C3. The proposed C3 checkpoint works through complement receptor 3 (CR3), which binds both C3d and iC3b. The CR3 conformations involved differ; the bent, cis-acting CR3 engages C3d, activating the immune cell expressing CR3; the extended, transacting CR3 conformer binds iC3b on another cell, inhibiting IRs. The CR3 complexes formed with iC3b and C3d vary greatly in size. Only bound C3d is small enough to fit within the synapse. It stimulates IRs by countering the inhibitory signals that iC3b generates at the synapse edge. The competition between C3d and iC3b dynamically determines whether or not an immune cell activates. Host cells use regulators of complement activation (RCA) to coat themselves with iC3b, silencing IRs against self by preventing synapse formation. Tumors exploit this process by overexpressing C3 and RCA to masquerade as 'super-self', with iC3b masking neoantigens. Enhancing synapse formation by specifically labeling cancer cells as nonself with targeted C3d therapeutics offers a new strategy for boosting tumor-specific immunity.

Complement C3 deficiency ameliorates aging related changes in the kidney.

AIMS: Complement C3 (C3) has been shown to be involved in the aging process. However, the role of C3 in kidney aging has not been fully elucidated. This study aimed to investigate the effect of C3 on senescence related kidney disorders in mice. MATERIALS AND METHODS: Two-, 8-, and 16-month-old C3-deficient male mice (KO) (n = 6) and age-, gender-, and strain- matched wild type (WT) C57BL/6 mice (n = 6) were selected to represent young, middle-aged and aging mice. Renal, blood and urine samples were collected. Hematoxylin-eosin (HE), Masson, and immunohistochemistry (IHC) staining as well as ELISA and Western blotting were used to explore the mechanisms involved in renal aging. KEY FINDINGS: The level of C3 was upregulated during aging in WT mice. The glomerular sclerosis index and tubulointerstitial fibrosis index were increased significantly in WT mice during aging. Renal function was not significantly different between the young and aged groups. Compared with those in WT mice, the levels of inflammation and fibrosis were decreased, while the expression of CD31 was significantly increased in the KO group. SIGNIFICANCE: Our data demonstrated that age-related changes in renal structure occur earlier than functional changes and that complement C3 is involved in aging-related kidney disorder.

Complement Component 3 Is Required for Tissue Damage, Neutrophil Infiltration, and Ensuring NET Formation in Acute Pancreatitis.

BACKGROUND: Neutrophil extracellular traps (NETs) are known to play an important role in the pathophysiology of acute pancreatitis (AP). Activation of the complement cascade has been shown to occur in AP. The aim of this study was to examine whether complement component 3 is involved in the generation of NETs in AP. METHODS: AP was induced in wild-type and C3-deficient mice by retrograde infusion of taurocholate into the pancreatic duct. Blood, lung, and pancreas tissue were collected and MPO activity was determined in lung and pancreas tissue. Histological examination of the inflamed pancreas was performed. Plasma levels of CXCL2, MMP-9, IL-6, and DNA-histone complexes as well as pancreatic levels of CXCL1 and CXCL2 were determined by use of enzyme-linked immunosorbent assay. NETs were detected in the pancreas by electron microscopy. The amount of MPO and citrullinated histone 3 in neutrophils isolated from bone marrow was examined using flow cytometry. RESULTS: In C3-deficient mice, challenge with taurocholate yielded much fewer NETs in the pancreatic tissue compared with wild-type controls. Taurocholate-induced blood levels of amylase, tissue injury, and neutrophil recruitment in the pancreas were markedly reduced in the mice lacking C3. Furthermore, MPO levels in the lung, and plasma levels of IL-6, MMP-9, and CXCL2 were significantly lower in the C3-deficient mice compared to wild-type mice after the induction of AP. In vitro studies revealed that neutrophils from C3-deficient mice had normal NET-forming ability and recombinant C3a was not capable of directly inducing NETs formation in the wild-type neutrophils. CONCLUSION: C3 plays an important role in the pathophysiology of AP as it is necessary for the recruitment of neutrophils into the pancreas and ensuring NETs formation. Targeting C3 could hence be a potential strategy to ameliorate local damage as well as remote organ dysfunction in AP.

Alternative pathway of complement activation has a beneficial role against Chandipura virus infection.

The complement system is a critical component of both innate and adaptive immune responses. It has both protective and pathogenic roles in viral infections. There are no studies regarding the role of complement system in Chandipura virus (CHPV) infection. The current study has investigated the role of complement pathways in the in vitro neutralization of CHPV in Vero E6 cells. Using normal human serum (NHS), heat-inactivated serum (HIS), human serum deficient of complement factor, respective reconstituted serum, assays like in vitro neutralization, real-time PCR, and flow cytometry-based tissue culture-based limited dose assay (TC-LDA) were carried out for assessing the activation of different complement pathways. NHS from 9/10 donors showed complement dependent neutralization, reduction in viral load and decrease in percentage of CHPV-positive cells compared to their HIS counterparts. EGTA or EDTA pretreatment experiments indicated that CHPV neutralization proceeds through the alternative pathway of the complement activation. Our data showed a strong dependence on C3 for the in vitro neutralization of CHPV. Disparity in CHPV neutralization levels between factor B-deficient and reconstituted sera could be attributed to amplification loop/"tick-over" mechanism. Assays using C3, C5, and C8 deficient sera indicated that complement-mediated CHPV neutralization and suppression of CHPV infectivity are primarily through C3 and C5, and not dependent on downstream complement factor C8. With no specific anti-viral treatment/vaccine against Chandipura, the current data, elucidating role of human complement system in the neutralization of CHPV, may help in designing effective therapeutics.

Intracellular cytosolic complement component C3 regulates cytoprotective autophagy in pancreatic beta cells by interaction with ATG16L1.

Complement component C3 is central to the complement system, a humoral effector mechanism of innate immune defense. When activated, C3 covalently binds to target particles, marking them for uptake and clearance by phagocytosis. We now show that C3 also exists within the cytosol where it interacts with ATG16L1, and is therefore involved in the intracellular clearance and recycling of material by macroautophagy/autophagy in pancreatic beta cells. C3 is highly expressed in isolated human islets, and its expression is upregulated in islets isolated from diabetic patients and rodents, and correlates with patient HBA1c and body mass index (BMI). Knockout of C3 in clonal beta cells leads to dysfunctional autophagy, and increased cell death after challenge with diabetogenic stresses, which are usually alleviated by increased autophagic turnover. However, autophagic degradation of INS (insulin) granules regulates total INS content, and increased autophagy due to C3 upregulation may deplete beta cell INS stores. C3 is therefore required for efficient autophagic turnover in beta cells, and is upregulated as a cytoprotective factor during diabetes.

Complement Components, C3 and C4, and the Metabolic Syndrome.

INTRODUCTION: Increased systemic inflammation plays a significant role in the development of adult cardiometabolic diseases such as insulin resistance, dyslipidemia, atherosclerosis, and hypertension. The complement system is a part of the innate immune system and plays a key role in the regulation of inflammation. Of particular importance is the activation of complement components C3 and C4. C3 is produced primarily by the liver but is also produced in adipocytes, macrophages and endothelial cells, all of which are present in adipose tissues. Dietary fat and chylomicrons stimulate C3 production. Adipocytes in addition to producing C3 also have receptors for activated C3 and other complement components and thus also respond to as well as produce a target for complement. C3adesArg, also known as acylation stimulation factor, increases adipocyte triglyceride synthesis and release. These physiological effects play a significant role in the development of metabolic syndrome. Epidemiologically, obese adults and non-obese adults with cardiometabolic disease who are not obese have been shown to have increased complement levels. C4 levels also correlate with body mass index. Genetically, specific C3 polymorphisms have been shown to predict future cardiovascular events and. D decreased C4 long gene copy number is associated with increased longevity. CONCLUSION: Future research is clearly needed to clarify the role of complement in the development of cardiovascular disease and mechanisms for its action. The complement system may provide a new area for intervention in the prevention of cardiometabolic diseases.

C1q restrains autoimmunity and viral infection by regulating CD8+ T cell metabolism.

Deficiency of C1q, the initiator of the complement classical pathway, is associated with the development of systemic lupus erythematosus (SLE). Explaining this association in terms of abnormalities in the classical pathway alone remains problematic because C3 deficiency does not predispose to SLE. Here, using a mouse model of SLE, we demonstrate that C1q, but not C3, restrains the response to self-antigens by modulating the mitochondrial metabolism of CD8+ T cells, which can themselves propagate autoimmunity. C1q deficiency also triggers an exuberant effector CD8+ T cell response to chronic viral infection leading to lethal immunopathology. These data establish a link between C1q and CD8+ T cell metabolism and may explain how C1q protects against lupus, with implications for the role of viral infections in the perpetuation of autoimmunity.

Classical and alternative complement activation on photoreceptor outer segments drives monocyte-dependent retinal atrophy.

Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.

Reduced bone loss in a murine model of postmenopausal osteoporosis lacking complement component 3.

The growing field of osteoimmunology seeks to unravel the complex interdependence of the skeletal and immune systems. Notably, we and others have demonstrated that complement signaling influences the differentiation of osteoblasts and osteoclasts, the two primary cell types responsible for maintaining bone homeostasis. However, the net effect of complement on bone homeostasis in vivo was unknown. Our published in vitro mechanistic work led us to hypothesize that absence of complement component 3 (C3), a central protein in the complement activation cascade, protects against bone loss in the ovariectomy-based model of postmenopausal osteoporosis. Indeed, we report here that, when compared to their C57BL/6J (WT) counterparts, ovariectomized C3 deficient mice experienced reduced bone loss at multiple sites and increased stiffness at the femoral neck, the latter potentially improving mechanical function. WT and B6;129S4-C3tm1Crr /J (C3-/- ) mice were either ovariectomized or sham-operated at 6 weeks of age and euthanized at 12 weeks. MicroCT on harvested bones revealed that the trabecular bone volume fraction in the metaphyses of both the proximal tibiae and distal femora of ovariectomized C3-/- mice is significantly greater than that of their WT counterparts. Lumbar vertebrae showed significantly greater osteoid content and mineral apposition rates. Mechanical testing demonstrated significantly greater stiffness in the femoral necks of ovariectomized C3-/- mice. These results demonstrate that C3 deficiency reduces bone loss at ovariectomy and may improve mechanical properties. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:118-128, 2018.

Complement Component C3 and Complement Factor B Promote Growth of Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common metastatic skin cancers with increasing incidence. We examined the roles of complement component C3 and complement factor B (CFB) in the growth of cSCC. Analysis of cSCC cell lines (n = 8) and normal human epidermal keratinocytes (n = 11) with real-time quantitative PCR and Western blotting revealed up-regulation of C3 and CFB expression in cSCC cells. Immunohistochemical staining revealed stronger tumor cell-specific labeling for C3 and CFB in invasive cSCCs (n = 71) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n = 11) than in cSCC in situ (n = 69), actinic keratoses (n = 63), and normal skin (n = 5). Significant up-regulation of C3 and CFB mRNA expression was noted in chemically induced mouse cSCCs, compared to benign papillomas. Knockdown of C3 and CFB expression inhibited migration and proliferation of cSCC cells and resulted in potent inhibition of extracellular signal-regulated kinase 1/2 activation. Knockdown of C3 and CFB markedly inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the roles of C3 and CFB in the development of cSCC and identify them as biomarkers and potential therapeutic targets in this metastatic skin cancer.

Proteomics of Urinary Vesicles Links Plakins and Complement to Polycystic Kidney Disease.

Novel therapies in autosomal dominant polycystic kidney disease (ADPKD) signal the need for markers of disease progression or response to therapy. This study aimed to identify disease-associated proteins in urinary extracellular vesicles (uEVs), which include exosomes, in patients with ADPKD. We performed quantitative proteomics on uEVs from healthy controls and patients with ADPKD using a labeled approach and then used a label-free approach with uEVs of different subjects (healthy controls versus patients with ADPKD versus patients with non-ADPKD CKD). In both experiments, 30 proteins were consistently more abundant (by two-fold or greater) in ADPKD-uEVs than in healthy- and CKD-uEVs. Of these proteins, we selected periplakin, envoplakin, villin-1, and complement C3 and C9 for confirmation because they were also significantly overrepresented in pathway analysis and were previously implicated in ADPKD pathogenesis. Immunoblotting confirmed higher abundances of the selected proteins in uEVs from three independent groups of patients with ADPKD. Whereas uEVs of young patients with ADPKD and preserved kidney function already had higher levels of complement, only uEVs of patients with advanced stages of ADPKD had increased levels of villin-1, periplakin, and envoplakin. Furthermore, all five proteins correlated positively with total kidney volume. Analysis in kidney tissue from mice with kidney-specific, tamoxifen-inducible Pkd1 deletion demonstrated higher expression in more severe stages of the disease and correlation with kidney weight for each protein of interest. In summary, proteomic analysis of uEVs identified plakins and complement as disease-associated proteins in ADPKD. These proteins are new candidates for evaluation as biomarkers or targets for therapy in ADPKD.

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.

Alzheimer's disease is associated with altered expression of genes involved in immune response and mitochondrial processes in astrocytes.

Alzheimer's disease (AD) is characterized by deficits in cerebral metabolic rates of glucose in the posterior cingulate (PC) and precuneus in AD subjects, and in APOEε4 carriers, decades before the onset of measureable cognitive deficits. However, the cellular and molecular basis of this phenotype remains to be clarified. Given the roles of astrocytes in energy storage and brain immunity, we sought to characterize the transcriptome of AD PC astrocytes. Cells were laser capture microdissected from AD (n = 10) and healthy elderly control (n = 10) subjects for RNA sequencing. We generated >5.22 billion reads and compared sequencing data between controls and AD patients. We identified differentially expressed mitochondria-related genes including TRMT61B, FASTKD2, and NDUFA4L2, and using pathway and weighted gene coexpression analyses, we identified differentially expressed immune response genes. A number of these genes, including CLU, C3, and CD74, have been implicated in beta amyloid generation or clearance. These data provide key insights into astrocyte-specific contributions to AD, and we present this data set as a publicly available resource.

The C3a receptor, caspase-1, and release of IL-1ß.

In this issue of Blood, Asgari et al report that engagement of the C3a receptor triggers interleukin-1b (IL-1b) processing and release via caspase-1 activation. The role of complement activation in IL-1 production has a long history; complement products function as "alarmins" during innate responses. For many years before the term "innate immune response" was coined, it was fully understood that a highly nonspecific event such as activation of complement would induce a highly nonspecific molecule such as IL-1; these 2 linked processes would then affect a highly specific event such antigen-driven lymphocyte activation, for example, polarization to a T helper 1 (Th1) or a Th17 response. In this issue, investigators link the generation of C3a to playing a role in the activation of caspase-1. A unique and unexpected finding of the study is that engagement of the C3a receptor results in phosphorylation of extracellular signal-regulated kinase-1 and 2 (ERK-1/2), which promotes the efflux of adenosine triphosphate (ATP) from the macrophage. Release of ATP is a rate-limiting step for activating caspase-1, as extracellular ATP triggers the P2X7 purinergic receptor to initiate oligomerization of NLRP3.

C3a modulates IL-1ß secretion in human monocytes by regulating ATP efflux and subsequent NLRP3 inflammasome activation.

Interleukin-1ß (IL-1ß) is a proinflammatory cytokine and a therapeutic target in several chronic autoimmune states. Monocytes and macrophages are the major sources of IL-1ß. IL-1ß production by these cells requires Toll-like receptor (TLR) and adenosine triphosphate (ATP)-mediated P2X purinoceptor 7 (P2X7) signals, which together activate the inflammasome. However, how TLR signals and ATP availability are regulated during monocyte activation is unclear and the involvement of another danger signal system has been proposed. Here, we demonstrate that both lipopolysaccharide (LPS) and the anaphylatoxin C3a are needed for IL-1ß production in human macrophages and dendritic cells, while in monocytes, C3a enhanced the secretion of LPS-induced IL-1ß. C3a and LPS-stimulated monocytes increased T helper 17 (Th17) cell induction in vitro, and human rejecting, but not nonrejecting, kidney transplant biopsies were characterized by local generation of C3a and monocyte and Th17 cell infiltration. Mechanistically, C3a drives IL-1ß production in monocytes by controlling the release of intracellular ATP into the extracellular space via regulation of as-yet unidentified ATP-releasing channels in an extracellular signal-regulated kinase 1/2-dependent fashion. These data define a novel function for complement in inflammasome activation in monocytes and suggest that C3aR-mediated signaling is a vital component of the IL-1ß-Th17 axis.