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Background and Objectives: Dental caries is one of the most common human pathological conditions resulting from the invasion of bacteria into the dentin. Current treatment options are limited. In many cases, endodontic therapy leads to permanent pulp tissue loss. Dentin-pulp complex regeneration involves dental pulp stem cells (DPSCs) that differentiate into odontoblast-like cells under an inflammatory context. However, limited information is available on how DPSC differentiation processes are affected under inflammatory environments. We identified the crucial role of complement C5a and its receptor C5aR in the inflammation-induced odontoblastic DPSC differentiation. Methodology: Here, we further investigated the role of a second and controversial C5a receptor, C5L2, in this process and explored the underlying mechanism. Human DPSCs were examined during 7-, 10-, and 14-day odontogenic differentiation treated with TNFα, C5L2 CRISPR, and tyrosine receptor kinase B (TrkB) antagonist [cyclotraxin-B (CTX-B)]. Results: Our data demonstrate that C5L2 CRISPR knockout (KO) enhances mineralization in TNFα-stimulated differentiating DPSCs. We further confirmed that C5L2 CRISPR KO significantly enhances dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) expression after 14-day odontoblastic DPSC differentiation, and treatment with CTX-B abolished the TNFα/C5L2 CRISPR KO-induced DSPP and DMP-1 increase, suggesting TrkB's critical role in this process. Conclusion and Key applications: Our data suggest a regulatory role of C5L2 and TrkB in the TNFα-induced odontogenic DPSC differentiation. This study may provide a useful tool to understand the mechanisms of the role of inflammation in dentinogenesis that is required for successful DPSC engineering strategies.

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The complement system plays crucial roles in a wide breadth of immune and inflammatory processes and is frequently cited as an etiological or aggravating factor in many human diseases, from asthma to cancer. Complement receptors encompass at least eight proteins from four structural classes, orchestrating complement-mediated humoral and cellular effector responses and coordinating the complex cross-talk between innate and adaptive immunity. The progressive increase in understanding of the structural features of the main complement factors, activated proteolytic fragments, and their assemblies have spurred a renewed interest in deciphering their receptor complexes. In this review, we describe what is currently known about the structural biology of the complement receptors and their complexes with natural agonists and pharmacological antagonists. We highlight the fundamental concepts and the gray areas where issues and problems have been identified, including current research gaps. We seek to offer guidance into the structural biology of the complement system as structural information underlies fundamental and therapeutic research endeavors. Finally, we also indicate what we believe are potential developments in the field.

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The complement fragment C5a is one of the most potent proinflammatory glycoproteins liberated by the activation of the biochemical cascade of the complement system. C5a is established to interact with a set of genomically related transmembrane receptors, like C5aR1 (CD88, C5aR) and C5aR2 (GPR77, C5L2) with comparable affinity. The C5aR1 is a classical G-protein-coupled receptor (GPCR), whereas C5aR2 is a nonclassical GPCR that tailors immune cell activity potentially through ß-arrestins rather than G-proteins. Currently, the exact function of the C5aR2 is actively debated in the context of C5aR1, even though both C5aR1 and C5aR2 are coexpressed on myriads of tissues. The functional relevance of C5aR2 appears to be context-dependent compared to the C5aR1, which has received enormous attention for its role in both acute and chronic inflammatory diseases. In addition, the structure of C5aR2 and its interaction specificity toward C5a is not structurally elucidated in the literature so far. The current study has attempted to close the gap by generating highly refined model structures of C5aR2, respectively in free (inactive), complexed to C-terminal peptide of C5a (meta-active) and the C5a (active), embedded to a model palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer. The computational modeling and the 1.5-µs molecular dynamics data presented in the current study are expected to further enrich the understanding of C5a-C5aR2 interaction compared to C5a-C5aR1, which will surely help in elaborating the currently debated biological function of C5aR2 better in the foreseeable future.

Asunto(s)

Complemento C5a , Genómica , Complemento C5a/genética , Complemento C5a/metabolismo , beta-Arrestinas , Receptor de Anafilatoxina C5a/genética

RESUMEN

Background and objectives: Overnutrition leads to a metabolic and inflammatory response that includes the activation of Complement. Properdin is the only amplifier of complement activation and increases the provision of complement activation products. Its absence has previously been shown to lead to increased obesity in mice on a high fat diet. The aim of this study was to determine ways in which properdin contributes to a less pronounced obese phenotype. Materials and Methods: Wild type (WT) and properdin deficient mice (KO) were fed a high-fat diet (HFD) for up to 12 weeks. Results: There was a significant increase in liver triglyceride content in the KO HFD group compared to WT on HFD. WT developed steatosis. KO had an additional inflammatory component (steatohepatitis). Analysis of AKT signalling by phosphorylation array supported a decrease in insulin sensitivity which was greater for KO than WT in liver and kidney. There was a significant decrease of C5L2 in the fat membranes of the KO HFD group compared to the WT HFD group. Circulating microparticles in KO HFD group showed lower presence of C5L2. Expression of the fatty acid transporter CD36 in adipose tissue was increased in KO on HFD and was also significantly increased in plasma of KO HFD mice compared to WT on HFD. CD36 was elevated on microparticles from KO on HFD. Ultrastructural changes consistent with obesity-associated glomerulopathy were observed for both HFD fed genotypes, but tubular strain was greater in KO. Conclusion: Our work demonstrates that complement properdin is a dominant factor in limiting the severity of obesity-associated conditions that impact on liver and kidney. The two receptors, C5L2 and CD36, are downstream of the activity exerted by properdin.

Asunto(s)

Dieta Alta en Grasa , Resistencia a la Insulina , Animales , Dieta Alta en Grasa/efectos adversos , Ratones , Ratones Noqueados , Obesidad , Properdina/genética

RESUMEN

INTRODUCTION: We recently reported that caries-associated C5a receptor (C5aR) expression and activation result in up-regulation of brain-derived neurotropic factor secretion by pulp fibroblasts inducing prominent neurite outgrowth toward the carious site. Our data further showed a negative regulation of this brain-derived neurotropic factor secretion by C5L2, another C5aR. C5L2 was considered a nonfunctional receptor and thus has received much less attention than C5aR. The aim of this study was to identify the role of C5L2 in pulp fibroblast-mediated neurite outgrowth. METHODS: In this study, lipoteichoic acid (LTA) was used to mimic dental caries-like inflammation. To evaluate the role of C5L2 in pulp neurite outgrowth, human pulp fibroblasts were C5L2 small interfering RNA silenced and cocultured with human neurons in a nerve growth assay system. RESULTS: C5L2 silencing drastically increased the neurite outgrowth toward the LTA-stimulated pulp fibroblasts. The number of neurites detected was increased in the LTA-treated pulp fibroblasts. CONCLUSIONS: Our results show that C5L2 constitutes a negative regulator of the neurite outgrowth under LTA stimulation. Of the events occurring during dentin-pulp regeneration, nerve regeneration is the key factor for maintaining tooth viability after infection or injury. Our study provides a foundation for creating therapeutic tools that target pulp fibroblasts during pulp/nerve regeneration.

Asunto(s)

Pulpa Dental/citología , Fibroblastos , Silenciador del Gen , Lipopolisacáridos/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , ARN Interferente Pequeño/genética , Receptores de Quimiocina/genética , Ácidos Teicoicos/farmacología , Células Cultivadas , Expresión Génica , Humanos , Receptor de Anafilatoxina C5a , Receptores de Quimiocina/fisiología

RESUMEN

Obesity closely correlates with metaflammation and characterizes with systemic-chronic-low inflammation. This study aims to evaluate effects of C5a on the inflammatory response and insulin resistance in 3T3-L1 adipocytes. 3T3-L1 pre-adipocytes were induced to the mature 3T3-L1 adipocytes. Then, 3T3-L1 were intervened with anaphylatoxin C5a, lipopolysaccharide (LPS) and C5a + LPS, respectively. Levels of Omentin, Chemerin, Vaspin and Apelin 12 in supernatants of medium were examined using ELISA. C5L2, C5a receptor (C5aR), I kappa B (IkB), IkB kinase (IKK), insulin receptor substrate 1 (IRS-1), IRS-2, PI3 K, p-PI3 K and ß-actin were examined using RT-PCR and western blot assay, respectively. C5L2-C5aR colocalization was identified using immunofluorescence double label. NF-kB expression or activity was evaluated using electrophoretic mobility shift assay (EMSA), dual luciferase assay and immunofluorescence assay, respectively. The glucose uptake and insulin sensitivity were also evaluated. Results showed that C5a intervention significantly enhanced inflammatory molecule levels in supernatants of 3T3-L1 adipocytes. IKK inflammatory signaling pathway participated in C5a induced inflammation of 3T3-L1 adipocytes. C5a triggered the colocalization of C5L2 and C5aR and activated the NF-kB inflammatory signaling pathway. C5a intervention in 3T3-L1 adipocytes decreased the glucose uptake and resulted in reduction of insulin sensitivity. Insulin signaling pathway participated in C5a caused insulin sensitivity reduction. C5a intervention triggered the phosphorylation of PI3 K. In conclusion anaphylatoxin C5a induced inflammatory response by activating TLR4/NF-kB signaling pathway and generating C5L2-C5aR dimer, and caused insulin sensitivity reduction by activating PI3 K signaling pathway.

Asunto(s)

Adipocitos/inmunología , Complemento C5a/inmunología , Resistencia a la Insulina/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor Toll-Like 4/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , Complemento C5a/farmacología , Inflamación , Lipopolisacáridos/farmacología , Ratones , FN-kappa B , Obesidad/inmunología , Obesidad/metabolismo , Transducción de Señal

RESUMEN

Complement factor C5a has two known receptors, C5aR, which mediates proinflammatory effects, and C5L2, a potential C5a decoy receptor. We previously identified C5a/C5aR signaling as a potent profibrotic pathway in the kidney. Here we tested for the first time the role of C5L2 in renal fibrosis. In unilateral ureteral obstruction (UUO)-induced kidney fibrosis, the expression of C5aR and C5L2 increased similarly and gradually as fibrosis progressed and was particularly prominent in injured dilated tubules. Genetic deficiency of either C5aR or C5L2 significantly reduced UUO-induced tubular injury. Expression of key proinflammatory mediators, however, significantly increased in C5L2- compared with C5aR-deficient mice, but this had no effect on the number of renal infiltrating macrophages or T cells. Moreover, in C5L2-/- mice, the cytokine and matrix metalloproteinase-inhibitor tissue inhibitor of matrix metalloproteinase-1 was specifically enhanced. Consequently, in C5L2-/- mice the degree of renal fibrosis was similar to wild type (WT), albeit with reduced mRNA expression of some fibrosis-related genes. In contrast, C5aR-/- mice had significantly reduced renal fibrosis compared with WT and C5L2-/- mice in UUO. In vitro experiments with primary tubular cells demonstrated that deficiency for either C5aR or C5L2 led to a significantly reduced expression of tubular injury and fibrosis markers. Vice versa, stimulation of WT tubular cells with C5a significantly induced the expression of these markers, whereas the absence of either receptor abolished this induction. In conclusion, in experimental renal fibrosis C5L2 and C5aR both contribute to tubular injury, and, while C5aR acts profibrotic, C5L2 does not play a role in extracellular matrix accumulation, arguing against C5L2 functioning simply as a decoy receptor.

Asunto(s)

Complemento C5a/metabolismo , Fibrosis/inmunología , Enfermedades Renales/inmunología , Receptores de Quimiocina/metabolismo , Animales , Complemento C5a/inmunología , Fibrosis/genética , Riñón/inmunología , Riñón/patología , Enfermedades Renales/patología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Ratones , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología

RESUMEN

C5a anaphylatoxin is a component of the complement system involved in the modulation of T-cell polarization. Herein we investigated whether C5a receptors, C5aR and C5L2, modulate the cytokine profiles induced by Mycobacterium tuberculosis (Mtb). We analyzed the impact of both receptors on T helper cell polarization induced by the multidrug resistant outbreak strain named M, which is a poor IFN-γ inducer compared with the laboratory strain H37Rv. To this aim, we first blocked C5aR or C5L2 of peripheral blood monocytes (Mo) from patients with tuberculosis and healthy donors, then we stimulated the Mo either with H37Rv or the M strain, and finally we analyzed cytokine profiles of Mo/macrophages (MΦ) and CD4+ T-cells. We found that: (i) Mtb modulated the expression of both C5a receptors, (ii) C5aR inhibited the expansion of CD4+IFN-γ+ lymphocytes stimulated by the M strain but not by H37Rv, (iii) both receptors modulated the Mo/MΦ cytokine expression induced by Mtb. We conclude that C5aR, but not C5L2, plays a role in T helper cell polarization induced by Mtb and that this effect is strain- and donor-dependent. We speculate that the epidemiologically successful M strain takes advantage of this C5aR-mediated inhibition of Th1 polarization to survive within the host.

Asunto(s)

Citocinas/inmunología , Brotes de Enfermedades , Mycobacterium tuberculosis/inmunología , Receptor de Anafilatoxina C5a/inmunología , Células TH1/inmunología , Tuberculosis Resistente a Múltiples Medicamentos/inmunología , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Citocinas/metabolismo , Femenino , Genotipo , Interacciones Huésped-Patógeno , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/microbiología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Fenotipo , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Células TH1/metabolismo , Células TH1/microbiología , Factores de Tiempo , Tuberculosis Resistente a Múltiples Medicamentos/metabolismo , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Adulto Joven

RESUMEN

BACKGROUND: C5aR-like receptor 2 (C5L2) has been identified as a receptor for the inflammatory factor Complement 5a (C5a) and acylation-stimulating protein (ASP). ASP binding to C5L2 leading to a net accumulation of TG stores and glucose transporter. The aim of the present study is to evaluate the association of the SNPs of C5L2 gene with coronary artery disease (CAD) in a Chinese population. METHODS: We examined the role of the tagging single nucleotide polymorphisms (SNPs) of C5L2 gene for CAD using a case-control design. We determined the prevalence of C5L2 genotypes in 505 CAD patients and 469 age and sex-matched healthy control subjects of Han population. RESULTS: There was significant difference in genotype distributions of rs2972607 and rs8112962 between CAD patients and control subjects. The rs2972607 was found to be associated with CAD in a dominant model (AA vs. AG + GG, P<0.001). Similarly, the rs8112962 was found to be associated with CAD in a dominant model (TT vs CT + CC, P=0.016). The difference remained statistically significant after multivariate adjustment (OR =1.401, 95% confidence interval [CI]:1.026~1.914, P=0.034; OR = 1.541, 95%CI:1.093~ 2.172, P=0.014; respectively). CONCLUSION: The results of this study indicate that both rs2972607 and rs8112962 of C5L2 gene are associated with CAD in a Han population of China.

Asunto(s)

Enfermedad de la Arteria Coronaria/genética , Polimorfismo de Nucleótido Simple , Receptores de Quimiocina/genética , Anciano , Pueblo Asiatico/genética , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , China , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/etnología , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Oportunidad Relativa , Fenotipo , Receptor de Anafilatoxina C5a , Factores de Riesgo

RESUMEN

Objective To investigate the prognostic value of C5L2 in patients with hepatocellular carcinoma (HCC).Methods The data of 175 patients with HCC who underwent curative resection at Zhongshan Hospital,Fudan University from Oct.,2012 to Sep.,2013 were analyzed retrospectively.The correlation between C5L2 and clinicopathologic characteristics were explored.COX regression model was used to determine the influence of clinical parameters on predicting recurrence,and the prognostic value of combined application of C5L2 and AFP were evaluated by Kaplan-Meier method.In vitro,the expression of C5L2 were tested in 5 HCC cell lines,and Hep3B and Huh7 were chosen for down-regulation and up-regulation of C5L2,respectively,the abilities of invasion and migration were examined by transwell and the potential mechanism was explored.Results The C5L2 expression was correlated to gender,tumor size and recurrence,and the recurrence rate of low C5L2 expression group was higher.Also,the multivariate analysis showed that C5L2 low expression was an independent risk factor for recurrence.Moreover,the combined application of C5L2 and AFP could estimate prognosis more effectively.Knockdown of C5L2 in Hep3B promoted the invasiveness and motility,and increased the level of β-catenin and MMP2;conversely,overexpression of C5L2 in Huh7 inhibited the invasiveness and motility,and decreased the level of β catenin and MMP2.Conclusions C5L2 could be regarded as an auxiliary indicator for prognosis of HCC,thereby the evaluation of C5L2 could help with making effective and comprehensive management for HCC patients.

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LPS-induced lung injury in the mouse is one of the most robust experimental models used for studies of acute lung injury (ALI) and acute respiratory distress syndrome in humans. Prior clinical and experimental studies support an important role for complement activation, particularly production of C5a, in the pathophysiology of human ALI/acute respiratory distress syndrome. In the mouse model, however, the precise role of C5a and its receptors is unclear. C5L2, an enigmatic second receptor for C5a, has been characterized, and results have generated substantial debate regarding its in vivo function. Our previous work with human neutrophils revealed a unique role for C5L2 in negatively modulating C5a-C5a receptor (C5aR)-mediated cellular activation, in which antibody-mediated blockade of C5L2 resulted in augmented C5a-C5aR responses. Here, we demonstrate that C5L2-/- mice (BALB/c background) administered intranasal LPS exhibit significantly more airway edema and hemorrhage than do wild-type animals. Bronchoalveolar lavage fluid and lung homogenates have significantly more neutrophils and myeloperoxidase activity, as well as proinflammatory cytokines and chemokines. When a blocking antibody against the C5aR was administered before LPS administration, the increased neutrophilic infiltration and cytokine levels were reversed. Thus, our data show not only that C5a contributes significantly to LPS-induced ALI in the mouse, but also that C5L2 plays an important antiinflammatory role in this model through actions resulting at least in part from negative modulation of C5a receptor activation.

Asunto(s)

Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Receptores de Quimiocina/metabolismo , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/fisiopatología , Administración Intranasal , Animales , Líquido del Lavado Bronquioalveolar , Quimiocinas/genética , Quimiocinas/metabolismo , Edema/complicaciones , Edema/patología , Edema/fisiopatología , Regulación de la Expresión Génica , Hemorragia/complicaciones , Hemorragia/patología , Hemorragia/fisiopatología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/administración & dosificación , Pulmón/patología , Ratones Endogámicos BALB C , Neutrófilos/metabolismo , Peroxidasa/metabolismo , Receptor de Anafilatoxina C5a , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Quimiocina/deficiencia , Mecánica Respiratoria , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo

RESUMEN

OBJECTIVE: This study was to analyze the acylation-stimulating protein (ASP) (301T>C) and C5a-like receptor 2 (C5L2) (698C>T) gene polymorphisms in Han and Hui populations, and investigate their association with coronary heart disease (CHD). METHODS: 245 Han CHD patients and 110 Hui CHD patients from Shandong, Jinan, China were included in this study. Biochemical analysis was performed to assess the blood sugar and lipid levels in these patients, and the TaqMan genotyping assay was used to determine the genotype distribution. RESULTS: Our results showed that the C allele frequency in the ASP (301T>C) polymorphism in the Hui population was significantly higher than normal controls, while no significant differences were observed in the Han population, which might contribute to the genetic susceptibility of CHD in the Hui population. Moreover, for C5L2 (698C>T) gene polymorphism in both Han and Hui populations, the frequencies of the C/T genotype and T allele were significantly higher in the CHD patients compared with normal controls. Moreover, there were slight differences in the association of ASP and C5L2 gene polymorphisms with blood sugar and lipid levels between Han and Hui populations. CONCLUSIONS: Our results suggest differential ASP and C5L2 genotype distributions between Han and Hui patients, which might be associated with the different CHD-related genetic susceptibilities in these populations. These findings might contribute to a better understanding of the etiology and pathogenesis of CHD in different regions and populations.

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Acute lung injury ( ALI) is a type of pulmonary excessive inflammation with high morbidity and mortality.It can be induced by multiple causes.There are currently no successful therapeutic or preventive measures because the patho-genesis of ALI has not been completely clarified.Many studies have shown that the activation of complement 5a (C5a) and its receptors is necessary in the process of ALI.Drug development targeting on C5a and its receptors may bring new hope to the treatment of ALI.In this article, the experimental evidence and possible mechanisms are summarized to reveal the rela-tionship between C5a and ALI.

RESUMEN

C5L2 is a receptor that binds to C5a and belongs to the family of G protein-coupled receptors, but its role in physiological C5a-mediated responses remains under debate. Here we show that, like the canonical C5a receptor C5aR, C5L2 plays a pro-inflammatory role in a murine model of acute experimental colitis. We demonstrate that C5L2 physically interacts with C5aR and is required for optimal C5a-mediated C5aR internalization and associated ERK activation. Abrogation of C5a-induced receptor internalization by treatment with the dynamin inhibitor dynasore(TM) impaired C5a-induced MEK and ERK signaling. Although the presence of C5aR alone was sufficient to recruit the scaffold protein ß-arrestin1 to the cell membrane in response to C5a stimulation, it was inadequate to mediate AP2 recruitment and subsequent C5aR internalization. Expression of C5L2 allowed normal internalization of C5aR in response to C5a stimulation, followed by normal ERK signaling. Thus, our work reveals an essential role for C5L2 in C5a-triggered, AP2-dependent C5aR internalization and downstream ERK signaling.

Asunto(s)

Colitis Ulcerosa/inmunología , Complemento C5a/inmunología , Endocitosis/inmunología , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Quimiocina/inmunología , Animales , Arrestinas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Complemento C5a/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Dinaminas/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Humanos , Hidrazonas/farmacología , Inflamación/inmunología , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neutrófilos/inmunología , Fosfatidilinositol 3-Quinasa/metabolismo , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/inmunología , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/genética , Transducción de Señal , beta-Arrestinas

RESUMEN

Recent studies suggested that the immunometabolic receptors; C5aR and C5L2, constitutively self-associate into homo-/heterodimers and that acylation stimulating protein (ASP/C3adesArg) or C5a treatment of adipocytes increased their colocalization. The present study evaluates the C5aR contribution in adipocytes to the metabolic and immune responses elicited by ligand stimulation. The effects of C5a, ASP, and insulin on cytokine production, triglyceride synthesis (TGS), and key signaling pathways were evaluated in isolated primary adipocytes and cultured 3T3-L1 differentiated adipocytes. In addition, mRNA expression of IRS1 and PGC1α was compared in adipose tissue samples from WT vs. C5aRKO mice. Both C5a and ASP directly increased MCP-1 (238±4%; P<0.001, and 377±2% vs. basal 100%; P<0.001, respectively) and KC (413±11%; P<0.001, and 529±16%; P<0.001 vs. basal 100%, respectively) secretion, TGS (131±1%; P<0.001, and 152±6%; P<0.001, vs. basal 100% respectively), and Akt/NFκB phosphorylation pathways in adipocytes. However, in C5aRKO adipocytes, C5a effects were disrupted, while stimulatory effects of ASP were mostly maintained. Addition of C5a completely blocked ASP signaling and activity in both C5aRKO and WT adipocytes as well as 3T3-L1 adipocytes. Furthermore, C5aRKO adipocytes revealed impaired insulin stimulation of cytokine production, with partial impairment of signaling and TGS stimulation, consistent with decreased IRS1 and PGC1α mRNA expression in adipose tissue. These observations indicate the importance of C5aR in adipose tissue metabolism and immunity, which may be regulated through heterodimerization with C5L2.

Asunto(s)

Tejido Adiposo/inmunología , Tejido Adiposo/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Quimiocina/metabolismo , Células 3T3-L1 , Adipocitos/inmunología , Animales , Complemento C3 , Complemento C5a/metabolismo , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Modelos Biológicos , Transducción de Señal/inmunología

RESUMEN

Tissue loss after myocardial ischemia with reperfusion (MI/R) is in part conveyed by neutrophil recruitment to post-ischemic myocardium. Strategies to prevent reperfusion injury would help to limit myocardial damage. The receptor for activated complement factor 5 (C5aR) plays a prominent role in inflammation. We examine the effects of C5aR-deficiency on reperfusion injury after MI/R. C5aR(-/-)-mice and their C57BL/6- (WT) littermates underwent transient myocardial ischemia followed by different time points of reperfusion. Infarct size and leukocyte infiltration were determined. Expression of C5aR, inflammatory cytokines and adhesion molecules were analyzed by real-time RT-PCR. Leukocyte-endothelial interactions were assessed by low-shear adhesion- and transmigration-assays in vitro. Myocardial C5aR mRNA expression was 2.8-fold increased by ischemia. Infarct size per area-at-risk and leukocyte recruitment into infarctions were reduced in C5aR(-/-)-compared to WT-mice as well as in WT mice treated with the C5aR-antagonist JPE1375. IL-6, IL-1ß, ICAM-1 and VCAM-1 expression were not different, while TNFα expression was reduced in C5aR(-/-)-mice after MI/R. In vitro, C5aR on leukocytes is required for effective transendothelial migration but not adhesion. Expression of MMP9 and JAM-A, molecules that are involved in leukocyte transmigration, were reduced in C5aR(-/-) mice in vivo. Genetic C5aR deficiency blunts the inflammatory response in murine MI/R resulting in reduced inflammatory cell recruitment, which is due to a C5aR-dependent effect on leukocyte transmigration across inflamed endothelium into the ischemic myocardium. This effect could be related to MMP9- and JAM-A expression in response to ischemia and reperfusion.

Asunto(s)

Infarto del Miocardio/inmunología , Daño por Reperfusión Miocárdica/complicaciones , Miocardio/metabolismo , Neutrófilos/inmunología , Receptor de Anafilatoxina C5a/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/genética , Células Cultivadas , Regulación de la Expresión Génica/genética , Humanos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/complicaciones , Daño por Reperfusión Miocárdica/etiología , Miocardio/inmunología , Miocardio/patología , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Migración Transendotelial y Transepitelial/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

The innate immune response is a complex process involving multiple pathogen-recognition receptors, including toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors. Complement is also a critical component of innate immunity. While complement is known to interact with TLR-mediated signals, the interactions between NOD-like receptors and complement are not well understood. Here we report a synergistic interaction between C5a and Nod2 signaling in RAW 264.7 macrophages. Long-term treatment with muramyl dipeptide (MDP), a NOD2 ligand, enhanced C5a-mediated expression of chemokine mRNAs in RAW 264.7 cells. This response was dependent on NOD2 expression and was associated with a decrease in expression of C5L2, a receptor for C5a which acts as a negative modulator of C5a receptor (C5aR) activity. MDP amplified C5a-mediated phosphorylation of p38 MAPK. Treatment of RAW264.7 cells with an inhibitor of p38 attenuated the synergistic effects of C5a on MDP-primed cells on MIP-2, but not MCP-1, mRNA. In contrast, inhibition of AKT prevented C5a stimulation of MCP-1, but not MIP-2, mRNA, in MDP-primed cells. Taken together, these data demonstrated a synergistic interaction between C5a and NOD2 in the regulation of chemokine expression in macrophages, associated with a down-regulation of C5L2, a negative regulator of C5a receptor activity.

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Objective To investigate the relationship between a novel polymorphism of C5L2 gene and type 2 diabetes mellitus (T2DM) in Uygur population from Xinjiang region.Methods A novel single nucleotide polymorphism(SNP),698C＞T(P233L) was found using a polymerase chain reaction direct-sequencing method.C5L2 gene 698C ＞ T variant from 252 patients with T2DM and 747 healthy control subjects was detected by polymerase chain reaction and restriction fragment length polymorphism.Result Heterozygote carriers of the 698CT genotype were more frequent among T2DM patients than that among controls (0.107 vs 0.036,x2 =18.576,P＜0.01) in the Uygur population. After adjustment of confounding factors such as sex,age,smoking,alcohol consumption,and hypertension,as well as serum levels of triglyceride,total cholesterol,low-density lipoproteincholesterol,and high-density lipoprotein-cholesterol,the difference remained significant ( P＜0.01,OR =3.373,95％ CI 1.736-6.553 ).Conclusion The CT genotype of the C5L2 gene might be a risk factor of T2DM in Uygur nationality population in Xinjiang.

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Objective To investigate the mutual effect between G protein-coupled receptors(GPCR)including C5a receptor(C5aR),C5a like receptor 2(C5L2)and chemotaxis inhibitory protein(CHIPs)secreted by Staphylococcus aureus.Methods The purified CHIPs was incubated with HEK 293T cells overexpressing C5aR,C5L2 and C-X-C chemokine receptor type 3(CXCR3).Binding signal was detected by Western blot.Results HEK 293T cells overexpressing C5aR can efficiently bind to CHIPs.No apparent relation between CHIPs and C5L2 or CXCR3 was identified.Conclusion C5aR but not C5L2 is a membrane receptor for binding CHIPs,suggesting a difference between C5aR and C5L2 in association with binding CHIPs.