The CTRP3-AdipoR2 Axis Regulates the Development of Experimental Autoimmune Encephalomyelitis by Suppressing Th17 Cell Differentiation.

C1q/TNF-related proteins (CTRP) including CTRP3 are a group of secreted proteins which have a complement C1q-like domain in common, and play versatile roles in lipid metabolism, inflammation, tumor metastasis and bone metabolism. Previously, we showed that the expression of C1qtnf3, encoding CTRP3, is highly augmented in joints of autoimmune arthritis models and CTRP3-deficiency exacerbates collagen-induced arthritis in mice. However, the mechanisms how CTRP3-deficiency exacerbates arthritis still remain to be elucidated. In this study, we showed that CTRP3 was highly expressed in Th17 cell, a key player for the development of autoimmune diseases, and Th17 cell differentiation was augmented in C1qtnf3-/- mice. Th17 cell differentiation, but not Th1 cell differentiation, was suppressed by CTRP3 and this suppression was abolished by the treatment with a receptor antagonist against AdipoR2, but not AdipoR1, associated with suppression of Rorc and Stat3 expression. Furthermore, AdipoR1 and AdipoR2 agonist, AdipoRon suppressed Th17 cell differentiation via AdipoR2, but not AdipoR1. The development of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis was enhanced in C1qtnf3-/- mice associated with increase of Th17 cell population. CTRP3 inhibited MOG-induced IL-17 production from T cells by affecting both T cells and dendritic cells. These results show that CTRP3 is an endogenous regulator of Th17 differentiation, suggesting that the CTRP3-AdipoR2 axis is a good target for the treatment of Th17 cell-mediated diseases.

An adiponectin receptor agonist antibody stimulates glucose uptake and fatty-acid oxidation by activating AMP-activated protein kinase.

Adiponectin (Ad) is a representative adipocytokine that regulates energy homeostasis including glucose transport and lipid oxidation through activation of AMP-activated protein kinase (AMPK) pathways. Plasma levels of Ad are reduced in obesity, which contributes to type 2 diabetes. Therefore, agents that activate the Ad signaling pathway could ameliorate metabolic diseases such as type 2 diabetes. Here, we report the identification of a high-affinitive agonist antibody against Ad receptors. The antibody was selected by using phage display of human combinatorial antibody libraries. The selected antibody induced phosphorylation of the acetyl-CoA carboxylase (ACC) and AMPK in skeletal muscle cells and stimulated glucose uptake and fatty-acid oxidation (FAO) in myotubes. In addition, the antibody significantly lowered blood glucose levels during a glucose challenge in normal mice as well as basal blood glucose levels in a type 2 diabetic mouse model. Taken together, these results suggest that the agonist antibody could be a promising therapeutic agent for the treatment of metabolic syndrome such as type 2 diabetes.

Adiponectin Receptors and Pro-inflammatory Cytokines Are Modulated in Common Variable Immunodeficiency Patients: Correlation With Ig Replacement Therapy.

Adiponectin exerts beneficial pleiotropic effects through three receptors, AdipoR1, AdipoR2, and T-cadherin; it also exerts immunomodulatory effects. We previously demonstrated that adiponectin levels are altered in common variable immunodeficiency disease (CVID). The purpose of the present study was to investigate further the specific involvement of adiponectin in CVID by characterizing (i) the expression profile of adiponectin receptors on peripheral blood mononuclear cells; (ii) the levels of another relevant adipokine, namely leptin; (iii) the levels of five other cytokines (IL-2, IL-6, IL-10, TNFα, and IFNγ) in 24 patients on maintenance therapy, in 18 treatment-naïve patients (before and 24 h after the first Ig infusion) and in 28 healthy controls. We found that (i) adiponectin was down-expressed in patients on maintenance therapy and in treatment-naïve patients, and that it increased in treatment-naïve patients 24 h after the first Ig infusion; (ii) leptin expression did not differ between maintenance patients and controls either before or after the first Ig infusion; (iii) AdipoR1 expression was significantly higher on B lymphocytes, monocytes and NK cells of CVID patients than in controls; (iv) the expression of AdipoR1 and AdipoR2 on B lymphocytes, monocytes and NK cells was higher after the first Ig infusion than in treatment-naïve patients; (v) T-cadherin expression did not differ between treatment- naïve CVID patients and controls, and was not affected by Ig infusion; and (vi) IL-6, IL-8, IL-10, and TNFα levels were differently expressed in CVID patients on therapy maintenance and were not affected by the first Ig replacement therapy. This is the first study to demonstrate that the expression of AdipoRs in peripheral blood mononuclear cells from CVID patients differs from that of controls, and changes after the first Ig infusion. The specificity of adiponectin involvement in CVID is supported by the absence of changes in leptin levels and in the levels of the cytokines investigated. Taken together, these results suggest that the adiponectin system plays an important and specific role in CVID. A better understanding of adiponectin as a link in the cross-talk between the immune system and adipose tissue may provide additional benefits for the management of CVID patients.

Adiponectin regulates psoriasiform skin inflammation by suppressing IL-17 production from γδ-T cells.

Accumulating epidemiologic evidence has revealed that metabolic syndrome is an independent risk factor for psoriasis development and is associated with more severe psoriasis. Adiponectin, primarily recognized as a metabolic mediator of insulin sensitivity, has been newly drawing attention as a mediator of immune responses. Here we demonstrate that adiponectin regulates skin inflammation, especially IL-17-related psoriasiform dermatitis. Mice with adiponectin deficiency show severe psoriasiform skin inflammation with enhanced infiltration of IL-17-producing dermal Vγ4+γδ-T cells. Adiponectin directly acts on murine dermal γδ-T cells to suppress IL-17 synthesis via AdipoR1. We furthermore demonstrate here that the adiponectin level of skin tissue as well as subcutaneous fat is decreased in psoriasis patients. IL-17 production from human CD4- or CD8-positive T cells is also suppressed by adiponectin. Our data provide a regulatory role of adiponectin in skin inflammation, which would imply a mechanism underlying the relationship between psoriasis and metabolic disorders.

Adiponectin and its receptor signaling: an anti-cancer therapeutic target and its implications for anti-tumor immunity.

INTRODUCTION: Adiponectin (APN), produced by adipocytes, has direct anti-diabetic, anti-atherogenic and anti-inflammatory properties. Circulating APN levels are lower in obesity, a disease state that is often associated with several malignancies. AREA COVERED: Increasingly, clinical data suggests that serum APN may have an important protective role in carcinogenesis. Certain cancer cell types express APN receptors and their downstream signaling pathways may influence cancer biology, possibly by regulating cell proliferation and inducing apoptosis. However, APN's role in the immune system, in particular to the anti-tumor response, remains elusive. Therefore, this review critically addresses all controversies associated with the effect of APN on the immune system. EXPERT OPINION: Currently, the promise of interfering with APN and its receptor axis as a novel anti-cancer therapeutic target is rather encouraging. Greater understanding of the immunological side effects following this interference is crucial for the development of effective therapeutic strategies against obesity-associated malignancies. APN receptor signaling on immune cells can blunt anti-tumor immunity and induce tumor-specific tolerance. This may also have far-reaching consequences on the application of APN as an anti-cancer agent.

C1q/Tumor Necrosis Factor-Related Protein 9 Protects against Acute Myocardial Injury through an Adiponectin Receptor I-AMPK-Dependent Mechanism.

Obesity is a risk factor for cardiovascular disease. C1q/tumor necrosis factor-related protein 9 (CTRP9) is an adipokine that is downregulated by obesity. We investigated the role of CTRP9 in cardiac injury with loss-of-function genetic manipulations and defined the receptor-mediated signaling pathway downstream of this adipokine. CTRP9-knockout (CTRP9-KO) mice at the age of 12 weeks were indistinguishable from wild-type (WT) mice under basal conditions. CTRP9-KO mice had exacerbated contractile left ventricle dysfunction following intraperitoneal injection of lipopolysaccharide (LPS) compared to WT mice. Administration of LPS to CTRP9-KO mice also resulted in increased expression of proinflammatory cytokines and oxidative stress markers in the heart compared to WT mice. Likewise, CTRP9-KO mice showed increased myocardial infarct size and elevated expression of inflammatory mediators in ischemic heart following ischemia and reperfusion compared to WT mice. Treatment of cardiac myocytes with CTRP9 protein led to suppression of LPS-induced expression of proinflammatory genes, which was reversed by blockade of AMPK or ablation of adiponectin receptor I (AdipoR1). Systemic delivery of CTRP9 attenuated LPS-induced cardiac dysfunction in WT mice but not in muscle-specific transgenic mice expressing dominant-negative mutant form of AMPK or in AdipoR1-knockout mice. CTRP9 protects against acute cardiac damage in response to pathological stimuli by suppressing inflammatory reactions through AdipoR1/AMPK-dependent mechanisms.

Adiponectin inhibits tumor necrosis factor-α-induced vascular inflammatory response via caveolin-mediated ceramidase recruitment and activation.

RATIONALE: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. OBJECTIVE: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin's vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. METHODS AND RESULTS: Adiponectin significantly reduced tumor necrosis factor (TNF)α-induced intercellular adhesion molecule-1 expression and attenuated TNFα-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin-induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNFα-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNFα-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. CONCLUSIONS: These results demonstrate for the first time that adiponectin inhibits TNFα-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.

Adiponectin attenuates human eosinophil adhesion and chemotaxis: implications in allergic inflammation.

OBJECTIVE: Growing evidence has shown an association between obesity and asthma. Adiponectin, an adipocyte-derived cytokine, is known to have anti-inflammatory effects with reduced concentrations in obese subjects. Recent findings raised the intriguing possibility that adiponectin might play a role in allergic inflammation, although the mechanistic basis for their relationship remains unclear. The purpose of this study was to examine whether adiponectin might affect functions of eosinophils, which play an important role in the pathogenesis of asthma. METHODS: Human peripheral blood eosinophils were purified to study expression of adiponectin receptors AdipoR1 and AdipoR2 using RT-PCR and flow cytometry. The effect of adiponectin on eosinophil survival was investigated using annexin V and propidium iodide staining. Eotaxin-induced cell adhesion was investigated using ICAM-1-coated plates. A Boyden chamber and real-time horizontal migration system were used for eotaxin-directed chemotaxis assay. Expression of eotaxin receptor CCR3 and intracellular calcium influx were assessed by flow cytometry. RESULTS: AdipoR1 and AdipoR2 were expressed in human eosinophils. Adiponectin did not affect eosinophil survival or CCR3 expression; however, eotaxin-enhanced adhesion was inhibited by pretreatment with adiponectin. Adiponectin also diminished eotaxin-directed chemotactic responses by disturbing both velocity and directionality. Calcium influx in response to eotaxin was attenuated by adiponectin. CONCLUSIONS: These results indicate that adiponectin attenuates the eosinophil functions induced by eotaxin without affecting cell viability. The inhibitory effect was associated with diminished calcium signaling rather than altering of surface receptor expression. Increasing circulating adiponectin might be a novel therapeutic modality for treatment of asthma, especially in obese asthmatics.

Inhibition of islet immunoreactivity by adiponectin is attenuated in human type 1 diabetes.

CONTEXT: Adiponectin is an adipocyte-derived cytokine with insulin-sensitizing and antiinflammatory properties. These dual actions have not previously been examined in the context of human disease. OBJECTIVES: Our objective was to examine the adiponectin axis in type 1 diabetes (T1D). T1D is an autoimmune inflammatory disease resulting from pancreatic ß-cell destruction, in which insulin resistance associates with progression to disease. DESIGN, PATIENTS, AND INTERVENTIONS: We measured circulating adiponectin and adiponectin receptor expression on blood-immune cells from 108 matched healthy, T1D, and type 2 diabetic subjects. We tested adiponectin effect on T cell proliferation to islet antigens and antigen-presenting function of monocyte-derived dendritic cells (mDCs). Lastly, we assessed the effect of a 3-week lifestyle intervention program on immune cell adiponectin receptor expression in 18 healthy subjects. RESULTS: Circulating concentrations of adiponectin were not affected by T1D. However, expression of adiponectin receptors on blood monocytes was markedly reduced and inversely associated with insulin resistance. Reduced adiponectin receptor expression resulted in increased T cell proliferation to islet-antigen presented by autologous mDCs. We demonstrated a critical role for adiponectin in down-regulating the costimulatory molecule CD86 on mDCs, and this function was impaired in T1D. We proceeded to show that lifestyle intervention increased adiponectin receptor but reduced CD86 expression on monocytes. CONCLUSIONS: These data indicate that T cells are released from the antiinflammatory effects of adiponectin in T1D and suggest a mechanism linking insulin resistance and islet immunity. Furthermore, we suggest that interventions that reduce insulin resistance could modulate the inflammatory process in T1D.

Adiponectin is a negative regulator of antigen-activated T cells.

Adiponectin (APN), a cytokine constitutively produced in fat tissue, has been shown to exert anti-inflammatory effects in various disease models. While the influence of APN on monocytic cells has been extensively studied in vitro, little is known about its role in T cells. In this study, we show that while <10% of human peripheral blood T cells express adiponectin receptors (AdipoRs) on their surface, most T cells store AdipoRs in intracellular compartments. AdipoRs colocalized with immune regulatory molecules CTLA-4 and TIRC7 within clathrin-coated vesicles. After stimulation, the expression of adiponectin receptor 1 (AdipoR1) and AdipoR2 was upregulated on the surface of antigen-specific T cells, as determined by tetramer or CD137 staining, and AdipoR1 and AdipoR2 coexpressed with CTLA-4. Addition of APN resulted in a significant diminution of antigen-specific T-cell expansion. Mechanistically, APN enhanced apoptosis and inhibited proliferation of antigen-specific T-cell lines. Further, APN directly inhibited cytokine production in response to antigen stimulation. In line with the in vitro data, APN-deficient (knockout, KO) mice had higher frequencies of CD137(+) T cells upon Coxsackie B virus infection. Altogether, our data suggest that APN is a novel negative T-cell regulator. In contrast to the CTLA-4 ligand B7 only expressed on APCs, APN is abundant in human plasma.

Adiponectin induces NF-kappaB activation that leads to suppression of cytokine-induced NF-kappaB activation in vascular endothelial cells: globular adiponectin vs. high molecular weight adiponectin.

Adiponectin circulates in plasma as various isoforms. However, the biological activity of each isoform has not been firmly established. High molecular weight (HMW) adiponectin may be the active form of adiponectin, while a proteolytic cleavage product of adiponectin, known as globular adiponectin (gAd), has recently been shown to activate vascular endothelial cells. We compared HMW adiponectin with gAd to investigate whether they could activate nuclear factor kappa B (NF-kappaB) and suppress cytokine-induced NF-kappaB activation in vascular endothelial cells. HMW adiponectin was found to activate NF-kB modestly compared to the activation observed with gAd. HMW adiponectin requires a shorter incubation period to demonstrate inhibition against tumour necrosis factor alpha (TNFalpha)-induced NF-kappaB activation, compared with gAd. gAd strongly activates NF-kappaB, thereby inducing the expression of various pro-inflammatory and adhesion molecule genes, and requires a longer incubation period to show inhibition against cytokine-induced NF-kappaB activation. Thus, HMW adiponectin might function to protect against inflammatory stimuli, while cleavage of adiponectin at inflammatory sites might enhance the inflammatory process.

[Bioinformatics analysis of mouse adiponectin receptor-1 and its antibody preparation].

To establish a method for preparation of anti-mouse adiponectin receptor-1 (AdipoR-1) polyclonal antibody, the polypeptide antigen corresponding to AdipoR-1 was designed by bioinformatics analysis. The possible physicochemical property and trans-membrane structure were predicted by ExPASy and TMHMM, respectively. The antigen epitopes of mouse AdipoR-1 and its immunogenicity were analyzed by Antigenic Prediction and AntigenProfiler, respectively. According to the similarity analysis between AdipoR-1 and AdipoR-2 by Clustal W, a 16-amino acid polypeptide was designed as the antigen corresponding to AdipoR-1. To ensure the specificity of the polypeptide antigen, similarity search was run in the protein databases such as SWISS-PROT, PDB and Prosite databases. The polypeptide synthesized by solid-phase synthesis was used as immunogen to immunize rats to obtain anti-mouse AdipoR-1 polyclonal antibodies, the specificity and titer of which was identified by Western blot and indirect ELISA. The antibodies were applied to detect the AdipoR-1 expression in the muscle tissue in normal and cholesterolemic mice. The results from bioinformatics analysis showed that the similarity of amino acid sequences between AdipoR-1 and AdipoR-2 in mouse was 66%, and the designed polypeptide antigen corresponding to AdipoR-1 exhibited excellent immunogenicity (score=3.1). Using the polypeptide as antigen for immunization, anti-mouse AdipoR-1 polyclonal antibodies with high titer and good specificity were obtained. The results of Western blot demonstrated that there was no statistical difference in AdipoR-1 expression in muscle tissue between normal (1.80±0.06) and cholesterolemic mice (1.71±0.11). These results suggest that the antigen epitopes of mouse AdipoR-1 are well predicted by bioinformatics analysis, and successful preparation of the specific anti-AdipoR-1 polyclonal antibodies provides a useful tool for identification and further functional study of AdipoR-1.