Alexander Jenke

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 protects against Toll-like receptor 4-mediated cardiac inflammation and injury.

AIMS Adiponectin (APN) is an immunomodulatory and cardioprotective adipocytokine. Toll-like receptor (TLR) 4 mediates autoimmune reactions that cause myocarditis resulting in inflammation-induced cardiac injury. Here, we investigated whether APN inhibits inflammation and injury in autoimmune myocarditis by interfering with TLR4 signalling. METHODS AND RESULTS APN overexpression in murine experimental autoimmune myocarditis (EAM) down-regulated cardiac expression of TLR4 and its downstream targets tumour necrosis factor (TNF)α, interleukin (IL)-6, IL-12, CC chemokine ligand (CCL)2, and intercellular adhesion molecule (ICAM)-1 resulting in reduced infiltration with cluster of differentiation (CD)3+, CD14+, and CD45+ immune cells as well as diminished myocardial apoptosis. Expression of TLR4 signalling pathway components was unchanged in hearts and spleens of APN-knockout (APN-KO) mice. In vitro APN had no effect on TLR4 expression in cardiac and immune cells but induced dissociation of APN receptors from the activated TLR4/CD14 signalling complex. APN inhibited the expression of a TLR4-mediated inflammatory phenotype induced by exogenous and endogenous TLR4 ligands as assessed by attenuated nuclear factor (NF)-κB activation and reduced expression of TNFα, IL-6, CCL2, and ICAM-1. Accordingly, following TLR4 ligation, splenocytes from APN-KO mice showed enhanced expression of TNFα, IL-6, IL-12, CCL2, and ICAM-1, whereas dendritic cells (DCs) from APN-KO mice demonstrated increased activation and T-cell priming capacity. Moreover, APN diminished TLR4-mediated splenocyte migration towards cardiac cells as well as cardiomyocyte apoptosis after co-cultivation with splenocytes. Mechanistically, APN inhibited TLR4 signalling through cyclooxygenase (COX)-2, protein kinase A (PKA), and meiosis-specific serine/threonine kinase (MEK)1. CONCLUSION Our observations indicate that APN protects against inflammation and injury in autoimmune myocarditis by diminishing TLR4 signalling thereby attenuating inflammatory activation and interaction of cardiac and immune cells.

Adiponectin expression in patients with inflammatory cardiomyopathy indicates favourable outcome and inflammation control

AIMS Circulating adiponectin (APN) is an immunomodulatory, pro-angiogenic, and anti-apoptotic adipocytokine protecting against acute viral heart disease and preventing pathological remodelling after cardiac injury. The purpose of this study was to describe the regulation and effects of APN in patients with inflammatory cardiomyopathy (DCMi). METHODS AND RESULTS Adiponectin expression and outcome were assessed in 173 patients with DCMi, 30 patients with non-inflammatory DCM, and 30 controls. Mechanistic background of these findings was addressed in murine experimental autoimmune myocarditis (EAM), a model of human DCMi, and further elucidated in vitro. Adiponectin plasma concentrations were significantly higher in DCMi compared with DCM or controls, i.e. 6.8 ± 3.9 µg/mL vs. 5.4 ± 3.6 vs. 4.76 ± 2.5 µg/mL (P< 0.05, respectively) and correlated significantly with cardiac mononuclear infiltrates (CD3+: r(2)= 0.025, P= 0.038; CD45R0+: r(2)= 0.058, P= 0.018). At follow-up, DCMi patients with high APN levels showed significantly increased left ventricular ejection fraction improvement, decreased left ventricular end-diastolic diameter, and reduced cardiac inflammatory infiltrates compared with patients with low APN levels. A multivariate linear regression analysis implicated APN as an independent prognostic factor for inhibition of cardiac inflammation. In accordance with these findings in human DCMi, EAM mice exhibited elevated plasma APN. Adiponectin gene transfer led to significant downregulation of key inflammatory mediators promoting disease. Mechanistically, APN acted as a negative regulator of T cells by reducing antigen specific expansion (P< 0.01) and suppressed TNFα-mediated NFκB activation (P< 0.01) as well as release of reactive oxygen species in cardiomyocytes. CONCLUSION Our results implicate that APN acts as endogenously upregulated anti-inflammatory cytokine confining cardiac inflammation and progression in DCMi.

High leptin and resistin expression in chronic heart failure: adverse outcome in patients with dilated and inflammatory cardiomyopathy.

The expression of leptin and resistin is known to be positively correlated with the incidence of chronic heart failure (CHF). Both adipokines have been implicated in immunomodulation and cardiac remodelling. Therefore, we performed for the first time a clinical study to elucidate the effects of leptin and resistin on progression of CHF in patients with non‐ischaemic dilated (DCM) and inflammatory (DCMi) cardiomyopathy.

Adiponectin modulates NK-cell function

Adiponectin (APN) has been shown to exert antiinflammatory effects in various disease models but little is known concerning its regulation of NK‐cell function. Here, we show that the majority of human CD56dim NK cells express surface Adiponectin receptor (AdipoR) 1 and 2 while most CD56high NK cells are AdipoR‐negative. Toll‐like receptor (TLR) ligand‐induced IFN‐γ production was diminished by APN while it had no influence on NK‐cell cytotoxicity. In contrast only a small subpopulation of murine NK cells expresses surface AdipoRs, but about 90% store them intracellularly. APN‐deficient knockout (KO) mice had elevated frequencies of NK cells. However, cytotoxic degranulation of NK cells was decreased in APN knockout (APN‐KO) animals. Accordingly, frequencies of CD11bhighCD27high and CD94high effector NK cells and expression of NKG2D were lower in APN‐KO mice. Upon CVB3 infection NK‐cell function was restored in APN‐KO mice. Our data suggest that in addition to its antiinflammatory effects APN also influences the numerical and differentiation status of NK cells, which may further impact the outcome of immune‐mediated diseases in APN‐KO mice.

Adiponectin promotes coxsackievirus B3 myocarditis by suppression of acute anti-viral immune responses

Adiponectin (APN) is an immunomodulatory adipocytokine that improves outcome in patients with virus-negative inflammatory cardiomyopathy and mice with autoimmune myocarditis. Here, we investigated whether APN modulates cardiac inflammation and injury in coxsackievirus B3 (CVB3) myocarditis. Myocarditis was induced by CVB3 infection of APN-KO and WT mice. APN reconstitution was performed by adenoviral gene transfer. Expression analyses were performed by qRT-PCR and immunoblot. Cardiac histology was analyzed by H&E-stain and immunohistochemistry. APN-KO mice exhibited diminished subacute myocarditis with reduced viral load, attenuated inflammatory infiltrates determined by NKp46, F4/80 and CD3/CD4/CD8 expression and reduced IFNβ, IFNγ, TNFα, IL-1β and IL-12 levels. Moreover, myocardial injury assessed by necrotic lesions and troponin I release was attenuated resulting in preserved left ventricular function. Those changes were reversed by APN reconstitution. APN had no influence on adhesion, uptake or replication of CVB3 in cardiac myocytes. In acute CVB3 myocarditis, cardiac viral load did not differ between APN-KO and WT mice. However, APN-KO mice displayed an enhanced acute immune response, i.e. increased expression of myocardial CD14, IFNβ, IFNγ, IL-12, and TNFα resulting in increased cardiac infiltration with pro-inflammatory M1 macrophages and activated NK cells. Up-regulation of cardiac CD14 expression, type I and II IFNs and inflammatory cell accumulation in APN-KO mice was inhibited by APN reconstitution. Our observations indicate that APN promotes CVB3 myocarditis by suppression of toll-like receptor-dependent innate immune responses, polarization of anti-inflammatory M2 macrophages and reduction of number and activation of NK cells resulting in attenuated acute anti-viral immune responses.

The forkhead transcription factor Foxo3 negatively regulates natural killer cell function and viral clearance in myocarditis

Aims Foxo3 is a transcription factor involved in cell metabolism, survival, and inflammatory disease. However, mechanistic insight in Foxo3 effects is still limited. Here, we investigated the role of Foxo3 on natural killer (NK) cell responses and its effects in viral myocarditis. Methods and results Effects of Foxo3 on viral load and immune responses were investigated in a model of coxsackie virus B3 myocarditis in wild-type (WT) and Foxo3 deficient mice. Reduced immune cell infiltration, viral titres, and pro-inflammatory cytokines in cardiac tissue were observed in Foxo3-/- mice 7 days post-infection (p.i.). Viral titres were also attenuated in hearts of Foxo3-/- mice at Day 3 while interferon-γ (IFNγ) and NKp46 expression were up-regulated suggesting early viral control by enhanced NK cell activity. CD69 expression of NK cells, frequencies of CD11b+CD27+ effector NK cells and cytotoxicity of Foxo3-/- mice was enhanced compared to WT littermates. Moreover, microRNA-155 expression, essential in NK cell activation, was elevated in Foxo3-/- NK cells while its inhibition led to diminished IFNγ production. Healthy humans carrying the longevity-associated FOXO3 single nucleotide polymorphism (SNP) rs12212067 exhibited reduced IFNγ and cytotoxic degranulation of NK cells. Viral inflammatory cardiomyopathy (viral CMI) patients with this SNP showed a poorer outcome due to less efficient virus control. Conclusion Our results implicate Foxo3 in regulating NK cell function and suggest Foxo3 playing an important role in the antiviral innate immunity. Thus, enhanced FOXO3 activity such as in the polymorphism rs12212067 may be protective in chronic inflammation such as cancer and cardiovascular disease but disadvantageous to control acute viral infection.

Adiponectin attenuates profibrotic extracellular matrix remodeling following cardiac injury by up‐regulating matrix metalloproteinase 9 expression in mice

Adiponectin (APN) is a multifunctional adipocytokine that inhibits myocardial fibrosis, dilatation, and left ventricular (LV) dysfunction after myocardial infarction (MI). Coxsackievirus B3 (CVB3) myocarditis is associated with intense extracellular matrix (ECM) remodeling which might progress to dilated cardiomyopathy. Here, we investigated in experimental CVB3 myocarditis whether APN inhibits adverse ECM remodeling following cardiac injury by affecting matrix metalloproteinase (MMP) expression. Cardiac injury was induced by CVB3 infection in APN knockout (APN‐KO) and wild‐type (WT) mice. Expression and activity of MMPs was quantified by qRT‐PCR and zymography, respectively. Activation of protein kinases was assessed by immunoblot. In cardiac myocytes and fibroblasts APN up‐regulates MMP‐9 expression via activation of 5′ adenosine monophosphate‐activated protein kinase (AMPK) and extracellular signal‐regulated kinase (ERK)1/2 which function as master regulators of inflammation‐induced MMP‐9 expression. Correspondingly, APN further increased up‐regulation of MMP‐9 expression triggered by tumor necrosis factor (TNF)α, lipopolysaccharide (LPS) and R‐848 in cardiac fibroblasts. In vivo, compared to WT mice cardiac MMP‐9 activity and serum levels of carboxy‐terminal telopeptide of type I collagen (ICTP) were attenuated in APN‐KO mice in subacute (day 7 p.i.) CVB3 myocarditis. Moreover, on day 3 and day 7 post CVB3 infection splenic MMP‐9 expression was diminished in APN‐KO mice correlating with attenuated myocardial immune cell infiltration in subacute CVB3 myocarditis. These results indicate that APN attenuates adverse cardiac remodeling following cardiac injury by up‐regulating MMP‐9 expression in cardiac and immune cells. Thus, APN mediates intensified collagen cleavage that might explain inhibition of LV fibrosis and dysfunction.

FOXO3A DEFICIENCY PROTECTS FROM COXSACKIEVIRUS B3 MYOCARDITIS: ROLE OF NK CELL FUNCTION

Coxsackievirus B3 (CVB3) myocarditis can lead to dilative cardiomyopathy and heart failure. Cardiac injury is mediated by direct viral damage and host immune response. FOXO transcription factors play key roles in immunoregulation and stress resistance, including anti-inflammatory effects. We

ADIPONECTIN PROMOTES CARDIAC REMODELING IN INFLAMMATORY HEART DISEASE BY INDUCTION OF MATRIX METALLOPROTEINASE-9 ACTIVITY

Background: Adiponectin (APN) is an immunomodulatory adipocytokine that has been shown to attenuate left ventricular hypertrophy and to diminish fibrosis after myocardial infarction. Coxsackievirus B3 (CVB3) causes severe myocarditis, which might progress to dilated cardiomyopathy. Here, we investigated whether APN modulates cardiac remodeling in CVB3 myocarditis by affecting matrix metalloproteinase (MMP) activity.