Eduardo J. Folco

Interferon-γ, a Th1 Cytokine, Regulates Fat Inflammation. A Role for Adaptive Immunity in Obesity

Adipose tissue (AT) can accumulate macrophages and secrete several inflammatory mediators. Despite its pivotal role in the progression of chronic inflammatory processes such as atherosclerosis, the adaptive role of immunity in obesity remains poorly explored. Visceral AT of diet-induced obese C57BL/6 mice had higher numbers of both CD4+ and CD8+ T cells than lean controls, monitored by flow cytometry. When stimulated in vitro, T cells from obese AT produced more interferon (IFN)&ggr; than those from controls. AT from obese animals also had more cells expressing I-Ab, a mouse class II histocompatibility marker implicated in antigen presentation, as determined by immunostaining. Differentiated 3T3-L1 cells stimulated with recombinant IFN&ggr; or T-helper 1–derived supernatant produced several chemokines and their mRNAs. Obese IFN&ggr;-deficient animals had significantly reduced AT expression of mRNA-encoding inflammatory genes such as tumor necrosis factor-&agr; and monocyte chemoattractant protein-1, decreased AT inflammatory cell accumulation, and better glucose tolerance than control animals consuming the same diet. Obese mice doubly deficient for IFN&ggr; receptor and apolipoprotein (Apo)E on a mixed 129SvEv/C57BL/6 (129/B6) genetic background, despite exhibiting similar AT mRNA levels of tumor necrosis factor-&agr; and monocyte chemoattractant protein-1 as 129/B6-ApoE−/− controls, had decreased expression of important T cell–related genes, such as IFN&ggr;-inducible protein-10 and I-Ab, and lower plasma triglycerides and glucose. These results indicate a role for T cells and IFN&ggr;, a prototypical T-helper 1 cytokine, in regulation of the inflammatory response that accompanies obesity.

Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome

Long QT syndrome (LQTS) is a heritable disease associated with ECG QT interval prolongation, ventricular tachycardia, and sudden cardiac death in young patients. Among genotyped individuals, mutations in genes encoding repolarizing K+ channels (LQT1:KCNQ1; LQT2:KCNH2) are present in approximately 90% of affected individuals. Expression of pore mutants of the human genes KCNQ1 (KvLQT1-Y315S) and KCNH2 (HERG-G628S) in the rabbit heart produced transgenic rabbits with a long QT phenotype. Prolongations of QT intervals and action potential durations were due to the elimination of IKs and IKr currents in cardiomyocytes. LQT2 rabbits showed a high incidence of spontaneous sudden cardiac death (>50% at 1 year) due to polymorphic ventricular tachycardia. Optical mapping revealed increased spatial dispersion of repolarization underlying the arrhythmias. Both transgenes caused downregulation of the remaining complementary IKr and IKs without affecting the steady state levels of the native polypeptides. Thus, the elimination of 1 repolarizing current was associated with downregulation of the reciprocal repolarizing current rather than with the compensatory upregulation observed previously in LQTS mouse models. This suggests that mutant KvLQT1 and HERG interacted with the reciprocal wild-type alpha subunits of rabbit ERG and KvLQT1, respectively. These results have implications for understanding the nature and heterogeneity of cardiac arrhythmias and sudden cardiac death.

Adiponectin Inhibits the Production of CXC Receptor 3 Chemokine Ligands in Macrophages and Reduces T-Lymphocyte Recruitment in Atherogenesis

Obese individuals often have low plasma adiponectin and concomitant chronic inflammation with a predisposition to metabolic and cardiovascular diseases. The present study reports a novel antiinflammatory action of adiponectin in human monocyte-derived macrophages (M&PHgr;) suppressing T-lymphocyte accumulation in atherogenesis. RNA profiling of lipopolysaccharide-stimulated human M&PHgr; identified CXC chemokine ligands (CXCLs), such as IP-10 (interferon [IFN]-inducible protein 10) (CXCL10), I-TAC (IFN-inducible T-cell α chemoattractant) (CXCL11), and Mig (monokine induced by IFN-γ) (CXCL9), T-lymphocyte chemoattractants associated with atherogenesis, among the top 14 transcripts suppressed by adiponectin. Real-time quantitative RT-PCR and ELISA verified that adiponectin inhibited expression of these chemokines at both the mRNA and protein levels in a concentration-dependent manner. Adiponectin reduced the release by lipopolysaccharide-stimulated M&PHgr; of chemoattractant activity for CXC chemokine receptor 3–transfected (receptor for IP-10, Mig, and I-TAC) lymphocytes. Adiponectin decreased lipopolysaccharide-inducible IP-10 promoter activity in promoter-transfected THP-1 M&PHgr; but did not change IP-10 mRNA stability. In lipopolysaccharide-stimulated M&PHgr;, reduction of IFN-β by adiponectin preceded inhibition of IP-10 mRNA expression. Immunoblot and chromatin immunoprecipitation analyses demonstrated that adiponectin attenuated activation of the transcription factor IFN regulatory factor 3, involved in the MyD88-independent pathway of Toll-like receptor 4 signaling, and subsequent IFN regulatory factor 3 binding to IFN-β promoter. In vivo studies further demonstrated that apolipoprotein E/adiponectin double-deficient (apoE −/−APN−/−) mice had increased plasma IP-10 levels, accelerated T-lymphocyte accumulation in atheromata, and augmented atherogenesis compared with apoE single-deficient (apoE−/−APN+/+) mice. This study establishes that low levels of adiponectin associated with obesity, the metabolic syndrome, and diabetes favor T-lymphocyte recruitment and contribute to adaptive immune response during atherogenesis.

Indocyanine Green Enables Near-Infrared Fluorescence Imaging of Lipid-Rich, Inflamed Atherosclerotic Plaques

Indocyanine green, a clinically approved near-infrared fluorescence imaging agent, rapidly targets atheromas for in vivo detection of lipid-rich, inflammatory plaques. Greenify Your Arteries Many have taken it upon themselves to “go green,” perhaps by turning down the thermostat, swapping out old light bulbs, or even buying a hybrid car. But who would have thought that even your heart doctor can take part in this green initiative? As described by Vinegoni et al., going green may be just what you and your arteries need to detect atherosclerotic plaques residing within them. Indocyanine green (ICG) is a Food and Drug Administration–approved dye for imaging the vascular system at near-infrared (NIR) wavelengths (~800 nm)—wavelengths that boast limited photon absorption by blood and low tissue autofluorescence. ICG is also quickly absorbed by lipid-rich plaques and cells, making it a potentially useful plaque-imaging agent. Vinegoni and colleagues decided to test this hypothesis in rabbit models of atherosclerosis. Lipid-rich, inflamed atheromas were induced in 19 cholesterol-fed rabbits with balloon injury of the aorta. Eight weeks after injury, rabbits received an injection of ICG. Only 45 min later, the animals were killed for fluorescence imaging, which showed strong focal signals in the abdominal aorta and iliac arteries—areas that colocalized with atherosclerotic plaques. Conversely, control animals showed minimal NIR fluorescence signal. The authors then performed in vivo NIR fluorescence imaging of ICG in live animals. Using a clinical-type intravascular guidewire and a previously described “pullback” technique, they were able to sense atheroma in the coronary arteries of five rabbits. The location of these plaques was confirmed by x-ray angiography and intravascular ultrasound. Furthermore, ICG localization to human atheroma was confirmed ex vivo with freshly resected carotid endarterectomy specimens from four patients. Together, these animal and human data suggest direct translation to the clinic and highlight the potential application of ICG as a routine, green screening tool for atherosclerosis. New high-resolution molecular and structural imaging strategies are needed to visualize high-risk plaques that are likely to cause acute myocardial infarction, because current diagnostic methods do not reliably identify at-risk subjects. Although molecular imaging agents are available for low-resolution detection of atherosclerosis in large arteries, a lack of imaging agents coupled to high-resolution modalities has limited molecular imaging of atherosclerosis in the smaller coronary arteries. Here, we have demonstrated that indocyanine green (ICG), a Food and Drug Administration–approved near-infrared fluorescence (NIRF)–emitting compound, targets atheromas within 20 min of injection and provides sufficient signal enhancement for in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. In vivo NIRF sensing was achieved with an intravascular wire in the aorta, a vessel of comparable caliber to human coronary arteries. Ex vivo fluorescence reflectance imaging showed high plaque target-to-background ratios in atheroma-bearing rabbits injected with ICG compared to atheroma-bearing rabbits injected with saline. In vitro studies using human macrophages established that ICG preferentially targets lipid-loaded macrophages. In an early clinical study of human atheroma specimens from four patients, we found that ICG colocalized with plaque macrophages and lipids. The atheroma-targeting capability of ICG has the potential to accelerate the clinical development of NIRF molecular imaging of high-risk plaques in humans.

Adiponectin Inhibits Pro-inflammatory Signaling in Human Macrophages Independent of Interleukin-10

Macrophages participate pivotally in the pathogenesis of many chronic inflammatory diseases including atherosclerosis. Adiponectin, a vasculoprotective molecule with insulin-sensitizing and anti-atherogenic properties, suppresses pro-inflammatory gene expression in macrophages by mechanisms that remain incompletely understood. This study investigated the effects of adiponectin on major pro-inflammatory signaling pathways in human macrophages. We demonstrate that pretreatment of these cells with adiponectin inhibits phosphorylation of nuclear factor κB inhibitor (IκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), induced by either lipopolysaccharide (LPS) or tumor necrosis factor (TNF) α, as well as STAT3 phosphorylation induced by interleukin-6 (IL6). Antagonism of IL10 by either neutralizing antibodies or siRNA-mediated silencing did not abrogate the anti-inflammatory actions of adiponectin, indicating that the ability of adiponectin to render human macrophages tolerant to various pro-inflammatory stimuli does not require this cytokine. A systematic search for adiponectin-inducible genes with established anti-inflammatory properties revealed that adiponectin augmented the expression of A20, suppressor of cytokine signaling (SOCS) 3, B-cell CLL/lymphoma (BCL) 3, TNF receptor-associated factor (TRAF) 1, and TNFAIP3-interacting protein (TNIP) 3. These results suggest that adiponectin triggers a multifaceted response in human macrophages by inducing the expression of various anti-inflammatory proteins that act at different levels in concert to suppress macrophage activation.

Inflammatory concepts of obesity.

Obesity, long considered a condition characterized by the deposition of inert fat, is now recognized as a chronic and systemic inflammatory disease, where adipose tissue plays a crucial endocrine role through the production of numerous bioactive molecules, collectively known as adipokines. These molecules regulate carbohydrate and lipid metabolism, immune function and blood coagulability, and may serve as blood markers of cardiometabolic risk. Local inflammatory loops operate in adipose tissue as a consequence of nutrient overload, and crosstalk among its cellular constituents-adipocytes, endothelial and immune cells-results in the elaboration of inflammatory mediators. These mediators promote important systemic effects that can result in insulin resistance, dysmetabolism and cardiovascular disease. The understanding that inflammation plays a critical role in the pathogenesis of obesity-derived disorders has led to therapeutic approaches that target different points of the inflammatory network induced by obesity.

Prostaglandin E Receptor Type 4-associated Protein Interacts Directly with NF-κB1 and Attenuates Macrophage Activation

Macrophage activation participates pivotally in the pathophysiology of chronic inflammatory diseases, including atherosclerosis. Through the receptor EP4, prostaglandin E2 (PGE2) exerts an anti-inflammatory action in macrophages, suppressing stimulus-induced expression of certain proinflammatory genes, including chemokines. We recently identified a novel EP4 receptor-associated protein (EPRAP), whose function in PGE2-mediated anti-inflammation remains undefined. Here we demonstrate that PGE2 pretreatment selectively inhibits lipopolysaccharide (LPS)-induced nuclear factor κB1 (NF-κB1) p105 phosphorylation and degradation in mouse bone marrow-derived macrophages through EP4-dependent mechanisms. Similarly, directed EPRAP expression in RAW264.7 cells suppresses LPS-induced p105 phosphorylation and degradation, and subsequent activation of mitogen-activated protein kinase kinase 1/2. Forced expression of EPRAP also inhibits NF-κB activation induced by various proinflammatory stimuli in a concentration-dependent manner. In co-transfected cells, EPRAP, which contains multiple ankyrin repeat motifs, directly interacts with NF-κB1 p105/p50 and forms a complex with EP4. In EP4-overexpressing cells, PGE2 enhances the protective action of EPRAP against stimulus-induced p105 phosphorylation, whereas EPRAP silencing in RAW264.7 cells impairs the inhibitory effect of PGE2-EP4 signaling on LPS-induced p105 phosphorylation. Additionally, EPRAP knockdown as well as deficiency of NF-κB1 in macrophages attenuates the inhibitory effect of PGE2 on LPS-induced MIP-1β production. Thus, PGE2-EP4 signaling augments NF-κB1 p105 protein stability through EPRAP after proinflammatory stimulation, limiting macrophage activation.

Adiponectin Induces Pro-inflammatory Programs in Human Macrophages and CD4+ T Cells

Background: Adiponectin modulates inflammatory diseases and dysmetabolism, conditions associated with immune cell activation. Results: Adiponectin elicits a pro-inflammatory response in human macrophages and promotes TH1 differentiation of isolated CD4+ T cells. Conclusion: The limited program of inflammatory activation induced by adiponectin likely desensitizes cells to further pro-inflammatory stimuli. Significance: Interplay of signals regulated by adiponectin may determine its net effect as an inflammatory modulator. Abundant experimental and clinical data support a modulatory role for adiponectin in inflammation, dysmetabolism, and disease. Because the activation of cells involved in innate and adaptive immunity contributes to the pathogenesis of diseases such as atherosclerosis and obesity, this study investigated the role of adiponectin in human macrophage polarization and T cell differentiation. Examination of the adiponectin-induced transcriptome in primary human macrophages revealed that adiponectin promotes neither classical (M1) nor alternative (M2) macrophage activation but elicits a pro-inflammatory response that resembles M1 more closely than M2. Addition of adiponectin to polyclonally activated CD4+ T lymphocytes did not affect cell proliferation but induced mRNA expression and protein secretion of interferon (IFN)-γ and interleukin (IL)-6. Adiponectin treatment of CD4+ T cells increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and signal transducer and activation of transcription (STAT) 4 and augmented T-bet expression. Inhibition of p38 with SB203580 abrogated adiponectin-induced IFN-γ production, indicating that adiponectin enhances TH1 differentiation through the activation of the p38-STAT4-T-bet axis. Collectively, our results demonstrate that adiponectin can induce pro-inflammatory functions in isolated macrophages and T cells, concurring with previous observations that adiponectin induces a limited program of inflammatory activation that likely desensitizes these cells to further pro-inflammatory stimuli.

Identification of two alkaline proteases and a trypsin inhibitor from muscle of white croaker (Micropogon opercularis)

Extracts from white croaker skeletal muscle showed two alkaline proteases and a trypsin inhibitor when they were chromatographed in DEAE‐Sephacel. The activity against azocasein was maximal at pH 8.5 and 9.1 for proteases I and II, respectively. Both enzymes showed optimum activity at 60° C. The molecular masses were found to be 132 kDa for protease 1,363 kDa for protease II, and 65 kDa for the inhibitor. Protease I showed the characteristics of a trypsin‐like enzyme, and protease II those of a SH‐enzyme. These proteins may play important roles in mechanisms of cellular proteolysis.

A Cellular Model for Long QT Syndrome TRAPPING OF HETEROMULTIMERIC COMPLEXES CONSISTING OF TRUNCATED Kv1.1 POTASSIUM CHANNEL POLYPEPTIDES AND NATIVE Kv1.4 AND Kv1.5 CHANNELS IN THE ENDOPLASMIC RETICULUM

We demonstrated that overexpression of a cRNA encoding a truncated potassium channel polypeptide that contains the NH2 terminus and the first transmembrane segment (Kv1.1N206Tag) abolished the expression of Kv1.1 and Kv1.5 outward currents in Xenopus oocytes (Babila, T., Moscucci, A., Wang, H., Weaver, F. E. & Koren, G. (1994) Neuron12, 615–626). Recently, we showed that expression ofKv1.1N206Tag in the heart of transgenic mice resulted in the creation of mice with prolongation of the surface electrocardiogram’s QT interval (London, B., Han, X., Folco, E. & Koren, G. (1996) Biophys. J. 70, A2601). To study the dominant negative mechanism of Kv1.1N206Tag, we overexpressed it in GH3 cells, a pituitary cell line expressing Kv1.5 and Kv1.4. RNase protection analysis comparing the steady-state levels of native Kv1.5 and Kv1.1N206Tag transcripts revealed an excess of Kv1.1N206Tag transcript. Immunoprecipitation analysis using 12CA5 monoclonal antibody detected a 25-kDa polypeptide in the transfected cells. The half-life of Kv1.1N206Tag was 2.6 h. Subcellular fractionation of cell lysates labeled with [35S]methionine revealed that Kv1.1N206Tagpolypeptide is detectable in the particulate (membranous) fraction, but not in the soluble (cytosol) fraction. A series of double immunoprecipitations with 12CA5 and polyclonal antibodies against Kv1.5 and Kv1.4 revealed that Kv1.1N206Tag forms heteromultimeric complexes with the native Kv1.4 and Kv1.5 polypeptides. The steady-state levels of Kv1.5 were not affected by the overexpression of Kv1.1N206Tag. Immunofluorescence colocalization and confocal microscopy analyses revealed thatKv1.1N206TagFlag did not reach the plasma membrane, and its distribution pattern was characteristic to that of a resident endoplasmic reticulum polypeptide. Our observations establish that the negative effect of Kv1.1N206Tag is mediated by the formation of heteromultimeric complexes with the native channels and by the retention of these complexes in the endoplasmic reticulum.