Association of TIM-3 expression with glucose metabolism in Jurkat T cells.

BACKGROUND: T cell activation is associated with increase in glycolysis and glutaminolysis. T cell immunoglobulin and mucin domain containing protein-3 (TIM-3), a T cell surface molecule, downregulates T cell activation and leads to insufficient immunity in cancer and chronic infection. TIM-3 regulates T cell activation possibly through alterations in metabolism; however, the relationship between TIM-3 expression and T cell metabolic changes has not been well studied. RESULTS: We investigated the association between TIM-3 expression and metabolic changes by analyzing glucose metabolism, glutamine metabolism, and mitochondrial function in TIM-3 overexpressing or knockout Jurkat T cell lines relative to their control cell lines. Glucose uptake and consumption, and lactate release were downregulated by TIM-3 expression but upregulated by TIM-3 knockout. Concomitantly, the expression of the glucose transporter, Glut1, but not Glut2, 3, or 4 was altered by TIM-3 expression. However, TIM-3 expression alone could not account for the change in glutamine consumption, glutamate release, and mitochondrial mass, ROS production or membrane potential in these cell lines. CONCLUSION: Our results show the association of TIM-3 expression with T cell glucose metabolism. These results are significant in chronic infections and cancers where it is necessary to control TIM-3 expressing T cells.

Ginsenoside rb1 and rg3 attenuate glucocorticoid-induced neurotoxicity.

Glucocorticoid (GC) hormones, increased in response to stress, can cause neuronal loss. We tested the effect of GC hormone on cell viability of neural SHSY-5Y cells and protective effects of ginsenoside Rb1 and Rg3 on the action of GC. We treated SHSY-5Y cells with increasing concentrations of synthetic GC dexamethasone (DEX; 10, 25, 50, and 100 nM) for 24 and 48 h, and then determined cell viability by using MTT assay. We then treated SHSY-5Y cells with DEX (100 nM) with or without the ginsenosides to examine their preventive effects on the cytotoxicity. To explore the underlying molecular mechanisms, we measured mRNA expression of bax and bcl-2 by using reverse transcriptase real-time PCR. SHSY-5Y cells treated with DEX significantly reduced cell viability as compared with control cells. In the presence of Rb1 or Rg3, DEX-induced cytotoxicity was effectively blocked. DEX considerably increased pro-apoptotic bax mRNA expression as compared with control cells. However, Rb1 and Rg3 completely blocked DEX-mediated up-regulation of bax expression. DEX significantly increased neuronal death in organotypic hippocampal slice cultures of rat brain with enhanced generation of ROS, which was effectively inhibited by ginsenoside Rb1 and Rg3. This suggests a potential role of the ginsenosides to target GC action in the brain.

Mechanism of glucocorticoid-induced oxidative stress in rat hippocampal slice cultures.

Prolonged stress results in elevation of glucocorticoid (GC) hormones, which can have deleterious effects in the brain. The hippocampus, which has a high concentration of glucocorticoid receptors, is especially vulnerable to increasing levels of GCs. GCs have been suggested to endanger hippocampal neurons by exacerbating the excitotoxic glutamate-calcium-reactive oxygen species (ROS) cascade. In an effort to reveal the mechanisms underlying GC-mediated hippocampal neurotoxicity, we aimed to clarify the molecular pathway of GC-induced ROS increase by using organotypic hippocampal slice cultures. Assays for ROS, using 2',7'-dichlorodihydrofluorescein diacetate fluorescence, showed that treatment of synthetic GC, dexamethasone (DEX) significantly enhanced ROS levels. Time course and dose response analyses indicated that peak amount of ROS was generated at 4 h after treatment with 50 micromol/L DEX. By contrast, other steroid hormones, progesterone and estradiol did not influence ROS production. N-acetyl-L-cysteine completely suppressed ROS produced by DEX. Propidium iodide staining exhibited prominent cell death in the hippocampal layer after 96 h of DEX treatment. RU486, a GC receptor antagonist, almost completely blocked the effect of DEX on ROS production and cell death, indicating that DEX-induced ROS overproduction and hippocampal death are mediated via GC receptors. Real-time reverse transcriptase PCR analysis demonstrated that after DEX treatment the level of glutathione peroxidase mRNA was decreased whereas that of NADPH oxidase mRNA was significantly enhanced. These findings suggest that excess GCs cause hippocampal damage by regulating genes involved in ROS generation.

Regulation of TIM-3 expression in a human T cell line by tumor-conditioned media and cyclic AMP-dependent signaling.

T cell immunoglobulin and mucin domain-3 (TIM-3) expression increases in exhausted T cells, which inhibits T cell function. TIM-3 expression is supposedly up-regulated in tumor-bearing individuals via chronic antigenic stimulation of T cells. Considering the immunosuppressive nature of the tumor microenvironment, we investigated whether tumor-secreted molecules might enhance TIM-3 expression in Jurkat T cells. We observed that TIM-3 expression was increased by the activation of prostaglandin (PG) E2 and cyclic AMP (cAMP) signaling pathways. Adenylate cyclase activation led to protein kinase A (PKA)-dependent upregulation of the TIM-3 minimal promoter region and of upstream conserved non-coding sequences. TIM-3 expression in Jurkat T cells was increased by the exposure to breast tumor cell-conditioned media partially through the interaction between PGE2 and its receptor, EP4. Our results propose that tumor-secreted molecules such as PGE2, which activates PKA and EPAC, may regulate TIM-3 expression in T cells.

Silver nanoparticles induce reactive oxygen species-mediated cell cycle delay and synergistic cytotoxicity with 3-bromopyruvate in Candida albicans, but not in Saccharomyces cerevisiae.

Background: Silver nanoparticles (AgNPs) inhibit the proliferation of various fungi; however, their mechanisms of action remain poorly understood. To better understand the inhibitory mechanisms, we focused on the early events elicited by 5 nm AgNPs in pathogenic Candida albicans and non-pathogenic Saccharomyces cerevisiae. Methods: The effect of 5 nm and 100 nm AgNPs on fungus cell proliferation was analyzed by growth kinetics monitoring and spot assay. We examined cell cycle progression, reactive oxygen species (ROS) production, and cell death using flow cytometry. Glucose uptake was assessed using tritium-labeled 2-deoxyglucose. Results: The growth of both C. albicans and S. cerevisiae was suppressed by treatment with 5 nm AgNPs but not with 100 nm AgNPs. In addition, 5 nm AgNPs induced cell cycle arrest and a reduction in glucose uptake in both fungi after 30 minutes of culture in a dose-dependent manner (P<0.05). However, in C. albicans only, an increase in ROS production was detected after exposure to 5 nm AgNPs. Concordantly, an ROS scavenger blocked the effect of 5 nm AgNPs on the cell cycle and glucose uptake in C. albicans only. Furthermore, the growth-inhibition effect of 5 nm AgNPs was not greater in S. cerevisiae mutant strains deficient in oxidative stress response genes than it was in wild type. Finally, 5 nm AgNPs together with a glycolysis inhibitor, 3-bromopyruvate, synergistically enhanced cell death in C. albicans (P<0.05) but not in S. cerevisiae. Conclusion: AgNPs exhibit antifungal activity in a manner that may or may not be ROS dependent, according to the fungal species. The combination of AgNPs with 3-bromopyruvate may be more useful against infection with C. albicans.

The Suppressive Effect of Butyrate and Bromopyruvate on Inflammatory Cytokine Production and Short Chain Fatty Acid Receptor Expression by Blood Mononuclear Cells in Patients with Behçet's Disease.

BACKGROUND: Controlling inflammation is a therapeutic goal of various autoimmune/autoinflammatory diseases including Behçet's disease (BD). The immunomodulatory effect of metabolites or metabolic analogs such as butyrate and 3-bromopyruvate has been observed in animal disease models. OBJECTIVE: We attempted to evaluate the effect of butyrate and 3-bromopyruvate on the inflammatory cytokine production by peripheral blood mononuclear cells (PBMCs) isolated from patients with mucocutaneous involvement of BD. METHODS: PBMCs isolated from 11 patients with BD and 10 healthy controls were stimulated with lipopolysaccharide in the presence of butyrate or 3-bromopyruvate. Butyrate receptor and cytokine messenger ribonucleic acid (mRNA) expression was analyzed by real-time reverse transcription polymerase chain reaction. Cytokine secretion was assessed by enzyme-linked immunosorbent assay. PBMCs survival was analyzed by flow cytometry. RESULTS: Bromopyruvate or butyrate treatment suppressed inflammatory cytokine production in PBMCs from all our subjects. Bromopyruvate also reduced PBMCs survival while butyrate did not. As the effect of butyrate was slightly greater in BD patients than in healthy controls, we analyzed butyrate receptor expression and found that lipopolysaccharide-induced free fatty acid receptor 2 mRNA level in PBMCs was higher in BD patients than in controls. CONCLUSION: We propose bromopyruvate and butyrate as supplementary therapeutic candidates to control inflammation in patients with BD.

Activation of microglial cells by ceruloplasmin.

Ceruloplasmin (Cp) is the major copper transport protein in plasma and catalyzes the conversion of toxic ferrous iron to the safer ferric iron. As an acute-phase protein, Cp is induced during inflammation. It is synthesized primarily in the liver and is expressed in several other tissues, including the brain. Elevated Cp levels have been observed in the brain of patients with neurodegenerative conditions, including Alzheimer's, Parkinson's, and Huntington's diseases. However, the exact role(s) of Cp in inflammatory and neuropathological conditions remains unclear. Microglia are the prime effector cells involved in immune and inflammatory responses in the central nervous system (CNS). They are activated during pathological conditions to restore CNS homeostasis, but chronic microglial activation endangers neuronal survival. Consequently, it is important to identify the regulators of microglial activation and the underlying mechanisms. We sought to examine whether Cp might modulate microglial activation. We observed that Cp induced nitric oxide (NO) release and inducible NO synthase mRNA expression in BV2 microglial cells and rat brain microglia. Cp also increased levels of mRNAs encoding tumor necrosis factor-alpha, interleukin-1beta, cyclooxygenase-2, and NADPH oxidase. Treatment of BV2 cells and primary microglia with Cp induced phosphorylation of p38 MAP kinase. Moreover, Cp induced nuclear factor (NF)-kappaB activation, showing a more sustained pattern than seen with bacterial lipopolysaccharide. Cp-stimulated NO induction was significantly attenuated by a p38 inhibitor, SB203580, and the NF-kappaB inhibitor SN50. Cp induced secretion of TNF-alpha and prostaglandin E(2) in primary microglial cultures. These results suggest that Cp may play an important role in neuropathological conditions by stimulating various proinflammatory and neurotoxic molecules in microglia.

The water extract of adlay seed (Coix lachrymajobi var. mayuen) exhibits anti-obesity effects through neuroendocrine modulation.

To find out whether the immunohistochemical expression of neuropeptid Y (NPY) and leptin receptor (LR) in the rat hypothalamus is influenced by adlay seed water extract (adlay), obesity in rats was induced by high fat diet (HFD) for 8 weeks; these rats were injected with 50 mg/100 g body weight adlay daily for 4 weeks. The results showed that the optical density of NPY immunoreactivity in paraventricular nucleus of rats increased approximately by 3.4 fold in HFD group compared to the normal diet group. Conversely, that of HFD + adlay group was about 2.6 fold lower than HFD group. The pattern of LR expression was similar to that of NPY. Both of NPY and LR mRNA levels, determined by real time PCR, in HFD + adlay group were decreased compared to those of HFD group, but there were no significant changes in the level of LR. These results suggest that adlay may regulate neuroendocrine activity in the brain. Accordingly, administration of adlay may be considered for therapies targeting obesity.

Transcription Factors Regulating Inflammatory Cytokine Production Are Differentially Expressed in Peripheral Blood Mononuclear Cells of Behçet Disease Depending on Disease Activity.

BACKGROUND: Behçet disease (BD) is a relapsing inflammatory disease with increased production of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs); however, the underlying molecular mechanisms are not well known. OBJECTIVE: To analyze whether the differential expression of transcription factors is involved in the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by PBMCs of BD patients compared to healthy controls (HCs). METHODS: Expression of transcription factors was examined by real-time reverse transcriptase-polymerase chain reaction and western blotting. Cytokine production by CD11b+ cells transfected with siRNAs against transcription factors was measured by enzyme-linked immunosorbent assay. RESULTS: In the absence of lipopolysaccharide stimulation, the transcript level of CCAAT-enhancer-binding proteins (C/EBP) ß was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBPδ transcript level was higher in PBMCs from patients with active BD than in those from HCs. The activating transcription factor 3 (ATF3) transcript level was increased in PBMCs from patients with stable BD compared to that in PBMCs from HCs. siRNAs targeting C/EBPß and C/EBPδ significantly reduced the production of IL-6 and TNF-α in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs. CONCLUSION: We found differential expression of C/EBPß, C/EBPδ, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.

MicroRNAs differentially expressed in Behçet disease are involved in interleukin-6 production.

BACKGROUND: Behcet's disease (BD) is characterized by systemic recurrent inflammation with increased production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 by peripheral blood mononuclear cells (PBMCs). To gain insight into the underlying mechanisms of this disease, the expression levels of distinct microRNAs in PBMCs of BD patients were determined and their association with TNF-α and IL-6 production was evaluated. FINDINGS: The expression levels of microRNAs, miR-638 and miR-4488, were reduced in patients with stable BD in comparison with healthy controls. In addition, the expression of miR-3591-3p was increased in patients with active BD when compared to patients with stable BD. Transfection of miR-638 and miR-4488 inhibitors, together with miR-3591-3p mimics, increased IL-6 mRNA levels in THP-1 cells in response to LPS stimulation. CONCLUSIONS: We observed differential expression of microRNAs associated with increased production of IL-6 in BD patients.

Silver nanoparticles affect glucose metabolism in hepatoma cells through production of reactive oxygen species.

The silver nanoparticle (AgNP) is a candidate for anticancer therapy because of its effects on cell survival and signaling. Although numerous reports are available regarding their effect on cell death, the effect of AgNPs on metabolism is not well understood. In this study, we investigated the effect of AgNPs on glucose metabolism in hepatoma cell lines. Lactate release from both HepG2 and Huh7 cells was reduced with 5 nm AgNPs as early as 1 hour after treatment, when cell death did not occur. Treatment with 5 nm AgNPs decreased glucose consumption in HepG2 cells but not in Huh7 cells. Treatment with 5 nm AgNPs reduced nuclear factor erythroid 2-like 2 expression in both cell types without affecting its activation at the early time points after AgNPs' treatment. Increased reactive oxygen species (ROS) production was detected 1 hour after 5 nm AgNPs' treatment, and lactate release was restored in the presence of an ROS scavenger. Our results suggest that 5 nm AgNPs affect glucose metabolism by producing ROS.

The regulation of TIM-3 transcription in T cells involves c-Jun binding but not CpG methylation at the TIM-3 promoter.

Tim-3 is an immunomodulatory protein that is expressed constitutively on monocytes but is induced in activated T cells. The mechanisms involved in the regulation of TIM-3 transcription are poorly understood. In the present study, we investigated whether methylation of the TIM-3 promoter is involved in regulatingTIM-3 transcription in T cells, and identified a transcription factor that regulates TIM-3 transcription by associating with the TIM-3 minimal promoter region. Pyrosequencing of the TIM-3 promoter up to -1440bp revealed 11 hypermethylated CpG sites and 4 hypomethylated CpG sites in human CD4(+) T cells as well as in CD11b(+) cells. Dimethylation of histone H3 lysine 4 (H3K4), a mark of transcriptional activation, was predominantly found in the proximal TIM-3 promoter -954 to -34bp region, whereas trimethylation of H3K9 and H3K27, which are markers of transcriptional suppression, were mostly observed in the distal promoter -1549 to -1048bp region in human CD4(+) T cells and CD11b(+) cells. However, no change in the methylation status of CpG sites and the histone H3 in the TIM-3 promoter was found during induction of TIM-3 transcription in T cells. Finally, AP-1 involvement in TIM-3 transcription was shown in relation with the TIM-3 minimal promoter -146 to +144bp region. The present study defines the minimal TIM-3 promoter region and demonstrates its interaction with c-Jun during TIM-3 transcription in CD4(+) T cells.

Induction of TGF-beta-inducible gene-h3 (betaig-h3) by TGF-beta1 in astrocytes: implications for astrocyte response to brain injury.

Transforming growth factor (TGF)-beta-inducible gene-h3 (betaig-h3) product is a secreted protein that is induced by TGF-beta in several cell types and implicated in various tissue pathologies. The aims of this study were to determine the effect of TGF-beta1 on betaig-h3 expression in cultured astrocytes and to examine whether betaig-h3 is expressed in the brain after traumatic injury. The results showed that betaig-h3 mRNA and protein increased in response to TGF-beta1 in U87 human astrocytoma cells and mouse cortical astrocytes. Treatment with other cytokines, including tumor necrosis factor-alpha and fibroblast growth factor-2, did not enhance the expression of betaig-h3 in astrocytes. betaig-h3 was significantly expressed in reactive astrocytes at the site of a stab wound in the cerebral cortex of adult rats. These results provide an insight into understanding a novel role for betaig-h3 protein in the response of astrocytes to brain injury.

Down-regulation of the expression of rat inhibitor-of-apoptosis protein-1 and -3 during transforming growth factor-beta1-mediated apoptosis in rat brain microglia.

Transforming growth factor (TGF)-beta1 is a key regulator of brain response to injury and inflammation. It exerts anti-inflammatory roles by inhibiting microglial proliferation and free radical induction. TGF-beta1 is known to induce apoptotic cell death of microglia in a Bcl-2-independent pathway. The purpose of this study was to examine detailed mechanisms of TGF-beta1-induced microglial apoptosis. Assays for cell viability and DNA fragmentation demonstrated that TGF-beta1 induced apoptotic cell death in primary rat microglial cultures. Reverse transcription (RT)-PCR analysis showed that primary microglial cells expressed mRNAs for rat inhibitor-of-apoptosis protein (RIAP)-1 and RIAP-3 under normal culture conditions and that treatment with TGF-beta1 resulted in a significant reduction in the amounts of RIAP-1 and RIAP-3 mRNAs. Because IAPs are potent suppressor of apoptotic cell death, decrease in IAP expression might provide an important regulatory function in TGF-beta1-mediated microglial death and in attenuation of excessive microglial activation in pathological conditions.

Reduction but not cleavage of poly(ADP-ribose) polymerase during stress-mediated cell death in the rat hippocampus.

Sustained stress induces neuronal atrophy and death, especially in the hippocampus, which impairs hippocampal function. However, underlying mechanisms of stress-induced neuronal damage have not been precisely defined. We analyzed the molecular events related to apoptosis in the hippocampus of rats exposed to immobilization stress. Terminal dUTP nick end-labeling exhibited positive nuclei in the hippocampus of stressed rats, indicating DNA fragmentation. RT-PCR and Western blot analyses showed that immobilization stress increased and decreased the expression of pro-apoptotic gene bax and anti-apoptotic bcl-2 genes, respectively. Western blot analysis demonstrated that the characteristic 85 kDa apoptotic fragment of poly(ADP-ribose) polymerase (PARP) was not observed in the hippocampus subjected to immobilization stress. The amount of PARP protein was significantly reduced following stress. This study may provide a novel insight into molecular mechanisms implicated in hippocampal damage associated with stress.

Immobilization stress induces the expression of alphaB-crystallin in rat hippocampus: implications of glial activation in stress-mediated hippocampal degeneration.

Overexpression and abnormal aggregation of a small heat shock protein, B-crystallin, in reactive astrocytes has been implicated in various neurodegenerative diseases and brain tumors. To investigate the potential involvement of glial activation in stress-induced hippocampal damage, we analyzed B-crystallin gene expression in the hippocampus of immobilized rats. Male Sprague-Dawley rats were subjected to single or repeated immobilization stress. Both single and repeated immobilization markedly increased the level of B-crystallin mRNA in the hippocampus. Repeated immobilization notably accumulated B-crystallin protein, with a substantial portion in the insoluble fraction. Treatment of primary cortical astrocytes with stress-related glucocorticoid resulted in considerable elevation of B-crystallin expression. The accumulation of B-crystallin may serve as one of the important pathogenic mechanisms involved in hippocampal degeneration associated with stress.

Transforming growth factor-beta-inducible gene-h3 (beta(ig)-h3) promotes cell adhesion of human astrocytoma cells in vitro: implication of alpha6beta4 integrin.

Beta(ig)-h3 is a secretory protein that is induced by transforming growth factor (TGF)-beta. We have recently found that beta(ig)-h3 expression is induced in cultured astrocytes by TGF-beta1 and in rat cerebral cortex by stab wound. The purpose of this study was to examine the effect of the secreted beta(ig)-h3 on cell adhesion of astrocytes and the underlying mechanisms. When U87 human astrocytoma cells were seeded on dishes coated with recombinant beta(ig)-h3, cell adhesion was significantly enhanced. Blocking experiments using various antibodies to the integrin subunit suggested alpha6beta4 integrin could be involved in the beta(ig)-h3-mediated astrocyte cell adhesion. Cell adhesion to beta(ig)-h3 substrate was substantially blocked by preincubation with the inhibitor to the src kinase. When cells were plated on beta(ig)-h3-coated dishes, tyrosine phosphorylation of focal adhesion kinase was prominently increased within 20 min in a beta4 integrin-dependent manner. The results suggest that alpha6beta4 integrin-mediated interactions of astrocytes with beta(ig)-h3 transduce intracellular signals through the focal adhesion proteins, which may regulate certain aspects of astrocyte response to brain injury.

Effect of electroacupuncture on the stress-induced changes in brain-derived neurotrophic factor expression in rat hippocampus.

Stress induces neuronal atrophy and death especially in the hippocampus. Alterations in the expression of neurotrophic factors are implicated in stress-induced hippocampal degeneration. In the hippocampus, stress decreases brain-derived neurotrophic factor (BDNF) mRNA levels. In oriental medicine, acupuncture has long been employed as a treatment of numerous disorders. The objective of this study was to examine whether electroacupuncture (EA) stimulation can influence BDNF expression in the hippocampus of rats exposed to immobilization stress. Rats were immobilized in plastic bags, and then subjected to EA at ST36 Zusanli. After treatment, the animals were decapitated and the hippocampi were rapidly removed and processed for RNA isolation and reverse transcription. Real-time polymerase chain reaction analysis showed that EA stimulation significantly restored BDNF mRNA expression declined by immobilization stress. The results suggest that EA may relieve neuropathological effects of stress by modulating neurotrophic factor expression.

Hypolipidemic effects of crude extract of adlay seed (Coix lachrymajobi var. mayuen) in obesity rat fed high fat diet: relations of TNF-alpha and leptin mRNA expressions and serum lipid levels.

To find out whether the expressions of these adipocyte markers are influenced by oriental medicine, obesity rats induced by high fat diet (HFD) for 8 weeks were injected with 50 mg/100 g body weight adlay seed crude extract (ACE), daily for 4 weeks. The results are summarized as follows: HFD + ACE group significantly reduced food intakes and body weights. Weights of epididymal and peritoneal fat were dramatically increased in HFD groups compared with those of normal diet (ND) group but significantly decreased more in HFD + ACE group than those of HFD + saline group (sham). Those of brown adipocytes were increased in HFD + ACE group compared to ND and sham groups but there was no significant difference. The sizes in white adipose tissue (WAT) by microscope were markedly larger in HFD groups than ND group but considerably reduced in HFD + ACE group compared with sham group. The levels of triglyceride, total-cholesterol and leptin in blood serum were significantly decreased in HFD + ACE group compared to those of sham group. Leptin and TNF-alpha mRNA expressions in WAT of rats were remarkably increased more in sham group than in those of ND group. Those of HFD + ACE group were significantly decreased compared with those of sham group, especially. TNF-alpha mRNA expression in HFD + ACE group was declined more than that of ND group. In conclusion, treatments of ACE modulated expressions of leptin and TNF-alpha and reduced body weights, food intake, fat size, adipose tissue mass and serum hyperlipidemia in obesity rat fed HFD. Accordingly, the oriental medicine extract, adlay seed crude extract, can be considered for obesity therapies controlling.