Non-Selective Cannabinoid Receptor Antagonists,Hinokiresinols Reduce Infiltration of Microglia/Macrophages into Ischemic Brain Lesions in Rat via Modulating 2-Arachidonolyglycerol-Induced Migration and Mitochondrial Activity

Growing evidence suggests that therapeutic strategies to modulate the post-ischemic inflammatory responses are promising approaches to improve stroke outcome. Although the endocannabinoid system has been emerged as an endogenous therapeutic target to regulate inflammation after stroke insult, the downstream mechanisms and their potentials for therapeutic intervention remain controversial. Here we identified trans- and cis-hinokiresinols as novel non-selective antagonists for two G-protein-coupled cannabinoid receptors, cannabinoid receptor type 1 and type 2. The Electric Cell-substrate Impedance Sensing and Boyden chamber migration assays using primary microglial cultures revealed that both hinokiresinols significantly inhibited an endocannabinoid, 2-arachidonoylglycerol-induced migration. Hinokiresinols modulated 2-arachidonoylglycerol-induced mitochondrial bioenergetics in microglia as evidenced by inhibition of ATP turnover and reduction in respiratory capacity, thereby resulting in impaired migration activity. In rats subjected to transient middle cerebral artery occlusion (1.5-h) followed by 24-h reperfusion, post-ischemic treatment with hinokiresinols (2 and 7-h after the onset of ischemia, 10 mg/kg) significantly reduced cerebral infarct and infiltration of ED1-positive microglial/macrophage cells into cerebral ischemic lesions in vivo. Co-administration of exogenous 2-AG (1 mg/kg, i.v., single dose at 2 h after starting MCAO) abolished the protective effect of trans-hinokiresionol. These results suggest that hinokiresinols may serve as stroke treatment by targeting the endocannabinoid system. Alteration of mitochondrial bioenergetics and consequent inhibition of inflammatory cells migration may be a novel mechanism underlying anti-ischemic effects conferred by cannabinoid receptor antagonists.     

Tibolone Reduces Oxidative Damage and Inflammation in Microglia Stimulated with Palmitic Acid through Mechanisms Involving Estrogen Receptor Beta

High concentrations of palmitic acid in plasma increase both the inflammation associated with obesity and the susceptibility to develop a neurodegenerative event. In the brain, the inflammatory response is mediated by activated microglial cells, which undergo morphological and biochemical changes and can directly affect cell viability. Recent evidence shows that the use of estrogenic compounds can control microglia-induced inflammation with promising results. In this study, we explored the actions of the synthetic steroid tibolone on BV-2 microglia cells stimulated with palmitic acid. Our results demonstrated that tibolone increased cell viability and reduced nuclear fragmentation and the production of reactive oxygen species, as well as preserved mitochondrial membrane potential. These effects were accompanied by reduced nuclear translocation of NF-κB p65, upregulation of neuroglobin, and improved antioxidant defense. Furthermore, estrogen receptor beta (ERβ) inhibition partially dampened tibolone’s protective actions in BV-2 cells stimulated with palmitic acid. In conclusion, tibolone protects BV-2 cells by a mechanism involving ERβ and upregulation of neuroglobin.     

Pharmacologic Blockade of JAK1/JAK2 Reduces GvHD and Preserves the Graft-Versus-Leukemia Effect

We have recently reported that interferon gamma receptor deficient (IFNγR−/−) allogeneic donor T cells result in significantly less graft-versus-host disease (GvHD) than wild-type (WT) T cells, while maintaining an anti-leukemia or graft-versus-leukemia (GvL) effect after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that IFNγR signaling regulates alloreactive T cell trafficking to GvHD target organs through expression of the chemokine receptor CXCR3 in alloreactive T cells. Since IFNγR signaling is mediated via JAK1/JAK2, we tested the effect of JAK1/JAK2 inhibition on GvHD. While we demonstrated that pharmacologic blockade of JAK1/JAK2 in WT T cells using the JAK1/JAK2 inhibitor, INCB018424 (Ruxolitinib), resulted in a similar effect to IFNγR−/− T cells both in vitro (reduction of CXCR3 expression in T cells) and in vivo (mitigation of GvHD after allo-HSCT), it remains to be determined if in vivo administration of INCB018424 will result in preservation of GvL while reducing GvHD. Here, we report that INCB018424 reduces GvHD and preserves the beneficial GvL effect in two different murine MHC-mismatched allo-HSCT models and using two different murine leukemia models (lymphoid leukemia and myeloid leukemia). In addition, prolonged administration of INCB018424 further improves survival after allo-HSCT and is superior to other JAK1/JAK2 inhibitors, such as TG101348 or AZD1480. These data suggest that pharmacologic inhibition of JAK1/JAK2 might be a promising therapeutic approach to achieve the beneficial anti-leukemia effect and overcome HLA-barriers in allo-HSCT. It might also be exploited in other diseases besides GvHD, such as organ transplant rejection, chronic inflammatory diseases and autoimmune diseases.     

Interleukin-6 Receptor Blockade Selectively Reduces IL-21 Production by CD4 T Cells and IgG4 Autoantibodies in Rheumatoid Arthritis

Interleukin-6 (IL-6) levels are known to be increased in patients with rheumatoid arthritis (RA). Tocilizumab, a monoclonal antibody to the IL-6 receptor (IL-6R), reduces disease activity in RA, although its mechanisms of action remain unclear. Since IL-6 regulates cytokine production by CD4 T cells during activation, we investigated whether treatment with tocilizumab altered the phenotype and cytokine production by CD4 T cells in patients with rheumatoid arthritis. We show here that tocilizumab treatment does not change the production of cytokines by naïve CD4 T cells. However, tocilizumab treatment causes a selective decrease of IL-21 production by memory/activated CD4 T cells. Since IL-21 is known to promote plasma cell differentiation, we examined the effect of tocilizumab on the production of autoantibodies. We show that there is a decrease in the levels of IgG4 anti-CCP antibodies, but there is no effect on IgG1 anti-CCP antibodies. In addition, we show that IL-21 is a powerful inducer of IgG4 production by B cells. Thus, IL-6 contributes to the presence of IgG4-specific anti-CCP autoantibodies in RA patients, likely through its effect on IL-21 production by CD4 T cells, and IL-6R blockade down-regulates this pathway.     

α2-Adrenergic Receptor Blockade Markedly Potentiates Duloxetine- and Fluoxetine-Induced Increases in Noradrenaline, Dopamine, and Serotonin Levels in the Frontal Cortex of Freely Moving Rats

Abstract: Evidence exists that a reinforcement in monoaminergic transmission in the frontal cortex (FCX) is associated with antidepressant (AD) properties. Herein, we examined whether blockade of α₂-adrenergic receptors modified the influence of monoamine reuptake inhibitors on FCX levels of serotonin (5-HT), noradrenaline (NAD), and dopamine (DA). The selective α₂-adrenergic receptor agonist S 18616 (0.16 mg/kg, s.c.) suppressed extracellular levels of NAD, DA, and 5-HT (by 100, 51, and 63%, respectively) in single dialysates of FCX of freely moving rats. In contrast, the selective α₂-adrenergic receptor antagonists atipamezole (0.16 mg/kg, s.c.) and 1-(2-pyrimidinyl)piperazine (1-PP; 2.5 mg/kg, s.c.) increased levels of NAD (by 180 and 185%, respectively) and DA (by 130 and 90%, respectively), without affecting 5-HT levels. Duloxetine (5.0 mg/kg, s.c.), a mixed inhibitor of 5-HT and NAD reuptake, and fluoxetine (10.0 mg/kg, s.c.), a selective 5-HT reuptake inhibitor, both increased levels of 5-HT (by 150 and 120%, respectively), NAD (by 400 and 100%, respectively), and DA (by 115 and 55%, respectively). Atipamezole (0.16 mg/kg, s.c.) markedly potentiated the influence of duloxetine and fluoxetine on levels of 5-HT (by 250 and 330%, respectively), NAD (by 1,030 and 215%, respectively), and DA (by 370 and 170%, respectively). 1-PP similarly potentiated the influence of duloxetine on 5-HT, NAD, and DA levels (by 290, 1,320, and 600%, respectively). These data demonstrate that α₂-adrenergic receptors tonically inhibit NAD and DA and phasically inhibit 5-HT release in the FCX and that blockade of α₂-adrenergic receptors strikingly potentiates the increase in FCX levels of 5-HT, NAD, and DA elicited by reuptake inhibitors. Concomitant α₂-adrenergic receptor antagonism and inhibition of monoamine uptake may thus provide a mechanism allowing for a marked increase in the efficacy of AD agents.     

Momordica charantia (Bitter Melon) Reduces Obesity-Associated Macrophage and Mast Cell Infiltration as well as Inflammatory Cytokine Expression in Adipose Tissues

Obesity is a world-wide epidemic disease that correlates closely with type 2 diabetes and cardiovascular diseases. Obesity-induced chronic adipose tissue inflammation is now considered as a critical contributor to the above complications. Momordica charantia (bitter melon, BM) is a traditional Chinese food and well known for its function of reducing body weight gain and insulin resistance. However, it is unclear whether BM could alleviate adipose tissue inflammation caused by obesity. In this study, C57BL/6 mice were fed high fat diet (HFD) with or without BM for 12 weeks. BM-contained diets ameliorated HFD-induced obesity and insulin resistance. Histological and real-time PCR analysis demonstrated BM not only reduced macrophage infiltration into epididymal adipose tissues (EAT) and brown adipose tissues (BAT). Flow cytometry show that BM could modify the M1/M2 phenotype ratio of macrophages in EAT. Further study showed that BM lowered mast cell recruitments in EAT, and depressed pro-inflammatory cytokine monocyte chemotactic protein-1 (MCP-1) expression in EAT and BAT as well as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression in EAT. Finally, ELISA analysis showed BM-contained diets also normalized serum levels of the cytokines. In summary, in concert with ameliorated insulin resistance and fat deposition, BM reduced adipose tissue inflammation in diet-induced obese (DIO) mice.     

体外共培养模式下神经干细胞对视网膜小胶质细胞生物学功能的影响

目的:通过构建体外共培养体系,探讨神经干细胞(NSCs)对脂多糖(LPS)活化后的视网膜小胶质细胞(RMG)生物学功能的影响及TIMP1/MMP9途径在其中的可能作用。方法:采用振荡分离法获取C57/BL6小鼠原代RMG,通过免疫荧光技术检测细胞Iba1的表达对其进行鉴定。采用含有LPS的培养基(终浓度为1μg·mL~(-1))刺激RMG 24h后,将其分为LPS对照组、NSCs组、TB-NSCs组,其中NSCs组将RMG与NSCs共培养24 h,TB-NSCs组将RMG与用中和性抗体封闭TIMP1的NSCs共培养24h;同时,未予以LPS刺激的RMG作为空白对照组。采用免疫荧光技术检测各组RMG的Ki67表达情况,观察其增殖能力;TUNEL技术检测各组RMG凋亡情况;ELISA方法检测各组RMG上清液中TNF-α、IL~(-1)β的蛋白质量浓度。结果:采用振荡分离法获取的原代RMG经免疫荧光染色鉴定Iba1呈阳性。NSCs组Ki67阳性率较LPS对照组降低(P0.05),而TB-NSCs组Ki67阳性率较NSCs组升高(P0.05)。NSCs组TUNEL阳性率较LPS对照组明显升高(P0.05),而TB-NSCs组TUNEL阳性率与NSCs组间差异无统计学意义(P0.05)。空白对照组、LPS对照组、NSCs组、TB-NSCs组RMG上清液中TNF-α蛋白质量浓度分别为(2.10±0.65)、(25.69±2.01)、(20.01±1.63)、(23.76±1.45)ng·mL~(-1),总体比较差异显著(FTNF-α=302.65,PTNF-α0.05);IL~(-1)β蛋白质量浓度分别为(1.77±0.74)、(15.38±1.18)、(10.88±0.95)、(13.45±1.41)ng·mL~(-1),总体比较差异非常显著(FIL~(-1)β=179.84,PIL~(-1)β0.05);其中,NSCs组TNF-α及IL~(-1)β蛋白质量浓度均较LPS对照组显著降低(P0.05),TB-NSCs组TNF-α及IL~(-1)β蛋白质量浓度较NSCs组明显升高(P0.05)。结论:体外共培养模式下,NSCs可抑制RMG增殖能力,提高其凋亡水平,并抑制其分泌促炎因子TNF-α及IL~(-1)β,该效应可能与调控TIMP1/MMP9相关。     

Combining Regulatory T Cell Depletion and Inhibitory Receptor Blockade Improves Reactivation of Exhausted Virus-Specific CD8+ T Cells and Efficiently Reduces Chronic Retroviral Loads

Chronic infections with human viruses, such as HIV and HCV, or mouse viruses, such as LCMV or Friend Virus (FV), result in functional exhaustion of CD8⁺ T cells. Two main mechanisms have been described that mediate this exhaustion: expression of inhibitory receptors on CD8⁺ T cells and expansion of regulatory T cells (Tregs) that suppress CD8⁺ T cell activity. Several studies show that blockage of one of these pathways results in reactivation of CD8⁺ T cells and partial reduction in chronic viral loads. Using blocking antibodies against PD-1 ligand and Tim-3 and transgenic mice in which Tregs can be selectively ablated, we compared these two treatment strategies and combined them for the first time in a model of chronic retrovirus infection. Blocking inhibitory receptors was more efficient than transient depletion of Tregs in reactivating exhausted CD8⁺ T cells and reducing viral set points. However, a combination therapy was superior to any single treatment and further augmented CD8⁺ T cell responses and resulted in a sustained reduction in chronic viral loads. These results demonstrate that Tregs and inhibitory receptors are non-overlapping factors in the maintenance of chronic viral infections and that immunotherapies targeting both pathways may be a promising strategy to treat chronic infectious diseases.     

TRAIL/DR5 Signaling Promotes Macrophage Foam Cell Formation by Modulating Scavenger Receptor Expression

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been shown to have protective effects against atherosclerosis. However, whether TRAIL has any effects on expression of macrophage scavenger receptors and lipid uptake has not yet been studied. Macrophage lines RAW264.7 and THP-1, and mouse primary peritoneal macrophages, were cultured in vitro and treated with recombinant human TRAIL. Real-time PCR and western blot were performed to measure mRNA and protein expressions. Foam cell formation was assessed by internalization of acetylated and oxidized low-density lipoproteins (LDL). Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. We found that TRAIL treatment increased expression of scavenger receptor (SR)-AI and SR-BI in a time- and dose-dependent manner, and this effect was accompanied by increased foam cell formation. These effects of TRAIL were abolished by a TRAIL neutralizing antibody or in DR5 receptor-deficient macrophages. The increased LDL uptake by TRAIL was blocked by SR-AI gene silencing or the SR-AI inhibitor poly(I:C), while SR-BI blockade with BLT-1 had no effect. TRAIL-induced SR-AI expression was blocked by the inhibitor of p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2 or JNK. TRAIL also induced apoptosis in macrophages. In contrast to macrophages, TRAIL showed little effects on SR expression or apoptosis in vascular smooth muscle cells. In conclusion, our results demonstrate that TRAIL promotes macrophage lipid uptake via SR-AI upregulation through activation of the p38 pathway.     

Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

Adiponectin, an abundant adipose tissue-derived protein, exerts protective effect against cardiovascular disease. Adiponectin receptors (AdipoR1 and AdipoR2) mediate the beneficial effects of adiponectin on the cardiovascular system. However, the alteration of AdipoRs in cardiac remodeling is not fully elucidated. Here, we investigated the effect of angiotensin II (AngII) on cardiac AdipoRs expression and explored the possible molecular mechanism. AngII infusion into rats induced cardiac hypertrophy, reduced AdipoR1 but not AdipoR2 expression, and attenuated the phosphorylations of adenosine monophosphate-activated protein kinase and acetyl coenzyme A carboxylase, and those effects were all reversed by losartan, an AngII type 1 (AT1) receptor blocker. AngII reduced expression of AdipoR1 mRNA and protein in cultured neonatal rat cardiomyocytes, which was abolished by losartan, but not by PD123319, an AT2 receptor antagonist. The antioxidants including reactive oxygen species (ROS) scavenger NAC, NADPH oxidase inhibitor apocynin, Nox2 inhibitor peptide gp91 ds-tat, and mitochondrial electron transport chain complex I inhibitor rotenone attenuated AngII-induced production of ROS and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. AngII-reduced AdipoR1 expression was reversed by pretreatment with NAC, apocynin, gp91 ds-tat, rotenone, and an ERK1/2 inhibitor PD98059. Chromatin immunoprecipitation assay demonstrated that AngII provoked the recruitment of c-Myc onto the promoter region of AdipoR1, which was attenuated by PD98059. Moreover, AngII-induced DNA binding activity of c-Myc was inhibited by losartan, NAC, apocynin, gp91 ds-tat, rotenone, and PD98059. c-Myc small interfering RNA abolished the inhibitory effect of AngII on AdipoR1 expression. Our results suggest that AngII inhibits cardiac AdipoR1 expression in vivo and in vitro and AT1 receptor/ROS/ERK1/2/c-Myc pathway is required for the downregulation of AdipoR1 induced by AngII.     

Microglia with an Endothelin ETB Receptor Aggregate in Rat Hippocampus CA1 Subfields Following Transient Forebrain Ischemia

Abstract: We examined endothelin (ET) receptors in the hippocampus CA1 subfields of stroke-prone spontaneously hypertensive rats subjected to a 10-min bilateral carotid occlusion and reperfusion. When delayed neuronal death had occurred in the pyramidal cell layer at 7 days after transient forebrain ischemia, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of ¹²⁵I-ET-1 binding sites in the hippocampus CA1 subfields. The highest number of de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death. These binding sites were characteristically the ET_B receptor. The de novo ¹²⁵I-ET-1 binding was mainly present on microglia aggregating with a high density in the damaged pyramidal cell layer. As ET-1- and ET-3-like immunoreactivities were highly expressed within astrocytes in damaged neural tissue, the possibility that microglia with the ET_B receptor are activated to participate in the pathophysiology of ischemia-related neural tissue damage by astrocytic ET-1 and ET-3 produced in response to transient forebrain ischemia would have to be considered.     

BMP-7 Treatment Increases M2 Macrophage Differentiation and Reduces Inflammation and Plaque Formation in Apo E-/- Mice

Inflammation plays a fundamental role in the inception and development of atherosclerosis (ATH). Mechanisms of inflammation include the infiltration of monocytes into the injured area and subsequent differentiation into either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. We have previously published data suggesting bone morphogenetic protein-7 (BMP-7) enhances M2 macrophage differentiation and anti-inflammatory cytokine secretion in vitro. In this regard, we hypothesized BMP-7 would inhibit plaque formation in an animal model of ATH through monocytic plasticity mediation. ATH was generated in male and female Apo E^-/- mice via partial left carotid artery (PLCA) ligation and mice were divided into 3 groups: Sham, PLCA, and PLCA+BMP-7 (200ug/kg; i.v.). Our data suggest that BMP-7 inhibits plaque formation and increases arterial systolic velocity. Furthermore, we report inhibition of monocyte infiltration and a decrease in associated pro-inflammatory cytokines (MCP-1, TNF-α, and IL-6) in the PLCA+BMP-7 mice. In contrast, our data suggest a significant (p<0.05) increase in M2 macrophage populations with consequential enhanced anti-inflammatory cytokine (IL-1RA, IL-10, and Arginase 1) expression following BMP-7 treatment. We have also observed that mechanisms promoting monocyte into M2 macrophage differentiation by BMP-7 involve the upregulation and activation of the BMP-7 receptor (BMP-7RII). In conclusion, we report that BMP-7 has the potential to mediate cellular plasticity and mitigate the inflammatory immune response, which results in decreased plaque formation and improved blood velocity.     

慢性阻塞性肺病患者巨噬细胞功能与受体表达的关系研究

目的:研究慢性阻塞性肺病(COPD)患者气道巨噬细胞功能变化及其与受体表达的相关性。方法:将COPD患者84例按病情分为轻中度组44例,重度组40例,选取同期健康体检者40例作为对照组,获取3组诱导痰,分离痰巨噬细胞,检测3组吞噬荧光标记曲霉孢子的吞噬指数(PI),采用实时定量反转录PCR法检测3组吞噬相关受体的表达。结果:轻中度组与重度组细胞总数均多于对照组,而巨噬细胞比例却显著下降(P0.05);轻中度组与重度组巨噬细胞吞噬功能均受到抑制,3组PI比较差异有统计学意义(P0.05);3组巨噬细胞胶原结构清道夫系统(MARCO)、清道夫受体A1(SR-A1)表达量比较差异不明显(P0.05);轻中度组与对照组Toll样受体4(TLR4)表达量比较差异不明显,但重度组TLR4表达上调,与轻中度组、对照组比较差异有统计学意义(P0.05);3组MUC5A、AQP5表达量比较差异显著(P0.05);巨噬细胞PI与TLR4、黏蛋白5AC(MUC5A)表达量呈负相关(P0.05),与水通道蛋白5(AQP5)表达量呈正相关(P0.05)。结论:COPD患者巨噬细胞占细胞总数的比例下降,其吞噬功能也受到抑制,其机制可能与TLR4、MUC5A表达上调及AQP5表达下调等有关。     

Blockade of FG 7142-Induced Increased Dopamine Utilization by the Glycine/NMDA Receptor Antagonist (+)-HA 966

Abstract: Systemic administration of the anxiogenic benzodiazepine inverse agonist FG 7142 has been shown to increase selectively dopamine utilization in the medial prefrontal cortex and the shell, but not core, subregion of the nucleus accumbens. In the present study, we examined the functional interaction between benzodiazepine and N -methyl- d -aspartate receptor influences on dopamine utilization in these areas. Male Sprague-Dawley rats were pretreated with the glycine receptor antagonist (+)-HA 966 (15 mg/kg, i.p.) or saline 15 min before FG 7142 (20 mg/kg, i.p.) or vehicle administration. Subjects were killed 30 min later and assayed for tissue concentrations of dopamine and its major metabolite 3,4-dihydroxyphenylacetic acid in the core and shell subdivisions of the nucleus accumbens and the medial prefrontal cortex. (+)-HA 966 administration blocked FG 7142-induced increased dopamine utilization in both the medial prefrontal cortex and the shell subdivision of the nucleus accumbens. Results are discussed in terms of N -methyl- d -aspartate receptor influences on the response of mesoaccumbal dopamine neurons to stress.     

研究: 肺炎衣原体抑制LXRα-ABCA1途径促进巨噬细胞脂质蓄积

为探讨肝X受体α (LXRα)-三磷酸腺苷结合盒转运体A1 (ABCA1)途径在肺炎衣原体 (C. pneumoniae)促巨噬细胞脂质蓄积中的作用和机制,以THP-1巨噬细胞源性泡沫细胞为模型,采用高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量,液体闪烁计数器检测细胞内胆固醇流出,RT-PCR检测ABCA1和LXRα mRNA的表达,蛋白质印迹检测ABCA1和LXRα的蛋白质表达;使用LXRα的特异性激动剂T0901317对细胞进行预处理,再观察上述指标的变化．结果显示,C. pneumoniae可促进THP-1巨噬细胞源性泡沫细胞内总胆固醇、游离胆固醇和胆固醇酯含量增加,抑制胆固醇外流,降低细胞ABCA1和LXRα的表达;使用ABCA1激动剂8-溴-环磷酸腺苷预处理细胞或LXR激动剂T0901317预处理细胞后,可明显减弱C. pneumoniae对THP-1细胞ABCA1的表达抑制,促进细胞胆固醇流出,降低细胞内胆固醇的含量．结果提示,C. pneumoniae促进巨噬细胞脂质蓄积及胆固醇流出障碍,其机制可能与LXRα-ABCA1途径有关．     

Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production

Background

Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number {"type":"entrez-protein","attrs":{"text":"Q8BGW0","term_id":"81896053","term_text":"Q8BGW0"}}Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 ({"type":"entrez-protein","attrs":{"text":"Q91YX0","term_id":"73920022","term_text":"Q91YX0"}}Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages.

Methodology/Principal Findings

Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly I∶C). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-κB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase ({"type":"entrez-protein","attrs":{"text":"P25911","term_id":"2507208","term_text":"P25911"}}P25911), the Rho guanine nucleotide exchange factor, Vav ({"type":"entrez-protein","attrs":{"text":"P27870","term_id":"137483","term_text":"P27870"}}P27870), and the adaptor protein Grb2 ({"type":"entrez-protein","attrs":{"text":"Q60631","term_id":"2498425","term_text":"Q60631"}}Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo.

Conclusions/Significance

We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.     

Oncomodulin/Truncated Protamine-Mediated Nogo-66 Receptor Small Interference RNA Delivery Promotes Axon Regeneration in Retinal Ganglion Cells

The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ∼12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.     

Up-regulated Endogenous Erythropoietin/Erythropoietin Receptor System and Exogenous Erythropoietin Rescue Retinal Ganglion Cells after Chronic Ocular Hypertension

Aims Recent studies have showed that erythropoietin (EPO) is a neuroprotectant for central nerve system neurons in addition to being a hematopoietic cytokine in response to hypoxia. In this study, we investigate the role of the EPO/EPO receptor (EPOR) system in the rat retina after ocular hypertension injury that mimics glaucoma. Methods Elevated intraocular pressure was induced by laser coagulation of the episcleral and limbal veins. Expression of EPO and EPOR in the normal and glaucomous retinas was investigated by immunohistochemistry and Western blot. To examine the effects of endogenous EPO on the survival of retinal ganglion cells (RGCs) subjected to hypertensive injury, soluble EPOR was directly injected into the vitreous body. Recombinant human EPO was both intravitreally and systemically administrated to study the effect of exogenous EPO on the survival of RGCs after ocular hypertension injury. Results Immunohistochemistry studies identified Müller cells as the main source of EPO in the normal retina. Expression of EPO and EPOR proteins was increased significantly 2 weeks after ocular hypertension. RGCs, amacrine and bipolar cells all demonstrated an increased expression of EPOR after ocular hypertension. Neutralization of endogenous EPO with soluble EPOR exacerbated ocular hypertensive injury, suggesting a role of the EPO/EPOR system in the survival of RGCs after injury. Similarly, topical and systemic administration of recombinant human EPO rescues RGCs after chronic ocular hypertension. Conclusions These results indicate that an endogenous EPO/EPOR system participates in intrinsic recovery mechanisms after retina injury and RGCs might be rescued by exogenous administration of EPO.     

Blockade of Striatal 5-Hydroxytryptmine2 Receptors Reduces the Increase in Extracellullar Concentrations of Dopamine Produced by the Amphhetamine analogue 3,4-Methylenedioxymethamphetamine

Abstract: 5-Hydroxytryptamine₂ (5-HT₂) receptor antagonists have been shown to interfere with the stimulation of striatal dopamine synthesis and release produced by the amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA). To localize the receptors responsible for the attenuation of MDMA-induced release, 5-HT₂ receptor antagonists were infused via the microdialysis probe directly into the brains of awake, freely moving rats before the systemic administration of MDMA. Intrastriatal infusions of the selective 5-HT₂ antagonist MDL 100, 907 produced a concentration-dependent inhibition of MDMA-induced dopamine release. Similar results were observed with intrastriatal infusions of the 5-HT₂ antagonist amperozide. In contrast, infusion of MDL 100, 907 into the midbrain region near the dopaminergic cell bodies was with out effect on the MDMA-induced elevation of extracellular dopamine in the ipsilateral striatum. Neither antagonist attenuated basal transmitter efflux nor the MDMA-stimulated release of [³H]dopamine from striatal slices in vitro indicating that the in vivo effect of the antagonists was not due to inhibition of the dopamine uptake carrier. Intrastriatal infusion of tetrodotoxin reduced both basal and MDMA-stimulated dopamine efflux and eliminated the effect of intrastriatal MDL 100, 907. The results indicate that 5-HT₂ receptors located in the striatum augment the release of dopamine produced by high doses of MDMA. Furthermore, these 5-HT₂ receptors appear to be located on nondopaminergic elements of the striatum.