The lipopolysaccharide-activated toll-like receptor (TLR)-4 induces synthesis of the closely related receptor TLR-2 in adipocytes

The central regulatory role of the adipocyte in whole body energy homeostasis is well established. However, recent findings suggest that preadipocytes and adipocytes may play an important physiological role in the regulation of both the innate and adaptive immune response. To systematically characterize the molecular machinery of the adipocyte that mediates the recognition of pathogens, we have focused our analysis on the recently identified Toll-like receptors (TLRs). These receptors have been implicated as mediators of the cellular response to bacterial lipopolysacharides (LPSs). Here, we report the cloning and functional characterization of mouse TLR-2 from 3T3-L1 adipocytes. TLR-2 synthesis is strongly induced in the adipocyte by LPS, TNFalpha, and the yeast cell wall extract zymosan. TLR-2 undergoes a lengthy intracellular maturation process with a half-life of exit from the ER of approximately 3 h. Furthermore, LPS treatment of adipocytes results in dramatic changes at the level of gene expression, including the synthesis of a distinct set of secretory proteins such as interleukin-6. Our studies demonstrate the presence of a fully intact pathway of innate immunity in the adipocyte that can be activated by LPS binding to the cell surface and results in the secretion of immunomodulatory molecules.     

Signaling flux redistribution at toll-like receptor pathway junctions

Various receptors on cell surface recognize specific extracellular molecules and trigger signal transduction altering gene expression in the nucleus. Gain or loss-of-function mutations of one molecule have shown to affect alternative signaling pathways with a poorly understood mechanism. In Toll-like receptor (TLR) 4 signaling, which branches into MyD88- and TRAM-dependent pathways upon lipopolysaccharide (LPS) stimulation, we investigated the gain or loss-of-function mutations of MyD88. We predict, using a computational model built on the perturbation-response approach and the law of mass conservation, that removal and addition of MyD88 in TLR4 activation, enhances and impairs, respectively, the alternative TRAM-dependent pathway through signaling flux redistribution (SFR) at pathway branches. To verify SFR, we treated MyD88-deficient macrophages with LPS and observed enhancement of TRAM-dependent pathway based on increased IRF3 phosphorylation and induction of Cxcl10 and Ifit2. Furthermore, increasing the amount of MyD88 in cultured cells showed decreased TRAM binding to TLR4. Investigating another TLR4 pathway junction, from TRIF to TRAF6, RIP1 and TBK1, the removal of MyD88-dependent TRAF6 increased expression of TRAM-dependent Cxcl10 and Ifit2. Thus, we demonstrate that SFR is a novel mechanism for enhanced activation of alternative pathways when molecules at pathway junctions are removed. Our data suggest that SFR may enlighten hitherto unexplainable intracellular signaling alterations in genetic diseases where gain or loss-of-function mutations are observed.     

Comparative fine maps of bovine toll-like receptor 4 and toll-like receptor 2 regions

Abstract Toll-like receptors are cell-surface receptors that activate innate and adaptive immune responses. We have used a 5000-rad, whole-genome radiation hybrid panel to map Toll-like receptor 4 (TLR4) to the distal end of bovine Chromosome (Chr) 8, and Toll-like receptor 2 (TLR2) to the proximal end of bovine Chr 17. To facilitate comparative mapping and contig construction, we have also used 5000- and 12,000-rad, whole-genome radiation hybrid panels to produce fine maps of the regions surrounding these genes in cattle. These fine maps triple the number of available markers in the TLR4 region and more than double the number of available markers in the TLR2 region. Comparative analyses show gene order conservation between the bovine Chr 8 region and human Chr 9, and between the bovine Chr 17 region and human Chr 4. In addition, the bovine Chr 8 region refines an evolutionary chromosomal breakpoint from a 10-megabase region to a 2.5-megabase region, and the bovine Chr 17 map suggests a new evolutionary chromosomal breakpoint.     

Single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3

Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands. The degree of modulation can be altered by the length, base sequence, and modification state of the ssDNAs. An inhibitory ssDNA was found to colocalize with TLR3 in transfected cells and in a cell line that naturally expresses TLR3. The inhibitory ssDNAs can compete efficiently with dsRNA for binding purified TLR3 ectodomains in vitro, while noninhibitory nucleic acids do not. The ssDNAs also decrease the levels of several cytokines produced by the human bronchial epithelial cell line BEAS-2B and by human peripheral blood mononuclear cells in response to poly(I-C) stimulation of native TLR3. These activities indicate that ssDNAs could be used to regulate the inflammatory response through TLR3.     

Simple synthetic toll-like receptor 2 ligands

Stimulation of toll-like receptor 2 (TLR2) by bacterial lipoproteins induces fast non-specific immune responses against pathogens followed by slow but specific adaptive immune responses. Development of synthetic TLR2 agonists/antagonists would be useful in the prevention of different infectious and immunologic disorders. The current study reports synthesis and TLR2 activity of two simple TLR2 ligands, which feature minimal structural requirement for TLR2 activity (two long lipid chains) and stimulate agonistic activity at nanomolar concentration.     

Mouse toll-like receptor 4.MD-2 complex mediates lipopolysaccharide-mimetic signal transduction by Taxol

Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice but not in humans. Although Taxol is structurally unrelated to LPS, Taxol and LPS are presumed to share a receptor or signaling molecule. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4); therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS responsiveness on TLR4. To determine whether TLR4.MD-2 complex mediates a Taxol-induced signal, we constructed transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2, and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion, and then examined whether Taxol induced NFkappaB activation in these transfectants. Noticeable NFkappaB activation by Taxol was detected in Ba/F3 expressing mouse TLR4 and mouse MD-2 but not in the other transfectants. Coexpression of human TLR4 and human MD-2 did not confer Taxol responsiveness on Ba/F3 cells, suggesting that the TLR4. MD-2 complex is responsible for the species specificity with respect to Taxol responsiveness. Furthermore, Taxol-induced NFkappaB activation via TLR4.MD-2 was blocked by an LPS antagonist that blocks LPS-induced NFkappaB activation via TLR4.MD-2. These results demonstrated that coexpression of mouse TLR4 and mouse MD-2 is required for Taxol responsiveness and that the TLR4.MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice.     

Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by toll-like receptor 2.

The agent of Lyme disease, Borrelia burgdorferi, produces membrane lipoproteins possessing potent inflammatory properties linked to disease pathology. The recent association of toll-like receptors (TLR) 2 and 4 with LPS responses prompted the examination of TLR involvement in lipoprotein signaling. The ability of human cell lines to respond to lipoproteins was correlated with the expression of TLR2. Transfection of TLR2 into cell lines conferred responsiveness to lipoproteins, lipopeptides, and sonicated B. burgdorferi, as measured by nuclear translocation of NF-kappaB and cytokine production. The physiological importance of this interaction was demonstrated by the 10-fold greater sensitivity of TLR2-transfected cells to lipoproteins than LPS. Futhermore, TLR2-dependent signaling by lipoproteins was facilitated by CD14. These data indicate that TLR2 facilitates the inflammatory events associated with Lyme arthritis. In addition, the widespread expression of lipoproteins by other bacterial species suggests that this interaction may have broad implications in microbial inflammation and pathogenesis.     

Apoptosis induced by the toll-like receptor adaptor TRIF is dependent on its receptor interacting protein homotypic interaction motif

TLRs detect specific molecular features of microorganisms and subsequently engage distinct signaling networks through the differential use of Toll/IL-1R (TIR)-domain-containing adapter proteins. In this study, we investigated the control of apoptosis by the TIR domain-containing adapter proteins MyD88, TIR-domain containing adapter protein (TIRAP), TIR-domain-containing adapter-inducing IFN-beta (TRIF), TRIF-related adapter molecule (TRAM), and sterile alpha motifs and beta-catenin/armadillo repeats (SARM). Upon overexpression, TRIF was the sole TIR-adapter to potently engage mammalian cell death signaling pathways. TRIF-induced cell death required caspase activity initiated by the Fas/Apo-1-associated DD protein-caspase-8 axis and was unaffected by inhibitors of the intrinsic apoptotic machinery. The proapoptotic potential of TRIF mapped to the C-terminal region that was found to harbor a receptor interacting protein (RIP) homotypic interaction motif (RHIM). TRIF physically interacted with the RHIM-containing proteins RIP1 and RIP3, and deletion and mutational analyses revealed that the RHIM in TRIF was essential for TRIF-induced apoptosis and contributed to TRIF-induced NF-kappa B activation. The domain that was required for induction of apoptosis could activate NF-kappa B but not IFN regulatory factor-3, yet the activation of NF-kappa B could be blocked by superrepressor I kappa B alpha without blocking apoptosis. Thus, the ability of TRIF to induce apoptosis was not dependent on its ability to activate either IFN regulatory factor-3 or NF-kappa B but was dependent on the presence of an intact RHIM. TRIF serves as an adaptor for both TLR3 and TLR4, receptors that are activated by dsRNA and LPS, respectively. These molecular motifs are encountered during viral and bacterial infection, and the apoptosis that occurs when TRIF is engaged represents an important host defense to limit the spread of infection.     

Regulation of toll-like receptor 2 expression by macrophages following Mycobacterium avium infection

Recent studies have implicated Toll-like receptors (TLR), especially TLR2 and TLR4, as sentinel receptors that signal the interaction of macrophages with bacterial pathogens via a NF-kappaB-mediated pathway. The regulation of TLR gene expression, however, has not been intensively studied. Here, we report that TLR2 mRNA was induced following infection of murine macrophages with Mycobacterium avium. The changes in TLR2 mRNA correlated with an increase in TLR2 surface expression. Infection with M. avium resulted in a concomitant decrease in TLR4 mRNA. The effect of M. avium infection on TLR2 mRNA appeared to be mediated, in part, by TLR2 because the induction of the mRNA was partially blocked by preincubation of the macrophages with an anti-human TLR2 Ab. In contrast, the effect of LPS stimulation was mediated via TLR4 because infection of macrophages from LPS(d) mice, which do not express active TLR4, resulted in an increase in TLR2 mRNA, while treatment of macrophages from these mice with LPS failed to induce TLR2 mRNA. Several cytokines, including TNF-alpha, IL-1alpha, and GM-CSF, but not IFN-gamma, induced TLR2 mRNA. M. avium infection resulted in the induction of TLR2 mRNA by macrophages from both TNFRI knockout and NF-kappaB p50 knockout mice.     

miRNA-200b improves hepatic fibrosis induced by CCL4 by regulating toll-like receptor 4 in mice

To study the effect of miRNA-200b on hepatic fibrosis induced by CCl₄ in mice. The C59BL/6 mice were randomly divided into three groups (normal control [NC], CCLR model [Model], and CCl ₄ + miRNA-200b [miRNA]). The hepatic fibrosis was induced by CCl ₄ injected subcutaneously twice per week in Model and miRNA groups. After 6 weeks building model, the mice of miRNA group were injected the miRNA-200b from caudal vein twice per week. The mice of Model and miRNA groups were continuously fed for 3 weeks. The IL-1β, IL-6, and TNF-α concentrations of serum were measured by enzyme-linked immunosorbent assay. The hepatic tissues of difference groups were observed by hematoxylin and eosin (H&E) staining, sirius red staining, Masson staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay and measured toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) proteins expressions by western blot assay. The correlation between miRNA-200b and TLR4 were analyzed by dual luciferase target assay. Compared with NC group, the interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) concentrations of Model group were significantly upregulated (P < 0.05, respectively). With miRNA-200b overexpression, the IL-1β, IL-6, and TNF-α concentrations were significantly suppressed (P < 0.05, respectively). The pathologies were improved by H&E staining, sirius red staining, and Masson staining; meanwhile, the hepatic cell apoptosis rate was significantly suppressed (P < 0.05). The TLR4 and NF-κB protein expressions of miRNA group were significantly suppressed compared with the Model group (P < 0.05, respectively). By dual luciferase target assay, the TLR4 was a target gene of miRNA-200b. The miRNA-200b upregulation improved hepatic fibrosis induced by CCl ₄ via regulation of TLR4 in vivo.     

外周血Toll样受体2与早产的关系

目的:探讨早产产妇外周血Toll样受体2(toll-like receptor2,TLR2)mRNA的表达水平及与亚临床绒毛膜羊膜炎的关系.方法:选择早产自然分娩组15例,足月妊娠自然分娩组20例,足月要求剖宫产10例.RT.PCR检测母血中TLR2 mRNA的表达及与胎盘亚临床绒毛膜羊膜炎的关系.结果:①早产自然分娩组产妇TLR2高表达,明显高于足月自然分娩组,差异有统计学意义(P<0.05).②妊娠合并亚临床绒毛膜羊膜炎组TLR2较无亚临床绒毛膜羊膜炎组高,有统计学意义(P<0.05).结论:孕妇外周血表达TLR2,与分娩方式及感染情况有关;可能参与了分娩发动,尤其是早产分娩发动.     

Atherosclerosis induced by endogenous and exogenous toll-like receptor (TLR)1 or TLR6 agonists

Atherosclerosis is a chronic inflammatory vascular disease. Toll-like receptors (TLRs) are major initiators of inflammation. TLR2 promotes atherosclerosis in LDL receptor (LDLr)-deficient mice fed a high-fat diet (HFD). TLR2 forms heterodimers with TLR1 or TLR6 to enable inflammatory responses in the presence of distinct ligands. Here we asked whether TLR1 and/or TLR6 are required. We studied atherosclerotic disease using either TLR1- or TLR6-deficient mice. Deficiency of TLR1 or TLR6 did not diminish HFD-driven disease. When HFD-fed LDLr-deficient mice were challenged with Pam3 or MALP2, specific exogenous ligands of TLR2/1 or TLR2/6, respectively, atherosclerotic lesions developed with remarkable intensity in the abdominal segment of the descending aorta. In contrast to atherosclerosis induced by the endogenous agonists, these lesions were diminished by deficiency of either TLR1 or TLR6. The endogenous ligand(s) that arise from consumption of a HFD and promote disease via TLR2 are unknown. Either TLR1 or TLR6 are redundant for this endogenous ligand detection, or they are both irrelevant to endogenous ligand detection. However, the exogenous ligands Pam3 and MALP2 promote severe abdominal atherosclerosis in the descending aorta that is dependent on TLR1 and TLR6, respectively.     

Signaling events mediating activation of brain ethanolamine plasmalogen hydrolysis by ceramide.

Ceramide is a lipid second messenger that acts on multiple-target enzymes, some of which are involved in other signal-transduction systems. We have previously demonstrated that endogenous ceramide modifies the metabolism of brain ethanolamine plasmalogens. The mechanism involved was studied. On the basis of measurements of breakdown products, specific inhibitor effects, and previous findings, we suggest that a plasmalogen-selective phospholipase A2 is the ceramide target. Arachidonate-rich pools of the diacylphosphatidylethanolamine subclass were also affected by ceramide, but the most affected were plasmalogens. Concomitantly with production of free arachidonate, increased 1-O-arachidonoyl ceramide formation was observed. Quinacrine (phospholipase A2 inhibitor) and 1-O-octadecyl-2-O-methyl-rac-glycerol-3-phosphocholine (CoA-independent transacylase inhibitor) prevented all of these ceramide-elicited effects. Therefore, phospholipase and transacylase activities are tightly coupled. Okadaic acid (phosphatase 2A inhibitor) and PD 98059 (mitogen-activated protein kinase inhibitor) modified basal levels of ceramide and sphingomyelinase-induced accumulation of ceramide, respectively. Therefore, they provided no evidence to determine whether there is a sensitive enzyme downstream of ceramide. The evidence shows that there are serine-dependent and thiol-dependent enzymes downstream of ceramide generation. Furthermore, experiments with Ac-DEVD-CMK (caspase-3 specific inhibitor) have led us to conclude that caspase-3 is downstream of ceramide in activating the brain plasmalogen-selective phospholipase A2.     

Signaling transduction regulated by 5-hydroxytryptamine 1A receptor and orexin receptor 2 heterodimers

As G-protein-coupled receptors (GPCRs), 5-hydroxytryptamine 1A receptor (5-HT1AR) and orexin receptor 2 (OX2R) regulate the levels of the cellular downstream molecules. The heterodimers of different GPCRs play important roles in various of neurological diseases. Moreover, 5-HT1AR and OX2R are involved in the pathogenesis of neurological diseases such as depression with deficiency of hippocampus plasticity. However, the direct interaction of the two receptors remains elusive. In the present study, we firstly demonstrated the heterodimer formation of 5-HT1AR and OX2R. Exchange protein directly activated by cAMP (Epac) cAMP bioluminescence resonance energy transfer (BRET) biosensor analysis revealed that the expression levels of cellular cAMP significantly increased in HEK293T cells transfected with the two receptors compared with the 5-HT1AR group. Additionally, the cellular level of calcium was upregulated robustly in HEK293T cells co-transfected with 5-HT1AR and OX2R group after agonist treatment. Furthermore, western blotting data showed that 5-HT1AR and OX2R heterodimer decreased the levels of phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP-response element-binding protein (CREB). These results not only unraveled the formation of 5-HT1AR and OX2R heterodimer but also suggested that the heterodimer affected the downstream signaling pathway, which will provide new insights into the function of the two receptors in the brain.