Eupatorium makinoi suppresses toll-like receptor signaling pathways

Toll-like receptors (TLRs) recognize microbial molecules that are widely presented by pathogens and initiate the innate immune system. TLR signaling is divided into two different signaling pathways, the myeloid differential factor 88 (MyD88)- and Toll/interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent pathways. Eupatorium makinoi, a plant species in Asteraceae, is used for medicinal purposes in China, Korea, and Japan. Through our previous research, we found that an ethanol extract of E. makinoi (EEM) suppresses inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. In this study, we investigated the effect of EEM on TLRs signaling pathways. EEM suppresses NF-κB activation and iNOS and COX-2 expressions induced by TLR2 or TLR4 agonists. Also, EEM suppresses the activation of interferon (IFNs) regulatory factor 3 (IRF3) induced by TLR3 or TLR4 agonists. All results indicate that EEM suppresses myeloid differentiation primary-response protein 88 (MyD88) and TRIF-dependent signaling pathways of TLRs and the expressions of target genes derived from the activation of TLRs     

阿托伐他汀对脂多糖诱导人脐静脉内皮细胞Toll样受体4及其下游信号转导通路的影响

背景：研究表明Toll样受体4参与了动脉粥样硬化的发生和发展,目前Toll样受体4与MyD88依赖性或MyD88非依赖性信号转导通路在动脉粥样硬化发生和发展中的机制尚不明确。 目的：观察阿托伐他汀对脂多糖诱导的人脐静脉内皮细胞Toll样受体4及其下游信号转导通路主要元件MyD88、TRAF-6、TRAM及TRIF表达的影响,分析阿托伐他汀防治动脉粥样硬化的机制。 方法：体外培养人脐静脉内皮细胞,用脂多糖刺激并加入阿托伐他汀干预24 h,收集细胞,用荧光定量PCR方法测定TLR4、MyD88、TRAF-6、TRAM及TRIF mRNA表达;用Western blotting法测定TLR4、MyD88及TRAF-6蛋白表达。 结果与结论：用脂多糖刺激人脐静脉内皮细胞后,引起TLR4、MyD88、TRAF-6、TRAM和TRIF的高表达(P < 0.01),用阿托伐他汀干预后可显著抑制TLR4、MyD88及TRAF-6的表达(P < 0.01)。提示阿托伐他汀可阻断Toll样受体4高表达,同时阻断Toll样受体4胞内信号转导的MyD88依赖性途径,这可能是阿托伐他汀抗动脉粥样硬化的作用机制之一。     

TIRAP: an adapter molecule in the Toll signaling pathway

Mammalian Toll-like receptors (TLRs) recognize conserved products of microbial metabolism and activate NF-kappa B and other signaling pathways through the adapter protein MyD88. Although some cellular responses are completely abolished in MyD88-deficient mice, TLR4, but not TLR9, can activate NF-kappa B and mitogen-activated protein kinases and induce dendritic cell maturation in the absence of MyD88. These differences suggest that another adapter must exist that can mediate MyD88-independent signaling in response to TLR4 ligation. We have identified and characterized a Toll-interleukin 1 receptor (TIR) domain-containing adapter protein (TIRAP) and have shown that it controls activation of MyD88-independent signaling pathways downstream of TLR4. We have also shown that the double-stranded RNA-binding protein kinase PKR is a component of both the TIRAP- and MyD88-dependent signaling pathways.     

Containment of aerogenic Mycobacterium tuberculosis infection in mice does not require MyD88 adaptor function for TLR2, -4 and -9

The role of Toll-like receptors (TLR) and MyD88 for immune responses to Mycobacterium tuberculosis (Mtb) infection remains controversial. To address the impact of TLR-mediated pathogen recognition and MyD88-dependent signaling events on anti-mycobacterial host responses, we analyzed the outcome of Mtb infection in TLR2/4/9 triple- and MyD88-deficient mice. After aerosol infection, both TLR2/4/9-deficient and wild-type mice expressed pro-inflammatory cytokines promoting antigen-specific T cells and the production of IFN-gamma to similar extents. Moreover, TLR2/4/9-deficient mice expressed IFN-gamma-dependent inducible nitric oxide synthase and LRG-47 in infected lungs. MyD88-deficient mice expressed pro-inflammatory cytokines and were shown to expand IFN-gamma-producing antigen-specific T cells, albeit in a delayed fashion. Only mice that were deficient for MyD88 rapidly succumbed to unrestrained mycobacterial growth, whereas TLR2/4/9-deficient mice controlled Mtb replication. IFN-gamma-dependent restriction of mycobacterial growth was severely impaired only in Mtb-infected MyD88, but not in TLR2/4/9-deficient bone marrow-derived macrophages. Our results demonstrate that after Mtb infection neither TLR2, -4, and -9, nor MyD88 are required for the induction of adaptive T cell responses. Rather, MyD88, but not TLR2, TLR4 and TLR9, is critical for triggering macrophage effector mechanisms central to anti-mycobacterial defense.     

MyD88 and TRIF synergistic interaction is required for TH1‐cell polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant‐stable emulsion (GLA‐SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly‐functional T_H1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR‐domain‐containing adapter inducing IFN‐beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal T_H1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA‐SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild‐type mice. We find that both MyD88 and TRIF are necessary for GLA‐SE to induce a poly‐functional T_H1 immune response characterized by CD4⁺ T cells producing IFN‐γ, TNF, and IL‐2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA‐SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo T_H1‐skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.     

Monocyte and neutrophil recruitment during oral Salmonella infection is driven by MyD88‐derived chemokines

Oral Salmonella infection recruits phagocytes to Peyer's patches (PP) and MLN. The chemokines induced in infected PP and MLN, the cellular sources during infection and the TLR signaling pathways involved in vivo are not known. Here, we show that CCL2, CXCL9 and CXCL2 mRNA are up‐regulated in PP and MLN coincident with the first arrival of monocytes and neutrophils. Laser capture microdissection microscopy revealed that chemokine mRNA up‐regulation was differently distributed in PP. Despite this, recruited monocytes and neutrophils formed inflammatory cell clusters throughout PP. Monocytes and neutrophils purified from infected mice preferentially produced CXCL2 and small amounts of CCL2, and neutrophils from infected mice migrated towards CXCL2 and CCL3. Furthermore, phagocyte recruitment to PP and MLN was intact in mice lacking TLR4 alone and when signaling through TLR4 and TLR5 was simultaneously absent; however, recruitment was compromised in MyD88^?/? and more so in MyD88^?/?TLR4^?/? double knockout mice. Phagocyte release into the blood, however, was only marginally reduced in MyD88^?/?TLR4^?/? mice. Defective phagocyte recruitment to PP and MLN of MyD88^?/?TLR4^?/? mice was paralleled by low chemokine induction. These data provide insight into the chemokines and TLR signaling pathways that orchestrate the early phagocyte response to oral Salmonella infection.     

Role of the Toll-like receptor pathway in the recognition of orthopedic implant wear-debris particles

The inflammatory response to prosthetic implant-derived wear particles is the primary cause of bone loss and aseptic loosening of implants, but the mechanisms by which macrophages recognize and respond to particles remain unknown. Studies of innate immunity demonstrate that Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPS). All TLRs signal through myeloid differentiation factor 88 (MyD88), except TLR3 which signals through TIR domain containing adapter inducing interferon-beta (TRIF), and TLR4 which signals through both MyD88 and TRIF. We hypothesized that wear-debris particles may act as PAMPs/DAMPs and activate macrophages via TLRs. To test this hypothesis, we first demonstrated that inhibition of MyD88 decreases polymethylmethacrylate (PMMA) particle-induced production of TNF-α in RAW 264.7 macrophages. Next we compared particle-induced production of TNF-α among MyD88 knockout (MyD88(-/-)), TRIF knockout (TRIF(-/-)), and wild type (WT) murine macrophages. Relative to WT, disruption of MyD88 signaling diminished, and disruption of TRIF amplified the particle-induced production of TNF-α. Gene expression data indicated that this latter increase in TNF-α was due to a compensatory increase in expression of MyD88 associated components of the TLR pathway. Finally, using an in?vivo model, MyD88(-/-) mice developed less particle-induced osteolysis than WT mice. These results indicate that the response to PMMA particles is partly dependent on MyD88, presumably as part of TLR signaling; MyD88 may represent a therapeutic target for prevention of wear debris-induced periprosthetic osteolysis.     

Avian-specific TLRs and downstream effector responses to CpG-induction in chicken macrophages

Chickens possess toll-like receptor (TLR15), a pattern recognition receptor (PRR) absent in mammals. We characterized the regulation and mechanism of CpG responsiveness via TLRs in chicken macrophage HD11 cells. TLR15 was significantly upregulated after induction with B- and C-type CpG oligonucleotides (ODN), tripalmitoylated lipopeptide (PAM3CSK4), Escherichia coli- and Salmonella enteritidis-derived lipopolysaccharide (LPS). In response to CpG-ODN inhibitor, TLR15 and IL1B were downregulated, but TLR21 was upregulated. IL1B was upregulated with CpG-ODN and downregulated after inhibitor treatment. The results suggest that responsiveness to different types of CpG-ODN in chicken macrophages requires multiple receptors, each with unique variation in expression. We utilized RNA interference (RNAi) technology to examine myeloid differentiation primary response gene (MyD88) dependency of TLR15 and TLR21. HD11 macrophages transfected with multiple MyD88-target siRNAs exhibited 70% decrease in MyD88 mRNA expression. IL1B was upregulated with CpG induction in cells with no reduction of MyD88 mRNA levels, but not in cells with 70% MyD88 reduction. Therefore, induction through TLR15 in response to CpG-ODN operates via the MyD88-dependent pathway in chicken macrophages.     

Host MyD88 signaling protects against acute graft-versus-host disease after allogeneic bone marrow transplantation

Recent experimental strategies to reduce graft-versus-host disease (GVHD) have focused largely on modifying innate immunity. Toll-like receptor (TLR)-driven myeloid differentiation primary response 88 (MyD88)-dependent signalling pathways that initiate adaptive immune function are also critical for the pathogenesis of GVHD. This study aimed to delineate the role of host MyD88 in the development of acute GVHD following fully major histocompatibility complex-mismatched allogeneic bone marrow transplantation (BMT). When myeloablated BALB/c MyD88 knock-out recipients were transplanted with C57BL/6 (B6) donor cells, they developed significantly more severe GVHD than wild-type (WT) BALB/c hosts. The increased morbidity and mortality in MyD88^–/– mice correlated with increased serum levels of lipopolysaccharide and elevated inflammatory cytokines in GVHD target organs. Additionally, MyD88 deficiency in BMT recipients led to increased donor T cell expansion and more donor CD11c⁺ cell intestinal infiltration with apoptotic cells but reduced proliferation of intestinal epithelial cells compared with that in WT BMT recipients. Decreased expression of tight junction mRNA in epithelial cells of MyD88^–/– mice suggested that MyD88 contributes to intestinal integrity. Cox-2 expression in the GVHD-targeted organs of WT mice is increased upon GVHD induction, but this enhanced expression was obviously inhibited by MyD88 deficiency. The present findings demonstrate an unexpected role for host MyD88 in preventing GVHD after allogeneic BMT.     

Effect of lentivirus-mediated gene silencing,targeting toll-like receptor 2, on corneal allograft transplantation in rats

AimThe present work aims to assess the effectiveness of lentiviral vector (LV) mediated Toll-like receptor 2 (TLR2) gene silencing in the survival of transplanted corneal allografts, against immune rejection, in rats.MethodsLV mediated TLR2 small interference RNA (SiRNA) with enhanced green fluorescent protein (eGFP) [LV-TLR2-siRNA-eGFP] was realised and transfected to both rat corneal epithelial (EC) and stromal cells (SC). Multiplicity of infection (MOI) was optimized for transfection efficiency using flow cytometric (FCM) analysis. Viability of transfected cells and the success rate of TLR2 gene silencing were respectively determined by CCK-8 assay and western blot assay. The in-vivo experiments were subjected to a penetrating keratoplasty (PK) performed between host Sprague Dawley (SD) and donor Wistar/SD rats, randomly dividing them into 4 groups including the allograft, isograft, allograft treated with LV-eGFP (LV blank control) and allograft treated with LV-TLR2-siRNA-eGFP (LV treated group). The rejection index (RI) was then recorded under a slit lamp, every day following surgery. Expression of the TLR2 and Myeloid differentiation primary response gene 88 (MyD88) were detected by using immunohistochemistry on day 9 post-surgery, whereas grafts’s TLR2 and MyD88 mRNA were determined on day 5, 9, and 14 post-surgery performing RT-PCR and, normal rat corneas were included as additional controls.ResultsTransfected cells showed the strongest eGFP expression when MOI was 200 with an efficiency of 77.5% for EC and 76.3% for SC. CCK-8 assay, as measured at 72 and 96 h post transfection, showed no significant changes in the cell viability (both EC and SC) between the transfected and the control group (p > 0.05, p > 0.05). Western blot results demonstrated a successful down regulation of TLR2 expression by LV-TLR2-SiRNA-eGFP, in both EC and SC. In vivo, compared to allograft group, LV treated group demonstrated less edema, opacity and neovascularization. Immunohistochemical analysis revealed a lower expression of TLR2 and MyD88 in isograft and LV treated group as compared to allograft group. TLR2 and MyD88 mRNA were detected in all grafts, and increased over time. With its highest expression in allograft group (peak on day 9), the mRNA expression for TLR2 and MyD88 showed a significant difference amongst the groups, on both day 9 and 14 (p < 0.001).ConclusionsLV mediated TLR2 siRNA could effectively down regulate the TLR2 expression via RNA interference and prolong the survival of corneal grafts, although not necessarily able to prevent the rejection.     

Acute hypoxia-induced depletion of striatal nitric oxide synthase pathway

Hypoxia-induced alteration of nitric oxide (NO) production may lead to brain disease, especially in the areas most sensitive to oxygen deficiency, such as the striatum. To date, the behaviour of the striatal NO pathway under hypoxia/reoxygenation remains unknown and its elucidation constitutes the aim of this work.Wistar rats were submitted to hypoxia (20 min) and analyzed after 0 h, 24 h, and 5 days of reoxygenation. Expression, activity, and location of the NO synthase (NOS) isoforms (neuronal, endothelial, and inducible) as well as nitrated protein expression were analyzed in the rat striatum. NO levels were indirectly quantified as nitrates and nitrites (NO_x), which act as NO-generating molecules.NOS isoform mRNA levels remained unaltered in hypoxic groups vs. normoxic control. However, quantification of immunoreaction showed a significant decrease in eNOS and nNOS after hypoxia. While in situ NOS activity and NO_x levels fell, levels of nitrotyrosine-modified proteins rose throughout the reoxygenation period.Our data revealed the great complexity of the NO pathway, showing that both acute hypoxia and the successive recovery period down-regulated the NOS system in the rat striatum. However, under hypoxia/reoxygenation NO may be produced in a NOS-independent way from the NO-storage molecules, compensating for the hypoxia-reduced NOS activity.     

Down-regulation of endothelial TLR4 signalling after apo A-I gene transfer contributes to improved survival in an experimental model of lipopolysaccharide-induced inflammation

The protective effects of high-density lipoprotein (HDL) under lipopolysaccharide (LPS) conditions have been well documented. Here, we investigated whether an effect of HDL on Toll-like receptor 4 (TLR4) expression and signalling may contribute to its endothelial-protective effects and to improved survival in a mouse model of LPS-induced inflammation and lethality. HDL cholesterol increased 1.7-fold (p<0.005) and lung endothelial TLR4 expression decreased 8.4-fold (p<0.005) 2 weeks after apolipoprotein (apo) A-I gene transfer. Following LPS administration in apo A-I gene transfer mice, lung TLR4 and lung MyD88 mRNA expression, reflecting TLR4 signalling, were 3.0-fold (p<0.05) and 2.1-fold (p<0.05) lower, respectively, than in LPS control mice. Concomitantly, LPS-induced lung neutrophil infiltration, lung oedema and mortality were significantly attenuated following apo A-I transfer. In vitro, supplementation of HDL or apo A-I to human microvascular endothelial cells-1 24 h before LPS administration reduced TLR4 expression, as assessed by fluorescent-activated cell sorting, and decreased the LPS-induced MyD88 mRNA expression and NF-κB activity, independently of LPS binding. In conclusion, HDL reduces TLR4 expression and signalling in endothelial cells, which may contribute significantly to the protective effects of HDL in LPS-induced inflammation and lethality.     

Comparative and evolutionary analysis of an adapter molecule MyD88 in invertebrate metazoans

The myeloid differentiation factor 88 (MyD88) is an essential adapter in Toll-like receptor (TLR) signalling pathways, with TLR the first pattern-recognition receptor (PRR) that was discovered in Drosophila. In the present study, a MyD88 gene was identified and characterized from a commercially important shellfish, Scapharca subcrenata, including a DEATH domain and TIR domain conserved within other molluscs. Furthermore, comparative genomic evidence revealed that MyD88 was of different lengths and contained quantitative exon and intron regions, which might be involved in specific mechanisms. To further explore the phylogenetic relationships of invertebrate metazoan MyD88, we applied MrBayes and PhyML software to construct phylogenetic trees using Bayesian and maximum likelihood approaches, respectively, which suggested that the MyD88 of Arthropoda was closely related to lower invertebrates, in contrast to morphological taxonomy. Finally, we investigated the evolutionary patterns and location of positive selection sites (PSSs) in the MyD88 gene from Arthropoda, Mollusca and Insecta using PAML software with the maximum likelihood method. The data showed that positive selection sites were detected in these groups, and partial sites were located in the TIR domain but were not found in the DEATH domain. To summarize, in this study, we report on the diversification of MyD88 in invertebrate metazoans, the specific evolutionary position of Arthropoda MyD88, and the positive selection pressures on MyD88 of Arthropoda, Mollusca and Insecta. These results are a valuable contribution to understand and clarify the evolutionary pattern of TLR/MyD88 signalling pathways in invertebrate and vertebrate taxa.     

Human skin endothelial cells can express all 10 TLR genes and respond to respective ligands

Breakdown of the skin barrier requires the recognition of and rapid responses to invading pathogens. Since wounding usually also affects endothelial intactness, the expression of receptors of the Toll-like family involved in pathogen recognition in human skin vessel endothelia was examined. We found that human skin-derived microvascular endothelial cells can express all 10 Toll-like receptors (TLRs) currently known and will respond to respective ligands. Using immortalized skin-derived (HMEC-1) and primary dermal endothelial cells (HDMEC), we screened for TLR expression by real-time PCR. Endothelial cells express 7 (for HDMEC) and 8 (for HMEC-1) of the 10 known human TLRs under resting conditions but can express all 10 receptors in proinflammatory conditions. To provide evidence of TLR functionality, endothelial cells were challenged with TLR ligands, and after the TLR downstream signaling, MyD88 recruitment as well as early (interleukin-8 [IL-8] release) and late immune markers (inducible nitric oxide synthase mRNA expression) were monitored. Surprisingly, the responses observed were not uniform but were highly specific depending on the respective TLR ligand. For instance, lipopolysaccharides highly increased IL-8 release, but CpG DNA induced significant suppression. Additionally, TLR-specific responses were found to differ between resting and activated endothelial cells. These results show that human skin-derived endothelial cells can function as an important part of the innate immune response, can actively sense pathogen-associated molecular patterns, and can mount an increased or reduced inflammatory signal upon exposure to any of the currently known TLR ligands. Moreover, we also show here that proinflammatory conditions may affect TLR expression in a specific and nonuniform pattern.     

人参二醇组皂苷对LPS致休克大鼠肝脏TLR2、TLR9 mRNA表达的影响

目的：观察人参二醇组皂苷(PDS)对TLR2和TLR9 mRNA表达的影响,探讨PDS抗休克的分子生物学机制。方法:大鼠随机分为对照(control)组、LPS休克(LPS)组、人参二醇组皂苷小剂量(LPS+PDSL)组和人参二醇组皂苷中剂量(LPS+PDSM)组。大鼠舌下静脉注射LPS(4 mg/kg)4 h后测定血清中NOS活性、NO含量，肝组织中LPO含量、SOD活性以及TLR2、TLR4 mRNA的表达。结果:LPS+PDSL组和LPS+PDSM组NOS活性、NO含量和LPO含量明显低于LPS组，SOD活性明显高于LPS组(P<0.05)；LPS+PDSL组和LPS+PDSM组TLR2 mRNA表达明显低于LPS组，TLR9 mRNA表达无变化。结论:PDS通过下调肝组织中TLR2 mRNA表达，降低NOS活性、NO含量，对肝脏有保护作用。