TIR domain--containing adaptors regulate TLR-mediated signaling pathways

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses via signaling pathways mediated by several Toll/IL-1R (TIR) domain-containing adaptors such as MyD88, TIRAP, and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4 that is responsible for type I interferon production in response to double-stranded RNA and LPS, respectively. TIRAP specifically participates in the MyD88-dependent pathways shared by TLR2 and TLR4, and TRAM is essential for the TLR4-mediated MyD88-independent pathway. Thus, TIR domain-containing adaptors play an important role in the TLR mediated signaling pathways.     

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.     

LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF

Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM) is the fourth TIR domain-containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-kappaB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-alpha/beta, regulated on activation, normal T cell expressed and secreted (RANTES), and gamma interferon-inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.     

Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6

Transition of immature antigen presenting cells (APCs) to the state of professional APCs is essential for initiation of cell-mediated immune responses to pathogens. Signal transduction via molecules of the Toll-like receptor (TLR)/interleukin 1 receptor (IL-1R) pathway is critical for activation of APCs either by pathogen-derived pattern ligands like lipopolysaccharides (LPS) or by CD40 ligation through T helper cells. The capacity of bacterial DNA (CpG-DNA) to induce APCs to differentiate into professional APCs represents an interesting discovery. However, the signaling pathways involved are poorly understood. Here we show that CpG-DNA activates the TLR/IL-1R signaling pathway via the molecules myeloid differentiation marker 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6), leading to activation of kinases of the IkappaB kinase complex and the c-jun NH(2)-terminal kinases. Moreover, cells of TLR2- and TLR4-deficient mice are activated by CpG-DNA, whereas cells of MyD88-deficient mice do not respond. The data suggest that CpG-DNA initiates signaling via the TLR/IL-1R pathway in APCs in a manner similar to LPS and to T helper cell-mediated CD40 ligation. Activation of the TLR/IL-1R signaling pathway by foreign bacterial DNA may be one way to initiate innate defense mechanisms against infectious pathogens in vivo.     

Identification of TLR downstream pathways in stroke patients

Objective

Toll-like receptors (TLRs) are important molecules for detecting both pathogen invasion and tissue damage. The expression of TLR4 is upregulated in ischemic stroke, at least in the subacute stage. However, the TLR downstream pathways in the context of stroke have not been well studied in previous research. The purpose of this study is to elucidate the TLR4 downstream pathways following ischemic stroke.

Design and methods

In this study, 12 ischemic stroke patients and 12 controls were selected from among 89 ischemic stroke patients and 166 controls. The chosen subjects had the highest levels of TLR4 mRNA in the peripheral blood. The differences in the TLR downstream signaling pathways, which were studied by using an RT2 Profiler TM PCR array system (Qiagen), were analyzed. The differentially expressed genes were analyzed by using GeneSpring GX and visualized based on the TLR pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG).

Results

The genes upregulated in stroke patients were found to be involved in the MyD88-independent pathway and in UBE2V1–TRAF6 ubiquitin-mediated proteolysis. The genes were more expressed in extracellular space, receptor binding, and cytokine receptor binding by use of gene ontology (GO) terms than in control patients.

Conclusions

We found that the MyD88-independent pathway and the ubiquitin-mediated proteolysis pathway, especially TRAF6, may be the most vital molecules among TLR downstream pathways in incidences of ischemic stroke.     

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24h,收集细胞,用荧光定量PCR方法测定TLR4、MyD88、TRAF-6、TRAM及TRIFmRNA表达;用Westernblotting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。     

Toll-Like Receptor 4 Signaling Contributes to Paclitaxel-Induced Peripheral Neuropathy

This paper tests the contribution of the toll-like receptors, TLR4 in particular, in the initiation and maintenance of paclitaxel-related chemotherapy-induced peripheral neuropathy. TLR4 and its immediate downstream signaling molecules—myeloid differentiation primary response gene 88 (MyD88) and toll/interleukin 1 receptor domain–containing adapter-inducing interferon-β (TRIF)—were found to be increased in the dorsal root ganglion (DRG) using Western blot by day 7 of paclitaxel treatment. The behavioral phenotype, the increase of both TLR4 and MyD88, was blocked by cotreatment with the TLR4 antagonist lipopolysaccharide–Rhodobacter sphaeroides during chemotherapy. A similar, but less robust, behavioral effect was observed using intrathecal treatment of MyD88 homodimerization inhibitory peptide. DRG levels of TLR4 and MyD88 reduced over the next 2 weeks, whereas these levels remained increased in spinal cord through day 21 following chemotherapy. Immunohistochemical analysis revealed TLR4 expression in both calcitonin gene-related peptide–positive and isolectin B4–positive small DRG neurons. MyD88 was only found in calcitonin gene-related peptide–positive neurons, and TRIF was found in both calcitonin gene-related peptide–positive and isolectin B4–positive small DRG neurons as well as in medium- and large-size DRG neurons. In the spinal cord, TLR4 was only found colocalized to astrocytes but not with either microglia or neurons. Intrathecal treatment with the TLR4 antagonist lipopolysaccharide–R. sphaeroides transiently reversed preestablished chemotherapy-induced peripheral neuropathy mechanical hypersensitivity. These results strongly implicate TLR4 signaling in the DRG and the spinal cord in the induction and maintenance of paclitaxel-related chemotherapy-induced peripheral neuropathy.PerspectiveThe toll-like receptor TLR4 and MyD88 signaling pathway could be a new potential therapeutic target in paclitaxel-induced painful neuropathy.     

FimH Can Directly Activate Human and Murine Natural Killer Cells via TLR4

Although the importance of natural killer (NK) cells in innate immune responses against tumors or viral infections are well documented, their ability to directly recognize pathogens is less well defined. We have recently reported FimH, a bacterial fimbrial protein, as a novel Toll-like receptor (TLR)4 ligand that potently induces antiviral responses. Here, we investigated whether FimH either directly or indirectly can activate human and murine NK cells. We demonstrate that FimH potently activates both human and murine NK cells in vitro to induce cytokines [interferon (IFN)-γ and tumor necrosis factor (TNF)-α] and cytotoxicity. Importantly, NK cells directly recognize FimH-expressing pathogens as FimH⁺, but not FimH⁻, bacteria were able to activate human NK cells. FimH activation of NK cells required TLR4 and MyD88 signaling, as NK cells from both TLR4^−/− and MyD88^−/− mice as well as human NK-92 cells, which lack TLR4, were all unresponsive to FimH. In addition, TLR4 neutralization significantly abrogated the response of human NK cells to FimH. Activation of purified NK cells by FimH was independent of lipopolysaccharide (LPS) or other bacterial contaminations. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to FimH via TLR4–MyD88 pathways to aid innate protection against cancer or microbial infections.     

Evidence for ProTα-TLR4/MD-2 binding: molecular dynamics and gravimetric assay studies

Objective: During preconditioning, lipopolysaccharide (LPS) selectively activates TLR4/MD-2/Toll/IL-1 receptor-domain-containing adaptor inducing IFN-β (TRIF) pathway instead of pro-inflammatory myeloid differentiation protein-88 (MyD88)/MyD88-adaptor-like protein (MAL) pathway. Extracellular prothymosin alpha (ProTα) is also known to selectively activate the TLR4/MD2/TRIF–IRF3 pathway in certain diseased conditions. In the current study, biophysical evidence for ProTα/TLR4/MD-2 complex formation and its interaction dynamics have been studied.

Research design and methods: Gravimetric assay was used to investigate ProTα/TLR4/MD-2 complex formation while molecular dynamics (MD) simulation was used to study its interaction dynamics.

Results: Through electrostatic interaction, full-length ProTα (F-ProTα) C-terminal peptide (aa 91 – 111) superficially interacts with similar TLR4/MD-2 (K_D = 273.36 nm vs 16.07 μg/ml [LPS]) conformation with LPS at an overlapping three-dimensional space while F-ProTα is hinged to the TLR4 scaffold by one-amino acid shift-Mosoian domain (aa-51 – 90). Comparatively, F-ProTα better stabilizes MD-2 metastable states transition and mediates higher TLR4/MD-2 interaction than LPS.

Conclusions: ProTα via its C-terminal peptide (aa 91 – 111) exhibits in vitro biophysical contact with TLR4/MD-2 complex conformation recognized by LPS at overlapping LPS-binding positions.     

Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis in mice

Mice deficient in the hemochromatosis gene, Hfe, have attenuated inflammatory responses to Salmonella infection associated with decreased macrophage TNF-α and IL-6 biosynthesis after exposure to LPS. In this study, we show that the abnormal cytokine production is related to impaired TLR4 signaling. Despite their abnormal response to LPS, Hfe KO macrophages produced amounts of TNF-α similar to those in WT cells after TLR2 stimulation. Consistent with this finding, LPS-induced activation of Mal/MyD88-dependent events was normal in the mutant macrophages. However, LPS-induced IFN-β expression, a TRAM/TRIF-dependent response activated by TLR4, was reduced by Hfe deficiency. This reduction could be replicated in WT macrophages with the use of iron chelators. In contrast, TLR3-activated expression of IFN-β, a TRIF-dependent response, was normal in Hfe KO macrophages and was unaffected by iron chelation. Our data suggest that low intracellular iron selectively impairs signaling via the TLR4/TRAM/TRIF pathway proximal to TRIF and results in reduced LPS-induced cytokine expression. Furthermore, by mimicking the altered iron metabolism associated with Hfe deficiency, we found that 3 different inhibitors of hepcidin attenuated Salmonella-induced and noninfectious enterocolitis. Thus, manipulation of iron homeostasis could represent a new therapeutic approach to controlling inflammation.     

Activation of RAW264.7 macrophage by Exopolysaccharide from Aphanothece halaphytica (EPSAH) and the underlying mechanisms

Exopolysaccharide from Aphanothece halophytica (EPSAH), a potent antitumor agent and immunological adjuvant, was investigated for the activation effect on RAW264.7 macrophages and the underlying mechanisms. EPSAH could significantly enhance macrophage phagocytosis and the secretion of nitric oxide, increase the mRNA expression levels of the pro-inflammatory cytokines (IL-1β, IL-6, IL-12, and TNF-α), anti-inflammatory cytokine IL-10, and chemokines (MCP-1 and MIP-1α). When RAW264.7 cells were treated with EPSAH, the mRNA expression of TLR4 and its downstream molecules TRAF6 and MyD88 were upregulated. When TLR4 was blocked using a TLR4-specific neutralizing antibody, nitric oxide secretion from the macrophages was significantly inhibited. EPSAH was further shown to induce phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK, JNK, and p38, and promote cytoplasmic IκB phosphorylation and increase nuclear NF-κB p65 levels remarkably in RAW264.7 cells. These data demonstrate the capacity of EPSAH to induce macrophage activation possibly via TLR4/MyD88 pathway, which leads to the activation of its main signaling downstream molecules MAPKs and NF-κB.     

Role of TLR4/MyD88/NF-κB signaling in heart and liver-related complications in a rat model of type 2 diabetes mellitus

AimsTo analyze expression of members of the Toll-like receptor (TLR)4/myeloid differentiation primary response 88 (MyD88)/nuclear factor (NF)-κB signaling pathway in the heart and liver in a rat model of type 2 diabetes mellitus (T2DM). Our overall goal was to understand the underlying pathophysiological mechanisms.MethodsWe measured fasting blood glucose (FBG) and insulin (FINS) in a rat model of T2DM. Expression of members of the TLR4/MyD88/NF-κB signaling pathway as well as downstream cytokines was investigated. Levels of mRNA and protein were assessed using quantitative real-time polymerase chain reaction and western blotting, respectively. Protein content of tissue homogenates was assessed using enzyme-linked immunosorbent assays.ResultsDiabetic rats had lower body weights, higher FBG, higher FINS, and higher intraperitoneal glucose tolerance than normal rats. In addition, biochemical indicators related to heart and liver function were elevated in diabetic rats compared with normal rats. TLR4 and MyD88 were involved in the occurrence of T2DM as well as T2DM-related heart and liver complications. TLR4 caused T2DM-related heart and liver complications through activation of NF-κB.ConclusionsTLR4/MyD88/NF-κB signaling induces production of tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1, leading to the heart- and liver-related complications of T2DM.     

Induction of IgG3 to LPS via Toll-Like Receptor 4 Co-Stimulation

B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR) and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs) also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise.     

Sexually Dimorphic Role of Toll-like Receptor 4 (TLR4) in High Molecular Weight Hyaluronan (HMWH)-induced Anti-hyperalgesia

High molecular weight hyaluronan (HMWH), a prominent component of the extracellular matrix binds to and signals via multiple receptors, including cluster of differentiation 44 (CD44) and toll-like receptor 4 (TLR4). We tested the hypothesis that, in the setting of inflammation, HMWH acts at TLR4 to attenuate hyperalgesia. We found that the attenuation of prostaglandin E₂ (PGE₂)-induced hyperalgesia by HMWH was attenuated by a TLR4 antagonist (NBP2-26245), but only in male and ovariectomized female rats. In this study we sought to evaluated the role of the TLR4 signaling pathway in anti-hyperalgesia induced by HMWH in male rats. Decreasing expression of TLR4 in nociceptors, by intrathecal administration of an oligodeoxynucleotide (ODN) antisense to TLR4 mRNA, also attenuated HMWH-induced anti-hyperalgesia, in male and ovariectomized female rats. Estrogen replacement in ovariectomized females reconstituted the gonad-intact phenotype. The administration of an inhibitor of myeloid differentiation factor 88 (MyD88), a TLR4 second messenger, attenuated HMWH-induced anti-hyperalgesia, while an inhibitor of the MyD88-independent TLR4 signaling pathway did not. Since it has previously been shown that HMWH-induced anti-hyperalgesia is also mediated, in part by CD44 we evaluated the effect of the combination of ODN antisense to TLR4 and CD44 mRNA. This treatment completely reversed HMWH-induced anti-hyperalgesia in male rats. Our results demonstrate a sex hormone-dependent, sexually dimorphic involvement of TLR4 in HMWH-induced anti-hyperalgesia, that is MyD88 dependent.PerspectiveThe role of TLR4 in anti-hyperalgesia induced by HMWH is a sexually dimorphic, TLR4 dependent inhibition of inflammatory hyperalgesia that provides a novel molecular target for the treatment of inflammatory pain.     

MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells

The transition of chronic pancreatic fibroinflammatory disease to neoplasia is a primary example of the paradigm linking inflammation to carcinogenesis. However, the cellular and molecular mediators bridging these entities are not well understood. Because TLR4 ligation can exacerbate pancreatic inflammation, we postulated that TLR4 activation drives pancreatic carcinogenesis. In this study, we show that lipopolysaccharide accelerates pancreatic tumorigenesis, whereas TLR4 inhibition is protective. Furthermore, blockade of the MyD88-independent TRIF pathway is protective against pancreatic cancer, whereas blockade of the MyD88-dependent pathway surprisingly exacerbates pancreatic inflammation and malignant progression. The protumorigenic and fibroinflammatory effects of MyD88 inhibition are mediated by dendritic cells (DCs), which induce pancreatic antigen-restricted Th2-deviated CD4(+) T cells and promote the transition from pancreatitis to carcinoma. Our data implicate a primary role for DCs in pancreatic carcinogenesis and illustrate divergent pathways in which blockade of TLR4 signaling via TRIF is protective against pancreatic cancer and, conversely, MyD88 inhibition exacerbates pancreatic inflammation and neoplastic transformation by augmenting the DC-Th2 axis.     

RIP links TLR4 to Akt and is essential for cell survival in response to LPS stimulation

Receptor-interacting protein (RIP) has been reported to associate with tumor necrosis-associated factor (TRAF)2 and TRAF6. Since TRAF2 and TRAF6 play important roles in CD40 signaling and TRAF6 plays an important role in TLR4 signaling, we examined the role of RIP in signaling via CD40 and TLR4. Splenocytes from RIP(-/-) mice proliferated and underwent isotype switching normally in response to anti-CD40-IL-4 but completely failed to do so in response to LPS-IL-4. However, they normally up-regulated TNF-alpha and IL-6 gene expression and CD54 and CD86 surface expression after LPS stimulation. RIP(-/-) splenocytes exhibited increased apoptosis and impaired Akt phosphorylation after LPS stimulation. These results suggest that RIP is essential for cell survival after TLR4 signaling and links TLR4 to the phosphatidylinositol 3 kinase-Akt pathway.     

VSV Oncolytic Virotherapy in the B16 Model Depends Upon Intact MyD88 Signaling

We show here, for the first time to our knowledge, that the antitumor therapy of oncolytic vesicular stomatitis virus (VSV) in the B16ova model depends upon signaling through myeloid differentiation primary response gene 88 (MyD88) in host cells. VSV-mediated therapy of B16ova tumors was abolished in MyD88^−/− mice despite generation of antigen-specific T cell responses similar to those in immune-competent mice. Mice defective in only toll-like receptor 4 (TLR4), TLR7, or interleukin 1 (IL-1) signaling retained VSV-induced therapy, suggesting that multiple, redundant pathways of innate immune activation by the virus contribute to antitumor immune reactivity. Lack of MyD88 signaling was associated with decreased expression of proinflammatory cytokines and neutrophil infiltration in response to intratumoral virus, as well as decreased infiltration of draining lymph nodes (LN) with plasmacytoid dendritic cells (pDCs) (CD11b⁻GR1⁺B220⁺) and myeloid-derived suppressor cells (CD11b⁺GR1⁺F4/80⁺). MyD88 signaling in response to VSV was also closely associated with a type I interferon (IFN) response. This inhibited virus replication within the tumor but also protected the host from viral dissemination from the tumor. Therefore, the innate immune response to oncolytic viruses can be, simultaneously, protherapeutic, antioncolytic, and systemically protective. These paradoxically conflicting roles need to be carefully considered in future strategies designed to improve the efficacy of oncolytic virotherapy.     

小儿脓毒症Toll样受体信号途径转导分子及调节因子的变化研究

目的 探讨Toll样受体(TLRs)信号途径转导分子及调节因子在小儿0脓毒症异常炎症免疫反应发病机制中的可能作用.方法 选择脓毒症患儿10例、严重脓毒症患儿13例及同期健康体检儿童17例.采用实时荧光定量聚合酶链反应(real-time PCR)检测前炎症细胞因子[肿瘤坏死因子-a(TNF-a)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)],以及TLRs信号途径转导分子和调节因子的mRNA表达;采用酶联免疫吸附法(ELISA)检测前炎症细胞因子蛋白水平.结果 与健康对照组比较,脓毒症组TNF-a、IL-1β和IL-6的mRNA和蛋白表达均增加,TLRs信号途径转导分子TLR2、TLR4、髓样细胞分化蛋白-88(MyD88)、TNF相关因子6(TRAF6)、IL-1受体相关激酶4(IRAK4)、转化生长因子-β活化激酶1(TAKl)、TAK1结合蛋白2(TAB2)的mRNA表达以及TLRs信号途径正性调节因子TLR4相关蛋白(PRAT4B)、信号转换接头蛋白2(STAP2)的mRNA表达均明显增高(P均<0.01),且严重脓毒症组较脓毒症组升高更显著(p均<0.01).脓毒症组TLRs信号途径负性调节因子IL-1受体相关激酶3(IRAK-M)、核转录因子-kB抑制性锌指蛋白(Triad3A)的mRNA表达均明显增高(P均<0.01),严重脓毒症组表达则明显低于脓毒症组(P均<0.01).结论 TLRs信号途径转导分子/调节因子异常表达可能是脓毒症时全身炎症反应的原因之一.     

Toll-Like Receptor-4 Coordinates the Innate Immune Response of the Kidney to Renal Ischemia/Reperfusion Injury

Toll-like receptors (TLRs) can detect endogenous danger molecules released upon tissue injury resulting in the induction of a proinflammatory response. One of the TLR family members, TLR4, is constitutively expressed at RNA level on renal epithelium and this expression is enhanced upon renal ischemia/reperfusion (I/R) injury. The functional relevance of this organ-specific upregulation remains however unknown. We therefore investigated the specific role of TLR4 and the relative contribution of its two downstream signaling cascades, the MyD88-dependent and TRIF-dependent cascades in renal damage by using TLR4−/−, MyD88−/− and TRIF-mutant mice that were subjected to renal ischemia/reperfusion injury. Our results show that TLR4 initiates an exaggerated proinflammatory response upon I/R injury, as reflected by lower levels of chemokines and infiltrating granulocytes, less renal damage and a more preserved renal function in TLR4−/− mice as compared to wild type mice. In vitro studies demonstrate that renal tubular epithelial cells can coordinate an immune response to ischemic injury in a TLR4-dependent manner. In vivo we found that epithelial- and leukocyte-associated functional TLR4 contribute in a similar proportion to renal dysfunction and injury as assessed by bone marrow chimeric mice. Surprisingly, no significant differences were found in renal function and inflammation in MyD88−/− and TRIF-mutant mice compared with their wild types, suggesting that selective targeting of TLR4 directly may be more effective for the development of therapeutic tools to prevent I/R injury than targeting the intracellular pathways used by TLR4. In conclusion, we identified TLR4 as a cellular sentinel for acute renal damage that subsequently controls the induction of an innate immune response.     

Role of TLR4 for paclitaxel chemotherapy in human epithelial ovarian cancer cells

Background  Paclitaxel has been reported to be a ligand to Toll like receptor 4 (TLR4). Myeloid differentiation factor 88(MyD88) was described as a myeloid differentiation primary response gene. TLR4 signalling owns two pathways: MyD88-dependent and MyD88-independent pathways. XIAP is a key member of the inhibitor of apoptosis protein family. Akt is a major downstream target of growth factor receptor tyrosine kinases, which negatively regulates apoptotic pathways through phosphorylation (pAkt). The aim of the present study is to investigate the role of TLR4 in paclitaxel resistance of ovarian cancer cells.
Materials and methods  We reconstructed the RNA interference expression vector, pGenesil-1-U6 specifically targeting TLR4 mRNA, which was stable transfected into the human ovarian cancer cell line SKOV3 (MyD88-positive expression) and A2780 (MyD88-negative expression). Cell proliferation, cell cycle distribution and cell apoptosis were assessed in the cells transfected with scramble control shRNA (SKOV3/shControl, A2780/shControl) and TLR4 shRNA (SKOV3/shTLR4, A2780/shTLR4) to explore the possible functions of TLR4 in ovarian cancer cells growth. The expression of TLR4, MyD88, XIAP, Akt and pAkt was analysed by Western blot analysis.
Results  A knockdown of TLR4 levels down-regulated the expression of XIAP and pAkt. And it restored the inhibitory effect of paclitaxel on cell proliferation and impeding cell cycle progression in SKOV3 cells.
Conclusions  It suggests that TLR4 negatively regulates paclitaxel chemotherapy and MyD88 is an essential downstream factor to TLR4 signalling for this resistance. Knockdown of TLR4 induces paclitaxel chemosensitivity which might depress the Akt pathway. The TLR4-MyD88 signalling represents an important source to promote tumour growth.