Toll样受体通路调节Tregs功能的研究进展

模式识别受体Toll样受体(Toll like receptors,TLRs)是固有免疫中免疫受体的代表,进化上十分保守,对生物体的生存极为重要。TLRs通过内源或外源的配体启动信号转导,激活下游一系列重要的基因表达与活化。研究表明调节性T细胞(Regulatory T cell,Treg)在维持机体外周免疫耐受和阻止移植排斥反应等方面发挥核心作用。Treg细胞表达某些TLRs,包括TLR2、TLR4、TLR5、TLR7、TLR8、TLR9等。TLRs的活化可能直接或间接地影响(主要是活化) Treg的增殖和免疫抑制功能,这种调节与感染、自身免疫病和癌症的发生密切相关。其中热休克蛋白作为TLRs配体分子对于Treg的调节发挥了重要的作用。因此,了解TLRs通路对研究Treg免疫调控机制、新药物研发和靶向治疗有重大意义。文中简要介绍了TLRs通路调节Treg免疫功能的相关研究进展。     

氧化性低密度脂蛋白对人单个核细胞Toll样受体表达及炎症介质的影响

目的:探讨氧化性低密度脂蛋白(ox-LDL)对单个核细胞Toll样受体1～10(TLR1～10)表达的影响及对炎症因子TNF-α、IL-6的调控作用。方法:用逆转录-聚合酶链反应检测健康人单个核细胞中TLR1～10 mRNA的组成型表达;用实时定量-聚合酶链反应检测ox-LDL刺激单个核细胞后,TLR1～10mRNA表达的变化;结合抗体阻断实验,用酶联免疫吸附法(ELISA)检测培养上清液中TNF-α和IL-6的含量。结果:人单个核细胞有TLR1～10 mRNA的组成型表达,ox-LDL刺激可上调人单个核细胞TLR2、TLR4 mRNA的表达。经ox-LDL刺激后,单个核细胞分泌TNF-α、IL-6上升,受体阻断剂阻断TLR2、TLR4后,可以减少ox-LDL诱导的TNF-α、IL-6分泌。结论:ox-LDL可能是TLRs的内源性配体,ox-LDL可通过TLR信号通路调节单个核细胞炎性细胞因子的生成。     

TLR4及其作用的研究进展

Toll样受体(toll-like receptors,TLRs)因其积极的研究成果而成为近年来广受关注的一种病原体识别受体,TLRs分布相对比较广泛,不但在小肠上皮、呼吸上皮细胞表达,同时也在血管内皮细胞、树突状细胞[1]、大鼠脾及心肌细胞[2]等细胞中表达.研究证实,它属于模式识别受体(pattern recognition receptors,PRRs),病原相关分子模式(pathogen-associated molecule pattern,PAMPs)可被其辨别,然后引发一系列的信号转导,TLRs 是备受关注的一种PRRs.Toll样受体4(toll-like receptor 4,TLR4)是TLRs家族中极为重要的成员,是天然免疫系统识别病原微生物的主要受体,在天然免疫反应中扮演着关键性作用.细菌脂多糖(lipopolysaccharide,LPS)作为一类受体,主要作用是介导信号跨膜转导,尤其对革兰氏阴性菌所引起的感染性炎症起着极为关键的作用.由于近年来对TLR4介导的信号转导及TLR4与疾病的关系研究成为热点,本文就TLR4的信号转导、TLR4与LPS的关系及TLR4信号通路调节进行综述如下.     

髓样分化蛋白-2在天然免疫中的作用

Toll样受体 (Toll likereceptor ,TLR)家族作为模式识别受体 ,在天然免疫中具有重要作用。髓样分化蛋白 2 (myeloiddifferentialprotein 2 ,MD 2 )可能含有两个相对独立的功能结构域 ,既能与Toll样受体家族中的TLR4、TLR2结合 ,也能与多种配体结合 (包括lipopolysaccharide ,LPS)。这种特殊的结构可能与其三方面的主要功能有关 :(1)MD 2与TLR4结合 ,赋予TLR4对各种配体 (包括LPS)的反应性 ;(2 )MD 2与TLR2结合 ,赋予TLR2对LPS的反应性 ,并增强TLR2对细菌及其胞壁成分的反应性 ;(3)MD 2能促进TLR4和TLR2的表达 ,并且与TLR4在细胞内的分布密切相关。这表明MD 2可以通过两种方式直接或间接调控TLRs的功能 :与TLR2 /TLR4结合 ,或调控TLR2 /TLR4的表达与分布。因而MD 2不仅仅是TLR4的辅助分子 ,而且还是天然免疫中的调控分子 ,可能在感染、炎症、免疫等病理生理过程中具有更广泛的生物学功能     

Toll样受体4与肝损伤的研究进展

Toll样受体4((toll like receptor4,TLR4)是内毒素(LPS)的关键受体,为Toll蛋白家族中的一个成员,是联系固有免疫和适应性免疫的纽带。TLR4主要表达于髓源性细胞,其启动的胞内信号转导在肝损伤的发生和发展过程中发挥重要作用。这一信号转导途径主要通过NF-κB、p38、JNK等的激活,使细胞产生炎症转录因子,介导肝脏炎症。TLR4与氧化应激的相互作用,使得肝脏对TLR4的配体及细胞因子的敏感性增加,从而加重肝脏损伤。随着TLR4在肝损伤中的作用进一步阐明,其在肝脏疾病中的治疗作用将会产生广阔的应用前景。     

TLR与足细胞损伤

TLRs（Toll—likereceptors）是一类重要的模式识别受体家族,主要调节灭然免疫反应。研究发现在。肾脏固有细胞及间质细胞都有TLR表达,其介导的炎性反应参与了许多肾脏疾病的发生。多种足细胞标志蛋白的发现加快了足细胞表面分子的研究进程。目前已发现足细胞表面有TLR的表达,且TLR的表达与足细胞的损伤有关。     

Toll样受体在双歧杆菌脂磷壁酸诱导结肠癌细胞凋亡中的作用

目的探讨双歧杆菌脂磷壁酸（LTA）对Toll样受体（TLRs）表达的影响及其与诱导结肠癌细胞凋亡之间的关系。方法用AnnexinV检测在双歧杆菌LTA处理前后结肠癌Lovo细胞凋亡的变化;流式细胞术检测Lovo细胞表面TLRs的表达,并用相应的TLRs封闭抗体作用后,AnnexinV检测经双歧杆菌LTA诱导的Lovo细胞凋亡的变化。结果经双歧杆菌LTA处理后,结肠癌Lovo细胞发生了明显的凋亡,并有一定的时间和剂量依赖关系;结肠癌Lovo细胞有TLR受体的基础表达,经双歧杆菌LTA处理后,TLR2和TLR4在Lovo细胞上的表达增加,其中尤以TLR2增加更为明显;用相应的TLRs抗体封闭作用后,双歧杆菌LTA诱导Lovo细胞凋亡的能力下降。结论双歧杆菌LTA能诱导肿瘤细胞凋亡,并且TLRs特别是TLR2在LTA诱导肿瘤细胞凋亡中可能发挥着主要作用,TLR4可能仅起着协同作用。     

病原真菌感染与TOLL样受体

TOLL样受体(TLR)是参与天然免疫的主要模式识别受体之一,与许多微生物病原体及其产物的病原相关分子模式PAMP结合后通过MyD88依赖性或非依赖性途径启动宿主胞内信号传导途径,引发一系列生物学效应。白色念珠菌表面的特征性糖磷脂甘露聚糖可被TLR2、TLR4识别,诱导前炎性细胞因子的释放及促进中性粒细胞的聚集等来介导宿主的抗真菌免疫反应。烟曲霉则可能利用表型转换(酵母样与菌丝态),通过不同TLRs逃避宿主天然免疫系统的识别。新型隐球菌的多糖荚膜成分葡糖醛氧化甘露聚糖GXM可与TLR2、TLR4、CD14结合,在单核细胞、巨噬细胞对GXM的内化、吞噬中起重要作用,而不是诱导细胞因子的分泌;酿酒酵母胞壁成分酵母多糖则可激活TLR2、TLR6异源二聚体。总之,TLR与真菌配体相互作用的具体机制及其活化后胞内信号传导调控机制的深入研究与分析,对临床真菌病的免疫调节及治疗具有重要意义。     

哺乳动物Toll样受体与组织再生修复

机体损伤后通过诱导组织细胞产生复杂而又相互调控的系列反应,来促进损伤组织的再生.不同细胞因子、生长因子及细胞之间的协调平衡对于组织再生的调节非常重要,免疫系统在此过程中起着极其重要的作用.Toll样受体(Toll-like receptors,TLRs)可识别微生物病原体,在触发机体防御性抗病原微生物免疫反应中发挥着重要作用,是先天免疫系统中必不可少的重要成分,TLRs内源性配体的存在提示TLRs不仅可诱导机体防御性的抗微生物免疫反应,同时还是机体损伤后启动组织再生修复的敏感监测系统.本文概述了TLRs及其内源性配体,以及TLRs在诱导损伤后组织再生中的作用.TLR内源性配体及其在组织再生过程中的作用为促进机体损伤组织的再生修复提供了新的思路策略.     

TLR9介导DNA病毒的免疫识别

Toll样受体(toll-like receptor,TLR)是免疫细胞表面的模式识别受体(patttern recognition receptoi,PRR),参与微生物病原体相关分子模式(pathogen associated molecular patterns,PAMPs)的识别,从而诱导天然免疫应答。迄今在人类已经确定了10个TLRs家族成员。不同的TLRs识别不同的PAMPs,如TLR9是免疫细胞识别病毒和细菌中非甲基化DNA的必需成分;     

Toll-like receptor expression in normal ovary and ovarian tumors

Recent studies have implicated inflammation in the initiation and progression of ovarian cancer, though the mechanisms underlying this effect are still not clear. Toll-like receptors (TLRs) allow immune cells to recognize pathogens and to trigger inflammatory responses. Tumor cell expression of TLRs can promote inflammation and cell survival in the tumor microenvironment. Here we sought to characterize the expression of TLRs in normal human ovaries, benign and malignant ovarian tumors from patients, and in established ovarian tumor cell lines. We report that TLR2, TLR3, TLR4, and TLR5 are strongly expressed on the surface epithelium of normal ovaries. In contrast to previous studies of uterus and endocervix, we found no cyclic variation in TLR expression occurred in murine ovaries. TLR2, TLR3, TLR4, and TLR5 are expressed in benign conditions, epithelial tumors, and in ovarian cancer cell lines. Variable expression of TLR6 and TLR8 was seen in benign and malignant epithelium of some patients, while expression of TLR1, TLR7, and TLR9 was weak. Normal and malignant ovarian stroma were negative for TLR expression. Vascular endothelial cells, macrophages, and occasional fibroblasts in tumors were positive. Functional activity for TLRs was demonstrated by stimulation of cell lines with specific ligands and subsequent activation and translocation of NFκB and release of the proinflammatory cytokines interleukin-6 and CCL-2. These studies demonstrate expression of multiple TLRs in the epithelium of normal ovaries and in ovarian tumor cells, and may indicate a mechanism by which epithelial tumors manipulate inflammatory pathways to facilitate tumor progression.     

Activation of human neutrophils with Helicobacter pylori and the role of Toll-like receptors 2 and 4 in the response

The innate immune response to Helicobacter pylori infection is beginning to be understood and recent works support a role for Toll-like receptors (TLRs). Our aim was to study the response of human neutrophils to H. pylori and to elucidate the role of TLR2 and TLR4. Neutrophils from healthy H. pylori-negative volunteers were cocultured with H. pylori 26695 strain. The release of IL-8, IL-1beta, tumor necrosis factor (TNF)-alpha and IL-10 was measured. The role of TLR2 and TLR4 was investigated with blocking assays using monoclonal antibodies against TLRs. Neutrophils produced a significant increase of IL-8, IL-1beta and TNF-alpha after 3, 6 and 24 h of H. pylori challenge, respectively, whereas IL-10 increased after 24 h. Helicobacter pylori challenge increased TLR2 and TLR4 expression; and antibodies against TLR2 and TLR4 diminished significantly the H. pylori-induced production of IL-8 and IL-10. In human neutrophils, H. pylori induces an early inflammatory response, partially mediated via TLR2 and TLR4 activation.     

Toll-like receptor 3 mediates expression of clusterin/apolipoprotein J in vascular smooth muscle cells stimulated with RNA released from necrotic cells

Clusterin/Apolipoprotein J is a protein that is upregulated in a broad spectrum of diverse pathological processes. The predominant form is a secreted glycoprotein (sCLU) with cytoprotective and anti-inflammatory properties which shows enhanced expression in vascular smooth muscle cells (VSMC) following aortic injury and in atherosclerotic disease. Recent evidence indicates that during atherosclerosis, Toll-like receptors (TLRs) are activated in vascular cells by endogenous ligands. Here, we analyzed whether CLU expression in VSMC is controlled by TLRs, and stimulated by factors associated with or released by necrotic cells. Activation of TLR3 by the synthetic RNA analogue polyinosinic-polycytidylic acid (poly(I:C)) in CRL2018 VSMC and in mice led to induction of CLU mRNA and protein synthesis, respectively. In TLR3-deficient 10A yolk sac cells, induction of CLU by poly(I:C) challenge depended on the ectopic expression of human TLR3. In mice lacking the TLR3-signaling adaptor protein TRIF (TIR-domain-containing adaptor protein inducing IFN-β) CLU induction by poly(I:C) was abrogated. In addition to poly(I:C) CLU gene expression in CRL2018 cells was induced by purified cellular RNA and RNA present in necrotic cell lysate.Our data indicate that cellular RNA following its release from necrotic cells in atherosclerotic lesions can act as an endogenous TLR3 ligand to induce CLU expression in VSMC and in vivo. Thus, they expand the view on TLR2 and TLR4 as known pro-atherosclerotic effectors toward TLR3. Conclusively, TLR3 activation induces expression of cytoprotective and anti-inflammatory CLU by VSMC and mice, to potentially counteract atherosclerotic pathology.     

Toll-like receptor 4 signaling plays a role in triggering periodontal infection

Toll-like receptors (TLRs) are a group of sensors on the surface of antigen-presenting cells, such as dendritic cells and macrophages, which recognize microbial pathogens and induce innate and adaptive immune responses. Periodontitis is an inflammatory disease characterized by the destruction of tooth-supporting structures. In order to address whether TLR4 signaling plays a role in periodontitis, we studied the gene expression change in human periodontal ligament cells (HPDLCs) in response to TLR4 ligand, lipopolysaccharide treatment by microarray analysis. Expression of TLR4 was detected in HPDLCs. Lipopolysaccharide treatment increased the expression of 12 genes (more than twofold), including TLR4, TLR5, TLR7, Pellino 1, colony stimulating factor 2 (CSF2) and IL-6. In addition, the expression of 15 genes (less than equal to twofold) was decreased, including Fos, LY64 and LY86. In addition, real-time PCR was used to confirm the change of gene expression of TLR4, IL-6 and Fos. We also showed that the upregulation of IL-6 by lipopolysaccharide treatment was TLR4-dependent. This pattern of gene expression indicates that pathogens may trigger TLR4 signaling and cause periodontitis. Manipulating TLR4 signaling may potentially become one of the recognized therapies for periodontitis.     

Toll-like receptors expressed in tumor cells: targets for therapy

Toll-like receptors (TLRs), mainly expressing in human immune related cells and epithelial cells, play an essential role in the host defense against microbes by recognizing conserved bacterial molecules. Recently, the expression or up-regulation of TLRs has been detected in many tumor cell lines or tumors, especially epithelial derived cancers. Although the TLR profile varies on different tumor cells, the current evidences indicate that the expression of TLRs is functionally associated with tumor progression. TLR expression may promote malignant transformation of epithelial cells. Engagement of TLRs increases tumor growth and tumor immune escape, and induces apoptosis resistance and chemoresistance in some tumor cells. These findings demonstrate that TLR is a promising target for the development of anticancer drugs and make TLR agonists or antagonists the potential agents for tumor therapy.     

Discovery of substituted 4-aminoquinazolines as selective Toll-like receptor 4 ligands

The Toll-like receptors (TLRs) are critical components of the innate immune system that regulate immune recognition in part through NF-κB activation. A human cell-based high throughput screen (HTS) revealed substituted 4-aminoquinazolines to be small molecular weight activators of NF-κB. The most potent hit compound predominantly stimulated through the human TLR4/MD2 complex, and had less activity with the mouse TLR4/MD2. There was no activity with other TLRs and the TLR4 activation was MD-2 dependent and CD14 independent. Synthetic modifications of the quinazoline scaffold at the 2 and 4 positions revealed trends in structure–activity relationships with respect to TLR dependent production of the NF-κB associated cytokine IL-8 in human peripheral blood mononuclear cells, as well as IL-6 in mouse antigen presenting cells. Furthermore, the hit compound in this series also activated the interferon signaling pathway resulting in type I interferon production. Substitution at the O-phenyl moiety with groups such as bromine, chlorine and methyl resulted in enhanced immunological activity. Computational studies indicated that the 4-aminoquinazoline compounds bind primarily to human MD-2 in the TLR4/MD-2 complex. These small molecules, which preferentially stimulate human rather than mouse innate immune cells, may be useful as adjuvants or immunotherapeutic agents.     

Alteration of Toll-like receptor 4 activation by 4-hydroxy-2-nonenal mediated by the suppression of receptor homodimerization

Toll-like receptors (TLRs) detect invading microbial pathogens and initiate immune responses as part of host defense mechanisms. They also respond to host-derived substances released from injured cells and tissues to ensure wound healing and tissue homeostasis. Dysregulation of TLRs increases the risk of chronic inflammatory diseases and immune disorders. Inflammatory events are often accompanied by oxidative stress, which generates lipid peroxidation products such as 4-hydroxy-2-nonenal (4-HNE). Therefore, we investigated if 4-HNE affects TLR activation. We found that 4-HNE blocked LPS (a TLR4 agonist)-induced activation of NFκB and IRF3 as well as expression of IFNβ, IP-10, RANTES, and TNFα. To investigate the mechanism of inhibition by 4-HNE, we examined its effects on TLR4 dimerization, one of the initial steps in TLR4 activation. 4-HNE suppressed both ligand-induced and ligand-independent receptor dimerization. The thiol donors, DTT and NAC, prevented the inhibitory effects of 4-HNE on TLR4 dimerization, and LC–MS/MS analysis showed that 4-HNE formed adducts with cysteine residues of synthetic peptides derived from TLR4. These observations suggest that the reactivity of 4-HNE with sulfhydryl moieties is implicated in the inhibition of TLR4 activation. Furthermore, inhibition of TLR4 activation by 4-HNE resulted in down-regulation of the phagocytic activity of macrophages. Collectively, these results demonstrate that 4-HNE blocks TLR4-mediated macrophage activation, gene expression, and phagocytic functions, at least partly by suppressing receptor dimerization. They further suggest that 4-HNE influences innate immune responses at sites of infection and inflammation by inhibiting TLR4 activation.     

Enteroendocrine cells express functional Toll-like receptors

Intestinal epithelial cells (IECs) provide a physical and immunological barrier against enteric microbial flora. Toll-like receptors (TLRs), through interactions with conserved microbial patterns, activate inflammatory gene expression in cells of the innate immune system. Previous studies of the expression and function of TLRs in IECs have reported varying results. Therefore, TLR expression was characterized in human and murine intestinal sections, and TLR function was tested in an IEC line. TLR1, TLR2, and TLR4 are coexpressed on a subpopulation of human and murine IECs that reside predominantly in the intestinal crypt and belong to the enteroendocrine lineage. An enteroendocrine cell (EEC) line demonstrated a similar expression pattern of TLRs as primary cells. The murine EEC line STC-1 was activated with specific TLR ligands: LPS or synthetic bacterial lipoprotein. In STC-1 cells stimulated with bacterial ligands, NF-kappaB and MAPK activation was demonstrated. Furthermore, the expression of TNF and macrophage inhibitory protein-2 were induced. Additionally, bacterial ligands induced the expression of the anti-inflammatory gene transforming growth factor-beta. LPS triggered a calcium flux in STC-1 cells, resulting in a rapid increase in CCK secretion. Finally, conditioned media from STC-1 cells inhibited the production of nitric oxide and IL-12 p40 by activated macrophages. In conclusion, human and murine IECs that express TLRs belong to the enteroendocrine lineage. Using a murine EEC model, a broad range of functional effects of TLR activation was demonstrated. This study suggests a potential role for EECs in innate immune responses.     

Toll-like receptor-3-induced mitochondrial dysfunction in cultured human hepatocytes

Several studies have shown the presence of liver mitochondrial dysfunction during sepsis. TLR3 recognizes viral double-stranded RNA and host endogenous cellular mRNA released from damaged cells. TLR3 ligand amplifies the systemic hyperinflammatory response observed during sepsis and in sepsis RNA escaping from damaged tissues/cells may serve as an endogenous ligand for TLR3 thereby modulating immune responses. This study addressed the hypothesis that TLR3 might regulate mitochondrial function in cultured human hepatocytes.HepG2 cells were exposed to TLR-3 ligand (dsRNA — polyinosine–polycytidylic acid; Poly I:C) and mitochondrial respiration was measured. Poly I:C induced a reduction in maximal mitochondrial respiration of human hepatocytes which was prevented partially by preincubation with cyclosporine A (a mitochondrial permeability transition pore-opening inhibitor). Poly-I:C induced activation of NF-κB, and the mitochondrial dysfunction was accompanied by caspase-8 but not caspase-3 activation and by no major alterations in cellular or mitochondrial ultrastructure.