Toll样受体4在急性肺损伤中的作用

目的观察Toll样受体4(toll-like receptor 4,TLR4)基因突变型小鼠与野生型小鼠内毒素攻击致急性肺损伤(acute lung injury,ALI)的差异,分析TLR4在ALI中的作用。方法采用TLR4基因突变小鼠(C3H/HeJ品系,TLR4~(-/-)及野生型小鼠(CBA品系,TLR4~( / )),用内毒素攻击复制小鼠ALI模型,用显微病理及肺干湿重比(W/D)分析肺损伤程度,检测肺组织髓过氧化物酶(MPO)水平,探讨损伤差异的可能机制。结果用1,5 mg·kg~(-1)LPS溶液经尾静脉注射复制ALI模型,结果示TLR4突变小鼠肺组织大体及显微病理损伤较野生型小鼠减轻,同时肺组织水肿(W/D)亦较野生小鼠减轻,尤其是在大剂量(5mg·kg~(-1))时,差异有显著性[(4．08±0．1)vs．(4．55±0．2),n=10,t=12．71,P＜0．01]。突变小鼠肺组织PMN浸润水平较野生型低[MPO：1 mg·kg~(-1)组,(20．73±4．58)vs．(39．97±3．66),n=10,t=13．43,P＜0．01];5 mg·kg~(-1)组,[(24．0±3．94)vs．(48．5±4．07),n=10,t=15．33,P＜0．01],且两者均较假手术组高。结论TLR4可能通过介导中性粒细胞肺内聚集而导致ALI的发生发展。     

MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4.

Toll-like receptor 4 (TLR4) is a mammalian homologue of Drosophila Toll, a leucine-rich repeat molecule that can trigger innate responses against pathogens. The TLR4 gene has recently been shown to be mutated in C3H/HeJ and C57BL/10ScCr mice, both of which are low responders to lipopolysaccharide (LPS). TLR4 may be a long-sought receptor for LPS. However, transfection of TLR4 does not confer LPS responsiveness on a recipient cell line, suggesting a requirement for an additional molecule. Here, we report that a novel molecule, MD-2, is requisite for LPS signaling of TLR4. MD-2 is physically associated with TLR4 on the cell surface and confers responsiveness to LPS. MD-2 is thus a link between TLR4 and LPS signaling. Identification of this new receptor complex has potential implications for understanding host defense, as well as pathophysiologic, mechanisms.     

Intracellular recognition of lipopolysaccharide by toll-like receptor 4 in intestinal epithelial cells

Toll-like receptor (TLR)4 has recently been shown to reside in the Golgi apparatus of intestinal crypt epithelial m-ICcl2 cells, colocalizing with internalized lipopolysaccharide (LPS). Here we demonstrate that disruption of the integrity of the Golgi apparatus significantly reduced LPS-mediated nuclear factor kappaB activation. Also, the TLR4 adaptor protein MyD88 and the serine/threonine kinase IRAK-1 were rapidly recruited to the Golgi apparatus upon stimulation. LPS-mediated activation required lipid raft formation and intact clathrin-dependent internalization. In contrast to macrophages, prevention of ligand internalization by use of LPS-coated beads significantly impaired recognition by epithelial cells. The localization of TLR4 to the Golgi apparatus was abrogated by expression of a genetically modified form of the TLR4 binding chaperone gp96. Thus, our data provide evidence that in contrast to the situation in macrophages, LPS recognition in intestinal epithelial cells may occur in the Golgi apparatus and require LPS internalization.     

Toll-like receptor 4 and atherogenesis

Toll-like receptor 4 (TLR4) is a pattern recognition receptor involved in the innate immune response to various microorganisms and other exogenous and endogenous stress factors. Recently, evidence emerged that important inflammatory processes implicit in human atherogenesis are mediated in part via the TLR4/nuclear factor-kappaB pathway. Polymorphisms of TLR4, which attenuate receptor signalling, enhance the risk of acute severe infections but may have opposite effects on atherogenesis. The aim of this review is to critically discuss current experimental and epidemiological evidence for a role of TLR4 in atherogenesis and to highlight the main controversies and perspectives in this emerging field of vascular biology.     

Toll-like receptor 4 and atherogenesis

Toll-like receptor 4 (TLR4) is a pattern recognition receptor involved in the innate immune response to various microorganisms and other exogenous and endogenous stress factors. Recently, evidence emerged that important inflammatory processes implicit in human atherogenesis are mediated in part via the TLR4/nuclear factor-κB pathway. Polymorphisms of TLR4, which attenuate receptor signalling, enhance the risk of acute severe infections but may have opposite effects on atherogenesis. The aim of this review is to critically discuss current experimental and epidemiological evidence for a role of TLR4 in atherogenesis and to highlight the main controversies and perspectives in this emerging field of vascular biology.     

脂多糖对间充质干细胞Toll样受体4基因的表达水平及活性的影响

目的 探讨脂多糖(LPS)对骨髓间充质干细胞(MSC)Toll样受体4(TLR-4)基因的表达及其功能的影响.方法 采用贴壁培养和密度梯度离心法从大鼠骨髓分离MSC,通过细胞形态、成骨分化潜能及流式细胞术检测表型以鉴定其纯度;半定量RT-PCR和流式细胞术分别检测MSC在不同浓度(1、10、100 μg/ml)LPS存在条件下培养24 h的TLR-4 mRNA相对表达量和共刺激分子(CD80、CD86、MHC-Ⅱ)的表达水平;ELISA法定量检测TNF-α的分泌水平.结果 骨髓MSC低表达TLR-4mRNA(相对表达量0.61±0.10),同时表达CD80[(9.56±0.69)%]、CD86[(22.03±2.03)%]、MHC-Ⅱ[(2.51±0.97)%],少量分泌TNF-α[(4.97±2.98)pg/ml].MSC经LPS处理后,其TLR-4mRNA、共刺激分子表达和TNF-oα分泌水平均升高,其中10 μg/ml LPS培养组升高显著,TLR-4 mRNA相对表达水平为1.55±0.02;CD80、CD86、MHC-Ⅱ阳性细胞率分别为(41.70±2.92)%、(59.72±2.00)%、(24.56±2.19)%;TNF-α分泌水平为(213.12±69.08)pg/ml,与对照组比较,P值均＜0.01.100μg/ml LPS处理组与10 μg/mlLPS处理组相比,各项检测指标均降低,但MHC-Ⅱ和TNF-α的降低水平无统计学意义(P＞0.05).结论 骨髓MSC低表达TLR-4,体外LPS可促进骨髓MSCTLR-4的表达,且与浓度相关.伴随TLR-4表达水平的升高,CD80、CD86、MHC-Ⅱ表达水平及TNF-α水平同时升高.     

Toll样受体4与急腹症

Toll样受体4(TLR4)为研究最广的先天性免疫受体,主要与免疫、感染相关;已证明与多种感染性疾病、急性脏器损伤及某些肿瘤相关。近年来发现TLR4与新生儿坏死性小肠结肠炎、急性胰腺炎、腹膜炎等急腹症密切相关。现对其研究进展加以综述,为急腹症进一步的研究提供向导。     

Toll样受体在脂多糖耐受性机制作用中的研究进展

脂多糖(LPS)耐受性是指机体或细胞经低剂量LPS刺激后，对LPS的再次刺激呈低反应或无反应状态，它与LPS信号转导变化密切相关。LPS细胞外信号转导是通过脂多糖结合蛋白(LBP)呈递给CD4^ 来实现的，但对LPS跨膜转导机制一直不明确。近年来Toll样受体(TLRs)的发现为此提供了新的线索。TLRs表达量和功能下调及TLR4信号通路中各个环节的功能缺陷，     

Toll-like receptors: their roles in bacterial recognition and respiratory infections

Although respiratory infections cause significant morbidity and mortality throughout the world, the immunologic factors that mediate host susceptibility to these infections remain poorly understood. The lung contains a vast surface at the host-environment interface and acts as a crucial barrier to invading pathogens. The lung is equipped with specialized epithelial and hematopoietic cells, which express pattern recognition receptors that act as both sentinels and mediators of pulmonary innate immunity. Toll-like receptors (TLRs) mediate a particularly critical role in pathogen recognition and subsequent initiation of the host immune response. In this review, we will summarize current knowledge of TLRs and their bacterial ligands and explore their role in respiratory infections. Moreover, we will highlight recent advances in the role of TLRs in pulmonary infections from a human immunogenetics perspective.     

Toll-like receptor 4 deficiency causes pulmonary emphysema

TLRs have been studied extensively in the context of pathogen challenges, yet their role in the unchallenged lung is unknown. Given their direct interface with the external environment, TLRs in the lungs are prime candidates to respond to air constituents, namely particulates and oxygen. The mechanism whereby the lung maintains structural integrity in the face of constant ambient exposures is essential to our understanding of lung disease. Emphysema is characterized by gradual loss of lung elasticity and irreversible airspace enlargement, usually in the later decades of life and after years of insult, most commonly cigarette smoke. Here we show Tlr4(-/-) mice exhibited emphysema as they aged. Adoptive transfer experiments revealed that TLR4 expression in lung structural cells was required for maintaining normal lung architecture. TLR4 deficiency led to the upregulation of what we believe to be a novel NADPH oxidase (Nox), Nox3, in lungs and endothelial cells, resulting in increased oxidant generation and elastolytic activity. Treatment of Tlr4(-/- )mice or endothelial cells with chemical NADPH inhibitors or Nox3 siRNA reversed the observed phenotype. Our data identify a role for TLR4 in maintaining constitutive lung integrity by modulating oxidant generation and provide insights into the development of emphysema.     

A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin

The molecular basis for Toll-like receptor (TLR) recognition of microbial ligands is unknown. We demonstrate that mouse and human TLR5 discriminate between different flagellins, and we use this difference to map the flagellin recognition site on TLR5 to 228 amino acids of the extracellular domain. Through molecular modeling of the TLR5 ectodomain, we identify two conserved surface-exposed regions. Mutagenesis studies demonstrate that naturally occurring amino acid variation in TLR5 residue 268 is responsible for human and mouse discrimination between flagellin molecules. Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition. These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand. These findings suggest that ligand binding at the beta sheets results in TLR activation and provide a new framework for understanding TLR-agonist interactions.     

Toll样受体4在免疫相关细胞中的作用

Toll样受体(TLRs)是参与固有免疫的重要蛋白分子,也是连接固有免疫和适应性免疫的桥梁。TLR4是TLRs家族的重要成员之一。TLR4通过调控中性粒细胞和单核巨噬细胞的活化,可促进细胞因子的释放,启动固有免疫应答。同时TLR4能促进树突状细胞的成熟,激活初始T细胞,启动适应性免疫应答。在T细胞和B细胞的适应性免疫应答中,TLR4可促进细胞分泌细胞因子,介导全身炎症反应,并调控记忆性细胞的生成。本文就TLR4在中性粒细胞、树突状细胞、单核巨噬细胞、T淋巴细胞和B淋巴细胞中的作用进行综述。     

Functional selectivity of melanocortin 4 receptor peptide and nonpeptide agonists: evidence for ligand-specific conformational states

Agonists of the melanocortin 4 (MC4) receptor have potential pharmaceutical benefit in the treatment of obesity and sexual dysfunction. In this study, we have compared the ability of a number of peptide and nonpeptide agonists to activate a FLAG-tagged human MC4 (FMC4) receptor, as measured by both cAMP accumulation and calcium mobilization using a fluorometric imaging plate reader (FLIPR). In addition, we have analyzed the ability of these agonists to cause receptor internalization, as measured by fluorescence-activated cell sorting analysis. The endogenous agonist alpha-melanocortin-stimulating hormone (alpha-MSH) increased cAMP accumulation, calcium mobilization, and receptor internalization in a dose-dependent manner in human embryonic kidney 293 cells expressing the FMC4 receptor. The activity of the other agonists varied considerably in these assays, and overall, the potency and intrinsic activity of the agonists in the cAMP accumulation assays did not correlate with their potency or intrinsic activity in either the FLIPR or receptor internalization assays. Agonists could be clearly separated into two functional classes based on their structure. Peptide agonists beta-MSH, des-acetyl-alpha-MSH, and [Nle(4), D-Phe(7)]-alpha-melanocortin-stimulating hormone exhibited 80 to 112% of the maximal alpha-MSH response in cAMP accumulation and 62 to 96% in FLIPR assays and were able to cause 75 to 118% of receptor internalization induced by alpha-MSH. Conversely, although the nonpeptide agonists exhibited 73 to 149% of the alpha-MSH response in the cAMP accumulation assays, they were significantly impaired in the FLIPR (7-40%) and receptor internalization (-5-38%) assays. These findings demonstrate an important difference in activation and internalization of the MC4 receptor by nonpeptide versus peptide agonists and provides evidence of agonist-specific conformational states.     

高氧致大鼠急性肺损伤Toll样受体2和4的变化及意义

目的 探讨Toll样受体(TLRs)在高氧致急性肺损伤(ALI)发病过程中的作用.方法 将32只SD大鼠按随机数字表法分为空气对照组和高氧暴露24、48、72 h组,每组8只.分别于相应时间点活杀8只大鼠,取肺组织标本,测定肺湿/干重(W/D)比值并行肺组织病理评分,用实时荧光定量逆转录-聚合酶链反应(RT-PCR)测定肺组织TLR2和TLR4的mRNA表达,用蛋白质免疫印迹法(Western blotting)测定肺组织TLR2和TLR4的蛋白表达,用酶联免疫吸附法(ELISA)测定肺组织匀浆中白细胞介素-6(IL-6)含量.结果 各高氧暴露组肺W/D比值均较空气对照组明显增高(P均<0.01).高氧暴露48 h、72 h肺组织病理评分[(2.69±0.53)分,(3.94±0.62)分]均明显高于空气对照组[(0.41±0.38)分,P均<0.01].RT-PCR结果显示,高氧暴露组肺组织TLR2和TLR4 mRNA表达均明显高于空气对照组(0.67±0.15和0.63±0.19),并均于24 h达高峰(1.82±0.33,1.35±0.26,P均<0.05).Western blotting结果显示,高氧暴露组TLR2和TLR4蛋白表达均较空气对照组[(7.20±0.51)%和(14.26±0.19)%]明显增高,分别于48 h、72 h达峰值[(28.12±0.24)%,(81.35±0.82)%,P均<0.05].ELISA结果显示,高氧暴露组肺组织匀浆IL-6含量较空气对照组[(639.38±95.24)pg/L]明显增高,于72 h达高峰[(1 300.58±442.24)pg/L,P<0.05].结论 在高氧引起的ALI中,TLR2和TLR4在肺组织炎症启动和维持中起重要作用.     

A bacterial carbohydrate links innate and adaptive responses through Toll-like receptor 2

Commensalism is critical to a healthy Th1/Th2 cell balance. Polysaccharide A (PSA), which is produced by the intestinal commensal Bacteroides fragilis, activates CD4+ T cells, resulting in a Th1 response correcting the Th2 cell skew of germ-free mice. We identify Toll-like receptors as crucial to the convergence of innate and adaptive responses stimulated by PSA. Optimization of the Th1 cytokine interferon-gamma in PSA-stimulated dendritic cell-CD4+ T cell co-cultures depends on both Toll-like receptor (TLR) 2 and antigen presentation. Synergy between the innate and adaptive responses was also shown when TLR2-/- mice exhibited impaired intraabdominal abscess formation in response to B. fragilis. Commensal bacteria, using molecules like PSA, potentially modulate the Th1/Th2 cell balance and the response to infection by coordinating both the innate and adaptive pathways.     

Sustained desensitization to bacterial Toll-like receptor ligands after resolution of respiratory influenza infection

The World Health Organization estimates that lower respiratory tract infections (excluding tuberculosis) account for approximately 35% of all deaths caused by infectious diseases. In many cases, the cause of death may be caused by multiple pathogens, e.g., the life-threatening bacterial pneumonia observed in patients infected with influenza virus. The ability to evolve more efficient immunity on each successive encounter with antigen is the hallmark of the adaptive immune response. However, in the absence of cross-reactive T and B cell epitopes, one lung infection can modify immunity and pathology to the next for extended periods of time. We now report for the first time that this phenomenon is mediated by a sustained desensitization of lung sentinel cells to Toll-like receptor (TLR) ligands; this is an effect that lasts for several months after resolution of influenza or respiratory syncytial virus infection and is associated with reduced chemokine production and NF-kappaB activation in alveolar macrophages. Although such desensitization may be beneficial in alleviating overall immunopathology, the reduced neutrophil recruitment correlates with heightened bacterial load during secondary respiratory infection. Our data therefore suggests that post-viral desensitization to TLR signals may be one possible contributor to the common secondary bacterial pneumonia associated with pandemic and seasonal influenza infection.     

紫癜性肾炎患者外周血单个核细胞Toll样受体3和4的信号转导途径

背景：目前关于Toll样受体3和Toll样受体4介导的信号转导通路在紫癜性肾炎的发病机制中的作用尚不清楚。 目的：分析Toll样受体3和Toll样受体4在过敏性紫癜和紫癜性肾炎发病机制中的作用。方法：选取过敏性紫癜患儿64例,分为过敏性紫癜无肾损害组36例及过敏性紫癜性肾炎组28例,另选健康儿童30例作为正常对照组。实时荧光定量PCR检测外周血单核细胞Toll样受体3、Toll样受体4、髓样分化蛋白2、髓样细胞分化因子88、白细胞介素1β、白细胞介素6、白细胞介素12 mRNA的基因相对表达量;应用流式细胞术检测外周血单核细胞Toll样受体3、Toll样受体4蛋白表达率。结果与结论：①过敏性紫癜患儿Toll样受体4 mRNA及蛋白表达显著高于正常对照组（P 〈 0.05）。紫癜性肾炎组Toll样受体4 mRNA及蛋白表达均显著高于紫癜无肾损害组（P 〈 0.05）。②过敏性紫癜组髓样分化蛋白2、髓样细胞分化因子88、白细胞介素1β、白细胞介素6 mRNA的表达均显著高于正常对照组（P 〈 0.05）,白细胞介素12 mRNA的表达显著低于正常对照组（P 〈 0.05）;紫癜性肾炎组髓样分化蛋白2、髓样细胞分化因子88、白细胞介素1β、白细胞介素6 mRNA的表达显著高于紫癜无肾损害组（P 〈 0.05）,紫癜性肾炎组白细胞介素12 mRNA的表达显著低于紫癜无肾损害组（P 〈 0.05）。③过敏性紫癜组患儿外周血单核细胞Toll样受体4 mRNA与蛋白表达呈正相关（r=0.60,P 〈 0.01）;过敏性紫癜患儿Toll样受体4 mRNA与髓样分化蛋白2、髓样细胞分化因子88、白细胞介素1β、白细胞介素6表达均呈正相关（P 〈 0.01）,与白细胞介素12 mRNA表达呈负相关（r=-0.66,P 〈 0.01）。提示Toll样受体4可能通过髓样细胞分化因子88依赖信号转导途径介导过敏性紫癜的免疫发病机制,Toll样受体4的过度活化可能与过敏性紫癜的肾损伤有关。     

Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) have been identified as a potent secretor of the type I interferons (IFNs) in response to CpG as well as several viruses. In this study, we examined the molecular mechanism of virus recognition by pDCs. First, we demonstrated that the CD11c+Gr-1intB220+ pDCs from mouse bone marrow secreted high levels of IFN-alpha in response to either live or UV-inactivated Herpes simplex virus-2 (HSV-2). Next, we identified that IFN-alpha secretion by pDCs required the expression of the adaptor molecule MyD88, suggesting the involvement of a Toll-like receptor (TLR) in HSV-2 recognition. To test whether a TLR mediates HSV-2-induced IFN-alpha secretion from pDCs, various knockout mice were examined. These experiments revealed a clear requirement for TLR9 in this process. Further, we demonstrated that purified HSV-2 DNA can trigger IFN-alpha secretion from pDCs and that inhibitory CpG oligonucleotide treatment diminished HSV-induced IFN-alpha secretion by pDCs in a dose-dependent manner. The recognition of HSV-2 by TLR9 was mediated through an endocytic pathway that was inhibited by chloroquine or bafilomycin A1. The strict requirement for TLR9 in IFN-alpha secretion was further confirmed by the inoculation of HSV-2 in vivo. Therefore, these results demonstrate a novel mechanism whereby the genomic DNA of a virus can engage TLR9 and result in the secretion of IFN-alpha by pDCs.     

Toll样受体4介导的信号转导与肠黏膜损伤

由肠黏膜机械性屏障受损引起的细菌/内毒素移位是导致肠源性感染,引起全身炎症反应综合征(SIRS)和多器官功能障碍综合征(MODS),最终发展为多器官功能衰竭(MOF)的重要原因之