Fucoidan对巨噬细胞RAW264.7的免疫调节作用

目的 观察褐藻多糖硫酸酯（Fucoidan）对巨噬细胞RAW264.7体外吞噬活性、细胞因子TNF-α和IL-6分泌,以及Toll样受体4（TLR4）mRNA表达的影响。方法 实验分对照组,Fucoidan高、中、低剂量组（浓度分别是200、400和800 μg/mL）。药物处理6~48 h后,MTT法检测RAW264.7细胞活力;中性红比色法检测细胞吞噬活性;ELISA法检测培养上清中TNF-α和IL-6的分泌水平;实时定量PCR检测Toll样受体4（TLR4）mRNA表达量。结果 与对照组相比,Fucoidan显著增强RAW264.7细胞代谢活力和吞噬能力（P＜0.01）,增加TNF-α和IL-6的分泌,上调TLR4的表达,呈剂量依赖关系。结论 Fucoidan可上调TLR4表达,增强巨噬细胞代谢和吞噬活性,增加TNF-α和IL-6的分泌,具有潜在的调节免疫作用。     

激活Toll样受体2可以促进胃癌细胞中的OXPHOS和糖酵解

目的:调查TLR家族中哪种TLR受体的配体依赖性激活可引起胃癌细胞的代谢重编程。方法:通过实时荧光定量PCR(RT-qPCR)和蛋白质印迹(WB)在一组人GC细胞中测量TLR家族成员的表达。通过进行Seahorse生物能测定以及测量L-乳酸和活性氧(ROS)的产生,确定激动剂对不同TLR(TLR2、4、9)诱导的人GC细胞的代谢变化;通过RT-qPCR在被刺激的GC细胞中分析了涉及氧化磷酸化和糖酵解的基因的表达;通过Western印迹表征SOD2的表达。结果:由合成分子或全病原体抗原激活的TLR2信号传导增强了胃癌细胞中高表达TLR2的细胞株的糖酵解活性和线粒体呼吸,而配体诱导的TLR4和TLR9活化抑制了线粒体呼吸或细胞外酸化率。同时,涉及葡萄糖代谢和氧化还原系统调节的基因,例如HIF1A,PFKFB3和SOD2,在TLRs下游被上调。结论:由配体诱导的特定TLRs的激活介导了人类GC细胞中不同的代谢表型。TLR2是唯一同时促进OXPHOS和糖酵解的家族成员,这可能导致肿瘤进展。     

组成型过量表达hTERT对Vero细胞体外培养生物学特性的影响

目的：考察组成型过量表达人端粒酶催化亚单位（hTERT）对Vero细胞在无血清培养体系中的细胞形态、生长和代谢的影响。方法：以组成型过量表达hTERT的Vero细胞系T1为研究对象,以活细胞密度和细胞活力为主要观察指标,结合细胞形态和贴附伸展动态,考察T1细胞和野生型Vero细胞在静止贴附培养、微载体固定化培养和悬浮培养体系中的细胞生长;以葡萄糖比消耗速率（qglc）、乳酸比生成速率（qlac）、乳酸转化率（Ylac/glc）和谷氨酰胺比消耗率（qgin）为反映细胞代谢的主要观察指标,考察T1细胞和野生型Vero细胞在静止贴附培养、微载体固定化培养的细胞代谢。结果：hTERT组成型过量表达在降低Vero细胞的贴附伸展能力和对血清的依赖程度的同时,提高了细胞无血清批次培养后期的细胞活力和活细胞密度,并赋予了T1非贴附依赖性生长的能力。hTERT组成型过量表达未对Vero细胞的代谢产生明显的影响。结论：hTERT组成型过量表达可降低Vero细胞的贴附生长依赖性和对血清的依赖程度,是有应用潜力的改良哺乳动物细胞体外培养性状的技术途径。     

TLR4在急性肝衰竭模型大鼠中的动态变化及意义

目的 探讨TLR4在D-Gal/LPS诱导的大鼠急性肝衰竭中的表达变化及其作用。方法 65只SD雄性大鼠随机分为正常对照组（n=5）、D-Gal/LPS模型组（n=25）、PDTC干预组（n=25）,于不同时间点检测肝功能,免疫组化观察肝组织TLR4的表达,TUNEL法观察肝脏细胞凋亡。结果 模型组大鼠4h开始ALT、AST明显升高,8h达峰值,4-24h ALT、AST明显高于正常对照组（P〈0.05）。模型组各时间点TLR4表达明显高于正常对照组（P〈0.05）。模型组凋亡的肝脏细胞数随时间的延长逐渐增多,均高于正常对照组（P〈0.05）。PDTC干预后ALT、AST水平,TLR4的表达及凋亡的肝脏细胞数低于模型组（P〈0.05）。结论 急性肝衰竭模型鼠TLR4表达增强,PDTC干预可下调其表达,提示TLR4在急性肝衰竭的发生发展中可能具有一定作用。     

自噬作用引起自身耐受

一个对自身组织耐受且功能完整的T细胞库的建立离不开对胸腺上皮细胞（thymic epithelial cells,TECs）提呈的主要组织相容性复合体Ⅱ（major histocompatibility complex class II,MHC Ⅱ）-自身抗原肽表位复合物的识别过程。已知在骨髓造血系统的抗原提呈细胞产生MHCⅡ-自身抗原肽复合物主要是通过细胞的内吞作用。TECs是非造血细胞中唯一能够持续表达MHCⅡ的细胞,属于专职的抗原提呈细胞（antigenpresenting cell,APC）。     

热激蛋白gp96免疫学功能及在主动免疫治疗肿瘤和传染病中的应用

热激蛋白gp96可特异性结合来源于肿瘤和病毒的抗原肽,与抗原呈递细胞表面CD91等受体作用进入胞内,并在内质网中将结合的抗原通过抗原呈递链呈递给MHCI类分子,激活特异性T细胞。同时,与细胞表面Toll样受体(TLR)TLR2、TLR4等相互作用,激活天然免疫。近期研究发现调节性T细胞(Treg)对gp96免疫功能有显著抑制作用,随着对影响gp96免疫功能的免疫抑制机制的深入了解,以及利用汉逊酵母表达有免疫活性的全长gp96蛋白体系的建立,gp96将在治疗肿瘤及传染性疾病中发挥更大的作用。     

结肠腺瘤息肉蛋白突变经PI3K/AKT 信号通路影响犬肾细胞MDCK的细胞粘附

结肠腺瘤息肉蛋白（APC）是一个肿瘤抑制因子,它不仅参与Wnt信号通路的传导,而且对细胞粘附、细胞骨架的组织和迁移等都有影响.APC突变发生于大多数结肠癌中.为了探讨APC突变对细胞粘附的影响及机制,本研究利用细胞粘附实验分析了MDCK-APC-N1和对照MDCK-GFP稳定表达细胞株系的细胞粘附情况.实验结果显示,在MDCK细胞中过表达APC-N1导致细胞-细胞间的粘附减少,细胞-基质间的粘附增加.荧光定量PCR和Western印迹实验表明,在MDCK-APC+N1细胞中,E-cadherin表达水平降低,CD29、P-FAK (Y397)、β-catenin和 P-AKT (T308)表达水平升高. 在MDCK-APC-N1细胞中,敲减β-catenin导致E-cadherin表达量升高,而CD29表达没有明显变化.进一步利用PI3K抑制剂LY294002处理MDCK-APC-N1细胞,结果发现,E-cadherin表达量明显升高,CD29表达量明显降低.这些结果揭示,APC-N1可活化 PI3K/AKT 信号通路,进而改变粘附蛋白E-cadherin和CD29影响细胞粘附.     

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。     

人类外周血单核细胞HLA-Ⅱ类抗原的表达及其与抗原提呈功能的关系

人类外周血单核细胞(HPMφ)是重要的抗原提呈细胞(APC)。APC在行使抗原提呈功能时,以其膜Ia抗原的表达为先决条件之一;但最强有力的APC是树突状细胞(DC)。部分HPMφ在一定条件下也可衍变为类似DC样的细胞。只有既是HLA—DR~+又是HLA—DQ~+的HPMφ才具有抗原提呈能力。人类Ia分子在抗原提呈中的作用是保护外来抗原,有利于T细胞的识别和激活,继而发生增殖反应。     

缺血后处理对大鼠局灶性脑缺血再灌注损伤时TLR4信号通路表达的影响

目的：观察缺血后处理对大鼠局灶性脑缺血再灌注损伤后TLR4通路表达的影响。方法：成年健康雄性SD大鼠110只,随机分为假手术组（sham组）（n=10）、缺血再灌注组（I／R组）和后处理组（IP组）,后两组又依据缺血再灌注6h、12h、24h、48h、72h不同的时间点再分五个亚组。对各组行神经行为学评分,脑组织梗死体积测量,TUNEL技术检测神经细胞凋亡的情况,免疫组织化学技术观察各组大鼠脑组织TLR4、NF—KB和TNF—a蛋白的表达,原位杂交方法检测各组大鼠脑组织TLR4mRNA、NF-KBmRNA的表达。结果：缺血后处理可下调TLR4、NF-KB、TNF-a细胞炎性因子的表达,抑制细胞凋亡、减少脑梗死体积,改善神经行为。结论：后处理可通过抑制TLR4信号通路表达,减少脑梗死体积,改善神经功能。     

How Location Governs Toll-Like Receptor Signaling

Toll-like receptors (TLRs) are a family of innate immune system receptors responsible for recognizing conserved pathogen-associated molecular patterns (PAMPs). PAMP binding to TLRs initiates intracellular signaling pathways that lead to the upregulation of a variety of costimulatory molecules and the synthesis and secretion of various cytokines and interferons by cells of the innate immune system. TLR-induced innate immune responses are a prerequisite for the generation of most adaptive immune responses, and in the case of B cells, TLRs directly regulate signaling from the antigen-specific B-cell receptor. The outcome of TLR signaling is determined, in part, by the cells in which they are expressed and by the selective use of signaling adaptors. Recent studies suggest that, in addition, both the ligand recognition by TLRs and the functional outcome of ligand binding are governed by the subcellular location of the TLRs and their signaling adaptors. In this review we describe what is known about the intracellular trafficking and compartmentalization of TLRs in innate system's dendritic cells and macrophages and in adaptive system's B cells, highlighting how location regulates TLR function.     

Innate immunity: structure and function of TLRs

The innate immune system provides the first line of defence against infection. Through a limited number of germline-encoded receptors called pattern recognition receptors (PRRs), innate cells recognize and are activated by highly conserved structures expressed by large group of microorganisms called pathogen-associated molecular patterns (PAMPs). PRRs are involved either in recognition (scavenger receptors, C-type lectins) or in cell activation (Toll-like receptors or TLR, helicases and NOD molecules). TLRs play a pivotal role in cell activation in response to PAMPs. TLR are type I transmembrane proteins characterized by an intracellular Toll/IL 1 receptor homology domain that are expressed by innate immune cells (dendritic cells, macrophages, NK cells), cells of the adaptive immunity (T and B lymphocytes) and non immune cells (epithelial and endothelial cells, fibroblasts). In all the cell types analyzed, TLR agonists, alone or in combination with costimulatory molecules, induce cell activation. The crucial role played by TLR in immune cell activation has been detailed in dendritic cells. A TLR-dependent activation of dendritic cells is required to induce their maturation and migration to regional lymph nodes and to activate na?ve T cells. The ability of different cell types to respond to TLR agonists is related to the pattern of expression of the TLRs and its regulation as well as their intracellular localization. Recent studies suggest that the nature of the endocytic and signaling receptors engaged by PAMPs may determine the nature of the immune response generated against the microbial molecules, highlighting the role of TLRs as molecular interfaces between innate and adaptive immunity. In this review are summarized the main biological properties of the TLR molecules.     

B细胞Toll样受体的表达和功能

Toll样受体(TLRs)广泛表达于固有免疫和获得性免疫系统.它们通过识别内外源性致病原含有的保守病原体相关模式分子,启动宿主防卫反应.TLRs也是沟通固有免疫和获得性免疫反应,尤其是T细胞介导的细胞免疫反应的重要桥梁.新近研究表明,几乎所有亚型TLR均表达在B淋巴细胞,不仅参与B细胞增殖、成熟和功能调节,而且在系统性红斑狼疮和慢性淋巴细胞白血病等疾病发生过程中发挥重要调节作用.以TLRs为靶点,调节B细胞介导的免疫反应,可能成为具有崭新应用前景的免疫治疗途径和方法.     

Insights into the role of Toll-like receptors in modulation of T cell responses

The innate immune receptors, such as Toll-like receptors (TLRs), are intimately involved in the early sensing of invading microorganisms or their structural components. Engagement of TLRs with their ligands results in activation of several downstream intracellular pathways leading to activation of innate and adaptive immune system cells. It was initially thought that TLRs are primarily expressed by antigen-presenting cells (APCs), such as macrophages and dendritic cells, and that interactions between microbial ligands and TLRs in these cells will indirectly result in activation of cells of the adaptive immune system, especially T cells. However, it has now become evident that TLRs are also expressed by various T cell subsets, such as conventional αβT cells, regulatory T cells, and γδT cells as well as natural killer T cells. Importantly, it appears that at least in some of these T cell subsets, TLRs are functionally active, because stimulation of these cells with TLR agonists in the absence of APCs results in exertion of effector or regulatory functions of T cells. The present review attempts to summarize the recent findings related to TLR expression in different T cell subsets and the direct role of TLRs in the induction and regulation of T cell responses, including those responses that occur at mucosal surfaces. In addition, the potential use of TLR agonists for steering T cell responses as a prophylactic or therapeutic strategy in the context of infectious, allergic or autoimmune diseases is explored.     

Saturated and polyunsaturated fatty acids reciprocally modulate dendritic cell functions mediated through TLR4

TLRs provide critical signals to induce innate immune responses in APCs such as dendritic cells (DCs) that in turn link to adaptive immune responses. Results from our previous studies demonstrated that saturated fatty acids activate TLRs, whereas n-3 polyunsaturated fatty acids inhibit agonist-induced TLR activation. These results raise a significant question as to whether fatty acids differentially modulate immune responses mediated through TLR activation. The results presented in this study demonstrate that the saturated fatty acid, lauric acid, up-regulates the expression of costimulatory molecules (CD40, CD80, and CD86), MHC class II, and cytokines (IL-12p70 and IL-6) in bone marrow-derived DCs. The dominant negative mutant of TLR4 or its downstream signaling components inhibits lauric acid-induced expression of a CD86 promoter-reporter gene. In contrast, an n-3 polyunsaturated fatty acid, docosahexaenoic acid, inhibits TLR4 agonist (LPS)-induced up-regulation of the costimulatory molecules, MHC class II, and cytokine production. Similarly, DCs treated with lauric acid show increased T cell activation capacity, whereas docosahexaenoic acid inhibits T cell activation induced by LPS-treated DCs. Together, our results demonstrate that the reciprocal modulation of both innate and adaptive immune responses by saturated fatty acid and n-3 polyunsaturated fatty acid is mediated at least in part through TLRs. These results imply that TLRs are involved in sterile inflammation and immune responses induced by nonmicrobial endogenous molecules. These results shed new light in understanding how types of dietary fatty acids differentially modulate immune responses that could alter the risk of many chronic diseases.     

Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs

Microglia are the resident macrophage-like population in the CNS. Microglia remain quiescent until injury or infection activates the cells to perform effector inflammatory and APC functions. Our previous studies have shown that microglia infected with a neurotropic strain of Theiler's murine encephalomyelitis virus secreted innate immune cytokines and up-regulated costimulatory molecules and MHC class II, enabling the cells to present viral and myelin Ags to CD4+ T cells. Recently, TLRs have been shown to recognize pathogen-associated molecular patterns and initiate innate immune responses upon interaction with infectious agents. We examined TLR expression on brain microglia and their functional responses upon stimulation with various TLR agonists. We report that mouse microglia express mRNA for all of the recently identified TLRs, TLR1-9, used for recognition of bacterial and viral molecular patterns. Furthermore, stimulation of quiescent microglia with various TLR agonists, including LPS (TLR4), peptidoglycan (TLR2), polyinosinic-polycytidylic acid (TLR3), CpG DNA (TLR9), and infection with viable Theiler's murine encephalomyelitis virus, activated the cells to up-regulate unique patterns of innate and effector immune cytokines and chemokines at the mRNA and protein levels. In addition, TLR stimulation activated up-regulation of MHC class II and costimulatory molecules, enabling the microglia to efficiently present myelin Ags to CD4+ T cells. Thus, microglia appear to be a unique and important component of both the innate and adaptive immune response, providing the CNS with a means to rapidly and efficiently respond to a wide variety of pathogens.     

Toll样受体直接调节Treg细胞抑制功能的研究进展

Treg细胞具有维持自身免疫耐受,调节免疫应答的作用。Toll样受体（Toll-like receptors,TLRs）家族可识别病原相关分子模式或内源性配体,启动固有和适应性免疫应答。Treg细胞选择性表达某些TLRs,TLRs活化可能直接增强或降低Treg的免疫抑制功能,这种调节可以影响对感染和肿瘤的免疫监视、移植免疫排斥和自身免疫病发生的进程。因此,了解两者的关系对发现新的治疗靶点和对策有重要的作用。简要综述TLRs对Treg细胞抑制功能直接调节作用的研究进展。     

Toll-like receptor ligands directly promote activated CD4+ T cell survival

Toll-like receptor (TLR) engagement by pathogen-associated molecular patterns (PAMPs) is an important mechanism for optimal cellular immune responses. APC TLR engagement indirectly enhances activated CD4(+) T cell proliferation, differentiation, and survival by promoting the up-regulation of costimulatory molecules and the secretion of proinflammatory cytokines. However, TLRs are also expressed on CD4(+) T cells, suggesting that PAMPs may also act directly on activated CD4(+) T cells to mediate functional responses. In this study, we show that activated mouse CD4(+) T cells express TLR-3 and TLR-9 but not TLR-2 and TLR-4. Treatment of highly purified activated CD4(+) T cells with the dsRNA synthetic analog poly(I:C) and CpG oligodeoxynucleotides (CpG DNA), respective ligands for TLR-3 and TLR-9, directly enhanced their survival without augmenting proliferation. In contrast, peptidoglycan and LPS, respective ligands for TLR-2 and TLR-4 had no effect. Enhanced survival mediated by either poly(I:C) or CpG DNA required NF-kappaB activation and was associated with Bcl-x(L) up-regulation. However, only CpG DNA, but not poly(I:C)-mediated effects on activated CD4(+) T cells required the TLR/IL-1R domain containing adaptor molecule myeloid differentiation factor 88. Collectively, our results demonstrate that PAMPs can directly promote activated CD4(+) T cell survival, suggesting that TLRs on T cells can directly modulate adaptive immune responses.     

抗病毒感染固有免疫应答的信号转导

入侵病毒的探知和适应性免疫应答启动均依靠固有免疫系统。三种模式识别受体(PRRs)在宿主防御系统第一线占据极其重要地位:Toll样受体、维甲酸诱导基因I样受体、核苷酸结合寡聚化结构域样受体。PRRs识别病原相关分子模式(PAMP)或危险信号分子模式(DAMPs)启动和调节固有免疫和适应性免疫应答。每种PRR都有单独的识别配体和细胞定位。激活的PRRs将信号分子传递给其配体分子(MyD88,TRIF,IRAK,IPS-1),配体活化后作为信使激活信号途径下游激酶(IKK复合物,MAPKs,TBK1,RIP-1)和转录因子(NF-κB,AP-1,IRF3),最终产生细胞因子、趋化因子、促炎细胞因子和I型干扰素。本文重点讨论PRRs信号通路及该领域取得的成果,以期为人类健康和免疫疾病防治提供策略。     

Cutting edge: TLR2 directly triggers Th1 effector functions

Toll-like receptors recognize pathogen-associated molecular patterns, activate innate immunity, and consequently modulate adaptive immunity in response to infections. TLRs are also expressed on T cells, and it has been shown that T cell activation is modulated by TLR ligands. However, the functions of TLRs on Th1 and Th2 effector cells and the molecular mechanisms underlying TLR-mediated activation are not fully understood. We analyzed TLR functions and downstream signaling events in both effector T cells. In mouse Th1 cells the stimulation by TLR2 but not by other TLRs directly induced IFN-gamma production, cell proliferation, and cell survival without TCR stimulation, and these effects were greatly enhanced by IL-2 or IL-12 through the enhanced activation of MAPKs. In contrast, no TLR affected the function of effector Th2 cells. These results identify TLR2 as a new specific activator of Th1 cell function and imply the involvement in Th1-mediated responses.