真菌细胞壁病原相关分子模式TLR/Dectin-1通路及相关细胞因子的免疫作用

侵袭性真菌感染(IFI)是致ICU患者死亡的重要原因之一。机体通过天然免疫重要的模式识别受体(PRR)Toll样受体(TLR)及C型凝集素受体(CLR)与病原相关分子模式(PAMP)识别后启动抗真菌免疫反应。目前明确磷脂甘露聚糖(PLM)的识别主要依赖TLR2和TLR4;β-葡聚糖经TLR2/树突状细胞相关性C型凝集素-1(Dectin-1)或只经Dectin-1识别。PLM和β-葡聚糖被识别后及经Dectin-1驱动获得性免疫后产生一系列下游细胞因子,一些下游细胞因子的免疫作用对不同组织有所不同、不确定或存在争议,一些免疫反应在细节上还没有详尽描述。因此,深入研究PAMP与PRR相互作用及其细胞因子对免疫调节影响,对IFI的预防治疗有重要意义。     

甘露聚糖结合凝集素诱导DC成熟机制分析

目的:探讨甘露聚糖结合凝集素(Mannan-binding lectin,MBL)诱导人外周血单核细胞(Mo)来源树突状细胞(DC)成熟的机制。方法:从健康成人外周血分离Mo,常规方法体外诱导未成熟DC(imDC),FACS分析DC成熟过程中MBL对CD分子表达的影响,进一步分析MBL与imDC的结合情况;RT-PCR分析DC表面模式识别分子Toll样受体-2(TLR2)和Toll样受体-4(TLR4)的表达;凝胶电泳迁移率法(EMSA)和Western blot分析NF-κB的活性及细胞核移位。结果:FCM分析表明,MBL能够使DC表面CD83、CD86及MHC分子表达升高;MBL能够以Ca2+浓度依赖关系与imDC细胞结合;RT-PCR结果表明MBL能够减少DC表面TLR2和TLR4的表达。EMSA和Western blot分析结果显示,MBL能够增强NF-κB活性及细胞核移位。结论:MBL可通过直接结合imDC表面分子来影响DC模式识别分子的表达并调节信号分子NF-κB活性,提示MBL能够通过配体结合调控DC分化成熟,揭示了MBL调节DC分化成熟有关信号途径。     

模式识别受体在抗真菌免疫中的作用

真菌感染机体后,通过模式识别作用及细胞内信号传导,启动固有免疫和适应性免疫应答.模式识别作用足抗真菌免疫的始动环节,Toll样受体(TLRs)和树突状细胞相关性C型凝集素-1(Dectin-1)是参与抗真菌免疫的主要模式识别受体(RPP).两者活化后启动机体免疫应答,产生各种细胞因子和趋化因子,并直接诱导巨噬细胞、自然杀伤细胞吞噬和杀灭病原体.TLRs和Dectin-1的信号传导、抗真菌感染的免疫机制以及两者之间的相互作用等已成为目前研究的热点.     

半乳糖凝集素对树突状细胞及T淋巴细胞功能影响的研究进展

半乳糖凝集素(Galectins)属于S型动物凝集素家族,是一种模式识别受体(PRRs),含有可以特异性地结合β-半乳糖苷的糖识别结构域(CRDs),该氨基酸序列在进化中高度保守.对于脊椎动物,Gal在固有免疫(病原体识别、吞噬和补体活化等)和适应性免疫系统(调节B/T淋巴细胞的数量、细胞因子的分泌和单核/巨噬细胞介导的炎症反应等)中发挥着重要作用.树突状细胞(DC)的成熟是启动免疫反应的关键因素,在其成熟活化的过程中合成并分泌Gal-1、Gal-3和Gal-9的量会发生显著的改变,进而会对DC及T淋巴细胞免疫功能产生影响.     

C型凝集素受体Dectin-2的生物学特性及功能

树突细胞相关凝集素2（Dectin-2）是一种新发现的C型凝集素受体,具有一个碳水化合物识别域CRD,可识别高甘露糖结构,广泛存在于树突状细胞等固有免疫细胞表面。由于其具有抗真菌病原体感染和紫外线诱导耐受等作用,因此在机体的免疫系统中发挥重要作用。对于Dectin-2等C型凝集素受体的深入研究将有利于我们对微生物感染、肿瘤免疫和自身免疫等方面有更深入的认识,从而对预防和治疗疾病起着指导作用。     

自然杀伤细胞受体介导抗病毒免疫的分子机制

自然杀伤(NK)细胞是参与固有免疫的主要成分,其表面可表达多种受体.识别和杀伤靶细胞与其表面受体特性密切相关.根据所介导的功能不同,将NK细胞受体分为抑制性受体和活化性受体.前者识别一定型别的MHC I类分子后,通过免疫受体酪氨酸抑制基序(ITIM)传递抑制信号,阻抑对相应靶细胞的杀伤.后者识别靶细胞表面的相应配体后,通过免疫受体酪氨酸活化基序(ITAM)传递活化信号,产生细胞毒活性和分泌细胞因子/趋化因子,从而发挥抗感染作用.细胞毒作用可能主要依赖于ERK途径,而细胞因子/趋化因子的分泌可能主要依赖于NF-κB和(或)p38MAPK或JUK/AP-1途径.     

C型凝集素受体CLEC-2的研究进展

C型凝集素样受体-2(CLEC-2)是一个非经典型的C型凝集素受体,主要表达在血小板、中性粒细胞等细胞表面,在马来西亚蝮蛇蛇毒蛋白rhodocytin和跨膜唾液酸糖蛋白poloplanin等相关配体的刺激下,能通过细胞质尾部的YXXL结构域形成二聚体,从而激活syk依赖的信号传导.最近有很多研究显示:CLEC-2在血小...     

蝙蝠蛾被毛孢菌丝体通过激活TLR2、TLR4和Dectin-1诱导Th1型免疫反应

目的:研究蝙蝠蛾被毛孢菌丝体(MHCS)对抗原呈递细胞树突状细胞(Dendritic cells,DCs)成熟和功能的调节作用.方法:利用流式细胞术、实时荧光定量PCR、Western blot和混合淋巴细胞培养等实验方法,检测了MHCS对DCs成熟和功能相关表面分子、细胞因子表达以及Toll样受体(TLR)2、TLR4和Dectin-1相关信号转导通路蛋白活性的调节作用.结果:MHCS显著上调DCs表面分子CD11c、MHCⅠ和MHC Ⅱ、辅助刺激分子CD40、CD80和CD86以及模式识别受体TLR2、TLR4和Dectin-1的表达;促进Th1型细胞因子IL-12产生,抑制Th2型细胞因子IL-10、IL-13和TGF-β1产生;诱导TAK1和IRF3磷酸化活性增加.MHCS也显著刺激幼稚型Th1细胞增殖,诱导幼稚型Th细胞向Th1方向分化.利用中和性抗体,分别阻断DCs细胞TLR2、TLR4或Dectin-1活性,可部分抑制MHCS诱导的DCs成熟.结论:MHCS能促进DO成熟,诱导Th1型免疫反应.MHCS的这些作用与其激活模式识别受体TLR2、TLR4或Dectin-1有关.     

Toll样受体与树突状细胞

树突状细胞是目前已知的功能最强的抗原递呈细胞 ,可通过表达的Toll样受体 (TLR )来识别病原微生物特有的保守成分 ,如脂多糖、细菌DNA中非甲基化的CpG序列和病毒的dsRNA等 ,并通过TLR介导的信号传导通路激活转录因子NF κB和Jun/Fos,引起炎性细胞因子的释放和促进树突状细胞成熟 ,并且可以影响免疫应答的类型。为制备树突状细胞疫苗提供了一条新思路     

过表达SIRT1抑制脂多糖诱导的胰岛β细胞炎症因子表达及NF-κB信号通路激活

目的研究沉默信息调节因子1(SIRT1)对脂多糖(LPS)诱导的胰岛β细胞炎症因子表达及核因子-κB(NF-κB)信号通路激活的影响。方法用LPS处理胰岛β细胞,qRT-PCR和Western blot测定细胞中SIRT1表达变化。用pcDNA3.1-SIRT1慢病毒感染胰岛β细胞,qRT-PCR检测细胞中白细胞介素-6(IL-6)mRNA和肿瘤坏死因子-α(TNF-α)mRNA表达水平,流式细胞术测定细胞凋亡变化,Western blot检测细胞中Bcl-2相关X蛋白(Bax)、活化型Caspase-3(cleaved Caspase-3)、核因子-κBp65亚型(NF-κBp65)、Toll样受体4(TLR4)蛋白表达水平。结果 LPS处理后的胰岛β细胞中SIRT1表达水平降低。pcDNA3.1-SIRT1慢病毒感染可以提高LPS条件下胰岛β细胞中SIRT1表达水平。LPS处理后的胰岛β细胞中IL-6 mRNA和TNF-αmRNA表达水平升高,凋亡率升高,细胞中Bax、Cleaved Caspase-3、NF-κBp65、TLR4蛋白表达升高。过表达SIRT1可以降低LPS条件下胰岛β细胞中IL-6 mRNA和TNF-αmRNA表达水平,抑制细胞凋亡,减少细胞中Bax、cleaved Caspase-3、NF-κBp65、TLR4蛋白表达。结论 SIRT1减少LPS诱导的胰岛β细胞炎症因子表达并下调细胞中NF-κB信号激活水平。     

Dectin-1 and Dectin-2 in innate immunity against fungi

Dectin-1 and Dectin-2 are type II transmembrane proteins of the C-type lectin family with single carbohydrate recognition domains (CRDs) in their extracellular region. They are expressed mainly in dendritic cells and macrophages. Dectin-1 recognizes β-glucans with its CRD and transduces signals through its immunoreceptor tyrosine-based activation motif (ITAM)-like motif in the cytoplasmic domain, whereas Dectin-2 recognizes α-mannans and transduces its signal through association with the ITAM-containing Fc receptor γ chain. Upon ligand binding, spleen tyrosine kinase is recruited to the ITAM and activates the caspase recruitment domain family member 9 (CARD9)-nuclear factor-κB axis, resulting in the activation of various genes including those encoding pro-inflammatory cytokines. Both β-glucans and α-mannans are major cell wall components of fungi including Candida albicans and Pneumocystis carinii. Recently, it was reported that Dectin-1 is important in protection against P. carinii by inducing reactive oxygen species, whereas both Dectin-1 and Dectin-2 play important roles in defense against C. albicans by preferentially inducing T(h)17 cell differentiation. In this review, we briefly revisit the structures, ligands, signal transduction and functional roles of Dectin-1 and Dectin-2 in host defense against fungal infection.     

CLEC-2 signaling via Syk in myeloid cells can regulate inflammatory responses

Myeloid cells express a plethora of C-type lectin receptors (CLRs) that can regulate immune responses. CLEC-2 belongs to the Dectin-1 sub-family of CLRs that possess an extracellular C-type lectin-like domain and a single intracellular hemITAM motif. CLEC-2 is highly expressed on mouse and human platelets where it signals via Syk to promote aggregation. We generated a monoclonal antibody (mAb) against mouse CLEC-2 and found that CLEC-2 is additionally widely expressed on leukocytes and that its expression is upregulated during inflammation. MAb-mediated crosslinking of CLEC-2 leads to hemITAM-dependent signaling via Syk, Ca(2+) and NFAT and, in myeloid cells, modulates the effect of toll-like receptor (TLR) agonists to selectively potentiate production of IL-10. A macrophage/dendritic cell-dependent increase in IL-10 is also observed in mice given anti-CLEC-2 mAb together with LPS. Collectively, these data indicate that CLEC-2 is expressed in myeloid cells and acts as a Syk-coupled CLR able to modulate TLR signaling and inflammatory responses.     

Syk-coupled C-type lectin receptors that mediate cellular activation via single tyrosine based activation motifs

Summary: Different dendritic cell (DC) subsets have distinct specialized functions contributed in part by their differential expression of pattern recognition receptors (PRRs). C-type lectin receptors (CLRs) are a group of PRRs expressed by DCs and other myeloid cells that can recognize endogenous ligands as well as a wide range of exogenous structures present on pathogens. Dual roles in homeostasis and immunity have been demonstrated for some members of this receptor family. Largely due to their endocytic ability and subset specific expression, DC-expressed CLRs have been the focus of significant antigen-targeting studies. A number of CLRs function on the basis of signaling via association with immunoreceptor tyrosine-based activation motif (ITAM)-containing adapter proteins. Others contain ITAM-related motifs or immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tails. Here we review CLRs that induce intracellular signaling via a single tyrosine-based ITAM-like motif and highlight their relevance in terms of DC function.     

The role of Syk/CARD9 coupled C-type lectins in antifungal immunity

Fungal infections are affecting an increasing number of people, and the failure of current therapies in treating systemic infection has resulted in an unacceptably high mortality rate. It is therefore of importance that we understand immune mechanisms operating during fungal infections, in order to facilitate development of adjunctive immunotherapies for the treatment of these diseases. C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that are critical for immune responses to fungi. Many of these receptors are coupled to Syk kinase, which allows these receptors to signal via CARD9 leading to NF-κB activation, which in turn contributes to the induction of both innate and adaptive immunity. Dectin-1, Dectin-2 and Mincle are all CLRs that share this common signalling mechanism and have been shown to play key roles in antifungal immunity. This review aims to update existing paradigms and summarise the most recent findings on these CLRs, their signal transduction mechanisms and the collaborations between these CLRs and other PRRs.     

Syk kinase-coupled C-type lectin receptors engage protein kinase C-σ to elicit Card9 adaptor-mediated innate immunity

C-type lectin receptors (CLRs) that couple with the kinase Syk are major pattern recognition receptors for the activation of innate immunity and host defense. CLRs recognize fungi and other forms of microbial or sterile danger, and they induce inflammatory responses through the adaptor protein Card9. The mechanisms relaying CLR proximal signals to the core Card9 module are unknown. Here we demonstrated that protein kinase C-δ (PKCδ) was activated upon Dectin-1-Syk signaling, mediated phosphorylation of Card9 at Thr231, and was responsible for Card9-Bcl10 complex assembly and canonical NF-κB control. Prkcd(-/-) dendritic cells, but not those lacking PKCα, PKCβ, or PKCθ, were defective in innate responses to Dectin-1, Dectin-2, or Mincle stimulation. Moreover, Candida albicans-induced cytokine production was blocked in Prkcd(-/-) cells, and Prkcd(-/-) mice were highly susceptible to fungal infection. Thus, PKCδ is an essential link between Syk activation and Card9 signaling for CLR-mediated innate immunity and host protection.     

C-Type Lectin-Like Receptors of the Dectin-1 Cluster: Ligands and Signaling Pathways

Innate immunity is constructed around genetically encoded receptors that survey the intracellular and extracellular environments for signs of invading microorganisms. These receptors recognise the invader and through complex intracellular networks of molecular signaling, they destroy the threat whilst instructing effective adaptive immune responses. Many of these receptors, like the Toll-like receptors in particular, are well-known for their ability to mediate downstream responses upon recognition of exogenous or endogenous ligands; however, the emerging family known as the C-type lectin-like receptors contains many members that have a huge impact on immune and homeostatic regulation. Of particular interest here are the C-type lectin-like receptors that make up the Dectin-1 cluster and their intracellular signaling motifs that mediate their functions. In this review, we aim to draw together current knowledge of ligands, motifs and signaling pathways, present downstream of Dectin-1 cluster receptors, and discuss how these dictate their role within biological systems.     

NKG2D-DAP10 triggers human NK cell-mediated killing via a Syk-independent regulatory pathway

The immune recognition receptor complex NKG2D-DAP10 on natural killer cells is stimulated by specific ligands carried on virus-infected and malignant cells. Because DAP10 does not have an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail, its ability to trigger killing has been debated. Here we show that a crucial Tyr-Ile-Asn-Met amino acid motif in the cytoplasmic tail of DAP10 couples receptor stimulation to the downstream activation of phosphatidylinositol 3-kinase, Vav1, Rho family GTPases and phospholipase C. Unlike that of ITAM-containing receptors, the activation of NKG2D-DAP10 proceeds independently of Syk family protein tyrosine kinases. Yet the signals initiated by NKG2D-DAP10 are fully capable of inducing killing. Our findings identify a previously unknown mechanism by which receptor complexes that lack ITAM motifs can trigger lymphocyte activation.