【作者】 张庆华；

【导师】 曹雪涛；

【作者基本信息】 第二军医大学 ， 免疫学， 2011， 博士

【摘要】 Notch基因首次发现于1919年,其突变可造成果蝇翅膀边缘的一些缺刻(Notchs)而得以命名。目前发现,Notch受体在不同物种之间(从果蝇到人)以及同一物种的不同成员之间都有高度的结构同源性。已知的Notch受体有4种,Notch1、Notch2、Notch3表达于多种组织器官,其中Notch1的表达更为广泛。脊椎动物Notch配体分为Delta-like与Jagged两类,前者包括delta-like1,delta-like3和delta-like4(又称Delta1,Delta3,Delta4或Dll1,Dll3,Dll4),后者包括Jagged (Serrate) 1和Jagged(Serrate) 2。这些配体与相同或不同的Notch受体结合,激活Notch信号通路,参与调控许多器官、组织、细胞的生长发育。目前认为,Notch信号途径的活化主要采用“三步蛋白质水解模式”。无活性的Notch前体从高尔基体中合成后,在向胞膜转运的过程中,经不同蛋白酶体的切割作用,释放胞内段NICD转入核内。NICD的RAM区可以结合DNA结合蛋白CSL,在Notch未活化的情况下,CSL蛋白常常作为转录抑制因子,与SMRT、SHARP、HDAC、CIR等蛋白组成共抑制复合物来抑制基因转录。Notch信号除了CSL依赖途径外,Notch信号还有CSL非依赖途径,这一途径包含一个细胞内的锌指蛋白Deltex。NICD与CSL结合后所产生的效应是多样的,CSL通过与不同的启动子结合可产生转录活化或抑制作用。由此可见,Notch在调节基因的表达中可产生协同或拮抗作用。Notch信号在神经系统发育中发挥着重要作用,Notch信号可关闭神经原细胞bHLH基因的表达使外胚层细胞不能继续向神经细胞分化,阻止了神经元的分化。Notch信号在肿瘤的形成、发展中也发挥着不同的作用:一方面,Notch可通过活化一些生长信号,促进一些肿瘤的生长。另一方面,目前也有很多研究发现Notch可通过抑制某些信号抑制肿瘤的生长。综上所述,Notch信号通路在细胞增殖分化及凋亡中有重要作用,是许多重要细胞信号通路的交汇点。目前已经有研究表明Notch信号在免疫细胞的分化和发育过程中也起决定性作用,但是,有关Notch信号与天然免疫的关系尚有待于深入研究。由此,我们提出的科学问题是:Notch信号在天然免疫识别与活化中的调控作用是什么?对Toll样受体(Toll-like receptors, TLRs)信号转导调控的深入研究是近几年来免疫学领域的研究热点之一,具有重要的理论意义和临床应用价值。TLR在识别PAMP中起重要作用,作为一种重要的模式识别受体,主要表达于巨噬细胞和树突状细胞(Dendritic cells, DCs)表面,构成机体抵御病原体入侵的第一道防线。大多数TLRs与特定的配体结合后,通过MyD88依赖途径即MyD88/IRAK/TRAF6级联反应来活化MAPK和NF-κB信号通路,促进相关基因的表达,启动天然免疫。而TLR3和TLR4还存在着MyD88非依赖途径,即通过接头分子TRIF或TRAM,最终引起NF-κB的晚期活化和IRF3的核转位,调控炎性因子和I型干扰素的表达。TLR不仅启动天然免疫应答,控制炎症反应的性质、强度和持续时间,而且调节获得性免疫应答的强度和类型,成为连接初始免疫应答和获得性免疫应答的枢纽。TLR信号过度活化或活化不足都会导致机体功能异常和疾病的发生,利用TLRs激动剂可增强机体免疫应答,有利于机体清除入侵病原微生物。而负相调控TLR信号通路,可抑制TLRs诱发的过强免疫应答,为感染性疾病、肿瘤和自身免疫性疾病,以及TLRs介导的免疫紊乱疾病如内毒素休克的防治提供新的思路。针对Notch信号在天然免疫识别与活化中的调控作用是什么的科学问题,我们想知道Notch信号对TLR信号转导调控究竟具有什么样的影响?大量研究表明Notch信号可与其它信号途径相互作用来调控免疫细胞的功能。Notch信号不但可调控Th1/Th2细胞的分化,Notch信号可调控T细胞中IFN-γ、IL-4、Il-10等细胞因子的分泌,调节免疫应答。近年来人们发现在单核细胞和巨噬细胞分化发育过程中,Notch受体、配体的表达模式具有时空性,这提示Notch信号可能参与了抗原递呈细胞介导的免疫应答。研究发现,巨噬细胞中过表达Notch1受体可增强IFN-γ诱导的Raw264.7细胞STAT1依赖的转录,上调IFN-γ调控因子,抑制ICAM1和MHCII分子的表达,继而参与调控一些与抗原递呈和杀伤相关基因的表达。最近有人发现TLR信号通路可上调巨噬细胞表面Notch1的表达,影响Th1/Th2细胞的分化,调控免疫反应; LPS刺激可上调Raw123细胞表面的Notch1表达,增强Notch信号的活化。那么,作为可被TLR诱导增加的Notch1是否可反过来调控TLR信号通路来影响免疫应答?如果可以,它又发挥着怎样的调控作用?其具体的调控机制如何?对于这些科学问题,未见研究报道。本课题针对Notch信号可能参与TLR信号触发的炎性细胞因子的产生这一设想进行研究,旨在探讨Notch信号对TLRs触发的炎性细胞因子产生的影响及与TLRs信号通路的交互作用点。该课题主要分三部分进行研究:TLRs触发的巨噬细胞中Notch分子的表达情况;Notch分子对TLR信号诱发的巨噬细胞炎性细胞因子产生的影响;Notch分子对巨噬细胞TLRs信号通路的调控机制。1、TLRs触发的巨噬细胞中Notch受体、配体表达的研究我们检测了静息的巨噬细胞(Raw264.7细胞及小鼠腹腔巨噬细胞)表面Notch受体及配体的表达以及经不同TLR配体(LPS, CpG ODN, poly I:C)刺激后,巨噬细胞中Notch1-4的表达变化。荧光定量PCR(Q-PCR)结果表明,Raw246.7和小鼠腹腔巨噬细胞均表达Notch1、Notch2和Notch14,而且Notch1的表达量最大,Notch 2次之,Notch 4最少,但不表达Notch3。在巨噬细胞中Notch的配体均有不同程度的表达,其中,Dll4和Jagged1的表达丰度高,其他的配体次之。100ng/ml LPS、0.33μM CpG和10 ng/ml poly I:C刺激可显著上调Raw246.7细胞和腹腔巨噬细胞中Notch1和Notch2的表达。2、Notch信号对TLR信号诱发的巨噬细胞炎性细胞因子产生的影响研究表明,过表达Notch1分子的胞内区(NICD)与Notch1配体激活Notch1后对靶细胞的作用是一致的。鉴于此,我们将组成性活化形式的NICD1、NICD2以及NICD1的PEST结构域缺失的突变(N1-6MT)载体,转染至原代腹腔巨噬细胞及Raw246.7细胞中,观察Notch信号对TLR信号通路的影响。首先,在原代腹腔巨噬细胞中研究Notch信号对TLR信号通路的影响。我们分离小鼠腹腔巨噬细胞,体外进行培养,观察过表达组成性活化的NICD1、NICD2和N1-6MT,LPS刺激后细胞因子的分泌情况。ELISA的结果表明,过表达NICD1和NICD2均可增强原代腹腔巨噬细胞IL-10的分泌,而下调炎性细胞因子IL-6、IL-1β、TNF-α、IFN-β的分泌。说明Notch信号活化抑制了炎性细胞因子的分泌,对TLR信号通路可能起负向调控作用。而过表达N1-6MT对以上细胞因子的分泌效应具有相反的效果,说明,NICD1的PEST结构域对炎性细胞因子分泌的抑制效应是至关重要的。其次,在Raw246.7细胞中研究了Notch1对TLR信号通路诱发的炎性细胞因子产生的影响。我们将组成性活化形式的NICD1和N1-6MT载体,转染至Raw246.7细胞中,经G418筛选出稳定表达ICN的细胞株和对照细胞株;观察经TLR配体刺激后Raw246.7-ICN和Raw246.7对照载体细胞中细胞因子分泌(IL-10、IL-6、IL-1β、TNF-α、IFN-β)的变化。Q-PCR及ELISA的结果发现:过表达NICD1可明显增强LPS诱导的IL-10的分泌,而下调炎性细胞因子IL-6、IL-1β、TNF-α、IFN-β的分泌。而过表达N1-6MT,对以上细胞因子的分泌效应正好具有相反效果。进而,在此基础上,我们利用Notch信号的化学阻断剂GSI处理Raw264.7细胞和小鼠的腹腔巨噬细胞,观察不同TLRs配体刺激对巨噬细胞分泌细胞因子(IL-10、IL-6、TNF-α)的变化。结果显示:阻断剂GSI处理巨噬细胞24h抑制了Notch信号后,再用LPS刺激,发现细胞因子IL-10的分泌减少,而IL-6、IL-1β、TNF-α、IFN-β的分泌增加。这一结果说明,Notch信号被阻断后,对TLR信号通路诱发的炎性细胞因子产生被逆转,从反面证明了Notch信号活化对TLR信号引起的细胞因子的调控作用。另一方面,我们通过Notch1特异性干扰RNA,检测Notch1表达的下调对LPS诱发的细胞因子分泌的影响。我们首先用Q-PCR及Western Blot检测了其特异性干扰RNA的干扰效率,在Notch1特异性干扰RNA的干扰效率达70%以上时,在小鼠腹腔巨噬细胞中,干扰Notch1的表达可下调LPS诱发的IL-10的分泌,而上调了IL-6、IL-1β、TNF-α、IFN-β的分泌。这一结果,与化学阻断剂GSI的作用效果相一致。因此,在该部分试验中,无论是Notch1信号的活化还是不同方法进行的Notch1信号的阻断,在原代腹腔巨噬细胞和Raw264.7细胞中均得到一致的结果。这说明Notch1信号确实参与了LPS诱发的炎性细胞因子的产生。3、Notch对巨噬细胞TLRs信号通路的调控机制首先,对信号通路进行Western Blot检测。结果显示,在原代腹腔巨噬细胞和Raw264.7细胞中过表达NICD1可明显降低LPS诱导的磷酸化ERK1/2的活化及NF–κB转录活性,而过表达N1-6MT,可明显增强LPS诱导的磷酸化ERK1/2的活化及NF–κB转录活性。我们进一步研究了GSI抑制剂及Notch1特异性干扰RNA在原代腹腔巨噬细胞和Raw264.7细胞中Notch1信号对LPS诱发的信号通路的影响,结果发现,GSI抑制剂和Notch1特异性干扰RNA抑制Notch信号后,LPS促发的ERK1/2的磷酸化明显增强。此外,我们还发现过表达N1-6MT质粒后,Notch信号失活,抑制IKBα的表达,增强IKBα的磷酸化水平,提示Notch信号活化可抑制NF-κB的转录活性。既然,在原代腹腔巨噬细胞和Raw264.7细胞中均能观察到Notch信号对LPS促发的ERK1/2的磷酸化明显变化,于是我们用MAPKK抑制剂,即MAPK kinase抑制剂或MEK抑制剂——PD 98059(可以选择性抑制MEK1,从而抑制ERK1/2的磷酸化和激活),来观察阻断p44/42 MAPK的磷酸化后细胞因子的分泌影响。ELISA的结果表明,PD 98 059可明显下调由Notch1信号活化所造成的细胞因子的变化。因此,我们认为Notch1可能通过ERK1/2信号途径来参与TLRs信号的调控。其次,我们用荧光素酶报告基因分析来研究Notch和NF-κB通路以及和TNF-α之间的关系。结果显示,过表达NICD1、NICD2可以显著降低由MyD88和TRAF6活化所引起的NF-κB以及TNF-α报告基因的活性。该结果说明,过表达NICD1、NICD2可以抑制NF-κB和TNF-α的转录活性,而过表达N1-6MT,可以逆转由NICD1对NF-κB的转录活性的抑制作用,说明NICD1的PEST结构域对NF-κB的转录活性以及TNF-α的分泌起到至关重要的抑制作用。最后,我们利用免疫共沉淀方法寻找能与Notch1胞内段结合的信号蛋白。在上述的研究中发现Notch信号可调控TLRs信号诱发的细胞因子的变化,在信号通路方面,Notch可降低LPS促发的ERK1/2信号的活化,那么Notch信号是怎样调控ERK1/2信号通路的呢?它们之间是否有直接的相互作用呢?于是我们利用免疫共沉淀方法寻找能与Notch1胞内段结合的信号蛋白。我们利用免疫沉淀检测发现:在Notch1的免疫沉淀物中检测不到ERK1/2,因此,我们认为Notch1与TLR信号的交互作用分子并非ERK1/2,可能直接或间接作用于其他靶分子调控了TLRs介导的细胞因子分泌。因此,我们认为Notch1并不是直接作用于ERK分子,可能通过其它分子起间接作用。综上所述,我们的研究表明, Notch1和Notch2分子参与巨噬细胞中TLR触发的炎性细胞因子产生的调控,其通过ERK1/2和/或NF-κB途径发挥了调控作用。其可能的原因有两种:1、Notch分别通过ERK1/2和NF-κB两条通路参与调控TLR触发的炎性细胞因子;2、ERK1/2和NF-κB信号在Notch调控TLRs的信号通路中,存在先后,这将在后续的实验中进行证实。另外,对于本文的研究还存在一些需要进一步探讨的问题: 1、Notch分子通过不同的信号通路,抑制了炎性细胞因子如IL-6、IL-1β、TNF-α、IFN-β等的分泌,而促进了IL-10的分泌;2、Notch分子先促进抗炎细胞因子IL-10的分泌,IL-10又进一步抑制了IL-6、IL-1β、TNF-α、IFN-β等促炎细胞因子的分泌。关于这一假设,其具体的靶点还未有充分的证据,机制尚不明确,可以用IL-10的阻断性抗体进行后续的深入的研究。总之,这一调控机制的发现,有助于深入阐明Notch信号途径在TLR信号途径中的作用,并可能为探明不同信号途径的相互作用在抗感染免疫中的作用提出新证据,该课题丰富了TLR信号转导的调控机制,将为感染、肿瘤等疾病的免疫学机理探究以及潜在的新型治疗手段的发现提供新思路。更多还原

【Abstract】 Notch proteins are a family of large transmembrane receptors that regulate cell fate decisions during many developmental processes. Signals transduced through Notch receptors during cell–cell interactions directly regulate the expression of genes that are important to cell fate choices and differentiation. To date, four Notch receptors (Notch1-4) and five ligands (Delta1, -3, -4 and Jagged1, -2) have been identified in mammalians. Notch proteins play a pivotal role in regulating interpretation of environmental signals in a wide variety of cell types and species. After ligand binding, the intracellular region of Notch is proteolytically cleaved from the transmembrane portion and translocated to the nucleus, where it interacts with a transcriptional repressor, known as CSL. After binding Notch, CSL becomes a transcriptional activator, and in conjunction with the activity of other cofactors, including SKIP, PCAF, and/or GCN5, induces transcription of downstream targets.The ability of the innate immune system to recognize and respond to microbial components has been largely attributed to Toll-like receptors (TLRs). As a kind of important pattern recognition receptors (PRRs), TLRs recognize pathogen-associated molecule patterns (PAMPs), leading to a variety of signaling events that initiate innate immunity and activate immune cells to produce proinflammatory cytokines and type I interferon (IFN). In addition, TLR signaling can induce the up-regulation of MHC II and co-stimulatory molecules on antigen-prsenting cells such as dendritic cells (DC), facilitates DC maturation, and instructs development of antigen-specific adaptive immunity, especially Th1 response. Various other signal pathways are involved in the tight regulation of TLR signaling to enhance or attenuate their activation, which maintains the immunological homeostasis.There is a substantial body of literature detailing the expression of Notch family members as well as Notch ligands in cells and tissues of the immune system, and these reports provide supportive evidence that endogenous Notch may mediate regulation of immune function. Activated Notch can regulate the differentiation of thymocytes into either CD4 or CD8 SP cells. Notch signaling may regulate apoptosis during thymocyte maturation by preventing death by neglect and/or negative selection in cells destined to die. Notch is essential for Th2 cell differentiation but can also influence a Th1 cell response. However, the role of Notch signaling in TLR-triggered innate immunity remains unclear. It’s been reported that LPS stimulation could increase the expression of Notch1, enhancing the Notch activation. Then, we wonder whether or not Notch receptors in turn affect the TLR-triggered signal pathway to regulate innate immunity? The aim of this study is to focus on the effect of activated Notch signaling on TLR-triggered production of inflammatory cytokines in macrophages and explore its underlying mechanisms.Part I. Notch expression in TLR-triggered macrophagesWe examined the expression of different Notch receptors in resting macrophages (Raw264.7 cells and mouse primary peritoneal macrophages) as well as macrophages after stimulation with different TLR ligands (LPS, CpG ODN or poly I:C). Fluorescence quota PCR (Q-PCR) result showed that both Raw246.7 cells and mouse peritoneal macrophages express Notch1, 2 and 4, but not Notch3. 100ng/ml LPS, 0.33M CpG and 10 ng/ml poly I:C stimulation could obviously increase the expression level of Notch1 in the Raw246.7 cells and mouse peritoneal macrophages. The results indicated that there may exist cross-regulation between the Notch and TLR signaling pathways. Part II. Effect of Notch signal on the TLR-triggered cytokine production in macrophagesConstitutive expression of active intracellular domain of Notch (NICD) in targeted cells could also result in an“activated”Notch phenotype. We constructed vectors expressing constitutively active intracellular domain of Notch1 (NICD1) and Notch2 (NICD2), as well as an inactive mutant of NICD1 (N1-6MT), in which the 3’PEST domain of NICD1 has been removed and replaced with 6 myc tags. The vectors were then transfected into mouse peritoneal macrophages and Raw 264.7 cells.Firstly, in mouse peritoneal macrophages, Q-PCR and ELISA results showed that overexpression of NICD1 and NICD2 can obviously strengthen LPS-induced secretion of IL-10, but decrease the secretion of inflammatory cytokines including IL-6,IL-1β, TNF-α, and IFN-β. But overexpression of the inactive form of NICD1, N1-6MT, had an opposite effects on cytokine secretion to that of NICD1 and NICD2, indicating that Notch signaling may inhibit TLR-triggered production of inflammatory cytokines in macrophages, and that the PEST domain of NICD1 may play an important role in the regulation of TLR-triggered cytokine secretion by NICD1.Secondly, the transfected Raw246.7cells were selected by resistance to neomycin. The stably transfected cells were designated as Raw246.7-NICD1, Raw246.7-NICD2 and Raw246.7-N1-6MT, respectively. We observed increased secretion of IL-10 but decreased secretion of inflammatory cytokines ( IL-6,IL-1β,TNF-αand IFN-β) after LPS stimulation in Raw246.7-NICD1 cells. But stable overexpression of N1-6MT results in opposite effect on cytokine production after TLR ligand stimulation. These results are consistent with those observed in mouse peritoneal macrophages, confirming the effect of Notch signaling on TLR-triggered cytokine production.Thirdly, Raw264.7 cells and mouse peritoneal macrophages were treated with Notch inhibitor, GSI, or transfected with Notch1-specific si-RNA, and then the secretion of cytokines (IL-10, IL-6, IL-1β, TNF-αand IFN-β) trigged by different TLR ligands were observed. The results showed that blockade of Notch1 signaling with GSI or inhibiting Notch1 expression by specific si-RNA could decrease IL-10 secretion, but increase IL-6, IL-1β, TNF-αand IFN-βsecretion induced by TLR ligands. The results further indicate the inhibitory effect of Notch signaling on TLR-triggered inflammatory cytokine production.Part III. The mechanisms for the regulation of TLR-triggered cytokine production in macrophages by Notch signalFirstly, we observed the effect of Notch signaling activation on the TLR signaling pathways in the macrophages. Western Blot result showed that overexpression of NICD1 could obviously suppress LPS-induced phosphorylation of ERK1/2, JNK p38 and I-κBαin Raw264.7 cells and mouse peritoneal macrophages. Accordingly, overexpression of N1-6MT had the opposite effect to that of NICD1. PD 98059 is a selective noncompetitive inhibitor of the MAPK pathway (Erk1/2, p-p44/42 MAPK). Pretreatment of macrophages with PD 98059 obviously antagonized the effect of Notch1 on TLR-triggered cytokine production. Therefore, the results suggest that Notch1 possibly participates in the TLR signaling through MAPK pathway.Secondly, we used the luciferase reporter gene assay to examine the effect of Notch signal on NF-κB as well as Notch and TNF-α. The results revealed that overexpression of NICD1 and NICD2 could obviously inhibit the reporter gene activities of NF-κB and TNF-αwhich activated by TRAF6 and MyD88; but overexpression of N1-6MT had a reverse effect on NF-κB and TNF-αreport gene activities.Thirdly, we used co-immunoprecipitation to identify target proteins that Notch1 interacted with in the TLR signaling pathways. We found that Notch1 promoted LPS-mediated phosphorylation of ERK1/2. But we detected no positive interaction between ERK1/2 and Notch1. So, it’s speculated that Notch1 may not directly interact with ERK members, and that Notch1 may regulate ERK1/2 phosphorylation in an indirect manner. In conclusion, here we demonstrate the negative regulation of TLR-triggered inflammatory cytokine production and signaling pathway in macrophages by Notch1/2 signaling. We showed that activation of Notch1/2 signaling could regulate TLR-stimulated secrection of cytokines through ERK1/2 and NF-κB pathway. There are two possible explanations which need to be addressed. On one hand, Notch activation promotes LPS-induced IL-10, however, suppresss IL-6,IL-1β,TNF-αand IFN-βsecretion through different pathways. On the other hands, Notch activation firstly promotes IL-10 section, and then IL-10 in trun suppresses secretion of inflammatory cytokine (IL-6,IL-1β,TNF-αand IFN-β). The data may help us to understand the mechanisms by which Notch signaling might be involved in the innate immunity and provide the experimental basis for looking for the approaches to the treatment of inflammation by regulating Notch signaling.更多还原