【作者】 章意亮；

【导师】 孙树汉；

【作者基本信息】 第二军医大学 ， 遗传学， 2008， 博士

【摘要】 识别并抵抗微生物的感染对于多细胞生物的生存是必须的。天然免疫应答是机体防御感染性疾病的第一防线,在天然免疫中,由于病原体不断地进化、突变会使识别的有效性逐步减弱。所以,对于宿主而言最大的挑战在于通过有限的受体迅速识别大量不同的病原体并作出应答。对Toll样受体(TLRs)及病原相关的分子模式(PAMPs)的发现彻底改变了这一看法。作为最具有代表性的模式识别受体,TLRs对PAMPs进行识别,引发的信号传导导致炎症介质的释放,在天然免疫防御中起重要作用,并且最终激活获得性免疫系统,因此TLRs控制着天然免疫和获得性免疫。近年来,越来越多的证据表明TLRs参与了天然免疫系统对病毒的识别,在抗病毒感染中发挥了重要的作用。病毒是一种相对简单的生物,由衣壳蛋白包裹核酸(DNA或RNA)组成。TLR4首先被发现可识别呼吸道合胞体病毒(RSV)的F蛋白并介导激活单核细胞。特别是最近的研究发现,对于病毒的核酸成分,除了提供遗传信息以外,还能够被TLR3、TLR9、TLR7/8这一类表达于细胞内体的TLRs所识别,诱导天然免疫反应并通过分泌的细胞因子控制获得性免疫的生成。TLR3识别病毒的双链RNA,介导激活树突状细胞(DC)、巨噬细胞和B细胞,导致了I-IFN的产生。TLR3缺失小鼠则丧失了这种功能,容易遭受病毒的感染。TLR9识别病毒非甲基化的CpG序列,特别是发现单纯疱疹病毒(HSV—2)感染骨髓细胞诱导了TLR9依赖性的IFN-a的产生,相似的结果也在HSV-1中发现。另一个确凿的证据是TLR9介导的抗鼠巨细胞病毒(MCMV)免疫。TLR9缺失小鼠则产生了较高的病毒滴度,并且死亡率大大增加。TLR7与TLR8在序列上的高度同源性使得它们识别的配体相同,均为单链RNA病毒。最初鉴定的TLR7/TLR8配体为咪唑喹啉家族,该合成物具有很强的抗病毒活性,这也许是因为它与核糖核酸在结构上的相似性。随后的研究证实了单链RNA为TLR7/TLR8的天然配体。基因缺失小鼠的研究表明,TLR7对于识别包括流感病毒(Ⅳ)和水泡性口炎病毒(VSV)在内的一些单链RNA病毒是必须的。对于Ⅳ或是HIV-1这些单链RNA病毒,即使在灭活的情况下也能激活pDCs,这似乎说明识别过程中并不要求活病毒的存在,不需要病毒复制过程,只要内体里存在病毒基因组的核酸成分就可以激活。然而另外一些单链RNA病毒,如RSV、VSV,灭活后就不能激活pDCs,需要活病毒的感染,感染后复制过程中产生的中间产物RNA通过自体吞噬作用进入内体,才能激活pDCs。合成的一些单链RNA寡核苷酸(ORN)可以模拟病毒RNA的作用,用脂质体包裹转染可以激活效应细胞,诱导分泌细胞因子。RNA40(GCCCGUCUGUUGUGUGACUC)是第一条从单链RNA病毒(HIV-1)中筛选并确认的RNA寡核苷酸,脂质体包裹后可刺激激活PBMC、pDCs和NK细胞等。能够诱导产生了Th1型细胞因子,细胞因子的产生导致T和B细胞的旁激活,并且,ORN能够促进抗原特异性的CTL和IgG2a型抗体反应。有些研究指出RNA寡核苷酸的免疫活性依赖于其碱基组成和结构特异性,目前,关于免疫刺激性RNA的序列特点没有明确的说法,不过,似乎U碱基对于RNA的活性是必须的,所以另一部分研究认为U的数量越多,免疫刺激活性越强。TLR7识别病毒的另一个研究方向在于,这种识别使得病毒在进化过程中调整它们的基因组组成以逃避这种识别,一些灭活的单链RNA病毒就不能激活免疫反应,病毒逃避TLR7识别的另一个机制也许在其基因组中存在抑制性的序列。我们首先研究的是TLR7在丙肝病毒(HCV)感染中所起的作用。机体被HCV感染后,55-85%的人发展成为慢性感染,大多数病人遭受了长期的肝脏炎症,从而导致肝纤维化或肝硬化。虽然诱导肝细胞死亡的任何因素都会建立NF-κB相关的炎症反应,但是确切的参与分子还不清楚。有研究显示,TLR7单核苷酸多态性(SNP)与HCV感染后引起的炎症反应密切相关,特殊位点的SNP对于肝纤维化的形成有显著的影响。我们的研究发现,与未感染者相比,HCV病染者外周血中促炎症因子(TNF-a)水平明显升高,并且检测发现,TLR7在PBMC和pDC中的表达量显著上调。鉴于HCV是一种单链RNA病毒,我们猜测很有可能TLR7介导了HCV的免疫反应。在体外,我们用HCV的RNA与pDCs共孵育,发现pDCs被激活并在培养上清中检测到IFN-a的分泌。为了说明HCV的核酸物质通过TLR7的介导激活了pDCs,我们从HCV基因组中筛选了一些RNA寡核苷酸,用脂质体包裹后刺激pDCs,发现在HCV核心区域上的一个RNA片段具有免疫活性,特别是HCV特有的Poly(U)尾,具有很强的免疫刺激性,诱导了大量的IFN-a产生。TLR7的抑制物破坏了RNA的免疫活性,说明了TLR7参与HCV-RNA刺激pDCs的介导作用。肝癌细胞系Huh-7细胞上表达有TLR7,免疫刺激性的HCV-RNA与Huh-7作用诱导了转录因子NF-κB的核转移,这些结果说明HCV-RNA在HCV持续感染期通过TLR7的介导作用,诱导了炎症反应。适当的天然免疫反应对于抵抗病原体的感染是必须的,但是如果这种反应过度,将会导致长期的炎症紊乱,造成病理性损伤。过度分泌的促炎症因子(尤其是TNF-a)在慢性HCV感染中很常见。因此,TLR7在HCV感染中的激活是一把双刃剑。那么,这种多余信号的清除就显得非常重要了。机体有很多手段来实现这种抑制,例如在蛋白水平,TIR8/SIGIRR通过与TLR受体竞争结合干扰下游信号从而限制过度炎症反应;例如在基因转录水平,Bcl-3与p50形成的同型二聚体与TLR信号激活的基因结合可以阻止其转录。我们的研究发现,HCV-RNA与效应细胞作用,诱导了miR-155的表达。MicroRNAs(miRNAs)是一种进化上保守、大小约21-23个碱基的非编码RNA,在转录后水平调控细胞的形成与发育,因此异常的miRNA水平与恶性肿瘤的形成有关。以前的研究提示,miR-155在天然免疫、炎症反应和肿瘤间发挥非常重要的作用。对于免疫反应的形成miR-155也是必不可少,并研究已经发现miR-155的靶基因,miR-155在炎症反应中表达上调也已有报道,只是在调节炎症反应中的靶基因还没有找到,我们的研究发现,miR-155可以与TLR7通路中的TAB2分子结合,从而抑制了TAB2的表达,进而对整个炎症反应起负调控作用。为了说明TLR7识别单链RNA病毒在不同物种间和不同病毒间具有广泛性,我们研究的第二个病毒是口蹄疫病毒(FMDV)。我们从FMDV基因组筛选得到了具有免疫活性的RNA序列,与PBMC作用诱导了IFN-a和IL-12p40的分泌。说明FMDV的核酸物质也能与免疫系统的效应细胞作用并激活这些细胞。接着克隆了FMDV的宿主(猪)TLR7基因(pTLR7),将pTLR7转染HEK293细胞构建细胞株,用FMDV-RNA刺激也能激活该细胞并诱导NF-κB的核转移和细胞因子的产生。FMDV-RNA与PBMC作用也导致了获得性免疫相关细胞的激活。这些结果说明,pTLR7也能够介导FMDV这种单链RNA病毒的免疫激活。有研究表明,ORN能以序列特异性的方式增强抗原的免疫反应,ORN的免疫刺激效果增强了抗原特异性的CTL和IgG2a型抗体反应,这提示ORN作为疫苗佐剂应用方面的前景,作为佐剂,ORN比CpG更有优势的地方在于,TLR7的表达比TLR9的表达范围更广,因此,ORN在增强疫苗效果方面显示出比CpG更好的效果。我们首先构建了以乙肝病毒的核心蛋白病毒样颗粒(HBc-VLP)为载体,在适当位点连有FMDV两个主要抗原表位(用柔性接头连接)的重组多肽疫苗。HBc-VLP具有较强的免疫原性,同时可以在颗粒表面很好的展示抗原,因此是一种理想的疫苗载体。由于外源表位的插入容易破坏HBc-VLP组装成颗粒,我们先对HBc进行了改造,分别在c/el区删除75-78位氨基酸(aa)、75-80aa、75-82aa,然后,将FMDV VP1上主要的B细胞表位与VP1主要的T细胞表位分别插入到修饰的HBc中,构建的重组质粒在大肠杆菌中表达可以形成VLP,能够形成VLP的重组疫苗易于被巨噬细胞吞噬,免疫动物诱导产生较强的肽特异性和病毒特异性的抗体,提高了IFN-γ、IL-4的分泌和淋巴细胞增殖反应,ORN增强该重组疫苗效果的实验正在评价当中。接下来,我们探讨了HBc-VLP能够快速激活免疫的机制,相比于不能形成VLP的蛋白,VLP易于被巨噬细胞、DC吞噬,从而可以快速激活免疫细胞,VLP与DC作用,使得Annexin aII的表达下调和生长因子受体结合蛋白2(GRB2)的上调,前者的下调有利于DC的快速活化,而GRB2的上调延长了DC活性时间,抑制了凋亡。综上所述,本研究首次证明了在HCV感染中,TLR7介导HCV的单链RNA激活免疫系统的效应细胞,诱导了炎症因子的表达;并且在肝癌细胞上也表达有TLR7,HCV单链RNA也能通过其TLR7的介导诱导NF-κB的核转移,活化生成炎症反应的信号通路;HCV-ssRNA的免疫刺激使得miR-155的表达上调,实验证明了miR-155可以与TLR7通路中TAB2分子作用,抑制TAB2蛋白的表达,从而负向调控了炎症因子的分泌。TLR7对单链RNA病毒的识别在各物种间是普遍的,实验证明了FMDV的单链RNA能够通过其宿主TLR7激活天然免疫反应。在抗病毒的疫苗研究上,我们构建了以乙肝病毒的核心蛋白病毒样颗粒(HBc-VLP)为载体,在适当位点连有FMDV两个主要抗原表位(用柔性接头连接)的重组多肽疫苗。这种VLP疫苗在体内诱导了增强的免疫应答反应,这主要是由于VLP易于被DC、巨噬细胞吞噬,同时VLP刺激DC也诱导了一些参与免疫激活分子的变化,增强了DC的活性。更多还原

【Abstract】 Sensing and defeating microbial infections is essential to the survival of metazoan species. Innate immune responses form the first line of defense against infective disease.Innate immunity once was thought as a low grade form of the immune system replies for external stimulus.However,the importance of the nonspecific immune system is accepted by more and more persons gradually with understanding in depth to immune system.In the innate immunity,the evolution and mutation of pathogen will make the validity of discerned microbe weaken progressively.So as to host,the greatest challenge lies in discerning a large amount of different causative agent and replying rapidly through the limited receptor.The finding of Toll-like receptors(TLRs) and pathogen-associated molecular patterns (PAMPs) changed completely this view.As the most representative pattern-recognition receptor,TLRs can recognize PAMPs and induce the release of inflammatory factors that play an important role in innate immunity and can further activate the adaptive immune responses. So TLRs control the innate immunity and adaptive immune responses.Recently,more and more evidences have suggested that TLRs are involved in the recognition of viruses by the innate immunity,and that play an important part in antivirus. Virus is a relatively simple organism that consists of nucleic acid(DNA or RNA) and capsid protein.TLR4 is firstly found to recognize the F protein of respiratory syncytial virus(RSV) and activate monocytes.Especially recent finding,the nuclear acid composition of the virus, besides offering hereditary information,can be recognized by TLR3,TLR7,TLR8 and TLR9 which are expressed in corpuscular endosome,induce innate immunity and activate adaptive immune responses by releasing cytokines.TLR3 recognize viral double-stranded RNA, activate dendritic cells,B cells and macrophages to produce I-IFN.However TLR3-defective mouse lost this kind of function and apt to suffer infection of virus.TLR9 recognize nonmethylated CpG motif,recent findings have showed that herpes simple virus(HSV-2) infect bone marrow cell induce TLR9-dependent production of IFN-a.The similar result was found in HSV-1 too.Another conclusive evidence is that TLR9 mediated the anti- mouse cytomegalovirus(MCMV) immunity.However,the TLR-defective mouse produced higher virus titer and mortality increases greatly.TLR7 and TLR8 discern identical ligands,single-stranded RNA virus,because of the height homology on the sequence.The imazaquin was firstly identified as TLR7/8 ligand,and The synthetical has very strong antiviral activity.Subsequent research verified the single-stranded RNA as the natural ligand of TLR7/TLR8.Imazaquin may be have the structural similarity with ribonucleic acid.Recent findings have confirmed that the sequence of containing in G-U rich can stimulate TLR7/TLR8.TLR7 is necessary to recognize ssRNA virus by the research of gene-defective mouse,such as vesicular stomatitis virus and influenza virus,pDCs play a important role in detecting virus via TLR7.Ultraviolet-irradiated HSV,heat-inactivated and UV-inactivated HIV-1 can induce IFN-a responses comparable to their live counterparts in pDCs.pDCs recognition of these ssRNA viruses occurs independently of fusion or replication but requires attachment and endocytosis showing that infection by live virus is not required, and that the presence of viral genomic nucleic acids within the edosomal/lysosomal compartments is sufficient to activate antiviral pathways.However,other ssRNA virus,for example,RSV and VSV required live virus infection and viral replication intermediates were being detected in pDCs by autophage.Synthetic ssRNA oligonucleotides(ORN) mimic the virus RNA and induce the production of cytokines. RNA40(GCCCGUCUGUUGUGUGACUC) is the first single-stranded RNA motif screened from HIV and can be recognized by TLR7.However,the specific RNA motifs constitute of immunostimulatory ORN is unclear.For example,the length,sequence composition and chemical composition are not known for ORN to strongly activate immune response.ORN is a strong activator of TLR7 and induces production of Thl-type cytokines both in vitro and vivo.Cytokine production led to bystander activation of T and B cells.Furthermore,ORN can trigger the generation of antigen-specific cytotoxic T cells and of an IgG2a-biased antibody response to antigen in a sequence-dependent manner.And so,ORN can simultaneously activate the innate and adaptive immune response like the single-stranded RNA virus.Another research direction of the discerns lies that the kind of discernment makes the virus to adjust the genome of them in order to escape this kind of discernment in evolution.What we studied at first is the role of TLR7 in(HCV) continuously.More than 170 million people are infected with hepatitis C virus(HCV) and 55-85%of patients become chronically infected.HCV infection leads to chronic liver inflammation in the majority of patients.A substantial proportion of patients develop fibrosis or cirrhosis.Fibrosis is the result of defective repair of liver damage resulting from inflammation caused by effector cells of the immune system.Although it is clear that any agent that induces hepatocyte death sets up an inflammatory state in the liver that is associated with NF-kB activation,the precise molecular players responsible for this have only just begun to be elucidated.Some researches showed that the prevalence of TLR7 single nucleotide polymorphisms(SNP) in the patients chronically infected with HCV and their effect on liver fibrosis,and showed specific TLR7 SNPs have a significant effect on fibrosis progression in patients with chronic HCV infection. In the current study,we have confirmed that HCV encodes immunostimulatory RNA oligonucleotides(ORNs) that activate immune and inflammatory response via Toll-like receptor 7.Moreover,HCV-derived ORNs also activated inflammation-related transcription factor NF-kB in Huh-7 cells.The ORNs stimulation of human monocytes and murine macrophages resulted in the up-regulation of miR-155.The change also occurred in vivo when Balb/C mice were i.p.injected with the ORNs.Crucially,the increase was correlated with a reduction in the production of TNF-a.miRNAs are an evolutionarily conserved class of endogenous "22-nt noncoding RNAs involved in posttranscriptional gene repression and play important roles in shaping cellular development and differentiation.Consequently, dysregulated miRNA levels are associated with several types of malignancies including pancreatic cancer and breast cancer and so on.miR-155 is extremely important molecule in innate immunity,inflammation and cancer.Although the precise roles for miR-155 in supporting or terminating the development of an innate immune response and inflammation have yet to be demonstrated,its relation to the development of B cell malignancies is documented.Therefore,miR-155 may provide a potential link between the inflammatory response and cancer.Luciferase reporter assays demonstrated that miR-155 directly targets 3’ untranslated region of MAP3K7IP2(TAB2) gene in TLR7 pathway.Functional studies demonstrated that transfection of miR-155 into RAW264.7 cells reduced the endogenous expression of the MAP3K7IP2 compared with no-transfected cells and decreased the production of TNF-a.These results suggest that HCV-encoded ORNs activated inflammatory response,which in turn was negatively regulated by miR-155,thus offering new target for prevention of hepatocellular carcinoma.Then,we investigated the interaction between the TLR7 and FMDV.Here we firstly cloned the porcine TLR7(pTLR7) cDNA encoding for 1050 amino acids.The pTLR7 exhibits 90%,87%,87%,86%,84%and 78%similarity to TLR7 from cattle,dog,horse,cat, human and mouse,respectively.RT-PCR data suggested that pTLR7 mRNA was mostly synthesized in secondary lymphoid tissue(spleen,lymph node and tonsil) and antigen-presenting cells(macrophage and B cell).The results of experiments in cells transfected with pTLR7 indicated that immunostimulatory RNA oligonucleotides(ORN) found in foot-and-mouth disease virus(FMDV) activated nuclear factor-kB and induced the expression of IFN-a via pTLR7.We showed that immunostimulatory ORN induced Thl-type cytokines and activated immunocytes in PBMC.The study provides foundation for further investigating pTLR7 interactions with its ligands and interactions between the porcine immune system and pathogens.The sequence-dependent adjuvant effect of ORN may play a role in enhancing the effect of mRNA-based vaccines.Furthermore,the addition of immunostimulatory RNA sequneces of mRNA vaccines could be used to enhance the potency of the vaccines.In addition,ORN show a therapeutic efficacy in vaccine formulations that is superior to TLR9 ligands because TLR7 are expressed in humans on a broad range of immune cells.Firstly,we investigated formation of virus-like particles by modified HBc fused with specified FMDV multiepitopes and evaluated their immune effects.Firstly,three HBc display vectors(pHBcl,pHBc2 and pHBc3) were constructed by deletions of different length within the HBc c/el region:75-78 amino acid,75-80 aa and 75-82 aa,respectively.Secondly,we inserted different compositions of FMDV multiepitopes,BT[VP 1(141-160)-VP4(21-40)]and BTB [VPI(141-160)-VP4(21-40)-VP1 (141-160)],into modified regions.As a result,only plasmid pHBc3-BTB of six recombinant vectors was expressed as soluble protein,which resulted in the formation of complete VLP confirmed by electron microscopy.Recombinant VLP could be uptaken by cells and presented in vitro and in vivo.Furthermore,the modified VLP displayed a significant stronger immunogenicity than other five recombinant proteins and GST-BTB with a higher titer of peptide-specific and virus-specific antibody,elevated IFN-γand IL-4 production,especially enhanced lymphocyte proliferation.The results encourage further work towards the development of FMDV vaccines using hepatitis B virus core particles fused with FMDV epitopes.VLP induced an acute activation of DC partly because the VLP is easy to be engulfed by DC.In addition,Proteomics was used for analysing the interaction of BMDCs with HBc-VLPs.Four conspicuous changed proteins from the comparison of HBc-pulsed-BMDCs and BMDCs were separated and identified by two-dimensional electrophoresis and mass spectrometry.Annexin A2(ANXA2) was found down-regulated expression and subsequently certified by western blotting;while the growth factor receptor bound protein 2(GRB2) was up-regulated.ANXA2,as the receptor of vitamin D3,could reduce the inhibition to DCs by down-regulation;while the up-regulated GRB2 might protect the DCs from apoptosis.So these proteins might play an important role in the cell vital process and enhance the activity of DCs.In conclusion,HBc-VLPs induced the strong potential of DCs antigen presentation,and the reason might due to the activity of DCs extended by HBc-VLPs.In summary,the study firstly identified the interaction between TLR7 and HCV.we have confirmed that HCV encodes immunostimulatory RNA oligonucleotides(ORNs) that activate immune and inflammatory response via Toll-like receptor 7.Moreover,HCV-derived ORNs also activated inflammation-related transcription factor NF-kB in Huh-7 cells.The ORNs stimulation of human monocytes and murine macrophages resulted in the up-regulation of miR-155.The change also occurred in vivo when Balb/C mice were i.p.injected with the ORNs.Crucially,the increase was correlated with a reduction in the production of TNF-a. Luciferase reporter assays demonstrated that miR-155 directly targets 3’ untranslated region of MAP3K7IP2(TAB2) gene in TLR7 pathway.Functional studies demonstrated that transfection of miR-155 into RAW264.7 cells reduced the endogenous expression of the MAP3K7IP2 compared with no-transfected cells and decreased the production of TNF-a. These results suggest that HCV-encoded ORNs activated inflammatory response,which in turn was negatively regulated by miR-155,thus offering new target for prevention of hepatocellular carcinoma.Meanwhile,other ssRNA virus have the same property.FMDV can be recognized by porcine TLR7 and activate immune responses.In research of antiviral vaccine,the VLP recombinant vaccine containing multi-epitopes demonstrates good antiviral function.更多还原