【作者】 吴亚男；

【导师】 曹雪涛；

【作者基本信息】 第二军医大学 ， 免疫学， 2009， 博士

【摘要】 上篇CMRF35样分子3(CLM3)促进TLR9触发的巨噬细胞产生炎性细胞因子及其相关分子机制研究免疫识别与免疫调节的细胞与分子机制是当前免疫学研究的重要领域。天然免疫系统是机体抗细菌、病毒等病原体入侵的“第一道防线”,而TOLL样受体(Toll-like receptors, TLRs)以及表达TLRs的巨噬细胞、树突状细胞等抗原提呈细胞是天然免疫的重要的组成部分,有关TLR参与巨噬细胞免疫识别的机制以及触发的免疫应答反应和信号转导过程的调节机制、特别是对于TLR信号转导的调控机制尚不十分清楚,因此,参与TLR信号转导的新的分子机制的研究备受瞩目。作为机体重要的模式识别受体(Pattern recognition receptors, PRR),TLRs主要表达于免疫细胞包括树突细胞、巨噬细胞和中性粒细胞、淋巴细胞表面。TLRs通过识别表达在细菌、病毒和真菌组成物上广谱的病原体相关分子模式,启动宿主抗感染的天然免疫反应。识别病原相关分子模式(Pathogen associated molecular patterns, PAMPs)后,TLRs招募胞浆内一系列含有TIR区域的接头蛋白,如MyD88、TIRAP (又称作MAL)、Trif (又称作TICAM1)和TRAM (又称作TICAM2)。除TLR3外,TLRs家族的所有成员都可以通过MyD88依赖途径活化,MyD88可通过活化下游的MAPKs和NF-κB来调控炎性细胞因子的分泌。TLR3和TLR4可通过活化TRIF依赖的途径活化NF-κB, MAPKs和IRF3,诱导TNF-α、IL-1β、IL-6和IFNβ等细胞因子。TLRs信号过度活化或负向调控不足会导致严重的免疫相关性疾病和炎症性疾病,如果活化不足将导致严重的免疫缺陷。目前已发现的一些正向调控因子,如DC-SIGN、Ras等,和负向调控因子,如A20、SHIP-1、ST2等,为维持TLRs介导的免疫应答稳定性发挥了重要的作用。由于TLRs信号转导对天然免疫及获得性免疫均具有重要的调节作用,TLRs信号转导机制的研究对于阐明机体免疫反应产生的机理、寻找免疫调节治疗的新途径具有重要意义,因此TLRs参与免疫识别与免疫调节的机制研究是当前免疫学研究的前沿热点。我们研究的CLM家族分子,其最早发现是源于CMRF35-A单抗识别的一个淋巴细胞表面抗原,该抗原分子属于IgSF超家族成员,含有单链的免疫球蛋白可变区的胞外段,跨膜段和胞内段,基因定位于人17号染色体。后来证明,此抗CMRF-35的单克隆抗体还能识别CMRF-35分子的同源分子,这些分子结构与CMRF-35相似,可变区与CMRF-35同源,在17号染色体上成簇分布(cluster)因而命名为CD300家族。其相应的小鼠同源分子命名为CLM(CMRF-35-Like Molecule,CLM)家族。鼠CMRF35样分子超家族含有9个成员,位于鼠第11号染色体上,CLM1-8非常紧密地位于11E2区的250kb左右的区域,而CLM9与其他家族成员分离,位于11D区。CLM家族成员与免疫应答、炎症及变态反应关系密切,首先, CLM家族成员主要表达在参与固有免疫应答的髓系免疫细胞表面,有些成员如CLM4在脾脏等免疫器官高表达,还有如CLM5在与外来抗原直接接触的气道和肺脏高表达;其次,多数活化性受体成员如CLM5、CLM7、CLM8在抗体交联活化后能促进中性粒细胞、肥大细胞、嗜酸性粒细胞或嗜碱性粒细胞分泌炎性因子,而抑制性受体成员如CLM1或CLM8则抑制炎性反应或免疫细胞应答;再次,许多受体成员如CLM1/5,CLM4/8,在炎症因子等外来刺激的细胞表面表达发生变化,提示在炎症反应过程中起一定的正向和负向调控作用。CLM分子参与TLRs免疫识别与应答的调节机制是值得研究的重要课题,目前基本上属于空白点。CLM3作为CLM家族的成员,其特征和功能都没有相关报道,本研究中我们从小鼠腹腔巨噬细胞中成功克隆了CLM3的全长基因,长738bp碱基,编码245个氨基酸(a.a)的蛋白质,推测分子量为27kDa。对其氨基酸序列分析表明,该蛋白含有长17a.a的信号肽和22a.a的疏水跨膜区,胞外段含有特征性的IgV功能域(domain),属于Ig超家族成员,CLM3定位于小鼠11号染色体的11E2区, CLM1-8非常紧密地位于该区,而CLM9与其他家族成员分离。同源性比对发现与CLM3同源性较高的均为CLM家族成员,其中与CLM5和CLM1同源性最高,胞外段一致性为85%。RT-PCR分析表明,CLM3广泛分布于小鼠正常组织,在心、肝、脾及淋巴结等组织均有不同程度表达,其中尤以淋巴结表达水平最高,在P815、Crip、RF、CT26、YAC-1、A20、3LL、Hepa、RAW264.7等细胞株中,CLM3仅在RAW264.7巨噬细胞株中表达,在新鲜分离的小鼠原代的细胞中,亦验证了此结果,CLM3仅在腹腔巨噬细胞中表达,这种在组织细胞的分布特点提示该分子在免疫防御中可能的功能。由于许多炎性因子可以通过影响具有调控功能的分子的表达进而调控细胞的应答,因此我们在本研究中观察了TLRs配体LPS、polyI:C、CpG ODN对CLM3分子表达的影响,然而LPS(0.1μg/ml剂量刺激)、polyI:C(0.1μg/ml剂量刺激)、CpG ODN(0.3μM剂量刺激)刺激至24小时,Raw264.7细胞中CLM3 mRNA的表达无明显变化。CLM3究竟有何功能?是否同其他CLM家族成员一样在免疫与炎症中发挥重要作用?是否也参与了TLRs的信号转导?我们发现,CLM3能促进CpG ODN诱导的炎性细胞因子(TNFα和IL6)的产生。我们将CLM3 siRNA转染小鼠腹腔巨噬细胞48小时,CpG ODN(0.3μM剂量刺激)刺激6小时后,收集上清采用ELISA试剂盒测定TNF-α和IL-6表达。结果显示,CLM3表达被干扰后,CpG ODN诱导的TNF-α和IL-6产生降低。将CLM3全长质粒转染RAW264.7细胞,操作步骤同上,结果显示,过表达CLM3后,CpG ODN诱导的TNF-α和IL-6产生增加。其它TLR配体如LPS刺激无明显变化。该结果证明CLM3参与并促进了TLR9触发的巨噬细胞的炎症反应过程。CLM3促进了TLR9触发的巨噬细胞的炎症反应的信号转导机制如何?我们将MyD88、IRAK1、TRAF6、CLM3质粒与TNF-α报告基因共转染HEK293细胞,24小时后检测TNF-α报告基因活化情况。结果显示,CLM3过表达增强了MyD88、IRAK1、TRAF6诱导的TNF-α活化;同时,我们应用TRIF、TAK1、CLM3质粒与TNF-α报告基因共转染HEK293,24小时后检测TNF-α报告基因活化情况。结果显示,CLM3过表达对TRIF、TAK1诱导的TNF-α活化没有显著影响。提示CLM3能通过MyD88、IRAK1、TRAF6途径促进TLR9的信号转导。经免疫共沉淀实验结果证实,CLM3通过与MyD88结合来促进后者的信号转导,提示CLM3可能通过促进IRAK1和TRAF6或TRAF6和TAK1复合物的形成来促进TLR9的信号传递。我们又检测了MAPK及NF-κB的活化情况,将CLM3全长质粒转染RAW264.7细胞,24小时后以CpG ODN刺激,观察MAPK的磷酸化及NF-κB的p65亚基的入核情况。应用CpG ODN刺激过表达CLM3的巨噬细胞后,JNK、ERK的磷酸化增加,NF-κB的P65亚基较对照组入核增加,提示CLM3通过活化MAPK和NF-κB来调控细胞因子的产生。综上所述,我们克隆了属于CLM家族的CLM3分子并对其的生物学特性及功能进行了研究。CLM3主要在小鼠腹腔巨噬细胞中表达,能促进CpG ODN刺激的腹腔巨噬细胞炎性细胞因子的分泌,免疫共沉淀证实CLM3通过与MyD88结合来促进信号转导,且CLM3增强了CpG ODN诱导的MAPK、NF-κB的活化。本课题探讨了CLM3对TLR9通路的细胞因子产生的影响并初步研究了其作用机制,为免疫识别以及抗感染治疗提供了一条新的途径。下篇CMRF35样分子5(CLM5)促进TLR和RLH触发巨噬细胞分泌I型干扰素和炎性细胞因子及其相关分子机制研究在抗病毒免疫应答中,RLHs(RIG-I-like Helicase,RLHs)和TLRs是识别RNA病毒感染的两大主要受体家族。迄今为止,人源TLRs家族发现了11个成员,鼠源TLRs家族发现了13个成员,它们通过识别病原体相关分子模式(pathogen-associated molecular patterns,PAMPs),而被活化,如TLR1, TLR2, TLR4, TLR6,和TLR10识别脂质,TLR3, TLR7, TLR8, TLR9识别病毒的RNA或DNA,因此,位于细胞内体的TLR3, TLR7, TLR8, TLR9被称为抗病毒的TLRs。受体活化胞内的级联信号通路后,诱导下游的TNF-α,IL-6,IL-1β等炎性因子的产生,且TLR3, TLR7, TLR8, TLR9能诱导抗病毒的I型干扰素产生。RLHs是细胞质螺旋酶,包括RIG-I、Mda5和LGP2。与膜定位的TLRs不同,RLH位于细胞质并且识别产生于胞质内的RNA。RIG-I和Mda5含有一个DExD/H盒RNA螺旋酶结构域和两个CARD样结构域,LGP2缺乏CARD样结构域。虽然RIG-I和Mda5分别与不同的配体特异性结合,但它们都能诱导I型干扰素和炎症性细胞因子的转录。RIG-I主要识别ssRNA病毒,包括副粘病毒、流感病毒、VSV和日本脑炎病毒(JEV),而Mda5主要识别微小RNA病毒,如EMCV、脑心肌炎病毒和鼠脑脊髓炎病毒,另外Mda5参与识别polyI:C。LGP2根据RNA病毒类型的不同正向或负向调控RIG-I和Mda5应答。RLR信号通路通过活化NF-κB、MAPK和IRFs诱导I型干扰素的产生。为维持机体正常的抗病毒免疫,RLR信号通路也被许多分子调控,例如TRIM25通过促进RIG-I的泛素化正向调控抗病毒应答(13),RNF125能与RIG-I、Mda5和IPS-I结合并促进他们的泛素化,促进RIG-I降解(14)。此外,Atg12–Atg5结合物直接与RIG-I和IPS-I的CARD样结构域结合,抑制I型干扰素的诱导(15)。CLM家族成员CLM5又名CD300d、DIgR1、MAIR-II、LMIR4。CLM5的跨膜区含有一个带负电荷的谷氨酸,胞内段很短且没有任何已知保守基序。RT-PCR证实CLM5主要表达在树突细胞、巨噬细胞和中性粒等髓系细胞中,但在淋巴系细胞中几乎没有表达。用Flag标记的CLM5转染RAW巨噬细胞后,抗FLAG抗体交联活化,引起受体下游P38、ERK1/2和JNK发生磷酸化,且交联活化的CLM5/RAW264.7细胞发生纤维化形态改变,由于CLM5能募集FcRγ,并且其它的FcRγ相关受体,如PIR-A和破骨细胞相关受体在抗体交联活化后也能增强破骨细胞的形成,因此提示CLM5可能也在破骨细胞的分化中发挥了作用。然而CLM5在细胞内的亚细胞定位情况如何?在炎性刺激时其细胞定位是否改变?其对巨噬细胞的细胞因子功能产生有何影响?是否参与了巨噬细胞的TLR和RLH的信号转导等等,这些问题尚未得到回答,也未见相关文献报道。通过GenBank数据库找到CLM5的全长mRNA序列(asscession No: AY457051),利用特异性引物,我们从小鼠腹腔巨噬细胞cDNA中克隆得到含666bp碱基的cDNA,预测其编码221氨基酸(a.a)的蛋白质,预计分子量为25kDa。对其氨基酸序列分析表明,该蛋白含有长17a.a的信号肽和23a.a的疏水跨膜区,胞外段含有特征性的IgV功能域(domain),属于Ig超家族成员,该受体胞内段很短,提示其可能对细胞功能起正向调控作用。通过搜索NCBI的基因组数据库,将CLM5定位于小鼠11号染色体的11E2区,染色体定位结果同CLM3,将CLM5蛋白序列在GenBank/EMBL数据库中作比较,发现与CLM5同源性较高的均为CLM家族成员,其中与CLM1同源性最高,胞外段IgV区域一致性为91%;与CLM家族蛋白质序列进行比对分析及进化树分析结果同CLM3。RT-PCR分析表明,CLM5广泛分布于小鼠正常组织,在心、脑、肝、脾及淋巴结、胎盘、睾丸等组织均有不同程度表达,其中尤以肝脏、脾脏和淋巴结表达水平最高,在P815、Crip、RF、CT26、YAC-1、A20、3LL、Hepa、RAW264.7等细胞株中,CLM5在RAW264.7巨噬细胞中表达,在新鲜分离的小鼠原代的细胞中, CLM5在腹腔巨噬细胞和树突状细胞中表达,CLM5的这种在组织的广泛分布特点提示该分子在天然免疫防御中可能扮演了重要的角色。那么CLM5的生物学功能究竟如何?实验中我们发现,CLM5表达下调能抑制TLRs配体诱导的IL6和IFN-β的产生,CLM5 siRNA转染小鼠腹腔巨噬细胞和RAW264.7细胞48小时,以LPS(0.1μg/ml剂量刺激)、polyI:C(10μg/ml剂量刺激)、CpG ODN(0.3μM剂量刺激)刺激6小时后,收集上清采用ELISA试剂盒测定IL-6、IFN-β表达,结果显示,CLM5表达被干扰抑制后,LPS、polyI:C、CpG ODN诱导的IL-6、IFN-β产生显著降低,提示CLM5参与了TLRs触发的巨噬细胞天然免疫应答过程及其信号转导的调控。以MOI 10的VSV感染干扰CLM5后的巨噬细胞,IFN-β产生显著降低,对CLM5的功能进行检测,发现干扰CLM5后其TCID50显著增高,提示CLM5参与了RLH触发的信号转导调控。我们进一步探讨CLM5对TLRs触发的IFN-β产生影响的作用机制,实验发现,CLM5促进TRIF、MDA5、RIG-I介导的IFNβ转录增强。将TRIF(或MDA5、RIG-I)、pCMV-CLM5质粒与IFN-β报告基因共转染HEK293细胞,24小时后检测IFN-β报告基因活化情况,结果显示,CLM5过表达增强了TRIF、MDA5、RIG-I诱导的IFN-β转录增强,并且,从结果中我们可以看出,单独转染CLM5与IFN-β报告基因,CLM5随转染剂量的增加,能呈递增趋势诱导IFN-β活化,提示CLM5能通过TRIF、MDA5、RIG-I途径促进IFN-β活化,其本身也可以通过以上三条途径以外的其它机制促进IFN-β活化。本实验提示,CLM5参与了RLH和TLRs的信号调控。免疫共沉淀证实,CLM5可以与含有ITAM基序的FcRγ相结合,FcRγ可能在CLM5对RLH和TLR信号转导的调控中发挥一定功能。综上所述,我们对CLM家族的CLM5的生物学特性及功能进行了研究,发现CLM5主要在小鼠腹腔巨噬细胞中表达,干扰CLM5表达之后能够降低TLRs配体刺激的腹腔巨噬细胞IFN-βand IL6的分泌,经VSV刺激后IFN-β产生也降低。进一步研究表明CLM5能增强TRIF、MDA5、RIG-I介导的IFNβ转录增加,可见CLM5可以促进TLR和RLH触发的巨噬细胞的天然免疫应答及其信号转导。更多还原

【Abstract】 CLM3 promotes TLR9-triggered production of proinflammatory cytokines in macrophages and the underlying mechanismsInnate immune system is the first line of host defense against pathogen infection. TLRs have emerged as the key sensors of microbial products, as they are expressed on sentinel cells in the immune system, most notably professional antigen-presenting cells (APC) including dendritic cells (DC) and macrophages, where they sense a range of chemicals produced by bacteria, viruses, fungi and protozoa. TLRs can be grouped into families according to the types of ligands they recognize. Lipid-based structures are recognized by TLR2 (in combination with TLR1 or TLR6 as heterodimers) and TLR4 (as a homodimer): the most studied examples of lipid-based recognition are bacterial or mycobacterial lipopeptides, or glycerophosphatidylinositol anchors from parasites, both of which are recognized by TLR2, and bacterial lipopolysaccharide (LPS), which is recognized by TLR4. Viral and/or bacterial nucleic acids are recognized by TLR3, TLR7, TLR8 and TLR9; the most characterized are the recognition of double stranded RNA (dsRNA) by TLR3 and recognition of CpG motifs in DNA by TLR9. Finally, TLR5 and TLR11 recognize proteins from pathogens (flagellin in the case of TLR5 and profilin in the case of TLR11. The events initiated inside cells when a given TLR is activated have been the subject of much investigation. Many biochemical details of these events have now been elucidated, including the description in the past years of novel adaptor proteins, protein kinases and transcription factors. However, the molecular mechanisms for the initiation and regulation of TLR response still remain to be fully understood now.In the TLR signaling, recognition of PAMPs by TLRs stimulates the recruitment of a set of intracellular TIR-domain- containing adaptors, including MyD88, TIRAP (also known as MAL), Trif (also known as TICAM1) and TRAM (also known as TICAM2) via TIR–TIR interactions. MyD88 is a universal adapter that activates inflammatory pathways, which is shared by all TLRs with the exception of TLR3. Recruitment of MyD88 leads to the activation of MAP kinases (MAPKs) (ERK, JNK, p38) and the transcription factor NF-κB to control the expression of inflammatory cytokine genes. TRIF is recruited to TLR3 and TLR4, and activates an alternative pathway (TRIF-dependent pathway) that culminates in the activation of NF-κB, MAPKs and the transcription factor IRF3. Activation of IRF3 is pivotal for induction of type I interferon (IFN), particularly IFN-β. The capacity not only to respond appropriately but also to self-regulate host responses to invading pathogens is critical to host immune system to mount an appropriate primary immune response against pathogens but avoid the pathogenesis of immune disorders. The continued focus of research on TLR signalling has provided a lot more information on pathways activated by TLRs, and new layers of complexity and regulation have been revealed, which present better understanding how TLR signaling is regulated and how to control TLR-mediated inflammatory diseases.Human CMRF35-A, previously isolated by recognition with the CMRF-35 monoclonal antibody, is a surface molecule with an IgV-like domain. It comprises a single extracellular Ig variable domain, a transmembrane domain and a cytoplasmic tail, located in chromosome 17. Anti-CMRF35 monoclonal antibody has been shown to be able to recognize other molecules which are homology of CMRF35, which located in chromosome 17 as cluster named CD300 family, and the mouse homology named as CMRF35-like molecules (CLM) family. The CLM family consists of nine members, which are encoded on mouse chromosome 11. CLM-1 to CLM-8 reside in close proximity in the ~250kb region of 11E2, whereas CLM-9 is separated from the other family members to be located in 11D. CLM family members have been proposed to play roles in the immune response, inflammatory and allergy. Firstly, CLM family members expressed on the surface of myeloid lineage cells. Secondly, the cross-linking with the active receptors such as CLM5、CLM7、CLM8 can promote neutrophilic leukocyte, mast cell, eosinophil, basophil degranulation. The cross-linking with inhibitory receptors such as CLM1 or CLM8 can inhibit inflammatory response or immune response. Thirdly, expression levels of CLM1/5, CLM4/8 are changed after stimulation with inflammatory factors. The investigations on the function and mechanism of CLM/CD300 family members will facilitate the development of new strategies to control inflammatory diseases, and also will contribute to the better understanding of immune recognition and immune regulation.CLM3 is a new member of CLM family, which has not been well investigated up to now. In this study, we cloned CLM3 full-length gene(accession No AY457049) from mouse abdominal cavity macrophages, which contains 738bp base, encoding 245 amino acids with a predicted molecular mass of 27 kD. The amino acid sequence begins with a hydrophobic signal peptide of 17 amino acids followed by an extracellular region composed of one IgV domains and a 22 amino acids transmember domain. CLM3 is located on mouse chromosome 11. CLM-1 to CLM-8 reside in close proximity in the region of 11E2. Comparative analysis indicated that CLM3 is closely related to some other CLM families, including CLM5 and CLM1 (85% identity with CLM3).Then we detected the expression patterns of CLM3 by RT-PCR. CLM3 mRNA was widely distributed in mouse tissues, including heart, spleen, liver, lymph nodes and testis, among which CLM3 is highest expressed in lymph nodes. RT-PCR analysis of cell lines and freshly isolated cells demonstrated that CLM3 mRNA was preferentially expressed in monocytes, and RAW264.7 macrophage cell lines.To explore the role of CLM3 in TLRs-triggered innate response in macrophages, we firstly examined whether the expression of CLM3 was changed in Raw264.7 cells after stimulated with TLR ligands. By using RT-PCR, we found that the mRNA expression of CLM3 was not changed after stimulation with different TLRs ligands during 24 hours.To identify the function of CLM3, we investigated the role of CLM3 in TLR-induced inflammatory response in macrophages. We found that CLM3 could promote CpG ODN-induced TNFαand IL6 production in macrophages. Mouse peritoneal macrophages were transfected with CLM3 siRNA. After 48h, the CLM3-silenced cells were treated with 0.3μM CpG ODN for 6 hrs. Then the concentrations of TNF-αand IL-6 in the supernatants were measured by ELISA. The productions of TNF-αand IL-6 were found to be decreased after silence of CLM3; Raw264.7 cells were transfected with CLM3 full-length cDNA. Then we found that overexpression of CLM3 promoted the production of TNF-αand IL-6 in macrophages in response to CpG ODN stimulation. These results indicate that CLM3 can promote TLR9-triggered proinfllamatory response in macrophages .To identify the mechanism of CLM3 involved in TLRs signaling, we cotransfected HEK293 with MyD88, IRAK1, TRAF6, TAK1, CLM3 plasmids and TNF-αluciferase reporter plasmid. After 24hrs of culture, luciferase activity was measured. Overexpression of CLM3 enhanced MyD88, IRAK1, TRAF6-induced but not TRIF, TAK1-induced TNF-αactivation, indicating that CLM3 promotes TLRs signaling through MyD88 pathway. Co-immunoprecipitation assays by cotransfection of HA-tagged MyD88, flag-tagged CLM3 plasmid in HEK293 cells, anti-flag antibody and anti-HA antibody proved that CLM3 could associate with MyD88, suggesting that CLM3 may influence TLR9 signalling via targeting IRAK1 and TRAF6 or TRAF6 and TAK1 complex formation.To analyze whether CLM3 could affect the activation of MAPK and NF-κB, CLM3 was transfected into RAW264.7 cells. After 24hrs of culture, the cells were treated with 0.3μM CpG ODN. The results showed that JNK and ERK phosphorylation and nuclear translocation of p65 subunit of NF-κB increased, compared with the negative control, indicating that CLM3 can enhance the TLR9-induced production of cytokines by activating MAPK and NF-κB pathway.In conclusion, we cloned CLM3 which belongs to CLM family. CLM3 is selectively expressed in lymph nodes and macrophages. CLM3 can promote TLR9-triggered production of inflammatory cytokines in macrophages. Co-immunoprecipitation experiments proved that CLM3 could promote TLRs signaling through association with MyD88 and enhance TLR9-induced activation of MAPK and NF-κB. We for the first time have provided the evidence that CLM3 promotes TLR9-triggered production of proinflammatory cytokines in macrophages though association with MyD88 and activation of MAPK, NF-κB pathways. Our findings provide new target and strategy to control inflammatory diseases. production of type I interferon and inflammatory cytokines in macrophages and the underlying mechanismsIn the immune response against viral invasion, RLHs (RIG-I-like Helicase) and TLRs are two major family of receptors which recognize RNA virus infection. To date, 11 human TLRs and 13 mouse TLRs have been identified. It has been shown that TLRs are activated by specific PAMPs and that the ability of specific TLRs to heterodimerize adds further to the diverse range of pathogens that may be recognized. For example, TLR1, -2, -4, -6, and -10 recognize lipids. TLR3, -7, -8, and -9, are endosomally localized, membrane-bound receptors which recognize virus RNA or DNA, so TLR3, -7, -8, -9 are named as anti-virus TLRs. After recognition of PAMPs, a cascade of intracellular signaling events is activated, which culminates in the induction of type I interferon (IFN) and proinflammatory cytokines such as tumor necrosis factor–α(TNF-α), interleukin (IL)-6, IL-1β.RIGI-like receptors (RLR), which are cytoplasmic RNA helicases, including retinoic acid-inducible gene-I (RIG-I), Mda5, and LGP2,Unlike membrane-bound TLRs, RLH reside in the cytoplasm and recognize RNA species produced in the cytoplasm. Whereas RIG-I and Mda5 contain a DExD/H box.RNA helicase domain and two caspase recruiting domain (CARD)-like domains required for eliciting downstream signaling pathways, but LGP2 lacks the CARD-like domains. RIG-I and Mda5 are able to interact with viral RNA, albeit with different ligand specificity, and elicit signaling leading to the transcription of type I IFN and inflammatory cytokines. RIG-I is essential for the recognition of a series of ssRNA viruses, which include paramyxoviruses, influenza virus, VSV, and Japanese encephalitis virus (JEV). Mda5 is required for the recognition of other RNA viruses, including picornaviruses such as EMCV, Mengo virus, and Theiler’s virus. Furthermore, Mda5, but not RIG-I, participates in the recognition of polyI:C. LGP2 negatively or positively regulates RIG-I and Mda5 responses, depending on types of RNA viruses. Although the important roles of RLH and CLM5 promotes TLR- and RLH-triggered TLR in the recognition of PAMP and initiation of immune response against pathogens have been extensively studied, the molecular mechanisms by which RLH and TLR effectively activate innate immune response and how the process can be controlled at the proper level remain to be fully understood, so as to avoid the immunological disorders happened once the response is over-activated or out of control. New regulators for the RLH and TLR-triggered innate immune response attract much attention in recent years.CLM family member CLM5 is also designated as CD300d,DIgR1,MAIR-II,LMIR4. CLM-5 has a short cytoplasmic tail without any known motif sequences. In addition, the transmembrane domain of CLM5 possesses a negatively charged glutamic acid. These characteristics suggest that CLM5 may be an activating receptor interacting with a signaling motif-bearing adaptor molecule. It has been shown that CLM5 is expressed in the myeloid lineage cells such as dendritic cells (DC), macrophages, neutrophilic leukocytes but not in lymphocytes. When FLAG-CLM-5 on CLM-5/RAW264.7 cells was cross-linked with anti-FLAG mAb followed by the F(ab9)2 fragment of goat anti-mouse IgG, tyrosine phosphorylation of p38, ERK1/2 and JNK was significantly enhanced, and CLM-5 cross-linking also induced a fibroblastic morphological change in CLM-5/RAW264.7 cells similar to that observed when RAW264.7 cells were stimulated with LPS. Other FcRc-associating molecules, PIR-A, and osteoclast-associated receptor also enhance osteoclast formation upon cross-linking with antibodies. So, CLM-5 has been proposed to be involved in the differentiation of osteoclasts. However, up to now, there is no report about the role of CLM5 in the RLH and TLR-triggered innate immune response against virus infection and the underlying mechanisms.We cloned CLM5 full-length gene (accession No AY457051) from mouse primary abdominal macrophages, which contains 666bp base, encoding 222 amino acids with a predicted molecular mass of 25 kD. The amino acid sequence begins with a hydrophobic signal peptide of 17 amino acids followed by an extracellular region composed of one IgV domains and a 23 amino acids transmember domain. CLM5 is located on mouse chromosome 11, in which CLM-1 to CLM-8 reside in close proximity in the region of 11E2. CLM-5 has a short cytoplasmic tail without any known motif sequences indicating its active function. Comparative analysis indicated that CLM5 is closely related to some other CLM families, including CLM1 and CLM3 (91% identity with CLM1; 85% identity with CLM3).Then we detected the expression profile of CLM5 by RT-PCR. CLM5 mRNA was widely distributed in mouse tissues, including heart, spleen, liver, lymph nodes, brain and testis, with highest level in spleen, liver and lymph nodes. RT-PCR analysis of cell lines and freshly isolated cells demonstrated that CLM5 mRNA was preferentially expressed in monocytes s, dendritic cells and RAW264.7 macrophage cell line. The selective expression of CLM5 in antigen-presenting cells (APC) indicated that CLM5 may be involved in the innate immune response against pathogens.To identify the function of CLM5, we investigated whether CLM5 could affect RLH and TLR-triggered type I IFN and proinflammatory cytokine production in macrophages. We found that CLM5 can promote TLRs ligand-induced IFN-βand IL-6 production in macrophages. After mouse peritoneal macrophages were transfected with CLM5 siRNA for 48h, the CLM5-silenced macrophages were treated with LPS(0.1μg/ml), polyI:C (10μg/ml), CpG ODN (0.3μM/L) for 6 hrs. Concentrations of IFN-βand IL-6 in the supernatants were measured by ELISA. The concentrations of IFN-βand IL-6 were found to be decreased after kncokdown of CLM5 in macrophages. These results indicate that CLM5 promotes TLR-triggered type I IFN and proinflammatory cytokine production in macrophages.As the same, The concentrations of IFN-βwas decreased after knockdown of CLM5 when infect with VSV for 24 hour,and TCID50 also increased.This indicate that CLM5 also participate in RLH-triggered signaling.To explore the underlying mechanism of CLM5 in the promotion of type I IFN and proinflammatory cytokine production, we cotransfected HEK293 with TRIF (or MDA5, RIG-I), pCMV-CLM5 plasmid and IFN-βluciferase reporter plasmid, and then the luciferase activity was measured after 24hrs. Overexpression of CLM5 enhanced TRIF, MDA5, RIG-I-induced IFN-βactivation, indicating that CLM5 positively participates in TLRs and RLH signaling. Co-immunoprecipitation experiments improved that CLM5 can associate with FcRγ,indicate the possible role of FcRγin CLM5 regulate RLH and TLR signaling.In conclusion, we cloned CLM5 which belongs to CLM family. CLM5 was selectively expressed in macrophages and dendritic cells. Knockdown of CLM5 in macrophages inhibited the production of IFN-βand IL6 induced by the stimulation with TLRs ligands,and the production of IFN-βwas also decreased after VSV infection. Furthermore, CLM5 enhanced TRIF, MDA5, RIG-I-induced IFNβactivation. Taken together, these results suggest that CLM5 may be a positive regulator of TLRs and RLH-triggered type I IFN and proinflammatory cytokine production in macrophages.更多还原