DNMT1和CLM-3在天然免疫应答中的功能及机制研究
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摘要
DNA甲基转移酶是一种在原核和真核生物高度保守的酶,能够在基因组中的DNA复制后对其胞嘧啶C5位点进行修饰,参与体内多种重要生理过程,主要包括:调节基因表达、基因印记、维持染色体完整性以及X-染色体灭活等。根据其结构和功能的不同,哺乳动物中DNA甲基转移酶(Dnmts)主要分为两大类: DNA甲基化维持酶Dnmt1以及DNA从头甲基化酶Dnmt3a、Dnmt3b和Dnmt3L等.
树突状细胞(dendritic cells，DC)是指具有树枝状突起和星状多形性，高表达MHCI类和I类分子，能有效摄取、加工和处理抗原并激活初始型T细胞的一类抗原提呈细胞(antigen presenting cell，APC)。DC是1973年由Steinman和Cohn首先发现的。作为目前发现的功能最强的APC，DC最大的特点是能够刺激初始型T细胞(naive Tcell)活化和增殖。DC是抗原特异性免疫应答的始动者，由于其在免疫应答中的独特地位，对DC的研究有助于深入了解机体免疫应答的产生和调控机制，对肿瘤、移植排斥、感染、自身免疫性疾病等的发生发展机制的认识有重要的理论意义。
免疫球蛋白超家族含有众多的膜上和膜内受体,参与天然免疫和获得性免疫中的各种免疫应答反应.小鼠CMRF-35样分子家族为免疫球蛋白超家族的成员之一.CLM家族成员在识别不同的配体后能够正向或负向调控免疫应答反应。本实验室通过小鼠树突状细胞cDNA文库大规模测序后，发现了一系列选择性地在树突状细胞和巨噬细胞等抗原提呈细胞上表达的CLM家族成员受体，包括树突状细胞来源的免疫球蛋白受体1(DC-derived Ig receptor1, DIgR1)/CLM-4,树突状细胞来源的免疫球蛋白受体2(DC-derived Ig receptor2, DIgR2)/CLM-1, CLM-5，以及本文中研究的CLM-3。
抗原递呈细胞能够通过模式识别受体(pattern recognition receptors, PRR)识别病原体相关分子模式(pathogen-associated molecular patterns, PAMP)来检测入侵宿主的病原微生物，然后促发免疫球蛋白超家族含有众多的膜上和膜内受体,参与天然免疫和获得性免疫中的各种免疫应答反应。活化的天然免疫细胞能够诱导促炎因子和I型干扰素的产生来清除入侵的病原微生物。Toll样受体(Toll-like receptors, TLRs)作为模式识别受体中最重要的一种，显著高表达于抗原递呈细胞，包括巨噬细胞，单核细胞以及树突状细胞。根据所有TLRs家族成员的亚细胞定位，TLRs分成两大类：细胞膜定位的TLRs和细胞内定位的TLRs。在所有TLRs中，内体定位的TLR9是唯一一种能够识别病原体来源DNA的TLR。一旦结合细菌中含有CpG岛的DNA或者人工合成的含有为甲基化CpG基序的寡聚脱氧核苷酸(CpG-ODN)，TLR9就能够直接活化抗原递呈细胞和天然免疫细胞分泌各种促炎因子。当CpG-ODN结合到TLR9时能够招募接头分子MyD88，进而激活下游MAPKs和NF-κB的磷酸化信号通路，最终引起促炎因子和I型干扰素的产生。研究证实TLR9的激动剂能够诱导强烈的Th1型免疫反应来保护宿主免受细菌和病毒的感染。因此，对于如何选择性的活化TLR9信号来起始保护性免疫应答及TLR9完全活化的潜在机制仍需进一步研究。
基于以上研究进展和现状分析，我们利用现代实验技术对人单核细胞来源的树突状细胞和小鼠腹腔巨噬细胞中的DNMT1在天然免疫应答方面的功能进行了研究；同时对于CLM-3对于Toll样受体的功能影响进行了深入的探讨。
第一部分人单核细胞来源的树突状细胞中与DNMT1相互作用蛋白的探讨
随着小鼠骨髓来源树突状细胞在免疫应答中功能研究的深入，我们开始关注人单核细胞来源的树突状细胞的功能。作为重要的DNA甲基转移酶DNMT1功能的不断发现，使得我们开始探寻DNMT1在人单核细胞来源树突状细胞中的作用。
我们使用蛋白质免疫共沉淀技术、质谱兼容银染以及LS-MS/MS的手段，发现了一系列在人单核细胞来源的树突状细胞中与DNMT1相互作用的蛋白，这些蛋白主要集中在翻译起始因子eIF家族，主要包括eIF3A、eIF3H；以及细胞周期和凋亡调节蛋白1（cell cycle and apoptosis regulator protein1, CARP1)。
鉴于目前的研究报道，DNMT1主要是在核内发挥其对DNA的甲基化修饰功能，而eIF3A和eIF3H则主要是在胞浆中发挥蛋白的翻译。这就促使我们去探寻DNMT1是否在胞浆中也发挥其非经典功能，而参与了其他一些生物学功能。通过免疫共沉淀，我们发现了DNMT1确实存在于胞浆中，而且能够与同在胞浆中发挥作用的eIF3A和eIF3H相互结合，而在胞核中则为发现eIF3A和eIF3H与DNMT1的结合。对于DNMT1在胞浆中具体发挥怎么样的功能还有待于进一步的深入探讨。
对于同期发现的细胞周期和凋亡调节蛋白CARP1，研究发现DNMT1与CARP1两者之间主要在细胞核内相互作用，这提示我们CARP1可能参与了DNMT1的经典功能，作为一个新的可能参与DNA甲基化的蛋白，其具体功能有待于进一步深入的研究。
第二部分DNMT1在巨噬细胞天然免疫功能中的作用研究
TLR是研究最早的模式识别受体，其在激活机体的固有免疫应答和调控获得性免疫应答，抵御各种病原体感染中发挥重要的作用。它的活化不足导致机体不能有效地清除病原体，而过度的激活又会引发一系列的免疫损伤如脓毒症休克等。因此TLR信号通路必须受到严格的精密调控，TLR信号通路同其他信号通路之间的交叉联系也是近年来免疫学的研究热点之一。
对于DNA甲基转移酶DNMT1在TLR信号通路中的作用还没有相关的研究报道，我们通过干扰小鼠腹腔巨噬细胞中的DNMT1后，用各种TLR配体进行刺激，进而研究DNMT1是否参与TLR信号通路。研究发现，在DNMT1得到有效干扰后并不能够显著影响TLR3、TLR4以及TLR9活化产生的IFN-beta和TNF-alpha。
基于巨噬细胞在抗病毒的过程中也发挥着重要作用，我们观察了干扰DNMT1对于VSV和SeV感染的影响。研究发现DNMT1的干扰能够影响VSV和SeV引起的IFN-beta的产生，DNMT1能够促进IFN-beta的产生，这提示我们DNMT1可能参与RIG-I的信号通路，对于其具体机制，有待于进一步的研究和探索。
第三部分CLM-3调控TLR9触发的巨噬细胞天然免疫功能及其机制研究
CLM-3(CMRF-35like molecule3)作为CLM家族成员之一，与CLM-5关系密切，同属于活化性受体。研究表明CLM-5能够通过与含有免疫受体酪氨酸活化基序(immunoreceptor tyrosine based-activating motif， ITAM)的FcRγ相互作用，进而转导活化信号。CLM-3同样也能够与FcRγ相互作用，但是其在天然免疫应答中的功能还未得到确认。
本实验室早期的研究发现CLM-3选择性地表达于巨噬细胞中，CLM-3能够通过与MyD88结合以激活MAPK、NFkB通路从而促进了TLR9触发的巨噬细胞产生炎性细胞因子TNFa和IL-6，研究提示，CLM-3与带有ITAM基序的FcRγ相互作用,但是，CLM-3调控TLR9信号通路的具体分子机制尚不清楚。
通过研究证实，CLM-3选择性地表达于巨噬细胞中，为内体/溶酶体定位的免疫受体；在TLR9配体CpG-ODN刺激下，CLM-3在巨噬细胞中的表达被下调；CLM-3作为一个能够在巨噬细胞中正向调控TLR9信号转导的受体，主要通过增强TRAF6(TNFR-associated factor6)的泛素化，进而促进下游MAPKs和NF-κB的信号转导，最终使得TLR9引发的天然免疫应答反应全面活化。研究主要发现通过与CLM-3的交互作用能够促进TLR9引发的天然免疫反应，最终有利于宿主清除外来入侵病原体。
我们通过激光共聚焦检测发现在Raw264.7细胞系中CLM-3能够与LAMP1阳性的细胞器和早期内体标记Rab5共定位，而对于高尔基体的代表性标记GM130则没有明显共定位。因此，CLM-3不像其他细胞膜定位的CLM家族成员，而是选择性的定位于膜类细胞器内体及溶酶体。从CLM-3在巨噬细胞中定位内体及溶酶体，我们推测CLM-3可能在TLR9触发的巨噬细胞活化过程中发挥作用。我们用CpG-ODN刺激siRNA干扰过的原代腹腔巨噬细胞，发现CLM-3被干扰后降低TLR9活化引起的TNF-α和IL-6表达，但是不影响Poly(I:C)或LPS引起的TNF-α和IL-6的表达。因此，我们证实CLM-3能够选择性地促进TLR9，而非TLR3或TLR4，引起的促炎因子的产生。
为验证CLM-3过表达在体内的作用,我们制备了CLM-3转基因小鼠(CLM-3transgenic mice, CLM-3TG mice)观察CLM-3在TLR9触发的天然免疫应答。Westernblot证实在原代腹腔巨噬细胞中高表达带HA标签的CLM-3。取自CLM-3TG mice的巨噬细胞相较于同窝未表达HA-CLM-3的小鼠的巨噬细胞，在CpG-ODN刺激后能够产生更多的TNF-α和IL-6，实验结果和在巨噬细胞中过表达CLM-3的效应相一致。在小鼠腹腔注射CpG-ODN后，观察血清中TNF-α和IL-6的表达水平也得出相似的结论，CLM-3TG mice较同窝对照小鼠产生更多的炎症因子。鉴于在浆样树突状细胞(plasmacytoid dendritic cell, pDC)中TLR9在I型干扰素的产生过程中发挥着重要作用，我们观察了从转基因小鼠脾脏来源的pDC在GpG-ODN刺激后诱导产生I型干扰素中的效应。研究发现CLM-3转基因小鼠和同窝对照小鼠来源的pDC在干扰素的产生上并无显著性差异。研究证实CLM-3作为一个能够正向调控TLR9触发的天然免疫应答主要在巨噬细胞中发挥作用。
已有研究报道证实，MAPKs和NF-κB是CpG-OND触发细胞因子产生的关键信号通路。在CLM-3转基因小鼠来源的巨噬细胞中，我们观察到CLM-3的过表达能够促进ERK1/2，JNK1/2，以及p38的磷酸化水平，相同的作用趋势发现在CLM-3过表达的Raw264.7细胞系中。在体外检测CLM-3转基因小鼠来源的腹腔巨噬细胞发现CpG-ODN刺激活化IKKα/β和IκBα的磷酸化水平明显强于同窝对照小鼠。上述实验结果证实，CLM-3能够促进CpG-ODN触发的TLR9信号通路活化。
通过前面的实验我们已经证实了CLM-3对于TLR9的正向调控作用，对于潜在的机制研究将使我们对于CLM-3的了解更为透彻。报道证实，CpG-ODN在结合到TLR9后，下游信号分子MyD88能够招募IRAK1(IL-1receptor associated kinase1)、IRAK2以及IRAK4。这些IRAK激酶的活化能够招募TRAF6进行泛素化，进而激活下游的MPAKs和NF-κB信号。TRAF6作为TLR9信号中起关键作用的分子，其泛素化是激活下游信号活化所必需的。因此，我们想证实一下CLM-3的过表达是否会增强TRAF6的泛素化。在原代腹腔巨噬细胞中用siRNA干扰CLM-3后，我们检测了CpG-ODN刺激后TRAF6的泛素化水平，发现CLM-3的沉默能够降低TRAF6的泛素化水平。同理，我们检测了来自于CLM-3转基因小鼠的原代腹腔巨噬细胞，发现CpG-OND刺激后，来自CLM-3转基因小鼠的腹腔巨噬细胞中的TRAF6的泛素化水平高于同窝对照小鼠来源的。
在前面的研究中，我们证实了CLM-3定位于内体/溶酶体。所以我们接着探索CLM-3是否能够直接和TLR9、MyD88或TRAF6相互作用。在进行免疫共沉淀实验中，我们发现来自于CLM-3转基因小鼠的原代腹腔巨噬细胞中的CLM-3能够和TLR9发生微弱的相互结合，但是和MyD88或TRAF6则没有相互结合。进一步的实验表明，在CpG-ODN刺激后CLM-3和TLR9之间的结合增强。这些结果提示我们在CpG-ODN刺激的作用下，CLM-3能够和TLR9的结合增强，进而通过促进TRAF6的泛素化来增强TRAF6的活性，最终强化了下游的信号通路，使更多的炎症因子得到表达。
通过以上三部分的研究，增进了我们对于天然免疫应答过程的了解，通过研究我们发现在人外周血单核细胞来源的树突状细胞中DNMT1能够与eIF3A、eIF3H以及CARP1相互结合，至于其潜在的作用还有待于进一步的研究。在小鼠腹腔巨噬细胞中观察到的DNMT1能够促进VSV和SeV引起的IFN-beta产生，使得我们明白DNMT1除了发挥其经典的DNA甲基化转移酶作用外，还有可能参与了天然免疫应答。通过研究CLM-3在小鼠巨噬细胞中对于Toll样受体的功能，发现了CLM-3通过促进TRAF6的泛素化增强巨噬细胞中CpG-ODN引起的TLR9免疫应答及机制，以及CLM-3能够在CpG-ODN的巨噬细胞中和TLR9的结合增强。同时发现CLM-3能够增强MAPKs的磷酸化以及NF-κB的活化。上述研究拓宽了我们对于DNMT1和CLM-3分子的性质与功能的认识，丰富了TLR免疫识别及其调控机制的研究内容，也有望为感染免疫和肿瘤的研究与治疗方法的寻找提供新的启示。
DNA methyltransferases are highly conserved in prokaryotic and eukaryoticenzymes, they modified cytosine C5locus after the genome DNA replication, whichexert a variety of important physiological processes in the body, including: regulation ofgene expression, gene imprinting, maintain chromosomal integrity, and X-chromosomeinactivation. According to their different structure and function, mammalian DNAmethyltransferase (Dnmts) are divided into two categories: DNA methylationmaintenance enzyme Dnmt1and de novo DNA methyltransferase Dnmt3a,, Dnmt3b andDnmt3L.
Dendritic cells (dendritic cells, DC) refers to a class of cells that process dendriticand stellate pleomorphic, high expression of MHC I and class I molecules, which caneffectively uptake, processing and handling of antigen to activate naive T cells. DC isfirst discovered by Steinman and Cohn in1973. As the most powerful professionalantigen-presenting cells (antigen presenting cell, APC), DC's biggest feature is the abilityto stimulate naive T cells (naive T cell) activation and proliferation. DC is the trigger ofantigen-specific immune response, due to its unique position, the studies on DC providedeep insight into the generation and regulation mechanisms of immune response, cancer,transplant rejection, infection, autoimmune occurrence and development of diseases.which have important theoretical significance.
Immunoglobulin superfamily (IgSF) consists of a large number of both membraneand intracellular receptors, which involve nearly every aspect of immune response bothin innate and adaptive immunity. The murine CMRF-35like molecule (CLM) family is apaired immunoreceptor family that belongs to the IgSF. Members of CLM familycontribute to both positive and negative regulation of immune responses uponrecognition of various ligands. We and others have identified several CLM receptors,such as DC-derived immunoglobulin receptor (DIgR)2/CLM-1/LMIR3/MAIR-V andDIgR1/CLM-4/LMIR2/MAIRII, which are preferentially expressed onantigen-presenting cells such as DCs and macrophages. We show that CLM-1, aninhibitory receptor, containing two ITIM motifs in the cytoplasmic region that associatedwith Src homology2-containting phosphatase-1(SHP-1), can negatively regulatesignaling of DC-initiated antigen-specific T-cell responses. Deficiency of CLM-1results in significantly enhanced nitric oxide and pro-inflammatory cytokine production inbone-marrow derived CD11c+cells, along with increased demyelination and worsenedclinical scores. To date, the roles of CLM members in the regulation of TLR signalingremain unclear.
Antigen-presenting cells can detect the invading pathogens by recognizingpathogen-associated molecular patterns (PAMPs) through pattern recognition receptors(PRRs), and then trigger innate immune responses and prime antigen-specific adaptiveimmunity. Activation of innate immune cells can induce production of pro-inflammatorycytokines and type I interferons (IFNs), ultimately eliminating the invading pathogenicmicroorganisms. As one of most important kinds of PRRs, Toll-like receptors (TLRs) arehighly expressed in antigen-presenting cells including macrophages/monocytes, dendriticcells (DCs). According to the subcellular location of all TLR family members, TLRscould be classified into two categories: cell membrane-localized TLRs and intracellularTLRs. Among all the TLRs, the endosome-localized TLR9is the only one thatrecognizes pathogen-derived DNA. Once binding of bacterial CpG DNA or syntheticoligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-ODN), TLR9can directly activate antigen-presenting cells and innate immune cells to secret variouspro-inflammatory cytokines. Ligation of CpG-ODN to TLR9results in recruitment of theadaptor protein MyD88and the subsequent activation of downstream signaling pathwayswith the phosphorylation of MAPKs and NF-κB, ultimately leading to production ofpro-inflammatory cytokines and type I IFNs. It is believed that TLR9agonists couldinduce strong Th1immune response to show protective activity against bacterial andviral infection. However, how to selectively activate TLR9signaling so as to initiateeffective immune response and the underlying mechanisms for full activation ofTLR9-triggered innate response remain to be fully understood.
Based on the recent research, we use modern experimental techniques to study therole of DNMT1in human monocyte-derived dendritic cells and mouse peritonealmacrophages during the immune response; and the function of CLM-3in the Toll-likereceptor signaling transduction during pathgen invading.
Part I Identification of proteins that interacting with DNMT1inhuman monocyte-derived dendritic cells
With the progress of studies on the role of mouse bone marrow-derived dendritic cells in the immune response, we began to focus on the function of humanmonocyte-derived dendritic cell. As a critical DNA methyltransferase, the functions ofDNMT1continue to be found, so we began to explore the role of DNMT1in humanmonocyte-derived dendritic cells.
We use protein co-immunoprecipitation techniques, mass spectrometry compatiblesilver staining and LS-MS/MS, and detected a few proteins that interacting with DNMT1in human monocyte-derived dendritic cells, which are mainly concentrated on thetranslation initiation factor eIF family, including eIF3A, eIF3H; and cell cycle andapoptosis regulatory protein1(cell cycle and apoptosis regulator protein1, CARP1).
As is reported, DNMT1mainly exerts the role of DNA methylation modification inthe nucleus, and eIF3A and eIF3H mainly iniate protein translation in the cytoplasm.These prompted us to explore whether DNMT1could play its non-classical features inthe cytoplasm and participated in several other biological activities. By usingco-immunoprecipitation, we found that DNMT1do exist in the cytoplasm, and interactedwith eIF3A and eIF3H, but we couldn't detect any interaction between DNMT1andeIF3A or eIF3H in the nucleus. While the specifical role of DNMT1in the cytoplasmneeds further investigation.
We also found that CARP1(cell cycle and apoptosis regulatory protein1) in the listthat maybe interact with DNMT1, after co-IP detection, we found that DNMT1couldmainly interact with CARP1in the nucleus, suggesting that CARP1might be involved inDNMT1classic features. CARP1could be a new molecule that participates in DNAmethylation, but its specific function needs further study.
Part II The role of DNMT1in the innate response of macrophages
TLRs (Toll like receptors) are the important pattern recognition receptors, whichparticipate in the activation of innate immune response and regulation of adaptiveimmune responses against various pathogens. Due to insufficient activation of immuneresponses, the body couldn't effectively remove invading pathogens, while duringexcessive immune activation would cause a series of injury, such as septic shock.Therefore, TLRs signaling pathway must be strictly regulated, and the cross-talk betweenTLR signaling pathway and other signaling pathways is one of the hotspots in recent years.
Whether DNA methyltransferase DNMT1participate in the TLR signalingpathways is still unknown yet. After silencing the DNMT1in murine peritonealmacrophages, we stimulated the macrophages with a variety of TLR ligands, and thenQ-PCR examize the mRNA level of cytokines. Finally we found that DNMT1is unableto affect TLR3, TLR4and TLR9activation, with no obivious change of IFN-beta andTNF-alpha production.
As previous reported, macrophages also plays an important role in the process ofanti-virus immune responses. We found that DNMT1interference can affect VSV andSeV induced IFN-beta production, DNMT1can promote the production of IFN-betaduring virus infection, suggesting that DNMT1might be involved in RIG-I signalingpathway, but its specific mechanism needs further research.
Part III The molecular mechanism of CLM-3to regulate TLR9signaling in macrophages
CLM-3, a member of CLM family, is closely related to CLM-5, an activatingreceptor that transmits an activating signal by interacting with FcR among adaptormolecules containing ITAM or the related activating motif-bearing molecules. LikeCLM-5, CLM-3could associate with FcR; while the role of CLM-3in innate immunityhas not yet been identified. In the previous study, we demonstrated that CLM-3, which ispreferentially expressed in macrophages, is an endosome/lysosome locatedimmunoreceptor. CLM-3expression was downregulated in macrophages following thetreatment with TLR9ligand CpG-ODN. CLM-3can function as a positive regulator ofTLR9signaling in macrophages. In this study we showed that CLM-3can promoteTLR9signaling by augmenting the ubiquitination of TRAF6and subsequently enhancingdownstream MAPKs and NF-κB signaling, thus leading to the full activation ofTLR9-triggered innate immune response. Therefore, the findings suggest that crosstalkwith CLM-3immunoreceptor can promote TLR9-triggered innate immune responses,which may benefit host to eliminate the invading pathogens more potently.
We observed the subcellular localization of CLM-3in Raw264.7cells, and foundthat CLM-3co-localized with lysosome-associated membrane protein1(LAMP1)-positive compartment and early endosome marker Rab5. No obvious co-localization with GM130, the representative marker of Gogi, was detected. Therefore,unlike other cell membrane-located CLMs, CLM-3was located in membrane-containingintracellular organelles endosome and lysosome.
The subcellular localization of CLM-3in endosome and lysosome of macrophagesinspired us to investigate the roles of CLM-3in TLR-triggered activation ofmacrophages. We stimulated macrophages with TLR4,3,9ligands and found thatCLM-3expression was apparently down-regulated by CpG-ODN treatment. However,CLM-3expression remained almost unchanged following Poly I:C or LPS stimulation,indicating that CLM-3might be potentially involved in CpG-ODN/TLR9response.Furthermore, given to the observation that CLM-3resides in the endosome, weinvestigated whether overexpression of CLM-3could render the inhibitory effect ofchloroquine on the activation of TLR9pathway by CpG-ODN, but no reverse effect wasobserved in primary peritoneal macrophages from CLM-3-TG mice. Consistent with thatCLM-3expression was only regulated in response to CpG-ODN stimulation, these datasuggest that CLM-3may preferentially promote pro-inflammatory cytokine production inmacrophages triggered by TLR9but not TLR4or TLR3.
The CLM-3transgenic mice (CLM-3-TG) were generated and used to observe therole of CLM-3in TLR9-triggered innate response. We identified that primary peritonealmacrophages derived from CLM-3-TG mice highly expressed HA tag-coupled CLM-3.In consistent with the in vitro observation that CLM-3overexpression could promoteCpG-ODN-induced TNF-α and IL-6expression, we found that the primary peritonealmacrophages derived from the CLM-3-TG mice could express higher level of TNF-α andIL-6, at both mRNA and protein level, than that from the littermate control mice inresponse to CpG-ODN stimulation. Coincidently, we observed more production ofTNF-α and IL-6in serum of CLM-3-TG mice after peritoneal injection ofCpG-ODN.These results further identified CLM-3as a positive regulator ofTLR9-triggered innate response.
MAPKs and NF-κB signaling are key pathways for the CpG-ODN-initiatedcytokine production. Our previous studies showed that CLM-3enhancedCpG-ODN-induced activation of ERK1/2, JNK1/2and p38, and phosphorylation ofIKKα/β and IκBα in macrophages. In consistent with the observation, in this study we found that the enhanced CpG-ODN-induced activation of ERK1/2, JNK1/2and p38, andphosphorylation of IKKα/β and IκBα were observed in macropahges derived fromCLM-3-TG mice when stimulated with CpG-ODN. Consistently, more phosphorylatedp65was detected in the nucleus of primary peritoneal macrophages from CLM-3-TGmice after stimulated with CpG-ODN in vitro. Furthermore, NF-κB luciferase reporterassay showed that CLM-3overexpression could promote MyD88-mediated NF-κBactivity in HEK293cells. These results suggested that CLM-3promoted theCpG-ODN-initiated TLR9signaling pathway.
After the engagement of TLR9by CpG-ODN, MyD88recruits the IL-1receptor-associated kinases IRAK1, IRAK2and IRAK4. Then IRAK activation recruitsTRAF6for downstream activation of the MAPKs and NF-κB. TRAF6is a key player inthe TLR9signaling pathway and ubiquitination of TRAF6is shown to be necessary foractivation of downstream signaling. So we wonder whether overexpression of CLM-3may enhance the ubiquitination of TRAF6. After silencing of CLM-3in primaryperitoneal macrophages, we detected the ubiquitination level of TRAF6in macrophagesonce stimulated with CpG-ODN, and found that silencing of CLM3reduced TRAF6ubiquitination. Coincidently, higher ubiquitination level of TRAF6in primary peritonealmacrophages from CLM-3-TG mice was observed once stimulated with CpG-ODN invitro. Then the subcellular location of CLM-3encouraged us to investigate whetherCLM-3could directly interact with TLR9, MyD88or TRAF6. We found CLM-3couldweakly interact with TLR9in primary peritoneal macrophages from CLM-3transgenicmice, but CLM-3could not interact with MyD88or TRAF6. Furthermore, the interactionof CLM-3and TLR9could be enhanced in macrophages after stimulation withCpG-ODN. These results suggested that CLM-3could promote the activation of TRAF6by promoting its ubiquitination through interaction with TLR9, ultimately enhancing thedownstream signaling pathways in response to CpG-ODN ligation.
In summary, three parts of the above studies will be helpful to better understand themolecular regulation of the innate immune response. We found that DNMT1couldinteract with eIF3A, eIF3H, and CARP1, in human peripheral blood monocyte-deriveddendritic cells, although its potential role needs further study. In murine peritonealmacrophages, we observed that DNMT1could promote VSV and SeV induced IFN-betaproduction, indicating DNMT1may promote RIG-I-triggered innate response, so it's thefirst time that DNMT1may be involved in the innate immune response, addition to its classic role as a DNA methyltransferase enzyme. By studying the role of CLM-3onTLR9signaling in macrophages, we found CLM-3could promote the ubiquitination ofTRAF6then enhance TLR9CpG-ODN induced immune responses. and upon stimulationof CpG-ODN, the binding between CLM-3and TLR9could be enhanced, thus providingthe mechanistic explanation for our previous observation that CLM-3could enhance thephosphorylation of MAPKs and NF-κB after CpG-ODN activation. The study onDNMT1and CLM-3broadens our view of innate immune recognition, which is alsoexpected to be applied in seeking treatment methods on infection immunity and cancer.
树突状细胞(dendritic cells，DC)是指具有树枝状突起和星状多形性，高表达MHCI类和I类分子，能有效摄取、加工和处理抗原并激活初始型T细胞的一类抗原提呈细胞(antigen presenting cell，APC)。DC是1973年由Steinman和Cohn首先发现的。作为目前发现的功能最强的APC，DC最大的特点是能够刺激初始型T细胞(naive Tcell)活化和增殖。DC是抗原特异性免疫应答的始动者，由于其在免疫应答中的独特地位，对DC的研究有助于深入了解机体免疫应答的产生和调控机制，对肿瘤、移植排斥、感染、自身免疫性疾病等的发生发展机制的认识有重要的理论意义。
免疫球蛋白超家族含有众多的膜上和膜内受体,参与天然免疫和获得性免疫中的各种免疫应答反应.小鼠CMRF-35样分子家族为免疫球蛋白超家族的成员之一.CLM家族成员在识别不同的配体后能够正向或负向调控免疫应答反应。本实验室通过小鼠树突状细胞cDNA文库大规模测序后，发现了一系列选择性地在树突状细胞和巨噬细胞等抗原提呈细胞上表达的CLM家族成员受体，包括树突状细胞来源的免疫球蛋白受体1(DC-derived Ig receptor1, DIgR1)/CLM-4,树突状细胞来源的免疫球蛋白受体2(DC-derived Ig receptor2, DIgR2)/CLM-1, CLM-5，以及本文中研究的CLM-3。
抗原递呈细胞能够通过模式识别受体(pattern recognition receptors, PRR)识别病原体相关分子模式(pathogen-associated molecular patterns, PAMP)来检测入侵宿主的病原微生物，然后促发免疫球蛋白超家族含有众多的膜上和膜内受体,参与天然免疫和获得性免疫中的各种免疫应答反应。活化的天然免疫细胞能够诱导促炎因子和I型干扰素的产生来清除入侵的病原微生物。Toll样受体(Toll-like receptors, TLRs)作为模式识别受体中最重要的一种，显著高表达于抗原递呈细胞，包括巨噬细胞，单核细胞以及树突状细胞。根据所有TLRs家族成员的亚细胞定位，TLRs分成两大类：细胞膜定位的TLRs和细胞内定位的TLRs。在所有TLRs中，内体定位的TLR9是唯一一种能够识别病原体来源DNA的TLR。一旦结合细菌中含有CpG岛的DNA或者人工合成的含有为甲基化CpG基序的寡聚脱氧核苷酸(CpG-ODN)，TLR9就能够直接活化抗原递呈细胞和天然免疫细胞分泌各种促炎因子。当CpG-ODN结合到TLR9时能够招募接头分子MyD88，进而激活下游MAPKs和NF-κB的磷酸化信号通路，最终引起促炎因子和I型干扰素的产生。研究证实TLR9的激动剂能够诱导强烈的Th1型免疫反应来保护宿主免受细菌和病毒的感染。因此，对于如何选择性的活化TLR9信号来起始保护性免疫应答及TLR9完全活化的潜在机制仍需进一步研究。
基于以上研究进展和现状分析，我们利用现代实验技术对人单核细胞来源的树突状细胞和小鼠腹腔巨噬细胞中的DNMT1在天然免疫应答方面的功能进行了研究；同时对于CLM-3对于Toll样受体的功能影响进行了深入的探讨。
第一部分人单核细胞来源的树突状细胞中与DNMT1相互作用蛋白的探讨
随着小鼠骨髓来源树突状细胞在免疫应答中功能研究的深入，我们开始关注人单核细胞来源的树突状细胞的功能。作为重要的DNA甲基转移酶DNMT1功能的不断发现，使得我们开始探寻DNMT1在人单核细胞来源树突状细胞中的作用。
我们使用蛋白质免疫共沉淀技术、质谱兼容银染以及LS-MS/MS的手段，发现了一系列在人单核细胞来源的树突状细胞中与DNMT1相互作用的蛋白，这些蛋白主要集中在翻译起始因子eIF家族，主要包括eIF3A、eIF3H；以及细胞周期和凋亡调节蛋白1（cell cycle and apoptosis regulator protein1, CARP1)。
鉴于目前的研究报道，DNMT1主要是在核内发挥其对DNA的甲基化修饰功能，而eIF3A和eIF3H则主要是在胞浆中发挥蛋白的翻译。这就促使我们去探寻DNMT1是否在胞浆中也发挥其非经典功能，而参与了其他一些生物学功能。通过免疫共沉淀，我们发现了DNMT1确实存在于胞浆中，而且能够与同在胞浆中发挥作用的eIF3A和eIF3H相互结合，而在胞核中则为发现eIF3A和eIF3H与DNMT1的结合。对于DNMT1在胞浆中具体发挥怎么样的功能还有待于进一步的深入探讨。
对于同期发现的细胞周期和凋亡调节蛋白CARP1，研究发现DNMT1与CARP1两者之间主要在细胞核内相互作用，这提示我们CARP1可能参与了DNMT1的经典功能，作为一个新的可能参与DNA甲基化的蛋白，其具体功能有待于进一步深入的研究。
第二部分DNMT1在巨噬细胞天然免疫功能中的作用研究
TLR是研究最早的模式识别受体，其在激活机体的固有免疫应答和调控获得性免疫应答，抵御各种病原体感染中发挥重要的作用。它的活化不足导致机体不能有效地清除病原体，而过度的激活又会引发一系列的免疫损伤如脓毒症休克等。因此TLR信号通路必须受到严格的精密调控，TLR信号通路同其他信号通路之间的交叉联系也是近年来免疫学的研究热点之一。
对于DNA甲基转移酶DNMT1在TLR信号通路中的作用还没有相关的研究报道，我们通过干扰小鼠腹腔巨噬细胞中的DNMT1后，用各种TLR配体进行刺激，进而研究DNMT1是否参与TLR信号通路。研究发现，在DNMT1得到有效干扰后并不能够显著影响TLR3、TLR4以及TLR9活化产生的IFN-beta和TNF-alpha。
基于巨噬细胞在抗病毒的过程中也发挥着重要作用，我们观察了干扰DNMT1对于VSV和SeV感染的影响。研究发现DNMT1的干扰能够影响VSV和SeV引起的IFN-beta的产生，DNMT1能够促进IFN-beta的产生，这提示我们DNMT1可能参与RIG-I的信号通路，对于其具体机制，有待于进一步的研究和探索。
第三部分CLM-3调控TLR9触发的巨噬细胞天然免疫功能及其机制研究
CLM-3(CMRF-35like molecule3)作为CLM家族成员之一，与CLM-5关系密切，同属于活化性受体。研究表明CLM-5能够通过与含有免疫受体酪氨酸活化基序(immunoreceptor tyrosine based-activating motif， ITAM)的FcRγ相互作用，进而转导活化信号。CLM-3同样也能够与FcRγ相互作用，但是其在天然免疫应答中的功能还未得到确认。
本实验室早期的研究发现CLM-3选择性地表达于巨噬细胞中，CLM-3能够通过与MyD88结合以激活MAPK、NFkB通路从而促进了TLR9触发的巨噬细胞产生炎性细胞因子TNFa和IL-6，研究提示，CLM-3与带有ITAM基序的FcRγ相互作用,但是，CLM-3调控TLR9信号通路的具体分子机制尚不清楚。
通过研究证实，CLM-3选择性地表达于巨噬细胞中，为内体/溶酶体定位的免疫受体；在TLR9配体CpG-ODN刺激下，CLM-3在巨噬细胞中的表达被下调；CLM-3作为一个能够在巨噬细胞中正向调控TLR9信号转导的受体，主要通过增强TRAF6(TNFR-associated factor6)的泛素化，进而促进下游MAPKs和NF-κB的信号转导，最终使得TLR9引发的天然免疫应答反应全面活化。研究主要发现通过与CLM-3的交互作用能够促进TLR9引发的天然免疫反应，最终有利于宿主清除外来入侵病原体。
我们通过激光共聚焦检测发现在Raw264.7细胞系中CLM-3能够与LAMP1阳性的细胞器和早期内体标记Rab5共定位，而对于高尔基体的代表性标记GM130则没有明显共定位。因此，CLM-3不像其他细胞膜定位的CLM家族成员，而是选择性的定位于膜类细胞器内体及溶酶体。从CLM-3在巨噬细胞中定位内体及溶酶体，我们推测CLM-3可能在TLR9触发的巨噬细胞活化过程中发挥作用。我们用CpG-ODN刺激siRNA干扰过的原代腹腔巨噬细胞，发现CLM-3被干扰后降低TLR9活化引起的TNF-α和IL-6表达，但是不影响Poly(I:C)或LPS引起的TNF-α和IL-6的表达。因此，我们证实CLM-3能够选择性地促进TLR9，而非TLR3或TLR4，引起的促炎因子的产生。
为验证CLM-3过表达在体内的作用,我们制备了CLM-3转基因小鼠(CLM-3transgenic mice, CLM-3TG mice)观察CLM-3在TLR9触发的天然免疫应答。Westernblot证实在原代腹腔巨噬细胞中高表达带HA标签的CLM-3。取自CLM-3TG mice的巨噬细胞相较于同窝未表达HA-CLM-3的小鼠的巨噬细胞，在CpG-ODN刺激后能够产生更多的TNF-α和IL-6，实验结果和在巨噬细胞中过表达CLM-3的效应相一致。在小鼠腹腔注射CpG-ODN后，观察血清中TNF-α和IL-6的表达水平也得出相似的结论，CLM-3TG mice较同窝对照小鼠产生更多的炎症因子。鉴于在浆样树突状细胞(plasmacytoid dendritic cell, pDC)中TLR9在I型干扰素的产生过程中发挥着重要作用，我们观察了从转基因小鼠脾脏来源的pDC在GpG-ODN刺激后诱导产生I型干扰素中的效应。研究发现CLM-3转基因小鼠和同窝对照小鼠来源的pDC在干扰素的产生上并无显著性差异。研究证实CLM-3作为一个能够正向调控TLR9触发的天然免疫应答主要在巨噬细胞中发挥作用。
已有研究报道证实，MAPKs和NF-κB是CpG-OND触发细胞因子产生的关键信号通路。在CLM-3转基因小鼠来源的巨噬细胞中，我们观察到CLM-3的过表达能够促进ERK1/2，JNK1/2，以及p38的磷酸化水平，相同的作用趋势发现在CLM-3过表达的Raw264.7细胞系中。在体外检测CLM-3转基因小鼠来源的腹腔巨噬细胞发现CpG-ODN刺激活化IKKα/β和IκBα的磷酸化水平明显强于同窝对照小鼠。上述实验结果证实，CLM-3能够促进CpG-ODN触发的TLR9信号通路活化。
通过前面的实验我们已经证实了CLM-3对于TLR9的正向调控作用，对于潜在的机制研究将使我们对于CLM-3的了解更为透彻。报道证实，CpG-ODN在结合到TLR9后，下游信号分子MyD88能够招募IRAK1(IL-1receptor associated kinase1)、IRAK2以及IRAK4。这些IRAK激酶的活化能够招募TRAF6进行泛素化，进而激活下游的MPAKs和NF-κB信号。TRAF6作为TLR9信号中起关键作用的分子，其泛素化是激活下游信号活化所必需的。因此，我们想证实一下CLM-3的过表达是否会增强TRAF6的泛素化。在原代腹腔巨噬细胞中用siRNA干扰CLM-3后，我们检测了CpG-ODN刺激后TRAF6的泛素化水平，发现CLM-3的沉默能够降低TRAF6的泛素化水平。同理，我们检测了来自于CLM-3转基因小鼠的原代腹腔巨噬细胞，发现CpG-OND刺激后，来自CLM-3转基因小鼠的腹腔巨噬细胞中的TRAF6的泛素化水平高于同窝对照小鼠来源的。
在前面的研究中，我们证实了CLM-3定位于内体/溶酶体。所以我们接着探索CLM-3是否能够直接和TLR9、MyD88或TRAF6相互作用。在进行免疫共沉淀实验中，我们发现来自于CLM-3转基因小鼠的原代腹腔巨噬细胞中的CLM-3能够和TLR9发生微弱的相互结合，但是和MyD88或TRAF6则没有相互结合。进一步的实验表明，在CpG-ODN刺激后CLM-3和TLR9之间的结合增强。这些结果提示我们在CpG-ODN刺激的作用下，CLM-3能够和TLR9的结合增强，进而通过促进TRAF6的泛素化来增强TRAF6的活性，最终强化了下游的信号通路，使更多的炎症因子得到表达。
通过以上三部分的研究，增进了我们对于天然免疫应答过程的了解，通过研究我们发现在人外周血单核细胞来源的树突状细胞中DNMT1能够与eIF3A、eIF3H以及CARP1相互结合，至于其潜在的作用还有待于进一步的研究。在小鼠腹腔巨噬细胞中观察到的DNMT1能够促进VSV和SeV引起的IFN-beta产生，使得我们明白DNMT1除了发挥其经典的DNA甲基化转移酶作用外，还有可能参与了天然免疫应答。通过研究CLM-3在小鼠巨噬细胞中对于Toll样受体的功能，发现了CLM-3通过促进TRAF6的泛素化增强巨噬细胞中CpG-ODN引起的TLR9免疫应答及机制，以及CLM-3能够在CpG-ODN的巨噬细胞中和TLR9的结合增强。同时发现CLM-3能够增强MAPKs的磷酸化以及NF-κB的活化。上述研究拓宽了我们对于DNMT1和CLM-3分子的性质与功能的认识，丰富了TLR免疫识别及其调控机制的研究内容，也有望为感染免疫和肿瘤的研究与治疗方法的寻找提供新的启示。
DNA methyltransferases are highly conserved in prokaryotic and eukaryoticenzymes, they modified cytosine C5locus after the genome DNA replication, whichexert a variety of important physiological processes in the body, including: regulation ofgene expression, gene imprinting, maintain chromosomal integrity, and X-chromosomeinactivation. According to their different structure and function, mammalian DNAmethyltransferase (Dnmts) are divided into two categories: DNA methylationmaintenance enzyme Dnmt1and de novo DNA methyltransferase Dnmt3a,, Dnmt3b andDnmt3L.
Dendritic cells (dendritic cells, DC) refers to a class of cells that process dendriticand stellate pleomorphic, high expression of MHC I and class I molecules, which caneffectively uptake, processing and handling of antigen to activate naive T cells. DC isfirst discovered by Steinman and Cohn in1973. As the most powerful professionalantigen-presenting cells (antigen presenting cell, APC), DC's biggest feature is the abilityto stimulate naive T cells (naive T cell) activation and proliferation. DC is the trigger ofantigen-specific immune response, due to its unique position, the studies on DC providedeep insight into the generation and regulation mechanisms of immune response, cancer,transplant rejection, infection, autoimmune occurrence and development of diseases.which have important theoretical significance.
Immunoglobulin superfamily (IgSF) consists of a large number of both membraneand intracellular receptors, which involve nearly every aspect of immune response bothin innate and adaptive immunity. The murine CMRF-35like molecule (CLM) family is apaired immunoreceptor family that belongs to the IgSF. Members of CLM familycontribute to both positive and negative regulation of immune responses uponrecognition of various ligands. We and others have identified several CLM receptors,such as DC-derived immunoglobulin receptor (DIgR)2/CLM-1/LMIR3/MAIR-V andDIgR1/CLM-4/LMIR2/MAIRII, which are preferentially expressed onantigen-presenting cells such as DCs and macrophages. We show that CLM-1, aninhibitory receptor, containing two ITIM motifs in the cytoplasmic region that associatedwith Src homology2-containting phosphatase-1(SHP-1), can negatively regulatesignaling of DC-initiated antigen-specific T-cell responses. Deficiency of CLM-1results in significantly enhanced nitric oxide and pro-inflammatory cytokine production inbone-marrow derived CD11c+cells, along with increased demyelination and worsenedclinical scores. To date, the roles of CLM members in the regulation of TLR signalingremain unclear.
Antigen-presenting cells can detect the invading pathogens by recognizingpathogen-associated molecular patterns (PAMPs) through pattern recognition receptors(PRRs), and then trigger innate immune responses and prime antigen-specific adaptiveimmunity. Activation of innate immune cells can induce production of pro-inflammatorycytokines and type I interferons (IFNs), ultimately eliminating the invading pathogenicmicroorganisms. As one of most important kinds of PRRs, Toll-like receptors (TLRs) arehighly expressed in antigen-presenting cells including macrophages/monocytes, dendriticcells (DCs). According to the subcellular location of all TLR family members, TLRscould be classified into two categories: cell membrane-localized TLRs and intracellularTLRs. Among all the TLRs, the endosome-localized TLR9is the only one thatrecognizes pathogen-derived DNA. Once binding of bacterial CpG DNA or syntheticoligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-ODN), TLR9can directly activate antigen-presenting cells and innate immune cells to secret variouspro-inflammatory cytokines. Ligation of CpG-ODN to TLR9results in recruitment of theadaptor protein MyD88and the subsequent activation of downstream signaling pathwayswith the phosphorylation of MAPKs and NF-κB, ultimately leading to production ofpro-inflammatory cytokines and type I IFNs. It is believed that TLR9agonists couldinduce strong Th1immune response to show protective activity against bacterial andviral infection. However, how to selectively activate TLR9signaling so as to initiateeffective immune response and the underlying mechanisms for full activation ofTLR9-triggered innate response remain to be fully understood.
Based on the recent research, we use modern experimental techniques to study therole of DNMT1in human monocyte-derived dendritic cells and mouse peritonealmacrophages during the immune response; and the function of CLM-3in the Toll-likereceptor signaling transduction during pathgen invading.
Part I Identification of proteins that interacting with DNMT1inhuman monocyte-derived dendritic cells
With the progress of studies on the role of mouse bone marrow-derived dendritic cells in the immune response, we began to focus on the function of humanmonocyte-derived dendritic cell. As a critical DNA methyltransferase, the functions ofDNMT1continue to be found, so we began to explore the role of DNMT1in humanmonocyte-derived dendritic cells.
We use protein co-immunoprecipitation techniques, mass spectrometry compatiblesilver staining and LS-MS/MS, and detected a few proteins that interacting with DNMT1in human monocyte-derived dendritic cells, which are mainly concentrated on thetranslation initiation factor eIF family, including eIF3A, eIF3H; and cell cycle andapoptosis regulatory protein1(cell cycle and apoptosis regulator protein1, CARP1).
As is reported, DNMT1mainly exerts the role of DNA methylation modification inthe nucleus, and eIF3A and eIF3H mainly iniate protein translation in the cytoplasm.These prompted us to explore whether DNMT1could play its non-classical features inthe cytoplasm and participated in several other biological activities. By usingco-immunoprecipitation, we found that DNMT1do exist in the cytoplasm, and interactedwith eIF3A and eIF3H, but we couldn't detect any interaction between DNMT1andeIF3A or eIF3H in the nucleus. While the specifical role of DNMT1in the cytoplasmneeds further investigation.
We also found that CARP1(cell cycle and apoptosis regulatory protein1) in the listthat maybe interact with DNMT1, after co-IP detection, we found that DNMT1couldmainly interact with CARP1in the nucleus, suggesting that CARP1might be involved inDNMT1classic features. CARP1could be a new molecule that participates in DNAmethylation, but its specific function needs further study.
Part II The role of DNMT1in the innate response of macrophages
TLRs (Toll like receptors) are the important pattern recognition receptors, whichparticipate in the activation of innate immune response and regulation of adaptiveimmune responses against various pathogens. Due to insufficient activation of immuneresponses, the body couldn't effectively remove invading pathogens, while duringexcessive immune activation would cause a series of injury, such as septic shock.Therefore, TLRs signaling pathway must be strictly regulated, and the cross-talk betweenTLR signaling pathway and other signaling pathways is one of the hotspots in recent years.
Whether DNA methyltransferase DNMT1participate in the TLR signalingpathways is still unknown yet. After silencing the DNMT1in murine peritonealmacrophages, we stimulated the macrophages with a variety of TLR ligands, and thenQ-PCR examize the mRNA level of cytokines. Finally we found that DNMT1is unableto affect TLR3, TLR4and TLR9activation, with no obivious change of IFN-beta andTNF-alpha production.
As previous reported, macrophages also plays an important role in the process ofanti-virus immune responses. We found that DNMT1interference can affect VSV andSeV induced IFN-beta production, DNMT1can promote the production of IFN-betaduring virus infection, suggesting that DNMT1might be involved in RIG-I signalingpathway, but its specific mechanism needs further research.
Part III The molecular mechanism of CLM-3to regulate TLR9signaling in macrophages
CLM-3, a member of CLM family, is closely related to CLM-5, an activatingreceptor that transmits an activating signal by interacting with FcR among adaptormolecules containing ITAM or the related activating motif-bearing molecules. LikeCLM-5, CLM-3could associate with FcR; while the role of CLM-3in innate immunityhas not yet been identified. In the previous study, we demonstrated that CLM-3, which ispreferentially expressed in macrophages, is an endosome/lysosome locatedimmunoreceptor. CLM-3expression was downregulated in macrophages following thetreatment with TLR9ligand CpG-ODN. CLM-3can function as a positive regulator ofTLR9signaling in macrophages. In this study we showed that CLM-3can promoteTLR9signaling by augmenting the ubiquitination of TRAF6and subsequently enhancingdownstream MAPKs and NF-κB signaling, thus leading to the full activation ofTLR9-triggered innate immune response. Therefore, the findings suggest that crosstalkwith CLM-3immunoreceptor can promote TLR9-triggered innate immune responses,which may benefit host to eliminate the invading pathogens more potently.
We observed the subcellular localization of CLM-3in Raw264.7cells, and foundthat CLM-3co-localized with lysosome-associated membrane protein1(LAMP1)-positive compartment and early endosome marker Rab5. No obvious co-localization with GM130, the representative marker of Gogi, was detected. Therefore,unlike other cell membrane-located CLMs, CLM-3was located in membrane-containingintracellular organelles endosome and lysosome.
The subcellular localization of CLM-3in endosome and lysosome of macrophagesinspired us to investigate the roles of CLM-3in TLR-triggered activation ofmacrophages. We stimulated macrophages with TLR4,3,9ligands and found thatCLM-3expression was apparently down-regulated by CpG-ODN treatment. However,CLM-3expression remained almost unchanged following Poly I:C or LPS stimulation,indicating that CLM-3might be potentially involved in CpG-ODN/TLR9response.Furthermore, given to the observation that CLM-3resides in the endosome, weinvestigated whether overexpression of CLM-3could render the inhibitory effect ofchloroquine on the activation of TLR9pathway by CpG-ODN, but no reverse effect wasobserved in primary peritoneal macrophages from CLM-3-TG mice. Consistent with thatCLM-3expression was only regulated in response to CpG-ODN stimulation, these datasuggest that CLM-3may preferentially promote pro-inflammatory cytokine production inmacrophages triggered by TLR9but not TLR4or TLR3.
The CLM-3transgenic mice (CLM-3-TG) were generated and used to observe therole of CLM-3in TLR9-triggered innate response. We identified that primary peritonealmacrophages derived from CLM-3-TG mice highly expressed HA tag-coupled CLM-3.In consistent with the in vitro observation that CLM-3overexpression could promoteCpG-ODN-induced TNF-α and IL-6expression, we found that the primary peritonealmacrophages derived from the CLM-3-TG mice could express higher level of TNF-α andIL-6, at both mRNA and protein level, than that from the littermate control mice inresponse to CpG-ODN stimulation. Coincidently, we observed more production ofTNF-α and IL-6in serum of CLM-3-TG mice after peritoneal injection ofCpG-ODN.These results further identified CLM-3as a positive regulator ofTLR9-triggered innate response.
MAPKs and NF-κB signaling are key pathways for the CpG-ODN-initiatedcytokine production. Our previous studies showed that CLM-3enhancedCpG-ODN-induced activation of ERK1/2, JNK1/2and p38, and phosphorylation ofIKKα/β and IκBα in macrophages. In consistent with the observation, in this study we found that the enhanced CpG-ODN-induced activation of ERK1/2, JNK1/2and p38, andphosphorylation of IKKα/β and IκBα were observed in macropahges derived fromCLM-3-TG mice when stimulated with CpG-ODN. Consistently, more phosphorylatedp65was detected in the nucleus of primary peritoneal macrophages from CLM-3-TGmice after stimulated with CpG-ODN in vitro. Furthermore, NF-κB luciferase reporterassay showed that CLM-3overexpression could promote MyD88-mediated NF-κBactivity in HEK293cells. These results suggested that CLM-3promoted theCpG-ODN-initiated TLR9signaling pathway.
After the engagement of TLR9by CpG-ODN, MyD88recruits the IL-1receptor-associated kinases IRAK1, IRAK2and IRAK4. Then IRAK activation recruitsTRAF6for downstream activation of the MAPKs and NF-κB. TRAF6is a key player inthe TLR9signaling pathway and ubiquitination of TRAF6is shown to be necessary foractivation of downstream signaling. So we wonder whether overexpression of CLM-3may enhance the ubiquitination of TRAF6. After silencing of CLM-3in primaryperitoneal macrophages, we detected the ubiquitination level of TRAF6in macrophagesonce stimulated with CpG-ODN, and found that silencing of CLM3reduced TRAF6ubiquitination. Coincidently, higher ubiquitination level of TRAF6in primary peritonealmacrophages from CLM-3-TG mice was observed once stimulated with CpG-ODN invitro. Then the subcellular location of CLM-3encouraged us to investigate whetherCLM-3could directly interact with TLR9, MyD88or TRAF6. We found CLM-3couldweakly interact with TLR9in primary peritoneal macrophages from CLM-3transgenicmice, but CLM-3could not interact with MyD88or TRAF6. Furthermore, the interactionof CLM-3and TLR9could be enhanced in macrophages after stimulation withCpG-ODN. These results suggested that CLM-3could promote the activation of TRAF6by promoting its ubiquitination through interaction with TLR9, ultimately enhancing thedownstream signaling pathways in response to CpG-ODN ligation.
In summary, three parts of the above studies will be helpful to better understand themolecular regulation of the innate immune response. We found that DNMT1couldinteract with eIF3A, eIF3H, and CARP1, in human peripheral blood monocyte-deriveddendritic cells, although its potential role needs further study. In murine peritonealmacrophages, we observed that DNMT1could promote VSV and SeV induced IFN-betaproduction, indicating DNMT1may promote RIG-I-triggered innate response, so it's thefirst time that DNMT1may be involved in the innate immune response, addition to its classic role as a DNA methyltransferase enzyme. By studying the role of CLM-3onTLR9signaling in macrophages, we found CLM-3could promote the ubiquitination ofTRAF6then enhance TLR9CpG-ODN induced immune responses. and upon stimulationof CpG-ODN, the binding between CLM-3and TLR9could be enhanced, thus providingthe mechanistic explanation for our previous observation that CLM-3could enhance thephosphorylation of MAPKs and NF-κB after CpG-ODN activation. The study onDNMT1and CLM-3broadens our view of innate immune recognition, which is alsoexpected to be applied in seeking treatment methods on infection immunity and cancer.
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83. Zarbock A., C. L. Abram, M. Hundt, A. Altman, C. A. Lowell, K. Ley.2008.PSGL-1engagement by E-selectin signals through Src kinase Fgr and ITAMadapters DAP12and FcR gamma to induce slow leukocyte rolling. J. Exp. Med.205:2339-2347.
[1] Kawai T, Akira S. The role of pattern-recognition receptors in innateimmunity: update on Toll-like receptors [J]. Nat Immunol.2010,11(5):373-384.
[2] Clark GJ, Ju X, Hart DN, et al. The CD300family of molecules areevolutionarily significant regulators of leukocyte functions [J]. Trends Immunol,2009,30(5):209-217.
[3] Kumagai H, Oki T, Kitamura T. et al. Identification and characterization ofa new pair of immunoglobulin-like receptors LMIR1and2derived from murinebone marrow-derived mast cells [J]. Biochem Biophys Res Commun,2003,307(3):719-729.
[4] An H, Hou J, Zhou J. et al. Phosphatase SHP-1promotes TLR-andRIG-I-activated production of type I interferon by inhibiting the kinase IRAK1[J].Nat Immunol,2008,9(5):542-550.
[5] Yang P, An H, Liu X. et al. The cytosolic nucleic acid sensor LRRFIP1mediates the production of type I interferon via a beta-catenin-dependent pathway[J]. Nat Immunol,2010,11(6):487-494.
[6] Abram CL, Lowell CA. Convergence of immunoreceptor and integrinsignaling [J]. Immunol Rev,2007,218(1):29-44.
[7] Roger T, Froidevaux C, Le Roy D. et al. Protection from lethalgram-negative bacterial sepsis by targeting Toll-like receptor4[J]. Proc Natl AcadSci,2009,106(7):2348-2352.
[8] Raby AC, Le Bouder E, Colmont C et al. Soluble TLR2reducesinflammation without compromising bacterial clearance by disrupting TLR2triggering [J]. J Immunol,2009,183(1):506-517.
[9] Coll RC, O'Neill LA.. New insights into the regulation of signalling bytoll-like receptors and nod-like receptors [J]. J Innate Immun,2010,2(5):406-21.
[10] Liu Y, Stewart KN, Bishop E. et al. Unique expression of suppressor ofcytokine signaling3is essential for classical macrophage activation in rodents invitro and in vivo [J]. J Immunol,2008,180(9):6270-6278.
[11] Hymowitz SG, Wertz IE. A20: from ubiquitin editing to tumoursuppression [J]. Nat Rev Cancer,2010,10(5):332-341..
[12] Ciarrocchi A, D'Angelo R, Cordiglieri C. et al. Tollip is a mediator ofprotein sumoylation [J]. PLoS One,2009,4(2): e4404.
[13] Wang C, Chen T, Zhang J. et al. The E3ubiquitin ligase Nrdp1'preferentially' promotes TLR-mediated production of type I interferon [J]. NatImmunol,2009,10(7):744-752.
[14] Chung DH, Humphrey MB, Daws MR. et al. CMRF-35-like molecule-1, anovel mouse myeloid receptor, can inhibit osteoclast formation [J]. J Immunol,2003,171(12):6541-6548.
[15] Sui L, Li N, Liu Q. et al. IgSF13, a novel human inhibitory receptor of theimmunoglobulin superfamily, is preferentially expressed in dendritic cells andmonocytes [J]. Biochem Biophys Res Commun,2004,319(3):920-928.
[16] Shi L, Luo K, Xia D. et al. DIgR2, dendritic cell-derived immunoglobulinreceptor2, is one representative of a family of IgSF inhibitory receptors andmediates negative regulation of dendritic cell-initiated antigen-specific T-cellresponses [J]. Blood,2006,108(8):2678-2686.
[17] Izawa K, Kitaura J, Kitamura T. et al. Functional analysis of activatingreceptor LMIR4as a counterpart of inhibitory receptor LMIR3[J]. J Biol Chem,2007,282(25):17997-18008.
[18] Izawa K, Kitaura J, Kitamura T. et al. An activating and inhibitory signalfrom an inhibitory receptor LMIR3/CLMs-1: LMIR3augments lipopolysaccharideresponse through association with FcR gamma in mast cells [J]. J Immunol,2009,183(2):925-936.
[19] Yotsumoto K, Okoshi Y, Shibuya A. et al. Paired activating and inhibitoryimmunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell andmacrophage activation [J]. J Exp Med,2003,198(2):223-233.
[20] Okoshi Y, Tahara-Hanaoka S, Shibuya A. et al. Requirement of thetyrosines at residues258and270of MAIR-I in inhibitory effect on degranulationfrom basophilic leukemia RBL-2H3[J]. Int Immunol,2005,17(1):65-72.
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[23] Nakahashi C, Tahara-Hanaoka S, Shibuya A. et al. Dual assemblies of anactivating immune receptor, MAIR-II, with ITAM-bearing adapters DAP12and FcRgamma chain on peritoneal macrophages [J]. J Immunol,2007,178(2):765-70.
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[1] Kawai T, Akira S. The role of pattern-recognition receptors in innateimmunity: update on Toll-like receptors [J]. Nat Immunol.2010,11(5):373-384.
[2] Clark GJ, Ju X, Hart DN, et al. The CD300family of molecules areevolutionarily significant regulators of leukocyte functions [J]. Trends Immunol,2009,30(5):209-217.
[3] Kumagai H, Oki T, Kitamura T. et al. Identification and characterization ofa new pair of immunoglobulin-like receptors LMIR1and2derived from murinebone marrow-derived mast cells [J]. Biochem Biophys Res Commun,2003,307(3):719-729.
[4] An H, Hou J, Zhou J. et al. Phosphatase SHP-1promotes TLR-andRIG-I-activated production of type I interferon by inhibiting the kinase IRAK1[J].Nat Immunol,2008,9(5):542-550.
[5] Yang P, An H, Liu X. et al. The cytosolic nucleic acid sensor LRRFIP1mediates the production of type I interferon via a beta-catenin-dependent pathway[J]. Nat Immunol,2010,11(6):487-494.
[6] Abram CL, Lowell CA. Convergence of immunoreceptor and integrinsignaling [J]. Immunol Rev,2007,218(1):29-44.
[7] Roger T, Froidevaux C, Le Roy D. et al. Protection from lethalgram-negative bacterial sepsis by targeting Toll-like receptor4[J]. Proc Natl AcadSci,2009,106(7):2348-2352.
[8] Raby AC, Le Bouder E, Colmont C et al. Soluble TLR2reducesinflammation without compromising bacterial clearance by disrupting TLR2triggering [J]. J Immunol,2009,183(1):506-517.
[9] Coll RC, O'Neill LA.. New insights into the regulation of signalling bytoll-like receptors and nod-like receptors [J]. J Innate Immun,2010,2(5):406-21.
[10] Liu Y, Stewart KN, Bishop E. et al. Unique expression of suppressor ofcytokine signaling3is essential for classical macrophage activation in rodents invitro and in vivo [J]. J Immunol,2008,180(9):6270-6278.
[11] Hymowitz SG, Wertz IE. A20: from ubiquitin editing to tumoursuppression [J]. Nat Rev Cancer,2010,10(5):332-341..
[12] Ciarrocchi A, D'Angelo R, Cordiglieri C. et al. Tollip is a mediator ofprotein sumoylation [J]. PLoS One,2009,4(2): e4404.
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[17] Izawa K, Kitaura J, Kitamura T. et al. Functional analysis of activatingreceptor LMIR4as a counterpart of inhibitory receptor LMIR3[J]. J Biol Chem,2007,282(25):17997-18008.
[18] Izawa K, Kitaura J, Kitamura T. et al. An activating and inhibitory signalfrom an inhibitory receptor LMIR3/CLMs-1: LMIR3augments lipopolysaccharideresponse through association with FcR gamma in mast cells [J]. J Immunol,2009,183(2):925-936.
[19] Yotsumoto K, Okoshi Y, Shibuya A. et al. Paired activating and inhibitoryimmunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell andmacrophage activation [J]. J Exp Med,2003,198(2):223-233.
[20] Okoshi Y, Tahara-Hanaoka S, Shibuya A. et al. Requirement of thetyrosines at residues258and270of MAIR-I in inhibitory effect on degranulationfrom basophilic leukemia RBL-2H3[J]. Int Immunol,2005,17(1):65-72.
[21] Ivashkiv LB. Cross-regulation of signaling by ITAM-associated receptors[J]. Nat Immunol,2009,10(4):340-347.
[22] Luo K, Zhang W, Sui L. et al. DIgR1, a novel membrane receptor of theimmunoglobulin gene superfamily, is preferentially expressed by antigen-presentingcells [J]. Biochem Biophys Res Commun,2001,287(1):35-41.
[23] Nakahashi C, Tahara-Hanaoka S, Shibuya A. et al. Dual assemblies of anactivating immune receptor, MAIR-II, with ITAM-bearing adapters DAP12and FcRgamma chain on peritoneal macrophages [J]. J Immunol,2007,178(2):765-70.
[24] Xi H, Katschke KJ Jr, van Lookeren Campagne M. et al. Negativeregulation of autoimmune demyelination by the inhibitory receptor CLMs-1[J]. JExp Med,2010,207(1):7-16.
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