【作者】 刘星光；

【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】 Toll样受体（Toll-like receptors, TLRs）作为一类重要的模式识别受体（Pattern recognition receptors, PRRs）主要表达于巨噬细胞和树突状细胞（Dendritic cells, DCs）表面,选择性地识别病原体相关分子模式（Pathogen-associated molecular patterns, PAMPs）,构成机体免疫系统抵御病原体入侵的第一道屏障。TLR是一类在各种生物体内高度保守的I型跨膜蛋白,目前已在哺乳动物中发现并克隆了12种。一旦识别了病原体中特定的分子结构,TLR就会激活其下游一系列的信号通路,活化天然免疫细胞产生炎性细胞因子和I型干扰素。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,还可以通过上调DC表面的MHC II类分子和共刺激分子,促进DC的成熟,指导抗原特异的免疫应答尤其是Th1型反应的产生,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桥梁。TLR信号过度活化或活化不足会导致机体功能异常和疾病的发生。许多的其他信号通路参与对TLR信号的严密调控。因此,对TLR信号转导调控的深入研究具有重要的理论意义和应用价值。Ca²⁺作为细胞内第二信使之一,调节很多重要的生理过程。钙/钙调素依赖性蛋白激酶II（Calcium/calmodulin-dependent protein kinase II,CaMKII）是钙信号下游的一种多功能丝/苏氨酸蛋白激酶,广泛分布于机体大部分重要器官组织中。它由四个独立的基因α、β、γ及δ编码,且存在着不同的剪切体,因而产生多达24种不同的同型物。CaMKII有着众多的作用底物,如转录因子CREB、ATF及其他信号分子p56lck、SHP-2、STAT1等。CaMKII可调节机体的多种重要生理功能,如突触的信号传递、记忆、物质的代谢、肌肉的收缩、基因的表达及细胞周期的调控。近年来研究表明CaMKII在免疫系统中发挥重要的调控功能,如T细胞的活化和记忆,DC的成熟和抗原提呈。LPS（TLR4配体）被证明可以促发小鼠巨噬细胞胞浆中的钙流,其对LPS诱导的TNF-α的产生有重要的作用,也有研究显示CaMKII可增强血小板激活因子（PAF）预刺激的THP-1细胞中LPS诱导的TNF-α的产生。这些研究提示Ca²⁺和CaMKII可能参与了TLR4信号。然而,钙和CaMKII在TLR配体诱导的炎性细胞因子和I型干扰素产生中的作用,以及Ca²⁺/CaMKII信号通路与TLR信号通路的交互作用及其相关机制目前尚不清楚（问题1）。MicroRNAs （miRNAs）是一类众多的内源性非编码小RNA,参与很多的生理病理过程。miRNAs在免疫系统功能调节中发挥重要的作用,包括调控巨噬细胞的对外来病原体的初始免疫应答,淋巴细胞的发育、分化和功能等等。然而,关于具有调控DC成熟和功能的作用的miRNA却很少报道。既然CaMKII被证明在DC的成熟和功能中发挥重要的作用,且目前有关miRNA与CaMKII的研究尚未见报道,因此,我们想探索是否存在以及哪些miRNAs能通过直接作用于CaMKII,从而在DC的成熟、活化以及刺激T细胞的增殖方面发挥调节作用（问题2）。以前的研究表明抗原刺激能诱导DC胞浆中钙流的上升,而钙和CaMKII均能调控DC的MHC II类分子的表达,表明Ca²⁺/CaMKII与MHC II类分子之间存在着相互调控。然而MHC II分子被抗原之外的其他分子交联后能否诱导CaMKII的活化尚不清楚（问题3）。MHC II类分子主要表达于DC、单核巨噬细胞及B淋巴细胞等专职抗原提呈细胞上,而这些细胞正是外来病原体成分等危险信号的主要感应细胞。一些研究表明MHC II类分子可介导逆向信号,影响和调节免疫细胞的很多生理过程。基于此,MHC II类分子是否能感应危险信号或者对危险信号激发的免疫反应起调控作用,目前尚不清楚（问题4）。我们对上述四个方面的科学问题及其可能的内在联系进行了系统性的研究,分别从四个方面探讨了CaMKII、靶向CaMKII的miRNA和MHC II类分子这三类分子的相互作用和对TLR触发的巨噬细胞与树突状细胞天然免疫应答反应的调控及其机制。一、钙/钙调素依赖性蛋白激酶II对TLR触发的巨噬细胞天然免疫应答反应的调控及其机制研究在硕士论文的研究中,我们已经发现LPS、CpG ODN和poly（I:C（）分别为TLR4、TLR9和TLR3配体）能够显著地诱导CaMKII的活化,表现为CaMKII（T286）磷酸化的增加和激酶活性的上升,同时利用CaMKII的特异抑制剂KN62抑制CaMKII的活性能显著降低TLR4、9、3配体诱导的炎性细胞因子IL-6和TNF-α以及IFN-β的产生,其下游信号机制是通过抑制MAPK、NF-κB和IRF3来介导。在本部分研究中,我们继续深入的探索了Ca²⁺/CaMKII对TLR触发的巨噬细胞天然免疫应答反应的调控及其机制,发现LPS、CpG ODN和poly（I:C）刺激均能诱导巨噬细胞胞浆游离钙的快速显著升高。利用特异靶向CaMKII-α的siRNA降低内源CaMKII-α的表达后,可显著抑制巨噬细胞中LPS,CpG ODN或poly（I:C）诱导的IL-6、TNF-α、IFN-α和IFN-β的产生。在巨噬细胞中瞬时转染组成性活化的CaMKII-α载体后,可加强MyD88和TRIF通路依赖的炎性细胞因子IL-6、TNF-α和I型干扰素的产生。同时,在信号通路方面,发现瞬时转染组成性活化的CaMKII-α载体后,能加强LPS诱导的MAPK、NF-κB的活化以及IRF3的磷酸化及核转位。免疫沉淀和GST-pull down试验表明CaMKII可以通过其调节区的N端部分直接结合TAK1和IRF3,体外激酶试验显示CaMKII可以直接磷酸化并活化TAK1和IRF3。腹腔预注射KN62后可显著抑制内毒素休克小鼠血清中IL-6、TNF-α和IFN-β的产生以及降低其死亡率。综上所述,TLR配体能诱导巨噬细胞胞内钙的释放以及CaMKII的活化,活化的CaMKII通过直接结合、磷酸化并活化TLR信号中重要的信号分子TAK1和IRF3,增强下游信号通路的活化,从而加强TLR配体诱导的炎性细胞因子IL-6、TNF-α和I型干扰素的产生,表明Ca²⁺/CaMKII信号通路与TLR信号通路之间的交互作用是巨噬细胞充分活化所必需的。本部分研究发现了CaMKII的新的免疫调控功能及其所作用的靶分子,另外也丰富了TLR信号转导调控的研究内容,有助于进一步深入认识机体在抵御外来病原体时的免疫应答反应及其精细调控机制。二、靶向CaMKII的miR-148/152对树突状细胞成熟和功能的影响DC的成熟与活化是初始免疫应答和获得性免疫应答的重要基础,多种调节因子参与维持DC的稳态。以前的研究表明CaMKII也是DC成熟和功能发挥的一重要调节分子,抑制CaMKII的活化可下调DC表面MHC II类分子的表达、IL-12和IFN-γ的分泌以及CD4+T细胞的增殖。我们前一部分的研究表明CaMKII可以加强TLR配体诱导的巨噬细胞炎性细胞因子IL-6、TNF-α和I型干扰素的产生。既然DC也表达CaMKII,那么DC中的TLR信号也应该受到CaMKII的正向调控。以前的研究表明dicer基因突变的果蝇缺乏内源性miRNAs,然而其CaMKII的表达显著上调。考虑到CaMKII是多个物种中高度保守的分子,那么这些研究表明CaMKII很可能受到miRNAs的负向调控。通过预测软件TargetScan 5.0和microrna.org,我们发现在CaMKII的3’-UTR区,有几个在脊椎动物中保守的miRNA的结合位点,包括miR-148/152, miR-217, miR-129-5p。在本部分的研究中,我们探索了以上miRNA是否可以调控DC的成熟和功能。实时荧光定量PCR检测显示miR-148/152的表达在TLR配体诱导的成熟及活化的小鼠骨髓来源的DC中显著上调,而miR-217, miR-129-5p无显著变化。通过转染miRNA的模拟物或抑制剂,发现miR-148/152能抑制LPS诱导的DC表面MHC II类分子的上调,且能降低LPS、CpG ODN和poly（I:C）诱导的炎性细胞因子和I型干扰素的产生,其中包括促进T细胞增殖的IL-12。更重要的是miR-148/152能抑制DC促发的抗原特异的T细胞增殖。进一步研究表明miR-148/152直接作用于靶分子CaMKII-α3’-UTR,显著抑制CaMKII-α的蛋白水平和mRNA水平的表达。综上所述,miR-148/152通过作用于靶分子CaMKII-α,从而负向调控TLR配体触发的DC初始免疫应答和抗原提呈功能。miR-148/152为一新的DC成熟和功能的负向调节因子,为免疫应答反应的调控提供了另一种新的方式。三、Ca²⁺/CaMKII与MHC II类分子之间的相互调控以前的研究表明抗原刺激能诱导DC胞浆中钙流的上升,而上调的钙信号是DC成熟和功能发挥所需的一种重要因素。CaMKII能调节DC的MHC II类分子的表达,抑制CaMKII的活化不但能增加MHC II类分子的溶酶体转运和降解,还能抑制MHC II mRNA的转录及稳定性。然而,除了抗原之外其他分子交联MHC II类分子后引起的胞浆钙流及CaMKII的活化尚不很清楚。我们发现,利用siRNA降低DC中CaMKII的表达可显著抑制LPS诱导的DC表面MHC II类分子的上调表达,而用抗体交联DC表面的MHC II类分子能增强LPS未诱导的和诱导的CaMKII的活化。因此,我们的研究进一步表明Ca²⁺/CaMKII与MHC II类分子之间存在着相互正向调节,在DC的成熟和功能中发挥重要的调控作用。四、MHC II类分子逆向信号促进TLR触发巨噬细胞与树突状细胞的天然免疫应答反应及其机制研究目前对于MHC II类分子的非经典功能的研究比较少。既然MHC II类分子与TLR均主要表达于专职抗原提呈细胞上,我们设想MHC II类分子能否感应或辅助感应危险信号,或者对危险信号激发的免疫反应具有调控作用呢?利用基因敲除小鼠及RNA干扰技术,我们对这一问题进行了研究。与野生型小鼠（MHC II+/+）相比,MHC II缺陷（MHC II-/-）小鼠腹腔巨噬细胞和骨髓来源的DC在LPS、CpG ODN和poly（I:C）刺激后,产生的IL-6、TNF-α、IL-12和IFN-β显著降低。在巨噬细胞和DC中干扰MHC II类分子的表达后,也明显降低了TLR配体诱导的上述细胞因子的分泌。预先用特异抗体交联MHC II类分子后可显著上调LPS诱导的炎性细胞因子和I型干扰素的产生。信号通路研究发现,MHC II类分子干扰后能抑制LPS诱导的MAPK、NF-κB的活化和IRF3的磷酸化。另外,在TLR配体活化的巨噬细胞和DC中MHC II类分子能招募活化的Lck。降低MHC II类分子的表达能抑制LPS诱导的Lck、PLCγ1和CaMKII的活化。钙流检测发现,MHC II类分子干扰后能显著降低LPS促发的巨噬细胞胞浆中的钙流。利用LPS诱导内毒素休克的体内实验显示,MHC II-/-小鼠的血清中IL-6、TNF-α、IL-12和IFN-β的水平较野生型小鼠明显降低。综上所述,我们发现MHC II类分子能辅助TLR配体诱导非受体型蛋白酪氨酸激酶Lck的活化及下游的PLCγ1的活化,维持胞浆钙流的充分上升及钙信号下游的CaMKII的活化,从而促进炎性细胞因子和I型干扰素的产生,表明MHC II类分子的逆向信号能对TLR触发的信号转导以及天然免疫应答产生正向的调控作用,从而提出了MHC II类分子新的非经典功能,即MHC II类分子在先天免疫应答中也起重要的作用。总之,我们四部分的内容研究了CaMKII、靶向CaMKIIα的miR-148/152以及MHC II类分子的相互作用及其对TLR信号转导的调控及相关的机制,证明了CaMKII和MHC II类分子是抗原提呈细胞—巨噬细胞和DC中TLR信号充分活化所必需的,同时发现了新的可以调控DC成熟和功能的miRNA（miR-148/152）,揭示了CaMKII和MHC II这两种重要的分子可以在两种主要的抗原提呈细胞—巨噬细胞和DC中相互调控,从而共同参与维持免疫系统的平衡和稳定。该研究结果拓宽了人们对于MHC II类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,也有望为免疫相关疾病发病机制的研究与治疗方法的寻找提供新的启示。更多还原

【Abstract】 The ability of the innate immune system to recognize and respond to microbial components has been largely attributed to Toll-like receptors （TLRs）. As an important kind of pattern recognition receptors （PRRs）, TLRs comprise a large family consisting of at least 12 members and are mainly expressed on antigen-presenting cells such as macrophages and dendritic cells （DCs）. Recognition of pathogen-associated molecule patterns （PAMPs） by TLRs leads 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, facilitate DC maturation and instruct development of antigen-specific adaptive immunity, especially Th1 response. TLRs play important roles in linking innate and adaptive immune responses. Less efficient activation of TLR response may not evoke potent anti-infection or anti-tumor immunity, however, excessive activation of TLR may also induce immunopathological process such as endotoxin shock and autoimmune diseases. Various other signal pathways are involved in the tight regulation of TLR signaling to enhance or attenuate their activation, which maintain the immunological balance. However, these regulatory mechanisms remain to be fully understood up to now.Calcium (Ca²⁺) functions as a major second messenger that regulates a broad range of important cellular processes. Many of the cellular responses to Ca²⁺ signal are induced or modulated by a family of multifunctional Ca²⁺/calmodulin dependent protein kinases （CaMKs）, among which CaMKII is a ubiquitous serine/threonine protein kinase encoded by 4 separate genes （α,β,γandδ）. CaMKII can phosphorylate up to 50 diverse substrates including enzymes, kinases, ion channels, and transcription factors, and is involved in a broad range of cellular functions, such as metabolism, neurotransmitter release, cell proliferation and gene expression. Previous studies showed that CaMKII-αplayed important roles in immune responses, included T cell activation, DC maturation and antigen presentation. LPS has been shown to elicit Ca²⁺ flux in murine macrophages, which is important for TNF-αproduction; CaMKII was also reported to enhance platelet-activating factor （PAF）-primed and LPS-induced TNF-αproduction in THP-1 cells , suggesting a possible involvement of Ca²⁺ flux and CaMKII in TLR4 signaling.However, the exact roles of Ca²⁺ and CaMKII in TLR-triggered production of proinflammatory cytokines and type I interferon, and the cross-talk between Ca²⁺/CaMKII pathway and TLR signaling are still poorly characterized.MicroRNAs （miRNAs） are short, endogenous, non-coding RNA involved in a range of cellular processes. miRNAs also play important roles in the regulation of immunological functions including innate immune responses of macrophages; development, differentiation and function of T cells and B cells. However, few miRNAs have been found to be able to regulate the innate response and antigen-presenting function of DCs to date. Since CaMKII has been shown to be an important regulator of the maturation and function of DCs, we wonder whether and what miRNAs can regulate the innate response and APC function of DCs by targeting CaMKII-α.Previous studies showed that antigen stimulation of DC induces an increase in cytosolic Ca²⁺. Ca²⁺ and CaMKII were shown to regulate the expression level of MHC II in DCs. These studies indicate that Ca²⁺/CaMKII can cross-talk with MHC II signaling. Howerer, whether ligation of MHC II by antibody can induce CaMKII activation needs further investigation.MHC II are predominantly expressed by APCs including dendritic cells, macrophages and B cells, while these APCs are the major responders to danger signals such as microbial components. Previous studies showed the reverse signal mediated by MHC II can regulate many immune responses in B cell and activated T cells. It is reasonably straightforward to propose that MHC II might be responsible for these signals or regulate the immune response initiated by danger signals, while which need to be confirmed.In this study, we integrated to think about the four questions raised above and investigated the regulation of TLR-triggered innate immune response of macrophages and DCs by CaMKII, CaMKII-targeting miRNAs and MHC II as well as the underlying mechanisms. The reciprocal interaction of Ca²⁺/CaMKII and MHC II has been also explored.Part I. CaMKII promotes TLR-triggered proinflammatory cytokine and type I interferon production and the underlying mechanisms.In the research papers for my master’s degrees, we have provided evidences that TLR4, 9, 3 ligands （LPS, CpG ODN and poly（I:C）, respectively） significantly induce the activation of CaMKIIαin macrophages. Selective inhibitor KN62 significantly suppresses TLR4, 9, 3-triggered production of IL-6, TNF-α, IFN-α/βin macrophages. CaMKII inhibition attenuates LPS-induced ERK, JNK, NF-κB and IRF3 activation in macrophages. Here, we further focus on the study about the regulation of TLR-triggered innate immune response by CaMKII. We demonstrate that TLR4, 9, 3 ligands markedly and quickly trigger the elevation of intracellular Ca²⁺. RNAi knockdown of CaMKII significantly suppresses TLR4, 9, 3-triggered production of IL-6, TNF-α, IFN-α/βin macrophages. Coincidently, overexpression of constitutively active CaMKIIαsignificantly enhances production of the above cytokines. In addition to the activation of MAPK and NF-κB pathways, CaMKII-αcan directly bind, phosphorylate and activate TGF-β-activated kinase 1 （TAK1） and interferon regulatory factor 3 （IRF3） （serine on 386） via its N-terminal part of regulatory domain. In vivo administration of CaMKII inhibitor substantially decreases production of serum proinflammatory cytokines, IFN-βand increases survival rate of lethal LPS-challenged mice.So, CaMKII can be activated by TLR ligands, and in turn, promotes both MyD88- and TRIF-dependent inflammatory responses by directly activating TAK1 and IRF3. The cross-talk with Ca²⁺/CaMKII pathway is needed for full activation of TLR signaling in macrophages.Part II. The regulation of innate response and antigen presentation of TLR-triggered DCs by miR-148/152 via targeting CaMKII-α.The maturation and activation of DCs are essential for innate and acquired immune responses, which are regulated by multiple factors. CaMKII has been shown to be an important regulator of the maturation and function of DCs. Inhibition of CaMKII in myeloid DCs resulted in reductions of antigen-induced surface expression of MHC Class II, secretion of IL-12, IFN-γ, and MHC Class II-restricted T cell proliferation. Recently, CaMKII-αwas demonstrated by us to promote TLR-triggered proinflammatory cytokine and type I interferon production by directly binding and activating TAK1 and IRF3 in macrophages. Since DCs also express CaMKII, we predicted that TLR signaling in DCs should be also positively regulated by CaMKII. Previous study showed that in dicer mutant Drosophila, which display defect in endogenous miRNA generation, CaMKII expression is significantly higher. Considering that CaMKII is a highly conserver molecule in many species, it is possible that CaMKII is regulated by miRNA. Since CaMKII plays important roles in DCs maturation and functions, we want to know whether or what miRNAs function in DCs via targeting CaMKII. By analyzing with TargetScan 5.0 and microrna.org, we found that CaMKII-α3’-UTR contains some miRNA-binding sites, among which, the conserver sites for miRNA families broadly conserved in vertebrates are as follows: miR-148/152, miR-217, and miR-129-5p. In the present study, we investigated which one（s） of the above miRNAs may be involved in DC maturation and function. By Q-PCR, we found the expression of miR-148a, miR-148b and miR-152, which belong to miR-148 family, were significantly increased in DCs stimulated with LPS for 6-24 hours, while the expression of miR-217 and miR-129-5p remained almost unchanged. we proved that miR-148/152 could inhibit LPS-induced up-regulation of MHC II expression on DCs. LPS, CpG ODN or poly（I:C）-induced DC production of cytokines, especially including IL-12, which could promote T cell proliferation, was inhibited by miR-148/152. More importantly, miR-148/152 impaired DC-initiated antigen-specific T cell proliferation. MiR-148/152 target CaMKII-αand down-regulated its expression via both translational inhibition and mRNA degradation.Therefore, our results demonstrate that miR-148/152 expression is up-regulated in DCs upon maturation and activation induced by TLR agonists, which in turn inhibit the up-regulation of MHC II expression, cytokine production and antigen presentation of DCs by targeting CaMKII-α. MiR-148/152 are negative regulators for the innate response and APC function of DCs.Part III. The reciprocal regulation of Ca²⁺/CaMKII and MHC II molecule.Previous studies showed that antigen stimulation induces an increase in cytosolic Ca²⁺ in DCs, which is a critical component of DC maturation and function. CaMKII was shown to regulate the expression of MHC II in DCs. Inhibition of CaMKII resulted in the enhanced lysosomal trafficking and degradation as well as significant reductions in the level and stability of MHC II mRNA. Whether ligation of MHC II can induce CaMKII activation remains unclear to date. We found that ligation of MHC II by specific antibody significantly enhances the activation of CaMKII in DCs stimulated with or without LPS. In addition, konckdown of CaMKII-αby siRNA inhibits LPS-induced MHC class II expression on DCs. We can conclude that Ca²⁺/CaMKII can reciprocally interact with MHC II, which play important roles in DC maturation and function.Part IV. The regulation of TLR-triggered innate immune response of macrophages and DCs by MHC II reverse signal and the underlying mechanisms.The non-canonical functions of MHC II molecule attract more and more attentions now. Since MHC II and TLR are both predominantly expressed on APCs, we suppose that MHC II might be involved in the recognition of danger signals or regulation of the immune response initiated by danger signals such as TLR ligands. Employing MHC II deficient mice （MHC II-/-） and RNA interfering, we investigated this hypothesis. Our results showed that LPS-, CpG ODN- or poly（I:C）-induced production of cytokines, including IL-6, TNF-α, IL-12 and IFN-βwas significantly decreased in macrophages and DCs from MHC II-/- mice as compared with that in macrophages and DCs from wild type mice. MHC II knockdown in macrophages and DCs also impaired TLR-triggered production of the above cytokines. Ligation of MHC II by specific antibody led to the increase in cytokine production in TLR-activated macrophages and DCs. We also went further to investigate the underlying mechanisms and found that MHC II deficiency or knockdown inhibited the phosphorylation of ERK, JNK, p38 and IκBαas well as IRF3 induced by the above TLR agonists. In addition, MHC II recruits activated Lck in macrophages stimulated with LPS. The activation of Lck, PLCγ1 and CaMKII induced by TLR agonists were also impaired in MHC II silenced-macrophages and DCs. Ca²⁺ detection indicated that cytosolic Ca²⁺ level in LPS-stimulated MHC II-silenced macrophages and DCs was significant decreased compared with that in MHC II wild type macrophages and DCs. The production of cytokines, including IL-6, TNF-α, IL-12 and IFN-βwas markedly decreased in serum of lethal LPS-challenged MHC II-deficient mice, further demonstrating that MHC II reverse signal is required for the TLR-triggered innate immune response.Thus, our results demonstrate that MHC II deficiency impairs the activation of Lck and PLCγ1, leads to the decreased Ca²⁺ level and CaMKII activation, which results in the decreased production of inflammatory cytokines and type I IFN. MHC II reverse signal can promote TLR-triggered innate immune response.In conclusion, taken together of the results from the above four parts of studies, we demonstrates that CaMKII and MHC II can interact and regulate their functions in the innate immunity reciprocally. We also discover the new miRNAs, miR-148/152, which can regulate DC maturation and function by targeting CaMKII. The cross-talk of Ca²⁺/CaMKII pathway and MHC II reverse signal is needed for full activation of TLR signaling in macrophages and DCs.更多还原