【作者】 吴景华；

【导师】 魏林；

【作者基本信息】 河北医科大学 ， 免疫学， 2014， 博士

【摘要】 目的:巨噬细胞是抗感染的重要力量，通过其表面模式识别受体（PRR）识别细菌表面的病原相关分子模式（PAMPs）而活化，TLR是重要的PRR，可以识别结合多种病原菌成分而活化巨噬细胞。当TLR与PAMPs结合后，受体发生二聚体化，招募MyD88分子，MyD88分子中的DD结构域招募其下游的IRAK并活化，从而激活TRAF6，引起NF-κB活化。被活化的NF-κB转位至细胞核内,与DNA序列上的特异蛋白结合，诱导特定基因mRNA转录，翻译产生蛋白质。巨噬细胞的活性还受到多种负调控蛋白的影响，其中细胞因子信号抑制蛋白（suppressor of cytokinesignaling，SOCS）即为可调节免疫细胞活化、以保证免疫应答适可而止的重要分子。在许多免疫细胞中都已发现SOCS巨大的负反馈调节作用。SOCS由至少8种成分组成，即SOCS1-7和CIS。其中，SOCS-1主要通过细胞因子-细胞因子受体结合，进而激活JAK/STAT途径产生。已有资料表明，一些细胞因子的活化，如IFN-，IL-10，IL-2和集落刺激因子都可以诱导SOCS-1的产生，其中干扰素受体途径是其激活的经典途径，当干扰素和其位于细胞表面的干扰素受体结合后，激活受体相关的酪氨酸激酶（JAK-1and TyK2），随后STAT1被磷酸化激活，并转位入核，活化SOCS-1靶基因，表达SOCS-1蛋白。近几年有资料报道，SOCS-1还可以通过TLR，Dectin-1等通路被诱导产生，LPS、CpG、酵母多糖等是它的刺激剂。目前，越来越多的研究提示，微生物及寄生虫的成分可以诱导SOCS蛋白的产生，并以此下调免疫应答反应，从而逃避机体的免疫攻击。A群链球菌（GAS）可以导致咽炎、软组织感染和其他许多呼吸道感染。GAS感染可以激活巨噬细胞膜表面的TLR2、TLR4等模式识别受体，诱导炎症细胞因子IL-1，IL-6和TNF-α及IFN-等产生，但GAS之所以能在人群中持续存在并引起一些严重的感染性疾病，与其具有逃避宿主免疫攻击的能力密切相关，一个常见的现象就是GAS感染的严重坏死部位的一线防御细胞数量极少。因此为了进一步了解GAS下调炎症因子的分泌的策略，从而探索SOCS-1的产生机制，因此，我们进行了以下实验：1、检测GAS感染小鼠巨噬细胞初期SOCS-1蛋白的表达情况。2、鉴于细胞因子受体活化通路是诱导SOCS-1的经典通路，通过放线菌酮（抑制真核细胞蛋白质合成）作用于巨噬细胞再行GAS感染，以此检测细胞因子对于SOCS-1产生的影响。3、细胞因子IFN-活化通路是诱导SOCS-1的经典通路,因此，用IFN-特异性中和抗体进行阻断，以此验证IFN-在GAS感染诱生SOCS-1蛋白发挥的作用。4、另外，已有许多研究证明通过TLR途径可以直接诱导SOCS-1的表达，但其机制并不清楚。因此，我们检测了GAS感染小鼠巨噬细胞后，细胞表面TLR2和TLR4的活化情况，进而研究TLR信号通路与SOCS-1产生的关系。5、利用抑制剂、基因敲除鼠等手段研究TLR4、MYD88、NF-κB等关键节点蛋白在GAS诱导SOCS-1产生中的作用。6、鉴于NF-κB是一种多向性基因调控蛋白，调节多种参与免疫反应及免疫相关分子的基因转录过程，因此，通过对NF-κB活性的阻断研究以此证明NF-κB在SOCS-1表达中的调控作用。本研究丰富了对细菌与免疫细胞相互作用方式、机制的认识，特别是丰富了对微生物诱发宿主细胞快速的、直接的表达负调控因子进行逃逸固有免疫机制的了解，有助于拓宽抗感染免疫措施的建立与发展。方法:1A族链球菌以MOI100:1感染鼠巨噬细胞RAW264.7\小鼠骨髓来源BMDMs，诱导产生SOCS-1的特征。2放线菌酮处理巨噬细胞后感染GAS菌株，初步确定GAS诱导产生SOCS-1与细胞因子途径的关系。2.1检测GAS组，热灭活GAS组的细胞因子IFN-表达情况（实时定量PCR进行检测）。2.2用IFN-的特异性抗体阻断其活化后，检测JAK/STAT通路及SOCS-1的产生情况。2.3放线菌酮提前作用巨噬细胞30分钟后，加入GAS作用1小时后，去除GAS，在放线菌酮作用下继续培养6小时，通过RT-PCR，Western blot检测SOCS-1的产生情况，以及JAK/STAT通路的活化情况。3确定GAS感染巨噬细胞引起SOCS-1表达与巨噬细胞TLR4受体的关系。3.1检测GAS组，热灭活GAS组的模式识别受体TLR2/TLR4表达情况（实时定量PCR进行验证）。3.2以TLR4特异性阻断剂提前30分钟作用巨噬细胞后，GAS MOI100：1感染巨噬细胞1小时，去除细菌，在特异性阻断剂存在的情况下继续培养6h后检测JAK/STAT通路的活化及SOCS-1的表达情况。3.3分离TLR4基因敲除鼠骨髓来源巨噬细胞(BMDMs),重复以上实验验证TLR4与SOCS-1产生的关系。4确定GAS诱导SOCS-1高表达与TLR4/MyD88传导通路的关系。4.1通过免疫荧光及免疫共沉淀技术检测巨噬细胞中MyD88，JAK1，STAT1蛋白存在的状态。4.2根据以上结果，在确定MyD88分子和JAK1，STAT1在巨噬细胞中以复合物的形式存在的前提下，用GAS感染时，用免疫荧光和免疫共沉淀的方法检测这个复合物被活化的情况。4.3用放线菌酮去除细胞因子的作用后，用免疫共沉淀的方法验证TLR4/MyD88直接通路的存在。4.4分离MYD88基因敲除鼠骨髓来源的巨噬细胞（BMDMs），GAS感染，进一步确证TLR4/MyD88直接通路的存在。5NF-κB活化对GAS诱导的SOCS-1表达中的影响作用研究5.1小鼠巨噬细胞感染GAS后，经过1h，2h，4h，6h检测NF-κB活化情况，并同时检测JAK/STAT通路蛋白的表达及活化情况，初步确定JAK/STAT通路的活化与NF-κB活化的关系。5.2根据以上结果，利用NF-κB活化的特异性阻断剂阻断后，检测JAK/STAT通路上JAK，STAT蛋白的表达及活化情况，以及SOCS-1的表达情况。结果:1GAS感染巨噬细胞早期诱导SOCS-1大量表达以MOI100：1菌量的A族链球菌（GAS）、热灭活的GAS（nonviableGAS）分别刺激小鼠来源的巨噬细胞系RAW264.7和BMDMs1小时，SOCS-1的表达特征：GAS感染RAW264.7巨噬细胞后2小时SOCS-1基因开始有明显的升高，6小时达到高峰，8小时开始下降；同时对照组热失活GAS刺激RAW264.7巨噬细胞SOCS-1mRNA则在感染过后6-8小时开始轻度升高，且升高的幅度明显小于GAS组。在GAS感染BMDM细胞时，SOCS-1mRNA的表达情况基本和RAW264.7细胞一致，只是反应的强度大于RAW264.7细胞。Western Blot检测GAS引起SOCS-1蛋白在感染6小时后开始表达，而热灭活得GAS基本在10h内没有表达。2GAS感染小鼠巨噬细胞早期引起SOCS-1表达部分依赖于细胞因子IFN-β途径2.1GAS感染RAW264.7细胞2小时IFN-β开始升高，6小时达到高峰。2.2通过anti-IFN-β与GAS共培养，检测SOCS-1，结果表明，IFN-β活化途径可以引起SOCS-1的产生，但其并不是唯一途径。2.3为了检测是否为其他细胞因子活化引起SOCS-1的表达，加入放线菌酮(抑制真核细胞蛋白质的翻译,阻断细胞因子分泌)共培养，结果表明加入CHX后STAT1依然能被磷酸化活化，表明GAS感染诱导SOCS-1表达产生除了部分依赖于细胞因子活化外，还存在一条直接刺激巨噬细胞产生SOCS-1的途径。3GAS感染巨噬细胞后TLR4活化情况GAS感染RAW264.7细胞后，位于细胞膜表面的TLR2和TLR4被活化。在感染发生后4小时，TLR4的表达升高到了峰值，为正常的8倍左右，在感染后6小时其表达开始下降；而对照组热灭活处理的GAS感染巨噬细胞6小时内TLR4的表达没有升高；而细胞膜表面的TLR2在感染发生6小时内GAS组和热失活GAS组基本没有区别，都在感染后4小时达到峰值，并且升高幅度基本一致。4TLR4参与了GAS感染引起的SOCS-1的表达4.1为了确定巨噬细胞表面TLR4的激活是否参与了SOCS-1的表达，我们用TLR4的中和抗体提前封闭巨噬细胞表面的TLR4，再行GAS感染，结果显示，封闭巨噬细胞TLR4后， GAS感染引起的SOCS-1表达量明显降低，由此证明位于巨噬细胞表面TLR4在GAS感染引起SOCS-1表达方面发挥了重要作用。4.2GAS感染TLR4-/-小鼠BMDMs细胞，结果进一步证实了TLR4在诱导SOCS-1表达中发挥的重要作用。5SOCS-1表达呈MyD88分子依赖性5.1免疫荧光染色和免疫共沉淀结果表明，在RAW264.7细胞中MyD88，JAK1，STAT1是以复合体的形式存在的。当GAS感染发生后，与MyD88结合的JAK1蛋白迅速发生磷酸化，在感染后1-2小时达到高峰，随后复合物中的STAT1开始发生磷酸化激活，在3小时达到峰值。5.2GAS刺激MyD88-/-BMDMs细胞，检测发现SOCS-1表达缺失。5.3CHX阻断细胞因子后，进行免疫共沉淀实验，结果证实结合于MyD88分子上的STAT1依然被活化，由此进一步证明了感染早期，GAS本身可以通过直接激活结合于MyD88分子上的STAT，从而引起SOCS-1蛋白的快速表达。6NF-κB信号通路通过调控STAT1表达影响SOCS-1蛋白6.1GAS刺激巨噬细胞后，引起NF-κB被活化及STAT1蛋白表达增加，且STAT1表达增加落后于NF-κB的活化，说明NF-κB的活化可能影响STAT1的表达。6.2通过NF-κB信号通路阻断剂JSH-23作用后检测发现,STAT1的表达明显降低，进而磷酸化STAT1及SOCS-1蛋白的表达水平大大降低。结论:1GAS感染巨噬细胞早期可诱导SOCS-1蛋白大量表达。2GAS感染小鼠巨噬细胞引起SOCS-1表达部分依赖于IFN-活化通路。3TLR4/MyD88直接通路在GAS感染早期诱导表达SOCS-1中起重要作用。4NF-κB信号通路通过调控JAK1/STAT1通路中STAT1蛋白的表达水平进而影响SOCS-1蛋白的表达更多还原

【Abstract】 Objectives: Macrophages are important to fight to infection, and it wasactivated combined with a variety of pathogenic bacteria through its surfacepattern recognition receptors (PRR),which could identificated of the pathogenassociated molecular patterns (PAMPs). TLR, is important PRR, dimers wassynthesized when combined with PAMPs.MyD88molecules was recruited,andactivated its downstream IRAK which can activate the TRAF6to activate NF-κB.Then NF-κB transposited to the nucleus,and combined with specificprotein on the DNA sequence to inducte the specific gene and proteintranslation.The activity of macrophages is influenced by a variety of negativeregulatory proteins, including suppressor of cytokine signaling (SOCS),whichis adjustable immune cell activation to ensure the moderation of immuneresponse.SOCS proteins play a role in negative feedback regulation of manyimmune cells. SOCS is composed of at least eight kinds of ingredients,SOCS1-7and CIS.And the SOCS-1was produced through cytokinescombined cytokines receptors to activate JAK/STAT pathway.Many studiessuggest that the activation of some cytokines, such as IFN-, IL-10, IL-2and colony stimulating factor, could induce the SOCS-1protein expression.Among these the IFN receptor pathway is the classic way to itsproduction.When interferon combined its receptors, the tyrosine kinase (JAK-1and TyK2) was activated.Then STAT1was phosphorylated and transferedinto the nucleus to activate genes SOCS-1,and produce SOCS-1protein.Recently,these was reported that SOCS-1could induce through theTLR, Dectin-1pathway,and its stimulants is LPS, CpG, ans yeastpolysaccharide and so on. At present, more and more studies suggest that the composition ofmicroorganism and parasite can also induce the SOCS expression.Group A streptococcus (GAS) could lead to pharyngitis, soft tissueinfection, and many other respiratory infections.GAS-infected macrophagescould induce inflammatory cytokine IL-1, IL6and TNF-a and IFN-through activate TLR2and TLR4located in the membrane.GAS has a abilityto evade the host immune attack which is the reason of its persist in the bodyand cause some serious infectious diseases,and another phenomenon is a fewcells in GAS-infection induced necrotic areas.So in order to further understand GAS strategy to evade themacrophages, and explore the generation mechanism of SOCS-1, weconducted the following experiments:1, SOCS-1proteins expression inGAS-infected macrophages were detected.2, In view of the cytokinesactivation pathway is a classic pathways to induce the SOCS-1expression,wemeasure the SOCS-1levels in coincubated with cycloheximide (inhibition ofeukaryotic cell protein synthesis).3, Because of the classic pathway ofSOCS-1induction, we use IFN-neutralizing antibodies to verify the role ofIFN-to induce SOCS-1.4, Many studies have shown that TLR pathway could directly induce theexpression of SOCS-1, but its mechanism is unclear. Therefore, we woulddetect TLR2and TLR4activation in GAS-infected macrophages to confirmthe relationship the TLR signaling pathway and SOCS-1proteins expression.5,The key proteins (TLR4、MYD88、NF-κB) in induction of SOCS-1will bestudy through using of inhibitors, knockout mice.6, NF-κB is a diversityproteins, regulate a variety of genes transcription involved in immuneresponse and immune regulation related molecules.So we decide to researchNF-κB regulation on SOCS1expression.This study will help to enrich the mechanism of the interact immune cellsand bacteria, especially rich the understanding of the mechanism of host cellsrapid, direct expression of negative regulation factor which could helpmicrobial to evade host immune systems.So it will broaden the establishment and development of anti-infection immunity.Methods:1GAS-infected macrophage RAW264.7\BMDMs(MOI100:1) were detectedthe SOCS-1expression.2The relations of cytokines and SOCS-1expression was definited in GASinfected macrophages coincubate cycloheximide.2.1After cycloheximide incubate with macrophages30minutes, themacrophages were infectec with GAS,after1hour remove GAS and continueto develop6hours. SOCS-1was detected by RT-PCR and Western blot, aswell as the activation of the JAK/STAT pathway was test meantime.2.2IFN-was detected in GAS, nonviable GAS infectedmacrophages(RT-PCR).2.3The JAK/STAT pathway activation and SOCS-1expression was detectedafter blocking IFN-with a specific antibody.3The relationship will be confirmed in SOCS-1expression and TLR4activation.3.1TLR2/TLR4expression were detected in GAS and inviable GAS infectedmacrophages (RT-PCR).3.2After blocking TLR4with macrophages30minutes, the macrophages wereinfectec with GAS,after1hour remove GAS and continue to develop6hours.SOCS-1was detected by RT-PCR and Western blot, as well as the activationof the JAK/STAT pathway was test meantime.3.3The relationship between TLR4signaling and SOCS-1expression wasconfirmed by BMDMs from TLR4-/-mice again.4To study the relationship between GAS-induced SOCS-1expression andTLR4/MyD88pathway.4.1MyD88,JAK1, STAT1protein were detected in macrophages byimmunofluorescence and immune coprecipitation.4.2According to the above results, if MyD88and JAK1, STAT1exis ascomplex of in macrophages, we will detect the complex activation byimmunofluorescence and immune co-precipitation. 4.3TLR4/MyD88direct pathway will be comfirmed by mmuneprecipitationwith when cytokines activation pathway was excluded throughcoincubated with cycloheximide.4.4TLR4/MYD88direct pathway was further confirmed in GAS infectionthrough MYD88-/-macrophages.5The impaction of NF-κB activation to GAS-induced SOCS-1expression willbe cofirmed.5.1The relationship between JAK/STAT pathway activation and NF-κBactivation will be confirmed through activation of JAK and STAT proteinsafter GAS infected1h,2h,4h,6h.5.2According to the above results, theregulation was further confirmed byblcoking to NF-κB signaling.Results:1GAS-induced SOCS-1expression in early stage in macrophagesGAS and nonviable GAS (MOI100:1) respectively stimulate RAW264.7macrophages and BMDMs1hour, characteristics of SOCS-1expression asfollows: The SOCS-1gene had significantly increased after4h of GASinfection,peaked at6h, begin to decline after8h.However, although theinviable GAS-induced SOCS1mRNA elevated moderately6-8hours afterinfection,its level increased much smaller than GAS group. In GAS-infectedBMDMs, SOCS-1mRNA expression is the same as in RAW264.7cells, onlygreater than RAW264.7cells. SOCS-1protein begin to express after6hoursin GAS-infected macrophages,but inviable GAS don’t induce expression in10h.2GAS infected macrophages induced SOCS-1expression partly depends onthe IFN-βactivation pathway2.1IFN-βbegan to rise in GAS infection RAW264.7macrophages at2h, andpeaked at6h.2.2The IFN-βactivation pathway is not the only way to induce the expressionof SOCS-1through coincubating anti-IFN-βand GAS.2.3In order to check the SOCS-1expression whether depend on the cytokines activation pathway, Cycloheximide (it could inhibit protein translation ofeukaryotic cells) was used to coincubate with macrophages.The results showthat STAT1proteins were still phosphorylated in the cycloheximide,and itreveals GAS itself could induce SOCS-1expression besides cytokinesactivation dependent pathway.3GAS-infection induced TLR4activationWhen macrophages was infected with GAS, TLR2and TLR4located incells surface were activated, and peaked at4h after infection, was about8times of normal, and begun to decline after6hours. While the macrophagesinfected by inviable GAS,the TLR4has not been activated; TLR2activation has no difference in GAS group and inviable GAS infection within6hours.4TLR4play an important role in GAS-induced SOCS-1expression4.1In order to further determine whether the expression of SOCS-1wasdepend on the TLR4activation, we use the TLR4neutralizing antibody blockdthe TLR4on the macrophages.And the results show that the SOCS-1expression was declined in the present of anti-TLR4.So it could confirm thatTLR4play a role in the GAS-induced SOCS-1expression.4.2It was further confirmed that the SOCS-1expression depend on the TLR4activation in theTLR4-/-BMDMs cells.5SOCS-1early expression depends on the MyD885.1Through immunofluorescence and immune precipitation,we could believethat Jak1, STAT1and MyD88exist in RAW264.7macrophages as a complexin macrophages. It was found that when JAK1associated with MyD88,phosphorylation was rapidly activated in macrophages that were post-infectedwith GAS at1-2hrs post-infection, and STAT1was activated andphosphorylated STAT1levels peaked at3to4hrs post-infection.5.2SOCS-1expression levels were diminished in MyD88-/-BMDMs.To further test whether STAT1was phosphorylated by GAS itself throughthe MyD88-JAK1-STAT1complex, we detected p-STAT1levels combinedwith MyD88in the presence of CHX by immunoprecipitation assay. The cells were infected with GAS, or with GAS plus CHX, and then thep-STAT/MyD88complex was detected by immunoprecipitation analysis.Without cytokine activation signaling, the Without cytokine activationsignaling, the p-STAT1levels combined with MyD88were unaffected. Thus,these findings suggested that in GAS-induced infection, GAS itself promptlyactivated STAT1through the TLR4/MyD88-JAK1/STAT1complex.6NF-κB activation regulated early expression of SOCS-1through reducedSTAT1expression levels6.1GAS induced rapid and potent activation of NF-κB and increased STAT1expression. The expression of STAT1increased following NF-κB activationand peaked at6hrs. Having established that GAS infection induces NF-κBactivation and leads to increased STAT1expression; we think there maybe alink between NF-κB activation and STAT1expression.6.2Through pretreated BMDMs with JSH-23(20uM)30min prior toinfection and incubated JSH-23for the indicated times,we found thatGAS-induced expression of STAT-1was strongly diminished, and that thephosphorylation of STAT-1was almost completely abolished in BMDMstreated with JSH-23.And GAS-induced expression of SOCS-1was completelyabolished in cells treated with JSH-23.Conclusion:1GAS-infected macrophages could express SOCS-1proteins in theearly stage.2GAS-induced expression of SOCS-1partly relies on the IFN-activated pathway.3TLR4/MyD88direct pathway plays an important role in GAS-inducedSOCS-1expression in the early stage.4NF-κB signaling could affect SOCS-1protein levels throughregulation to the STAT1protein expression.更多还原