【作者】 侯楠；

【导师】 马春红； 孙汶生；

【作者基本信息】 山东大学 ， 免疫学， 2010， 博士

【摘要】 冠心病是发达国家首位致死性病因,在我国的发病率和致死率也逐年升高。动脉粥样硬化(Atherosclerosis, AS)占冠状动脉性心脏病的绝大多数(95%-99%),而动脉粥样硬化不稳定斑块的破裂、血小板的聚集及血栓形成造成的冠状动脉急性狭窄和闭塞是导致急性冠状动脉综合征(acute coronary syndrome,ACS)的重要原因。目前研究认为动脉粥样硬化是一种多病因的慢性炎症性疾病,局部和全身的炎症免疫反应在动脉粥样硬化发生发展过程中起着重要作用,固有免疫和适应性免疫共同参与调节动脉粥样硬化病变。动脉粥样硬化斑块中存在着多种免疫细胞,其中以巨噬细胞和T细胞最为常见,此外还有少量的树突状细胞(Dentritic cell, DC)、自然杀伤细胞(natural killer cell, NK cell)和肥大细胞(mast cell)等,偶有B淋巴细胞。动脉粥样硬化的发生是多因素共同作用的结果,目前已发现的动脉粥样硬化危险性因素很多,但相关针对治疗及控制效果均不理想,降脂和抗炎治疗是目前最主要的治疗措施。越来越多的证据显示免疫反应参与动脉粥样硬化发展的各个环节。免疫缺陷小鼠与正常小鼠相比,动脉粥样硬化的严重程度减轻约70%；而血清胆固醇水平没有改变。因此炎症免疫调节机制成为目前研究的热点,对该机制的研究将为动脉粥样硬化的治疗开辟新的道路。T细胞免疫球蛋白粘蛋白分子3(T cell immunoglobulin-and mucin-domain-containing molecule-3, Tim-3)是2002年发现的新型免疫调节分子,选择性表达在分化终末期的Th1细胞上。Tim-3与其配体galectin-9结合后负向调控Th1细胞功能,并诱导Th细胞凋亡,介导免疫耐受的形成。大量研究报道Tim-3通过调节适应性免疫反应在多种炎症性疾病中发挥重要作用。进一步研究发现Tim-3在多种固有免疫细胞,包括NK细胞、巨噬细胞和树突状细胞上均有表达,但Tim-3在固有免疫中的作用机制目前尚不清楚。近来研究报道Tim-3能够影响巨噬细胞和树突状细胞的功能,且在柯萨奇病毒感染诱导的心肌炎中发挥重要作用,这些信息提示Tim-3可能参与动脉粥样硬化的发生发展。本课题利用临床研究、体外细胞实验和动物实验研究Tim-3在动脉粥样硬化中的表达和功能,为进一步深入阐明动脉粥样硬化免疫调节机制和Tim-3在固有免疫中的作用提供实验依据,并为动脉粥样硬化的抗炎治疗提供新的靶点。第一部分Tim-3在动脉粥样硬化中的表达多种免疫细胞参与动脉粥样硬化发生,而Tim-3在固有免疫和适应性免疫细胞上均有表达。为了证实Tim-3在动脉粥样硬化中的作用,我们首先检测了其在人类和小鼠动脉粥样硬化病变个体中的表达。1.ApoE-／-小鼠体内各脏器及主动脉斑块中Tim-3表达显著高于对照鼠ApoE基因敲除小鼠(apolipoprotein E gene knockout, ApoE-/-)是动脉粥样硬化易感动物模型。为研究Tim-3在斑块内的表达情况,我们收集14周龄高脂饲养6周的ApoE-／-小鼠及其背景小鼠C57BL／6小鼠的新鲜脏器,抽提RNA,RT-PCR检测Tim-3表达。结果显示免疫细胞含量丰富的小鼠脾脏中Tim-3表达最高,并且ApoE-／-小鼠的多种脏器,包括心脏、肝脏、脾脏和主动脉中Tim-3mRNA的表达水平都较C57小鼠高。为了研究斑块中免疫细胞Tim-3的表达情况,取高脂饲养的ApoE-/-小鼠主动脉,冰冻切片后进行免疫组化染色,结果显示动脉粥样硬化早期斑块中的细胞几乎全部呈现阳性染色,Tim-3在早期斑块中的表达非常丰富。大量研究证实动脉粥样硬化早期斑块成分几乎只有巨噬细胞和T细胞,提示斑块中的免疫细胞高表达Tim-3。2.AS患者外周血单核细胞、NK细胞中Tim-3表达较健康志愿者显著升高,且与多种致病因素相关本研究收集经冠脉造影确诊存在冠脉粥样硬化的AS患者97例,其中稳定性心绞痛(stable angina pectoris, SAP)患者15例,不稳定型心绞痛患者(unstable angina pectoris, UAP)82例,健康志愿者51例为对照,不稳定心绞痛患者组含24例心肌梗死myocardial infarction, MI)患者。流式细胞术检测Tim-3在各淋巴细胞亚群中的表达。结果显示,Tim-3在外周血CD4+和CD8+T细胞上低水平表达,而在单核细胞和NK细胞上高水平表达。且冠心病患者单核和NK细胞上Tim-3表达水平显著高于健康志愿者(单核细胞,p<0.0001；NK细胞,p=0.0005),但冠心病患者CD4+和CD8+T细胞上Tim-3的表达同健康志愿者无显著性差异。动脉粥样硬化的发生是多因素共同作用的结果,临床资料分析结果显示单核细胞上Tim-3表达水平同多种致病因素呈现良好的相关性,包括甘油三酯(p=0.0320)和收缩压(p=0.0016)等,NK细胞上Tim-3表达水平也同甘油三酯(p=0.0121)和胆固醇(p=0.0220)正相关。2.动脉粥样硬化患者Tim-3表达与炎症水平密切相关ELISA检测结果显示AS患者血清学炎症指标包括肿瘤坏死因子α(tumer necrosis factor-α, TNF-α)和C-反应蛋白(C-reactive protein, CRP)较健康对照显著升高(CRP,p=0.0237；TNF-α=0.0235)。统计学分析发现单核细胞和NK细胞上Tim-3的表达水平与TNF-α及CRP正相关,提示Tim-3与AS病人体内炎症水平相关。与此相符,分析显示炎症水平较高的患者其单核细胞和NK细胞上Tim-3表达水平也相应较高,说明Tim-3同动脉粥样硬化患者炎症水平密切相关,可能参与动脉粥样硬化患者炎症过程。以上研究结果提示,Tim-3参与动脉粥样硬化病变,可能主要通过调控NK细胞和巨噬细胞发挥作用。第二部分Tim-3对单核／巨噬细胞的调控及其在动脉粥样硬化中作用单核／巨噬细胞是AS的重要效应细胞。单核细胞迁移至血管内膜以及单核细胞源性巨噬细胞摄取氧化低密度脂蛋白是动脉粥样硬化的关键性起始因素。临床研究显示AS患者外周血单核细胞Tim-3表达显著升高,且与多种致病因素相关,强烈提示单核／巨噬细胞表达的Tim-3参与AS发生。有研究显示Tim-3组成性表达于单核／巨噬细胞,促进巨噬细胞炎性反应。然而Tim-3是否参与AS发生中单核／巨噬细胞介导的炎症反应,Tim-3影响单核／巨噬细胞的哪种效应,目前尚未见报道。为了研究Tim-3对动脉粥样硬化单核／巨噬细胞的调控作用,本研究利用小鼠单核／巨噬细胞系RAW264.7和AS患者外周血单核细胞进行了一系列体外研究。1. LPS/ox-LDL活化的鼠单核／巨噬细胞系RAW264.7中Tim-3表达显著升高炎症介质脂多糖(lipopolysaccharide, LPS)可以有效活化巨噬细胞,而氧化低密度脂蛋白(oxidized-low density lipoprotein, ox-LDL)是介导巨噬细胞泡沫化的重要致动脉粥样硬化因子。免疫组化和实时定量PCR结果显示,LPS和ox-LDL刺激都能够显著上调小鼠单核／巨噬细胞系RAW264.7中Tim-3的表达,而LPS诱导效果较ox-LDL更为强烈,约为ox-LDL诱导效果的两倍。2.Tim-3对单核/巨噬细胞功能的调控作用外周血中的单核细胞释放一氧化氮(nitric oxide, NO)参与血管舒张和重构等,在动脉粥样硬化中起到积极的保护作用。但斑块中单核细胞来源的巨噬细胞吞噬斑块中的脂质成为荷脂细胞后,释放大量促炎因子TNF-α、IL-1和IL-6等介导斑块局部的炎症反应,促进斑块形成和破裂。为了研究Tim-3对单核／巨噬细胞功能的调节作用,本研究利用Tim-3表达质粒pc-DN A3-mouse-Tim-3,脂质体瞬式转染RAW264.7细胞,构建了Tim-3过表达细胞模型；同时利用Tim-3阻断型抗体预处理单核／巨噬细胞,阻断Tim-3通路,从正反两方面研究Tim-3对LPS/ox-LDL介导的单核／巨噬细胞分泌细胞因子分泌功能和NO功能的影响。2.1 Tim-3促进单核／巨噬细胞分泌TNF-α, IL-1β和IL-6等促炎因子巨噬细胞吞噬ox-LDL后活化并分泌各种促炎因子是AS发生的重要机制,因此,我们首先研究Tim-3对单核／巨噬细胞分泌TNF-α, IL-1β和IL-6的影响。ELISA和RT-PCR结果显示,阻断Tim-3明显抑制LPS或ox-LDL诱导的RAW264.7中TNF-α的产生,而Tim-3过表达则促进了TNF-a产生。实时定量RT-PCR结果显示Tim-3对RAW264.7细胞分泌白细胞介素-1β(interleukin-1β,IL-1β)和IL-6有相似的调控作用。为进一步研究Tim-3对AS发生中单核／巨噬细胞分泌炎性细胞因子的调控,本研究收集了4例AS患者外周血,磁珠分选AS患者外周血CD14+单核细胞,用Tim-3融合蛋白体外阻断Tim-3的表达后给予LPS或ox-LDL刺激,收集上清ELISA检测了TNF-α水平。结果显示,同细胞系结果一致,Tim-3阻断后LPS或ox-LDL诱导的单核细胞TNF-α的分泌呈现降低趋势。以上结果显示,Tim-3可以促进LPS/ox-LDL介导的单核／巨噬细胞分泌炎性细胞因子能力。2.2Tim-3抑制巨噬细胞NO分泌功能外周血单核／巨噬细胞受到各种刺激,合成诱生性一氧化氮合成酶(inducible nitric oxide synase, iNOS),分泌NO,参与血管舒张和重塑,是动脉粥样硬化的有效保护因子。本研究利用Griess法检测29例AS患者和16例健康志愿者血清NO水平。结果显示,AS患者血清NO与正常对照没有统计学差异,但是AS患者血清NO水平离散系数是健康志愿者的两倍,提示动脉粥样硬化患者体内NO水平可能处于不平衡状态。进一步分析发现单核细胞Tim-3表达水平同血清NO水平负相关。于是我们在RAW264.7细胞中阻断或过表达Tim-3后检测上清中NO的水平,同时RT-PCR检测细胞iNOS mRNA水平。结果显示,Tim-3过表达明显抑制RAW264.7中iNOS mRNA表达及NO分泌；而阻断Tim-3可以有效逆转上述结果。为了验证上述实验结果,收集AS患者外周血,磁珠分选获得CD14+单核细胞,用Tim-3融合蛋白体外阻断Tim-3的表达后,收集上清检测NO的水平。结果显示,同细胞系结果一致,Tim-3阻断后LPS或ox-LDL诱导的单核细胞NO的分泌呈现升高趋势。上述结果提示,Tim-3的确能够有效抑制LPS/ox-LDL诱导的单核／巨噬细胞NO的分泌。3.Tim-3调控单核／巨噬细胞的信号通路为了研究Tim-3调控单核／巨噬细胞功能的分子机制,本研究利用阻断性抗体阻断Tim-3信号后,western blot检测LPS刺激的RAW264.7细胞中NF-κB(nuclear factor-kappa B)通路和MAPK (Mitogen-activated protein kinase)通路中的关键分子。3.1 Tim-3阻断抑制NF-κB通路的活化LPS刺激经Tim-3阻断性抗体预处理的RAW264.7细胞后,分别在0、15、30min收集细胞,Western blot检测I-κB/NF-κB通路信号分子表达。结果显示LPS刺激15min和30min时,Tim-3阻断组NF-κB p65磷酸化较同型对照组明显减少,而LPS刺激15min时可以检测到I-κB的增加。可见Tim-3信号的阻断抑制了I-κB/NF-κB通路的活化。3.2Tim-3阻断促进MAPK通路的活化Tim-3阻断型抗体预处理后LPS刺激RAW264.7细胞,于相应时间点收集细胞,Western blot检测MAPK通路关键信号分子表达。结果显示：在5min、15min和30min时间点,Tim-3阻断组(aT3:anti-Tim-3)磷酸化的P38和磷酸化的c-JunN-terminal kinase (JNK)较同型对照组(IgG)增强,尤以磷酸化的P38更为显著,而磷酸化的ERK(extracellular regulatedprotein kinases))没有明显变化,可见Tim-3信号的阻断抑制了JNK通路和P38通路的活化。综合上述结果,当单核／巨噬细胞受到炎症介质或动脉粥样硬化致病因素刺激活化后Tim-3表达升高,上调的Tim-3能够促进单核／巨噬细胞促炎因子(如TNF-α、IL-1β和IL-6)的分泌而抑制保护性因子NO的分泌,因此初步推测,AS患者单核／巨噬细胞Tim-3表达的上调可能促进动脉粥样硬化的发生发展。第三部分Tim-3对NK细胞的调控及其在动脉粥样硬化中作用动物实验及临床检测证明NK细胞存在于斑块形成的各个时期,提示NK细胞在AS发生中的作用。多篇文献报道在AS患者外周血中NK细胞的活性和数量显著下降,然而其机制尚未阐明。已有研究发现Tim-3与其配体结合可以介导Thl细胞凋亡,抑制Th1应答,同时文献证实,Tim-3组成性高表达于NK细胞,但Tim-3对NK功能的影响迄今尚未见报道。本课题第一部分临床研究发现,AS患者外周血NK细胞Tim-3表达显著升高。为了研究Tim-3对NK细胞的调控作用,本部分利用人NK92细胞系和纯化的人外周血NK细胞,体外研究Tim-3过表达或Tim-3阻断对NK细胞活性和数量的影响。1.Tim-3抑制NK细胞活性冠心病患病危险度与病原感染相关,而且存在多重感染是冠心病患者发生心肌梗死的独立危险因素。NK细胞是人体第一道防线,在对病原体,尤其是病毒的防御中起到重要作用。NK主要通过细胞毒作用和分泌细胞因子参与免疫反应。同时多篇文献报道动脉粥样硬化中NK细胞介导的杀伤导致血管细胞损伤,可能促进了斑块的形成。因此NK细胞的杀伤功能对动脉粥样硬化起到多重影响。本研究用Tim-3融合蛋白阻断NK92细胞Tim-3信号通路后,以肝癌细胞系HepG2和乙肝病毒感染的肝癌细胞系HepG2.2.15为靶细胞,分别按效靶比1：1,5：1,25：1共孵育,4小时后CCK-8检测杀伤效率,研究Tim-3对NK杀伤功能的调控作用。结果显示在效靶比5：1和25：1组,Tim-3阻断后显著上调NK细胞对靶细胞的杀伤活性,同时ELISA检测显示Tim-3阻断后NK92细胞干扰素-γ(interferon-γ,IFN-γ)分泌增加。说明NK细胞上表达上调的Tim-3可能抑制其杀伤活性和细胞因子的分泌。2.Tim-3对NK细胞数量的调节2.1 AS患者NK细胞表达的Tim-3与外周循环NK细胞数量负相关流式检测结果证明AS患者外周血中NK细胞数量较健康志愿者显著下降,(p=0.0016),与文献报道一致,并且炎症水平较高的UAP患者NK细胞Tim-3表达水平较SAP患者有降低趋势(p=0.0580)。相关性分析发现在AS患者NK细胞的数量同该细胞Tim-3的表达水平负相关(r=-0.6424,p<0.0001),提示Tim-3可能是动脉粥样硬化患者外周血NK细胞缺失的原因。AS患者NK细胞数量减少可能是NK增殖功能抑制或凋亡敏感度增加所致。2.2 Tim-3抑制NK细胞增殖本研究首先利用过表达实验研究Tim-3对NK92细胞增殖的影响。Tim-3表达质粒pcDN A3-human-Tim-3及其对照质粒pcDNA3转染NK92细胞48小时后,苔盼蓝染色法显示两组细胞活细胞比率没有显著差异(p=0.7310),但Tim-3过表达组细胞数较对照组降低了约30%(p=0.0002),进一步细胞周期分析结果显示过表达组细胞G1期细胞比例显著升高,说明Tim-3过表达引起细胞G1期阻滞。上述结果提示,Tim-3在体外可以有效抑制NK92细胞的增殖。2.3 Tim-3促进多种因素诱导的NK细胞凋亡AS患者外周血中的多种因素,如氧化应激和多种细胞因子影响NK细胞的存活。文献报道NK细胞对氧化的脂质,如ox-LDL诱导的凋亡非常敏感。本研究利用Tim-3融合蛋白阻断NK92细胞Tim-3信号通路后,流式检测ox-LDL诱导的凋亡。结果显示Tim-3阻断能够有效减少ox-LDL诱导的NK92细胞的凋亡。(p=0.0078)本研究第一部分临床研究证实AS患者血清TNF-α显著升高,且血清TNF-α水平同NK细胞Tim-3表达水平正相关。进一步的分析发现血清TNF-α水平同NK细胞数量负相关(pearson r=-0.3383,p=0.0230)已有研究证实,TNF-α在NK细胞凋亡中发挥着重要的作用。为研究Tim-3是否参与TNF-α介导的NK细胞凋亡,本研究利用Tim-3融合蛋白阻断NK92细胞Tim-3信号通路后,流式检测TNF-α诱导的凋亡。结果显示Tim-3阻断显著延缓了细胞的死亡(p<0.0001)。用磁珠分选纯化的新鲜外周血NK细胞重复上述实验,结果显示Tim-3阻断能够减少TNF-α诱导的AS患者NK细胞的凋亡(p=0.0165),但对健康志愿者的NK细胞没有明显影响(p=0.5958)。提示,Tim-3可能通过影响检测TNF-α介导的NK凋亡而调控动脉粥样硬化病人体内NK数目。2.4 AS患者外周血PBMC中galectin-9的表达同NK细胞的数量和NK细胞上Tim-3的表达都没有相关性Tim-3同其配体glalectin-9的结合能够诱导Th1细胞的凋亡,为了研究galectin-9在Tim-3对NK细胞凋亡的调节中是否发挥作用,本研究收集21例AS患者和22例健康志愿者外周血PBMC, RT-PCR检测galectin-9 mRNA的表达。结果显示galectin-9在AS患者PBMC中的表达同健康志愿者没有统计学差异(p=0.7339)。进一步的分析显示PBMC中galectin-9的表达同NK细胞的数量(p=0.0746)和NK细胞上Tim-3的表达(p=0.2591)都没有相关性。综合上述结果,体外研究证实Tim-3能够上调NK细胞杀伤活性,并通过抑制NK细胞增殖和促进NK细胞凋亡两个途径导致NK细胞数量的减少,这可能是AS患者外周循环中NK细胞活性降低和数量减少的重要机制。但是NK细胞在动脉粥样硬化中的作用目前还不明确,多篇文献认为NK细胞对病原体感染的清除对动脉粥样硬化起到保护性作用,但也有研究报道NK细胞介导的杀伤导致血管细胞损伤,同时NK细胞在不同的动物模型中对动脉粥样硬化的影响结论不同,因此NK细胞在动脉粥样硬化中的作用尚需进一步研究。第四部分Tim-3对ApoE-／-小鼠斑块形成影响的体内研究临床标本检测结果显示Tim-3在AS患者单核细胞和NK细胞上表达上调,体外细胞功能研究证实Tim-3能够促进单核／巨噬细胞介导的炎症反应并抑制其NO的分泌,同时Tim-3对NK细胞也具有重要的调节作用。为了验证Tim-3表达上调及其对细胞功能的调节在体内对动脉粥样硬化发生发展的影响,我们利用ApoE-/-小鼠,抑制其体内Tim-3的表达后,观察了主动脉斑块的形成情况。1. lentivirus-shRNA-Tim-3有效抑制Tim-3基因的表达我们首先在体外用lentivirus-shRNA-Tim-3及其对照病毒lentivirus-shRNA-Tim-3分别感染RAW264.7细胞,实时定量结果显示lentivirus-shRNA-Tim-3感染组RAW264.7细胞Tim-3表达水平较对照病毒感染细胞下调约70%(p<0.0001)。Western Blot检测结果显示lentivirus-shRNA-Tim-3病毒感染能够显著抑制RAW264.7细胞Tim-3蛋白水平的表达。然后我们进一步检测了病毒在体内的抑制效果。我们选取了8-10周龄,体重在18～22g的雄性ApoE-／-小鼠,高脂饲养两周后将lentivirus-shRNA-Tim-3及其对照病毒lentivirus-shRNA-Tim-3分别尾静脉注射ApoE-/-小鼠。高脂饲养8周后处死小鼠,取材检测分析。RT-PCR结果显示尾静脉注射lentivirus-shRNA-Tim-3病毒的ApoE-/-小鼠各脏器包括心脏、脾脏和肝脏内Tim-3表达较注射对照病毒的小鼠均显著抑制。2.Tim-3基因沉默显著减少动脉粥样硬化斑块形成上述小鼠在注射慢病毒后0周、4周和8周称重,并在处死前取血清。小鼠体重数据显示各时间点两组小鼠体重无统计学差异,同时血清学检测显示两组小鼠TG也无统计学差异。分离主动脉,油红O染色显示lentivirus-shRNA-Tim-3组小鼠斑块形成较对照病毒组小鼠显著减少。上述结果证实,Tim-3基因沉默在不明显影响小鼠生理状态的情况下能显著减少动脉粥样硬化病变,提示Tim-3作为动脉粥样硬化治疗靶点的重要价值。综上所述,本研究首次证实Tim-3在AS患者外周血单核／巨噬细胞和NK细胞上表达上调,并通过基因转染和抗体阻断技术,进一步研究了Tim-3对单核／巨噬细胞和NK细胞的调控作用,发现,Tim-3不仅促进单核／巨噬细胞释放促炎因子、抑制保护性NO产生,并且参与AS患者体内NK细胞丢失,体内实验证实,抑制Tim-3表达,可以显著减少E-／-小鼠体内斑块形成。这些研究结果为了解动脉粥样硬化发病机制以及阐明Tim-3在固有免疫中的作用奠定了良好的基础,为动脉粥样硬化的治疗提供了良好的靶点。更多还原

【Abstract】 Atherosclerosis with its clinical manifestations such as coronary artery disease (CAD), is an underlying cause of myocardial infarction (MI), stroke, and other cardiovascular diseases and has become the leading cause of death worldwide. Emerging evidences support the concept that atherosclerosis is a chronic inflammatory disease, in which innate and adaptive immunity operate together to influence lesion progress. Lymphocytes and monocytes are well known major immune cells responsible for atherosclerosis development. Besides these major cells, there are also many other immune cells, such as dentrtic cells (DCs), Natural killer (NK) cells and mast cells, which are becoming increasingly important in understanding the complexity of this disease process. However, sufficient knowledge to identify the molecules involved in atherosclerosis and develop targeted immunomodulatory strategies has not yet been obtained.T cell immunoglobulin-and mucin-domain-containing molecule-3 (Tim-3), originally identified as a Th1-specific cell surface molecule, has received much attention as negative regulator of Th1 mediated immune response. Engagement with its ligand galectin-9, Tim-3 induces Th1 cells apoptosis and regulates Th1/Th17 cytokine profiles which involve in various inflammatory diseases. Increased body of data have shown that Tim-3 also express abundantly on innate immune cells such as NK cells, macrophages and DCs. However, the effects of Tim-3 on innate immunity remain controversial. The precise roles and exact mechanisms of Tim-3 on innate immunity needs further study. Concerning the critical roles of Tim-3 on T cells and macrophage, the two major cells in atherogenesis, it is reasonable to suppose that Tim-3 may involve in atherosclerosis.In this study, we showed evidence that Tim-3 is upregulated on monocytes and NK cells in AS patients, then studied its roles in the regulation of monocytes/macrophages and NK cells, and verified the effect of Tim-3 on plaque formation in ApoE knockout (apolipoprotein E gene knockout, ApoE-/-) mice. Our work thus provides new insights into mechanism of innate immune regulation by Tim-3 in atherogenesis and also justified the possibility that Tim-3 could serve as a potential target for drug design.PART I The Expression of Tim-3 in AtherosclerosisTim-3 expressed on both adaptive and innate immune cells which were involved in atherosclerosis. In order to evaluate the involvement of Tim-3 in atherogenesis, we first detected the expression of Tim-3 in AS patients and ApoE-/- mice.1. Tim-3 expression is upregulated in organs and aorta plaques from ApoE-/-mouseWe next examined Tim-3 expression in organs and plaques from 14-weeks-old ApoE-/- mice and C57BL/6 mice, which were fed with high diet for 6 weeks. RT-PCR showed higher expression of Tim-3 mRNA in spleens, which contain abundant immune cells. Augment expression was detected in organs of ApoE-/- mice, including heart, liver, spleen and aorta, compared to C57 mice.Tim-3 positive staining was detected in aorta plaques of early stage by immunohistochemistry. It is reported that plaque of early stage purely contains macrophages and T cells. Our results showed that the cells in plaque of early stage were almost all positively stained, suggesting immune cells in plaques expressed Tim-3.2. Tim-3 expression is upregulated on monocytes and NK cells from AS patients and correlated with many etiological and aggravating elements of atherogenesisBlood samples from 97 AS patients, including 24 stable angina pectoris (SAP) patients and 73 unstable angina pectoris patients, which are angiographically documented atherosclerosis, and 51 healthy subjects were collected. There were 24 myocardial infarction patients in the UAP group. Flow cytometry analysis showed that Tim-3 expression was high on CD14+ monocytes and CD16/56+ NK cells, but low on both CD4+ and CD8+ T cells in human PBMCs. Further analysis showed significantly higher expression of Tim-3 on monocytes or NK cells from atherosclerotic patients than that of healthy controls. (Monocytes, p＜0.0001; NK cells,p=0.0005) No significant differences of Tim-3 expression were observed on either CD4+ or CD8+ T cells between the two groups. Atherosclerosis is a multifactor, highly complex disease, in which many etiological and aggravating elements operate together to influence lesion progress. Analysis showed Tim-3 expression on monocytes was correlated well with triglyceride (TG,p=0.0320) and systolic pressure (p=0.0016), while Tim-3 expression on NK cells was positively correlated with TG (p=0.0121) and cholesterol (Cho,p=0.0220)2. The correlation of Tim-3 with inflammation in atherosclerosisEnzyme-linked immunosorbent assay (ELISA) detection showed serum level of C-reactive protein (CRP) and tumor necrosis factor (TNF)-α, the two inflammatory markers, were much higher in AS patients, compared to healthy subjects. (CRP, p= 0.0237; TNF-a, p=0.0235) And Tim-3 is positively correlated with both of them. Further more, Tim-3 expression is higher in patients with more severe inflammation. These results confirmed that Tim-3 positively correlated with inflammatory severity in AS patients.All above results indicated that Tim-3 is involved in atherogenesis through regulating monocytes/macrophages and NK cells.PART II The Regulation of Tim-3 on Monocyts/Macrophages in AtherosclerosisMonocytes/macrophages is one of the most important effector cells in atherogenesis. Recruitment of monocytes from peripheral blood to the intima of the vessel wall is a primordial event in atherogenesis. The results in Part I showed Tim-3 expression was upregulated on monocytes from AS patients and correlated with not only etiological and aggravating elements but also inflammatory severity, strongly supporting the idea that Tim-3 is involved in atherosclerosis. Constitutive expression of Tim-3 was already found on monocytes/macrophages, but the influence of Tim-3 on monocytes/macrophage is still not clear. To reveal the role of Tim-3 on monocytes/macrophages functions, we carried on a series of assay in vitro using the mouse monocytes/macrophages cell line RAW264.7 and purifed peripheral blood monocytes from atherosclerosis patients.1. LPS/ox-LDL induces augment expression of Tim-3 in mouse monocytes/macrophages cell line RAW264.7Lipopolysaccharide (LPS) is recognized as a strong inflammatory inducer, and oxidized low density lipoprotein (ox-LDL) is a etiological and aggravating element of atherosclerosis. Immunocytochemical staining and real-time PCR both showed upregulation of Tim-3 expression on RAW264.7 induced by either LPS or ox-LDL. LPS treatment induced higher expression of Tim-3, approximately twice as much as ox-LDL.2. The regulation of Tim-3 on cell functions in RAW264.7 cellsPeripheral monocytes secrets nitric oxide (NO), a critically important protective signaling molecule of atherosclerosis which clearly has a critical role in the maintenance and repair of the vasculature. After migrating into the vessel wall, monocyte-derived macrophages are turned into fat-loaded macrophages by taking up modified, oxidized or acetylated LDL, residing in the vessel wall and furthering the local inflammatory response by secretion of proinflammatory cytokines, such as TNF-α, interleukin (IL)-1βand IL-6. To further demonstrate the view that whether Tim-3 regulates NO production and proinflammation cytokines secretion in monocytes/macrophages, anti-mouse Tim-3 antibody/Tim-3-Fc (blocking/neutralizing) incubation or pcDNA3-Tim-3 plasmid DNA transfection were introduced in cultured RAW264.7 cells or purified peripheral monocytes before stimulation with LPS or ox-LDL. Supernant were collected for ELISA and Griess assays and cells were used for RNA extraction and RT-PCR analysis.2.1 Tim-3 promotes secretion of proinflammatory cytokines in monocytes/macrophagesELISA and RT-PCR showed that compared to isotype controls, pretreatment of blocking anti-Tim-3 led to decrease of TNF-a production in RAW264.7 cells with or without LPS or ox-LDL treatment. Consistently, a increase was detected in pcDNA3-Tim-3-transfected RAW264.7 cells. Real-time PCR showed the similar effect of Tim-3 on production of IL-1βand IL-6.To further confirm the results in human cells, monocytes were freshly purified from PBMCs of atherosclerosis patients by positive selection using CD14+ microbeads. Cells were blocked using human Tim-3-Fc fusion proteins and then stimulated with LPS or ox-LDL. ELISA. Results showed that Tim-3 blocking decreased TNF-αsecretion, consisting with the results in RAW264.7 cells.These results indicate that induced by LPS or ox-LDL, Tim-3 promotes monocytes/macrophages mediated inflammation.2.2 Tim-3 inhibits NO secretion in monocytes/macrophages Results of colorimetric assay using Griess reagent showed plasma NO levels had no significant differences between 29 AS patients and 16 healthy subject, but NO level in AS patients had increased coefficient of variance (AS vs NOR,1.41 vs 0.74), indicating an imbalance of NO in AS patients. Further more, NO level in AS patients was positively correlated with Tim-3 expression on monocytes. An increase in NO production was detected in anti-Tim-3-treated RAW264.7 cells induced by either LPS or ox-LDL, compared to controls. Consistently, a decrease was detected in pcDNA3-Tim-3-transfected RAW264.7 cells. When induced nitric oxide synthase (iNOS) was analyzed by RT-PCR, similar results were got.To further confirm the results on human cells, monocytes were freshly purified from PBMCs of atherosclerosis patients by positive selection using CD14+ microbeads. Cells were blocked using human Tim-3-Fc fusion proteins and then stimulated with LPS or ox-LDL. The supernatant were collected for NO detection. Results showed that Tim-3 blocking increased NO secretion, consisting with the results in RAW264.7 cells.These results indicate that induced by LPS or ox-LDL, Tim-3 inhibits NO secretion in monocytes/macrophages.3. The signaling pathway in Tim-3-mediated monocytes/macrophages regulationTo study the signaling pathways in Tim-3-mediated monocytes/macrophages regulation, Tim-3 blocking antibody pretreated RAW264.7 were stimulated with LPS and western blot was used to detect expression of critical molecules in I-κB/NF-κB pathway and MAPK(Mitogen-activated protein kinase) pathway, the two classical pathways involved in cytokine and NO secretion.3.1 Tim-3 blockade inhibits I-κB/NF-κB pathway.Tim-3 blockade RAW264.7 cell were stimulated with LPS, and cells were collected at indicated time points. Western Blot showed enhanced phosphorylation of NF-κB p65 at 15min and 30min time points, and degradation of I-κB at 15min time piont in Tim-3 blockade group, compared to the control group, suggesting Tim-3 blockade inhibited I-κB/NF-κB pathway.3.2 Tim-3 blockade inhibits MAPK pathway.Tim-3 blockade RAW264.7 cell were stimulated with LPS, and cells were collected at indicated time points. Western Blot showed enhanced phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular regulatedprotein kinase (ERK) at 5min,15min and 30min time points in Tim-3 blockade group, compared to the control group, suggesting Tim-3 blockade inhibited MAPK pathway.Taken together, Tim-3 expression on monocytes were upregulated by etiological factor of atherosclerosis or mediators of inflammation. The upregulated Tim-3 in turn promotes the production of proinflammatory cytokines and inhibits NO secretion of monocytes. Thus, the increased Tim-3 expression on monocytes from AS patients might promotes atherogenesis.PARTⅢThe Regulation of Tim-3 on NK Cells in AtherosclerosisNatural killer cells have been found in every stages of plaques development, and has been shown to be reduced with a concomitant loss in function in CAD. However, the mechanisms remain unclear. It is reported that Tim-3 induced Thl cell apoptosis by binding with its ligand galectin-9, indicating the mechanism of Tim-3 in cell loss. Since NK cells were recently found to be one of the major cellular sources of Tim-3, we here investigated the expression of Tim-3 on NK cells and assessed its possible roles in NK cells in AS.1. Tim-3 inhibits NK cell activityPathogen infection is related to the risk of AS, and has become the independent risk factor of myocardial infarction. As the first line of the body, NK cells play important roles in defense against pathogens, especially virus, and display their functions by mediating cytotoxicity as well as augmenting immune responses as a result of cytokines production. However, many studies reported NK mediated cytotoxicity damaged vessel cells, and promotes atherogenesis. Thus, the effects of NK mediated cytotoxicity on atherogenesis are multiple. In order to analyze the role of Tim-3 on NK cytotoxicity, Tim-3-Fc fusion proteins were involved in cytotoxic assay in which NK92 cells as effectors and hepatic cell line HepG2 or hepatitis B virus infected HepG2.2.15 as targets. Cell Counting Kit-8 (CCK-8) assay showed that pre-incubation with Tim-3-Fc fusion proteins resulted in an increase in NK92 cytotoxicity and ELISA showed augment production of interferon(IFN)-y, suggesting that Tim-3 could suppress NK-cell functions.2. The regulation of Tim-3 on NK cell loss2.1 Tim-3 expression is negatively correlated with peripheral NK cell quantityConsisting to previous reports, our flow cytometry analysis showed reduced NK cell number in patients with AS compared with healthy controls, (p=0.0016) and significant reverse correlation between Tim-3 expression with NK cell number was found in all AS patients (r=-0.6424,p＜0.0001).2.2 Tim-3 inhibits NK cell proliferationTo verify the correlation between NK cell quantity and Tim-3 expression, we first detected the effect of Tim-3 on NK cell proliferation. We preformed Tim-3 overexpression in cultured NK92 cells by transfecting pcDNA3-human-Tim-3. Results showed Tim-3 overexpression decreased NK92 cell number significantly (p=0.0002), while the percentage of viable cells had no difference between the two group(p=0.7310). Cell cycle analysis revealed the accumulation in G1 phase. These results demonstrated that Tim-3 inhibited NK cells proliferation in vitro.2.3 Tim-3 promotes NK apoptosisThere are many factors influencing the survival of NK cells in AS, among which oxidative stress is particular important. Oxidation of circulating lipids is an important source of oxidative stress. Previous study reported that the susceptibility of NK cells to ox-LDL contribute to the reduced NK cell activity in AS. To verify whether Tim-3 is involved in ox-LDL induced NK cell loss in AS, we blocked Tim-3 pathway with human Tim-3-Fc in NK92 cells treated with ox-LDL. Results showed Tim-3 blockade lead to significant inhibition of ox-LDL-mediated cell apoptosis, (p=0.0078) suggesting Tim-3 might participate in oxidative stress-mediated NK cell loss in AS.Besides ox-LDL, TNF-αis one of cytokines produced by NK cells which plays important role in induction of NK cell apoptosis. Consistent with previous report, our ELISA results showed increased serum TNF-αin patients with AS. Further analysis showed serum TNF-αnegatively correlated with peripheral NK cell percentage (pearson r=-0.3383,p=0.0230). Blocking Tim-3 with human Tim-3-Fc fusion proteins effectively delayed TNF-αinduced NK92 cell death (p＜0.0001). Peripheral NK cells from fresh blood was isolated, and used for the same assay. Results showed Tim-3 blockade decreased TNF-a induced apoptosis of NK cell from AS patients(p=0.0165), but not from healthy subjects(p=0.5958).2.4 Galectin-9 expression of PBMC from AS patients has no correlation with either NK cell quantity or Tim-3 expression on NK cell.PBMCs were isolated from 21 AS patients and 22 healthy subjects. PT-PCR results showed galectin-9 expression had no significant differences between AS patients and healthy subjects. Further analysis showed no correlation with either NK cell quantity or Tim-3 expression on NK cell. Taken together, our data indicated Tim-3 might involve in atherogenesis by inhibiting cytotoxic activity and decreasing cell number through regulating proliferation and apoptosis in NK cells. However, the exact role of NK cells in atherogenesis is still controversial. The studies in different animal models led to different conclusions about depletion of NK cells activity and plaque formation. Thus what’s the role NK cell played in atherosclerosis needs further study.PARTⅣEffect of Tim-3 on Plaque Formation in ApoE-/- miceTo verify the effect of Tim-3 on plaque formation in vivo, we inhibited Tim-3 expression in ApoE-/- mice and observed the plaque formation in the aorta.1. lentivirus-shRNA-Tim-3 effectively knockdown Tim-3 expressionRAW264.7 cells were infected with lentivirus-shRNA-Tim-3, lentivirus-shRNA-NS as control. RT-PCR and western blot showed that Tim-3 expression in lentivirus-shRNA-Tim-3 infected cells was decrease approximately 70%, compared to control cells. We next proved the effect in vivo.8-10 weeks old, 18-22g male ApoE-/- mice were fed with high diet for two weeks before injected with lentivirus-shRNA-Tim-3 or lentivirus-shRNA-NS through tail vein. After high fat diet for two months, mice were sacrificed and dissected. RT-PCR showed Tim-3 expression in organs, including heart, spleen and liver, from the ApoE-/- mice injected with lentivirus-shRNA-Tim-3 was much lower than that of control mice.2. Tim-3 gene knockdown decreased plaque formationThese above mice were weighted up at different time points and serum was acquired before sacrificed. The weight and serum TG in the two groups had no statistical difference. We further analyzed aorta plaque formation. Aortas were obtained and stained with Oil red O. Significant decrease of lesion was detected in aorta from lentivirus-shRNA-Tim-3 mice, compared to lentivirus-shRNA-NS mice. These results demonstrated that Tim-3 alleviated atherogenesis while didn’t influence body weight and serum lipid level.In summary, this study firstly revealed Tim-3 was upregulated in monocytes and NK cells in AS patients. The augment expression of Tim-3 is positively correlated with inflammation severity in dieases. Blocking assay and overexpression assay showed that Tim-3 promotes proinflamatory cytokines production and inhibits NO production in monocytes/macrophages which are benefit to plague formation. Tim-3 expressed on NK cells suppress NK proliferation and enhance ox-LDL/TNF-a mediated NK apoptosis. Knockdown of Tim-3 in vivo decrease plaque formation in ApoE-/-mice. Our work contributed to the mechanism of immune regulation in atherogenesis and confirmed the idea that Tim-3 could serve as a potential target for drug design for atherosclerosis.更多还原