Th17细胞及效应分子IL-17A在动脉粥样硬化发生过程中的作用及机制研究
|
摘要
目的
动脉粥样硬化是发生于大中动脉的慢性炎症性病变,以大量脂质沉积到血管壁为主要特点,并伴随多种免疫细胞浸润及平滑肌细胞增生,其所诱发的心脑血管疾病是发达国家及发展中国家常见的死亡原因之一。目前研究认为固有免疫应答(巨噬细胞)和适应性免疫应答(CD4+T细胞)均参与动脉粥样硬化的发生和发展,近年来对于CD4+T细胞在该疾病中的作用受到了广泛的关注。CD4+T细胞分为多种亚群,主要包括Th1、Th2和调节性T细胞(regulatory T cells, Treg)以及近年来发现的Thl7细胞。Thl细胞促进动脉粥样硬化的发生,Th2细胞在该疾病中的作用还不是非常明确,尚存在争议。调节性T细胞在动脉粥样硬化中主要发挥了保护性的作用,能够抑制动脉粥样硬化斑块的形成。然而,Th17在动脉粥样硬化中的作用尚不明确。
Th17细胞是以分泌IL-17A为主要特征的CD4+T细胞群体,CD4+初始T细胞受抗原刺激后,在转录因子RORγt和RORα的调控下,IL-6和TGF-β联合作用即可促使初始T细胞分化成为Th17细胞。目前的研究认为Th17细胞及IL-17A参与多种自身免疫性疾病和炎症性疾病的发生,例如：自身反应性脑脊髓炎(EAE)、类风湿性关节炎(RA)和结肠炎等,动脉粥样硬化是发生在动脉血管壁的慢性炎症性疾病,前期有研究显示急性冠状动脉综合症患者外周血中Th17细胞的比例及IL-17A的水平较健康人明显升高,而且在人的动脉粥样硬化斑块局部检测到IL-17A的存在,这就提示Th17细胞及IL-17A很有可能参与该疾病的发生和发展。但是,Th17细胞及其效应分子IL-17A在动脉粥样硬化模型小鼠的发生和发展中变化如何?对动脉粥样硬化斑块的形成究竟发挥什么样的作用,尚存在争议。IL-17A能否作为动脉粥样硬化的治疗靶点之一?IL-17A在该疾病中的作用机制是什么?目前这些问题尚不清楚,都非常值得研究和探索。
为了明确Th17细胞及IL-17A在动脉粥样硬化发生发展中的作用,阐明其发挥作用的机制,为IL-17A的临床应用提供理论依据和实验基础,本论文拟从以下两部分进行研究：
一、通过体内外实验确定Th17细胞及其主要效应分子IL-17A在动脉粥样硬化发展中的促进作用：
(一)标准动脉粥样硬化小鼠模型和快速颈动脉损伤小鼠模型的建立：通过高脂饮食喂养建立标准动脉粥样硬化小鼠模型；高脂喂养联合颈动脉缩窄手术模拟血管狭窄,建立快速颈动脉粥样硬化小鼠模型,便于进行下一步研究。
(二)Th17细胞及IL-17A在动脉粥样硬化斑块局部及外周免疫器官的表达：通过多种方法检测动脉粥样硬化斑块局部存在IL-17A及转录因子RORγt的表达以及Th17细胞的存在；流式细胞术检测动脉粥样硬化发生过程中,Th17细胞的比例及IL-17A在脾脏中呈现明显上升的趋势,而且Th17细胞的比例与动脉粥样硬化的发病程度以及血脂水平(TCH)具有明显的正相关。
(三)Th17细胞及IL-17A对动脉粥样硬化斑块形成的影响：采用IL-17A中和性抗体或小鼠重组IL-17A对小鼠进行体内干预,从正反两个方面证实IL-17A能够促进动脉粥样硬化斑块的形成,并且发现IL-17A能够减少斑块局部平滑肌细胞的含量,提示IL-17A还能够促进斑块向不稳定方向发展。
二、从巨噬细胞内质网应激、细胞凋亡入手探讨IL-17A促进动脉粥样硬化发展的分子机制
(一)IL-17A对巨噬细胞凋亡的影响及作用机制：IL-17A在体外能够促进巨噬细胞的凋亡,进一步研究发现IL-17A在体外能够诱导小鼠及人来源的巨噬细胞发生内质网应激,而且在体内IL-17A也能够诱导斑块局部巨噬细胞发生内质网应激。体内用化学伴侣PBA干预小鼠能够缓解IL-17A诱导的巨噬细胞内质网应激,斑块局部凋亡的巨噬细胞亦明显减少,说明IL-17A能够通过诱导巨噬细胞内质网应激从而促进巨噬细胞凋亡。
(二)IL-17A加剧动脉粥样硬化的发生机制：IL-17A单独作用及IL-17A联合PBA体内干预,发现IL-17A能够通过促进斑块局部巨噬细胞发生内质网应激进而导致巨噬细胞凋亡发挥加速动脉粥样硬化斑块形成的作用。
(三)IL-17A诱导巨噬细胞内质网应激的机制：IL-17A能够上调巨噬细胞内脂质伴侣aP2的表达,而且IL-17A能够激活巨噬细胞NF-κB和MAPK通路(ERK, p38和JNK),进一步研究发现IL-17A是通过激活NF-κB通路和p38.ERK通路上调aP2的表达。最后我们采用aP2抑制剂证实IL-17A是通过aP2的上调诱导巨噬细胞内质网应激的发生。
方法
一、通过体内外实验确定Th17和其主要效应分子IL-17A在动脉粥样硬化发展中的促进作用
(一)标准动脉粥样硬化小鼠模型和快速颈动脉损伤小鼠模型的建立：
1.标准模型的建立：ApoE-/-雄性8周龄小鼠给予高脂饮食到小鼠16周和24周,同品系的C57BL/6野生型小鼠同时给予高脂喂养作为对照组。
2.快速颈动脉损伤模型的建立：ApoE-/-雄性8周龄小鼠给予高脂饮食两周,即小鼠10周时行左侧颈动脉缩窄手术模拟血管狭窄,小鼠继续给予高脂饮食分别至16周和24周,同品系的C57BL/6野生型小鼠作为对照组。
(二)Th17细胞及IL-17A在动脉粥样硬化斑块局部及外周免疫器官中的表达
1.取小鼠主动脉根部和左侧颈动脉制备连续冰冻切片,HE染色和油红O染色检测斑块的形成和脂质沉积情况；免疫组织化学染色检测斑块局部IL-17A和IFN-γ表达,并用免疫荧光双染的方法检测IL-17A的细胞来源。
2.取小鼠主动脉弓部抽提RNA,RT-PCR方法检测IL-17A,RORγt和IFN-γ,T-bet在斑块局部的表达。
3.取小鼠脾脏制备脾细胞悬液,采用流式细胞术检测Th1和Th17细胞的比例。
4.分析Th1和Th17细胞比例与动脉粥样硬化斑块大小的相关性以及与小鼠血
脂水平的相关性。
(三)Th17细胞及IL-17A对动脉粥样硬化斑块形成的影响
1.中和性IL-17A抗体干预
(1)ApoE-/-雄性8周龄小鼠20只给予高脂饮食两周,小鼠10周行左侧颈动脉缩窄手术,术后一周小鼠随机分为两组,实验组小鼠腹腔注射小鼠中和性IL-17A抗体(50μg/只),对照组小鼠腹腔注射同型对照。
(2)干预结束后一周微型超声观察斑块形成情况,取小鼠左侧颈动脉和主动脉根部制备连续冰冻切片,油红O染色检测斑块大小。
(3)免疫组化检测斑块局部巨噬细胞和平滑肌细胞的表达。
2.外源性小鼠IL-17A干预
(1) ApoE-/-雄性8周龄小鼠10只随机分为两组给予高脂饮食,小鼠11周时,实验组小鼠腹腔注射外源性小鼠重组IL-17A (2μg/只/次),对照组小鼠腹腔注射生理盐水。
(2)干预结束后一周,取小鼠主动脉根部制备连续冰冻切片,油红O染色检测斑块大小。
(3)免疫组化检测斑块局部巨噬细胞和平滑肌细胞的表达。
二、从巨噬细胞内质网应激、细胞凋亡入手探讨IL-17A促进动脉粥样硬化发展的分子机制
(一)IL-17A对巨噬细胞凋亡的影响及作用机制以及促进动脉粥样硬化斑块形成的机制
1.体外实验
(1)取小鼠腹腔巨噬细胞,加入IL-17A刺激后,Hoechst染色从细胞形态学上观察细胞凋亡,流式细胞术(Annexin V/PI染色)检测细胞凋亡的数量。
(2)小鼠巨噬细胞系RAW264.7及小鼠腹腔巨噬细胞,IL-17A刺激后收取细胞蛋白,Western Blotting检测内质网应激标志分子p-PERK, p-eIF2a和XBP1s的表达。
(3)人单核细胞来源细胞系Thp-1和人外周血单个核细胞,体外诱导成巨噬细胞,IL-17A刺激后收取细胞蛋白,Western Blotting检测内质网应激标志分子p-PERK, p-eIF2a和XBP1s的表达。
(4)小鼠腹腔巨噬细胞,加入IL-17A刺激后,Western Blotting检测(?)CHOP, caspase12和caspase3剪切体的表达。
2.体内试验
(1) ApoE-/-雄性8周龄小鼠30只给予高脂饮食,小鼠11周时随机分为三组,实验组其中一组给予腹腔注射小鼠重组IL-17A (2μg/只/次,每周一次),另外一组腹腔注射IL-17A(2μg/只/次,每周一次)和化学伴侣PBA(100mg/kg,每周两次),对照组小鼠腹腔注射生理盐水。
(2)干预结束后一周,取小鼠主动脉根部制备连续冰冻切片,免疫组织化学方法检测斑块局部Moma-2(巨噬细胞)、p-PERK和p-eIF2a的表达以及CHOP和caspasel2的表达；Tunnel染色检测斑块局部巨噬细胞凋亡。取小鼠主动脉弓部抽提蛋白,Western Blotting检测内质网应激标志p-PERK, p-eIF2α和XBP1s的表达以及CHOP, caspase12和caspase3剪切体的表达。
(3)油红O染色检测斑块大小。
(二)IL-17A诱导巨噬细胞内质网应激的机制
1.小鼠腹腔巨噬细胞,IL-17A刺激后RT-PCR和Western Blotting检测aP2的表达。
2.小鼠腹腔巨噬细胞在IL-17A作用前加入aP2抑制剂(BMS309403)刺激3小时,收取细胞蛋白,Western Blotting检测(?)p-eIF2α和XBPls的表达。
3.小鼠腹腔巨噬细胞,IL-17A刺激后Western Blotting检测(?)p-IκB, p-ERK, p-p38和p-JNK的表达；IL-17A刺激前分别加入NF-κB (PDTC), ERK (PD98059), JNK (SP600125)和p38(SB203580)抑制剂作用1小时,然后用Western Blotting方法检测aP2的表达。
结果一、通过体内外实验证实Th17及效应分子IL-17A能够促进动脉粥样硬化的发生(一)Th17细胞及IL-17A在动脉粥样硬化斑块局部及外周免疫器官中的表达
1.动脉粥样硬化斑块局部IL-17A和IFN-γ的表达
为了研究Th17细胞和IL-17A在动脉粥样硬化发生中的作用,我们利用ApoE-/-雄性小鼠建立了快速颈动脉粥样硬化模型和标准动脉粥样硬化小鼠模型,取小鼠左侧颈动脉和主动脉根部制备连续冰冻切片并进行HE染色和免疫组化染色。结果发现,ApoE-/-小鼠8周时,血管壁仅出现内膜略微增厚没有明显斑块的形成,此时在斑块局部几乎检测不到IL-17A和IFN-γ。高脂喂养到小鼠16周时,血管内膜有明显的增厚,平滑肌大量增生,形成较厚的纤维帽,大量有核细胞浸润到斑块局部,其斑块结构和组成属于早期动脉粥样硬化斑块,此时斑块局部能够检测到IL-17A和IFN-γ的表达,而对照组小鼠没有明显的斑块形成也检测不到IL-17A和IFN-γ的表达。小鼠继续高脂喂养到24周时,斑块大小较16周增加,纤维帽明显变薄,斑块表面仅有很薄的内皮细胞覆盖,平滑肌细胞减少,斑块内呈现多个空泡样的结构即坏死核,此时的斑块属于动脉粥样硬化晚期斑块,同龄的野生型C57BL/6小鼠此时也没有明显的斑块形成,免疫组化在斑块局部仅能检测到IL-17A,几乎检测不到IFN-γ的存在。
此外,我们还采用RT-PCR检测了主动脉弓部IL-17A和IFN-γ及转录因子RORγt和T-bet的表达,与免疫组化结果一致,在早期斑块和晚期斑块局部均能够检测到IL-17A的表达,且较对照组小鼠明显升高；但是IFN-γ仅在早期斑块局部能够检测到而在晚期则几乎检测不到。RORγt和T-bet分别调控Th17和Th1细胞的分化,在ApoE-/-小鼠8周时表达即明显高于对照组小鼠,而且其表达持续升高能够维持到动脉粥样硬化斑块晚期。为了确定IL-17A的细胞来源,我们采用免疫荧光双染的方法证实斑块局部的IL-17A由CD4+T细胞和巨噬细胞分泌。上述结果提示Th17和Th1细胞均参与动脉粥样硬化的发生,而且Th17细胞在动脉粥样硬化晚期发挥更重要的作用。
2.Th17和Th1细胞随动脉粥样硬化的发生在外周免疫器官中的变化
为了观察Th17和Th1细胞在外周免疫器官中的变化,我们采用流式细胞术的方法检测Th17(CD4+IL-17A+)细胞和Th1(CD4+IFN-γ+)细胞在脾脏中的比例,结果显示在小鼠8周时,即无动脉粥样硬化斑块形成的小鼠脾脏中,实验组和对照组Th17和Th1细胞无明显的差异,在斑块形成的早期,Th17和Thl细胞比例在ApoE-/-小鼠脾脏中较对照组小鼠明显升高；随着疾病的进展,Th17和Th1细胞在斑块形成的晚期进一步升高,此外在疾病晚期小鼠脾脏中还发现一群即能够分泌IL-17A,同时也分泌IFN-γ的CD4+T细胞(CD4+IL-17A+IFN-γ+)较对照组小鼠也明显升高。
进一步统计学分析显示Th17、Th1和CD4+IL-17A+IFN-γ+T细胞的变化与动脉粥样硬化斑块的大小均呈明显的正相关,而且与小鼠血清中总胆固醇(TCH)的水平也呈现明显的正相关,这就提示Th17和Th1细胞均可能参与动脉粥样硬化的发生。
(二)Th17细胞及IL-17A对动脉粥样硬化斑块形成的影响
为了证实Th17细胞及IL-17A在动脉粥样硬化发生中的作用,我们用小鼠中和性IL-17A抗体和外源性小鼠IL-17A分别干预ApoE-/-小鼠,微型超声和油红O染色结果显示抗IL-17A抗体能够明显抑制斑块的形成,而外源性小鼠IL-17A则明显促进动脉粥样硬化斑块的形成,免疫组织化学染色结果显示IL-17A干预组小鼠斑块局部巨噬细胞的比例与对照组小鼠无明显差异,而平滑肌细胞的比例较对照组小鼠明显增多。该结果从正反两个方面证实IL-17A能够促进动脉粥样硬化斑块的形成,而且有可能促进斑块向不稳定型斑块发展。
二、IL-17A通过激活NF-κB和p38, ERK通路上调aP2诱导巨噬细胞内质网应激并继发细胞凋亡进而加速动脉粥样硬化的发生和发展
(一)IL-17A通过诱导巨噬细胞内质网应激促进细胞凋亡
1.IL-17A促进巨噬细胞凋亡
大量巨噬细胞凋亡能够促进动脉粥样硬化发生发展,为了研究IL-17A促进动脉粥样硬化发生的机制,在体外巨噬细胞用IL-17A刺激后,Hoechst染色和Annexin V/PI染色结果显示,IL-17A刺激后,随浓度的增高和时间延长,巨噬细胞内凋亡小体明显增多。流式细胞术定量分析显示IL-17A能够明显促进巨噬细胞发生晚期凋亡。
2.IL-17A诱导小鼠及人来源的巨噬细胞发生内质网应激
内质网应激是动脉粥样硬化发生过程中促进巨噬细胞凋亡的重要原因之一,为了研究IL-17A促进巨噬细胞凋亡的机制,小鼠巨噬细胞系RAW264.7和腹腔巨噬细胞在体外用IL-17A刺激后,Western Blotting结果显示内质网应激标志分子p-PERK、p-eIF2α和XBP1s的表达明显升高,由此可见IL-17A能够诱导小鼠巨噬细胞发生内质网应激。为了观察IL-17A是否对人来源巨噬细胞也具有同样的作用,我们将单核细胞系Thp-1和人外周血单个核细胞(PBMC)体外诱导成巨噬细胞后,加入IL-17A刺激,Western Blotting结果显示p-PERK、p-eIF2α和XBP1s的表达同样明显升高。上述结果说明IL-17A在体外能够诱导人和小鼠来源的巨噬细胞发生内质网应激。
3. IL-17A诱导动脉粥样硬化斑块局部巨噬细胞发生内质网应激
为了验证是否在小鼠体内IL-17A也能诱导巨噬细胞发生内质网应激,我们首先用Western Blotting检测主动脉弓部p-PERK、p-eIF2α和XBP1s的表达,结果显示IL-17A干预组小鼠斑块局部内质网应激标志分子的表达均明显高于对照组小鼠。进一步我们采用免疫组织化学染色的方法定位检测斑块局部Moma-2, p-PERK和p-eIF2α的表达,结果显示在巨噬细胞表达部位,p-PERK和p-eIF2α的表达量占斑块面积的比例在IL-17A干预组小鼠明显高于对照组小鼠,该结果说明IL-17A不仅可以在体外,在斑块局部也能够诱导巨噬细胞发生内质网应激。
4.IL-17A通过诱导巨噬细胞内质网应激促进细胞凋亡
长期内质网应激能够导致细胞发生凋亡,内质网应激诱导细胞凋亡主要通过上调CHOP的表达、激活caspas12分子、激活JNK进而活化Bcl-2,最终通过活化caspase3诱导细胞发生凋亡。为了探索IL-17A是否通过诱导内质网应激促进巨噬细胞凋亡,我们首先在体外发现IL-17A能够促进CHOP和caspase3剪切体的表达,caspase12略有上调。为了在小鼠体内验证该结果,我们将ApoE-/-小鼠随机分为三组：对照组,IL-17A干预组,IL-17A联合PBA干预组。Western Blotting和免疫组化染色显示IL-17A干预组小鼠斑块局部CHOP表达明显上调,而caspasel2无明显的差异,caspase3剪切体的表达亦明显升高；而IL-17A联合PBA干预组,p-PERK和p-eIF2α的表达较IL-17A干预组明显减弱,说明PBA能够缓解IL-17A诱导的巨噬细胞内质网应激,同时CHOP和caspase3的表达较IL-17A干预组亦明显减弱,caspasel2无明显的差异。Tunnel染色结果显示IL-17A干预组小鼠斑块局部凋亡的巨噬细胞数量明显高于对照组小鼠,而IL-17A联合PBA干预组凋亡细胞则较IL-17A干预组明显减少。上述结果证实IL-17A通过诱导巨噬细胞内质网应激进而通过激活CHOP-caspase3通路促进巨噬细胞的凋亡。
(二)IL-17A通过内质网应激引起的巨噬细胞凋亡促进动脉粥样硬化的发生
ApoE-/-小鼠随机分为三组(对照组,IL-17A干预组,IL-17A联合PBA干预组)进行干预后,我们采用油红O染色的方法检测斑块的大小及脂质沉积,结果显示IL-17A干预组小鼠主动脉根部斑块的面积明显较对照组增大,而IL-17A联合PBA干预组小鼠的斑块面积则明显较IL-17A单独干预组减小,由此可见,IL-17A确实是通过诱导巨噬细胞内质网应激进而通过激活CHOP-caspase3通路促进细胞凋亡、加速动脉粥样硬化斑块的形成。
(三)IL-17A通过激活NF-κB和p38, ERK通路上调aP2诱导巨噬细胞内质网应激的发生
1.IL-17A上调aP2的表达诱导巨噬细胞内质网应激
有研究表明aP2表达增高能够促进巨噬细胞内质网应激的发生,为了研究IL-17A诱导巨噬细胞内质网应激的发生机制,在体外我们用RT-PCR和Western Blotting检测发现IL-17A能够在RNA和蛋白水平上调巨噬细胞内aP2的表达；此外在小鼠体内我们也发现IL-17A干预组小鼠斑块局部aP2的表达较对照组小鼠明显升高。为了证实IL-17A是通过上调aP2的表达诱导巨噬细胞内质网应激,在体外我们先用aP2抑制剂作用于巨噬细胞,然后再加入IL-17A刺激,Western Blotting检测结果显示p-eIF2a和XBP1s的表达较IL-17A单独刺激明显减弱,该结果证实IL-17A通过上调aP2的表达诱导巨噬细胞内质网应激的发生。
2.IL-17A激活NF-κB和p38、ERK通路上调aP2的表达
IL-17A与其受体结合后能够通过激活NF-κB和MAPK通路传递信号入细胞,诱导多种分子的表达。为了研究IL-17A上调aP2的机制,IL-17A刺激巨噬细胞后,我们采用Western Blotting检测发现P-IκB和p-p38, p-ERK, p-JNK的表达均明显升高。进一步我们分别用NF-κB和p38, ERK, JNK抑制剂作用巨噬细胞,然后用IL-17A刺激后检测aP2的表达,结果显示NF-κB和p38, ERK抑制作用后aP2的水平明显减弱。由此可以证明IL-17A通过激活NF-κB和p38、 ERK通路进而促进aP2的表达。
结论
1.Th17细胞及效应分子IL-17A具有促进动脉粥样硬化的作用动脉粥样斑块局部Th17细胞和IL-17A高表达,外周免疫器官中Th17细胞的数量随着动脉粥样硬化的发生和发展逐渐升高,并与动脉粥样硬化斑块的大小及总胆固醇的水平呈正相关,体内实验证实Th17细胞及效应分子IL-17A能够促进动脉粥样硬化斑块形成。
2.IL-17A诱导巨噬细胞发生内质网应激和继发的细胞凋亡是IL-17A加速动脉粥样硬化斑块形成的机制之一IL-17A在体外以及斑块局部均能够诱导巨噬细胞发生内质网应激,长期内质网应激可以进一步通过激活CHOP-caspase3通路促进巨噬细胞凋亡,进而加速动脉粥样硬化斑块的形成。
3.IL-17A诱导的aP2上调是其导致巨噬细胞发生内质网应激的机制之一IL-17A可通过激活NF-κB和p38、ERK通路上调脂肪酸结合蛋白aP2的表达,抑制剂阻断aP2的表达则明显抑制IL-17A诱导的巨噬细胞内质网应激。
创新性和意义
本研究通过体内外实验,系统研究了新的T细胞亚群Th17细胞及其主要效应分子IL-17A在动脉粥样硬化斑块形成中的作用及其机制,对揭示动脉粥样硬化的免疫机制、防治动脉粥样硬化相关的心血管疾病具有重要的理论价值和潜在的应用价值。本研究的主要创新点和意义如下：
1.证明Th17和IL-17A参与促进动脉粥样硬化的发生和发展,外源性IL-17A治疗明显促进动脉粥样硬化斑块的形成,而中和性抗IL-17A抗体的应用可有效缓解斑块的发展,为临床以IL-17A为靶点治疗动脉粥样硬化提供了有力的理论和实验依据。
2.在国内外首先发现IL-17A能诱导巨噬细胞发生内质网应激,长期内质网应激继发的细胞凋亡是IL-17A促进动脉粥样硬化发生的重要机制之一,用化学伴侣分子PBA阻断内质网应激可有效控制IL-17A介导的动脉粥样硬化病变的进展。
3.在国内外首先发现IL-17A可通过NF-κB和p38/ERK MAKP信号通路上调脂肪酸结合蛋白aP2,进而诱导内质网应激的发生；首次通过aP2将细胞因子和脂质代谢联系在一起,为研究炎症反应与脂质代谢疾病的相互关系及机制提供了新的思路。
4.本研究证实IL-17A中和性抗体、内质网应激抑制剂PBA能够抑制斑块的形成,aP2抑制剂能够缓解巨噬细胞内质网应激,为临床以炎性因子、细胞内质网应激及脂质代谢为靶点联合治疗动脉粥样硬化提供了有力的理论和实验依据。
研究的局限性
1.IL-17A诱导内质网应激发生的机制还有待于更深入的研究；
2.aP2影响IL-17A诱导的内质网应激和促进斑块作用的体内试验尚有待进一步的确定。
Objective
Atherosclerosis is the most common diseases of heart cerebrovascular system and the leading cause of mortablity in advanced and developing countries. Atherosclerosis is a slowly progressing chronic inflammatory disease occurring in large and medium-sized arteries and is characterized by an accumulation of lipids in the artery wall, together with infiltration of immunocytes, such as macrophages, T cells and mast cells, and the proliferation of vascular smooth muscle cells. Accumulate research showed that innate immune respons (macrophages) and adaptive immune response (CD4+T cells) participated in the development of atherosclerosis. Recently, the effect of CD4+T cells in this disease received widespread concerns. CD4+T cells contain different subsets and compose of conventional Th1and Th2cells and regulatory T cells (Treg) and new subsets Th17cells. Th1cells promoted the development of atherosclerosis. And the effect of Th2cells on the diseases remains controversial. Regulatory T cells play a protective role in atherogenesis and could inhibit the formation of atherosclerotic plaque. However, the function of Th17cells in the development of atherosclerosis remains undefined.
Th17cell is characterized by secreting IL-17A. Naive CD4+T cells stimulated by antigens and differentiated into Th17cells with the regulation of transcription factors RORγt and RORoα and in the presence of IL-6and TGF-β.It has been shown that Th17cells and effector IL-17A play a part in many kinds of chronic inflammation and organ-specific autoimmune disease in animals such as experimental autoimmune encephalomyelitis (EAE), rheumatoid arthritis (RA) and colitis. Atherosclerosis is a chronic inflammatory disease occurring in artery vascular walls, previous studies have shown that Th17cells and its major effector cytokine IL-17A are increased in the peripheral blood of patients with coronary atherosclerosis. And T cells infiltrated in the atherosclerotic plaque express IL-17A in human or in mouse models. These suggest that Th17cells and IL-17A probably participate in the development of atherosclerosis. However, whether Th17cells and IL-17A increased with the development of atherosclerosis? What roles Th17cells and effector IL-17A play in the formation and development of atherosclerosis remains debate. Whether could IL-17A as one of potential therapeutic targets for atherosclerosis? What is the mechanism of IL-17A in this disease? All these problems remain to be investigated.
To confirm the effect of Th17cells and IL-17A on the development of atherosclerosis, illustrate the mechanism, and provide theoretical basis and experimental data for clinical applications, this paper intends to study from the following two parts:
1. To confirm the aggravated role of Th17cells and effector IL-17A in the development of atherosclerosis.
1) The establishment of standard atherosclerotic mice model and rapid carotid lesions mice model:ApoE-/-mice fed with high cholesterol diet to establish standard atherosclerotic mice model. And high cholesterol diet combined with placed perivascular constrictive silica collars on the left common carotid artery to establish rapid carotid lesions mice model.
2) The expression of Th17cells and IL-17A in atherosclerotic plaque and peripheral immune organ:Results for RT-PCR and immunohistochemistry showed that Th17cells and IL-17A could be detected in atherosclerotic lesions. And flow cytometry analysis found that the percentage of Th17cells and IL-17A in spleen increased by the formation and development of atherosclerosis. Furthermore, the proportion of Th17cells was positive correlated with the area of the plaque and the level of serum lipids (TCH).
3) The effect of Thl7cells and IL-17A on the formation of atherosclerotic plaque:We treated ApoE-/-mice with anti-IL-17A neutralizing antibody or exogenous recombinant mouse IL-17A, and from both the positive and negative aspects, we confirmed that IL-17A could promote the formation of atherosclerotic plaque. Furthermore, IL-17A reduces the content of smooth muscle cells in lesions, suggesting increased instability of the plaques.
2. To explore the mechanism of IL-17A promote the development of atherosclerosis from macrophage ER stress and cell apoptosis
1) The effect and mechanism of IL-17A on macrophage apoptosis:IL-17A could promote macrophage apoptosis in vitro. Further mechanism study found that IL-17A induced ER stress in murine and human-derived macrophages in vitro. In addition, IL-17A could also induce ER stress in macrophage-dense areas of the atherosclerotic lesions in ApoE-/-mice. However, in vivo, blocking of ER stress by chemical chaperone (PBA) alleviated IL-17A-induced ER stress and reduced the numbers of apoptotic macrophages in lesions, suggesting IL-17A could promote macrophage apoptosis through inducing ER stress.
2) The mechanism of IL-17A aggravated the development of atherosclerosis:We treated ApoE-/-mice with IL-17A or IL-17A combined with PBA, and confirmed that IL-17A could promote macrophages apoptosis by activating ER stress response and accelerate the formation of atherosclerotic plaque.
3) The mechanism of IL-17A induced ER stress in macrophage:In vitro, IL-17A could up-regulate the expression of aP2and activate NF-κB and MAPK signal pathway (ERK, p38and JNK). Furthermore, we found that IL-17A up-regulated aP2through activating NF-κB and p38、ERK pathway. Finally, we confirmed that aP2mediated IL-17A-induced ER stress in macrophages.
Methods
1. To confirm the aggravated role of Th17cells and effector IL-17A in the development of atherosclerosis.
1) The establishment of standard atherosclerotic mice model and rapid carotid lesions mice model:
i) Standard mice model:8weeks old male ApoE-/-mice fed with high cholesterol diet to16weeks or24weeks old. And same strain C57BL/6mice also give high cholesterol diet as control.
ii) Rapid carotid lesions mice model:8weeks old male ApoE-/-mice fed with high cholesterol diet for2weeks, and then carotid atherosclerotic lesions were induced by perivascular constrictive silica collars placed on the left common carotid artery. Mice were going on high cholesterol diet to16weeks or24weeks old. And same strain C57BL/6mice as control.
2) The expression of Th17cells and IL-17A in atherosclerotic plaque and peripheral immune organ:
i) Separate the left common carotid arteries and the aortic root vessels and prepare serial cryosections, HE staining and Oil red O staining detected the formation of atherosclerotic lesions and the content of lipids. And immunohistochemistry detected the expression of IL-17A and IFN-y in plaque, and confirmed the resource of IL-17A with immunofluorescence double staining.
ii) Separate the aortic arch to extract RNA and detect the expression of IL-17A, RORyt and IFN-γ, T-bet in atherosclerotic plaque with RT-PCR.
iii) Splenocytes were prepared from different age mice and flow cytometry detected the percentage of Thl and Th17cells.
iv) To analyze the correlation of the proportion of Thl and Th17cells with the size of atherosclerotic plaque and the level of serum lipids.
3) The effect of Th17cells and IL-17A on the formation of atherosclerotic plaque:
i) Anti-IL-17A neutralizing antibody treatment:
a) Twenty8weeks old male ApoE-/-mice fed with high cholesterol diet for2weeks, and then carotid atherosclerotic lesions were induced by perivascular constrictive silica collars placed on the left common carotid artery. Five days later, mice were randomly divided into two groups and treated with anti-IL-17A neutralization monoclonal antibody (50μg/mouse/time) or isotype antibody (rat IgG2A), once a week for4weeks.
b) During this interventional period, plaque sizes were evaluated by Micro-ultrasound imaging, and then plaque morphology was detected by histopathology after mice were sacrificed at one week following the last challenge. Oil red O staining detected the size of lesions.
c) Immunohistochemistry detected the expression of macrophage and smooth muscle cells in lesions.
ii) Application of exogenous IL-17A:
a) Ten8weeks old male ApoE-/-mice were randomly divided into two groups and fed with high cholesterol diet, and11weeks old mice were given intraperitoneal injection with recombinant mice IL-17A (2μg/mice/time) once a week for5weeks and normal saline as control.
b) At one week following the last challenge, separate the aortic root vessels and prepare serial cryosections. Oil red O staining observed the size of plaque.
c) Immunohistochemistry detected the expression of macrophage and smooth muscle cells in lesions.
2. To explore the mechanism of IL-17A promote the development of atherosclerosis from macrophage ER stress and cell apoptosis
1) The effect and mechanism of IL-17A on macrophage apoptosis and the formation of atherosclerotic plaque:
i) Experiment in vitro
a) Isolating of mice peritoneal macrophages and stimulated with IL-17A, Hoechst staining observed cell apoptosis from cytomorphology and flow cytometry (Annexin V/PI) detected the numbers of apoptotic cells.
b) Mice macrophage cell line RAW264.7and peritoneal macrophages stimulated by IL-17A, then Western Blotting detected the expression of ER stress markers p-PERK, p-eIF2a and XBP1s.
c) Human monocyte cell line Thp-1and peripheral blood mononuclear cells were induced into macrophages and stimulated with human IL-17A, then Western Blotting detected the expression of ER stress markers p-PERK, p-eIF2α and XBP1s.
d) Mice peritoneal macrophages stimulated by IL-17A, then Western Blotting detected the expression of CHOP, caspase12and caspase3cleavage.
ii) Experiment in vivo
a) Thirty8weeks old male ApoE-/-mice fed with high cholesterol diet, and11weeks old mice were randomly divided into3group:Control (i.p. normal saline), IL-17A-treated group (i.p. mice IL-17A,2μg/mice/time, once a week), IL-17A combined with PBA treated group (i.p. IL-17A,2μg/mice/time, once a week and PBA,100mg/kg, twice a week).
b) At one week following the last challenge, separate the aortic root vessels and prepare serial cryosections. Immunohistochemistry detected the expression of Moma-2(for macrophage), p-PERK, p-eIF2α and CHOP, caspase12in atherosclerotic lesions. And Tunnel staining for apoptotic macrophages in lesions. Separate the aortic arch to extract proteins, Western Blotting detected the expression of ER stress markers (p-PERK, p-eIF2α and XBP1s) and CHOP, caspase12, and caspase3cleavage.
c) Oil red O observed the size of atherosclerotic plaque.
2) The mechanism of IL-17A induced ER stress in macrophage:
i) Mice peritoneal macrophages stimulated by IL-17A, RT-PCR and Western Blotting detected the expression of aP2.
ii) Mice peritoneal macrophages with aP2inhibitor (BMS309403)(50uM) pre-treated three hours and then stimulated by IL-17A, Western Blotting detected the expression of p-eIF2α and XBPls.
iii) Mice peritoneal macrophages stimulated by IL-17A, Western Blotting detected the expression of p-IκB, p-ERK, p-p38and p-JNK. Furthermore, mice peritoneal macrophages with NF-κB (PDTC), ERK (PD98059), JNK (SP600125) and p38(SB203580) inhibitor pre-treated one hour and then stimulated by IL-17A, Western Blotting detected the expression of aP2.
Results
1. We confirmed that Th17cells and effector IL-17A aggravated the development of atherosclerosis with experiment in vivo and in vitro
1) The expression of Th17cells and IL-17A in atherosclerotic plaque and peripheral immune organ
i) The expression of IL-17A and IFN-y in atherosclerotic plaque
To study the effect of Th17cells and IL-17A on atherogenesis, we established rapid carotid lesions mice model and standard atherosclerotic model with ApoE-/-mice. And we separated the left common carotid arteries and the aortic root vessels and prepare serial cryosections, HE staining, Oil red O staining and immunohisto-chemistry detected the formation of atherosclerotic lesions and the expression of IL-17A and IFN-y. Results showed that8week-old ApoE-/-mice had no significant plaque formation in carotids and vascular intima only appeared slightly thickening. At this time, no IL-17A and IFN-y was detected in lesions. When mice fed with high cholesterol diet for additional eight weeks,16weeks old ApoE-/-mice showed that vascular intima significantly thicken and proliferation of smooth muscle cells. Atherosclerotic plaque characterized by thicker fibrous cap and much of nucleated cells infiltrating and was defined as the early stage of atherosclerosis. At16weeks old ApoE-/-mice, substantial expression of IL-17A and IFN-y was detected. The plaque was more severe when ApoE-/-mice were continuously fed with high cholesterol diet for16weeks,24weeks old ApoE-/-mice showed that the size increased obviously, fibrous cap became thinner, the number of smooth muscle cells reduced and many vacuoles present in lesions, that was the late stage of atherosclerosis. This stage, we could detect IL-17A in lesions and almost no IFN-y was found. In contrast, the age-matched wild-type C57BL/6mice (fed with the same regimen as ApoE-/-mice) had no detectable plaque in carotids throughout the whole experimental periods.
In addition, we detected the expression of IL-17A, RORyt and IFN-γ, T-bet with RT-PCR. Consistent with the results from immunohistochemistry or immune-fluorescence staining, the expression of IL-17A in ApoE-/-mice with the early and late stage of plaque was markedly higher than that of wild-type control mice. However, IFN-y only was detected in early stage of plaque and could not be detected in late stage of plaque. Notably, the expression of RORyt and T-bet, the transcription factors controlling Th17and Thl cell differentiation respectively, was significantly higher in the vascular wall of ApoE-/-mice than in that of control mice at as early as8weeks. The levels of RORyt and T-bet continued to increase and were maintained at higher levels in ApoE-/-mice at the early and late stages of atherosclerotic plaque (16-24weeks). To confirm the cellular sources of IL-17A, we co-stained IL-17A with CD4+T cells and macrophages with immunofluorescence staining. IL-17A was mainly expressed in both CD4+T cells and macrophages infiltrated into the atherosclerotic plaque. The data suggest that both Th17and Thl cells were involved in the development of the atherosclerotic inflammation, and Th17play more important roles in late stage of atherosclerosis.
ii) Increased Th17and Thl cells in the spleen of ApoE-/-mice with atherosclerotic plaque
To observe the change of Th17and Thl cells in peripheral immune organ, we detected the number of Th17(CD4+IL-17A+) and Thl (CD4+IFN-γ+) in spleen with flow cytometry. Results showed that the pre-symptomatic ApoE-/-mice (8weeks-old) had minimal numbers of Th17cells with no statistical difference compared with control mice. Although the number of Thl cells was higher than Th17, there was no statistical difference between ApoE-/-and control mice. However, the frequencies of Th17and Thl cells in16week-old ApoE-/-mice with plaque was significantly higher than that in control mice, which were further increased in the ApoE-/-mice with advanced atherosclerotic plaque (24weeks-old). In addition, we observed a population of CD4+T cells expressing both IL-17+and IFN-γ+. The frequency of CD4+IL-17+IFN-γ+T cells significantly increased in the late stage atherosclerotic ApoE-/-mice compared with age-matched control mice.
Furthermore, simple linear regression analysis, we observed that the area of the plaque and the level of TCH was not only correlated with the proportion of Thl cells, but also closely associated with Th17cell subset and CD4+IL17+IFN-γ+cells. These results suggested that Th17and Thl cells both probably participated in atherogenesis.
2) The influence of Th17cells and IL-17A on the development of atherosclerosis
To confirm the role of Th17cells and IL-17A on the development of atherosclerosis, we treated ApoE-/-mice with anti-IL-17A neutralizing antibody and exogenous IL-17A. Results from micro-ultrasound imaging and Oil red O staining showed that neutralizing IL-17A antibody significantly inhibited the formation of atherosclerotic plaque, however, exogenous IL-17A obviously accelerated atherogenesis. Immunohistochemistry show that IL-17A treatment had no significant effect on the number of macrophage and it decreased the numbers of smooth muscle cells in plaques. These results confirmed from positive and negative aspects that IL-17A could promote the development of atherosclerosis and increased instability of the plaques.
2. IL-17A-induced ER stress in macrophage promote cell apoptosis and aggravate atherogenesis by up-regulating NF-κB and p38, ERK pathway mediated the expression of aP2
1) IL-17A promote macrophage apoptosis by inducing ER stress
i) IL-17A promote macrophage apoptosis
Macrophage apoptosis is an important cause for the development of atherosclerosis. To explore the mechanism of IL-17A accelerate the formation of atherosclerotic plaque, we stimulated macrophage with IL-17A in vitro. Results from Hoechst staining showed that macrophages treated by IL-17A revealed increase of apoptotic bodies. Next, results from flow cytometry showed that IL-17A promote the number of late apoptotic macrophages increasing markedly.
ii) IL-17A induced endoplasmic reticulum stress in murine and human-derived
macrophage in vitro
Since increasing evidence supports that ER stress is one important mechanism for macrophage apoptosis, to explore the mechanism of IL-17A promote macrophage apoptosis. Mice macrophage cell line RAW264.7and mice peritoneal macrophage were stimulated by IL-17A, Western Blotting analysis showed that the expression of ER stress markers p-PERK、p-eIF2α and XBP1s increased significantly. This suggested that IL-17A could induce ER stress in murine-derived macrophages. To observe whether IL-17A play the same roles on human-derived macrophage, we induced Thp-1and PBMC into macrophages and then stimulated with IL-17A, Western Blotting analysis showed that the expression of p-PERK、p-eIF2α and XBP1s increased significantly. These results indicated that IL-17A could induce ER stress in human and murine-derived macrophages in vitro.
iii) Exogenous IL-17A induced ER stress in macrophage-dense areas of the atherosclerotic lesions in ApoE-/-mice
To confirm the impact of IL-17A on ER stress in vivo, we treated ApoE-/-mice fed with high cholesterol diet utilizing exogenous IL-17A, Western blotting analysis showed that p-PERK, p-eIF2α and XBP-1s were significantly up-regulated in aortic vessels with atherosclerotic plaque in IL-17A-treated mice compared with control. Consist with results from Western Blotting, immunohistochemistry staining revealed that increased expression of p-eIF2a and p-PERK mainly located in macrophage-dense areas of the atherosclerotic lesions. These indicate that IL-17A is able to induce directly ER stress response in macrophage both in vitro and in vivo.
iv) IL-17A promote macrophage apoptosis by inducing ER stress
It has been shown that prolonged activation of ER-associated CHOP and caspase12pathways are involved in the ER stress-mediated apoptosis. To explore whether IL-17A promote macrophage apoptosis by inducing ER stress, we found that IL-17A significantly promote the expression of CHOP and caspase3cleavage, and slightly up-regulate caspasel2in vitro. To confirm the effect in vivo, we divided ApoE-/-mice into3groups:Control, IL-17A-treated group and IL-17A combined with PBA treated group. Western Blotting and immunohistochemistry showed that the expression of CHOP and caspase3cleavage up-regulated markedly in lesions of IL-17A-treated mice, and caspase12had no alteration. However, the expression of p-PERK and p-eIF2α diminished obviously in lesions of IL-17A combined PBA-treated mice, suggesting PBA could alleviate ER stress in lesions. At the same time, CHOP and caspase3cleavage reduced significantly and caspase12had no obvious change. Tunnel staining showed that IL-17A leads to a marked increase in apoptotic cells in atherosclerotic lesions and IL-17A comined with PBA could reduce apoptotic cells. Those indicated that IL-17A could induce apoptosis in macrophages mainly via ER stress-CHOP-caspase3axis.
2) IL-17A accelerated the progression of atherosclerosis via ER stress-induced apoptosis in macrophage
ApoE-/-mice divided into three groups:control, IL-17A-treated group and IL-17A combined with PBA treated group. Oil red O detected the size of the aortic root for lesions, results showed that mice receiving IL-17A and PBA treatment had an obvious reduction in plaque size compared with IL-17A-treated mice. It follows that IL-17A could induce apoptosis in macrophages mainly via ER stress-CHOP-caspase3axis by which it accelerated progression of atherosclerosis.
3) IL-17A up-regulated expression of aP2by activating NF-κB and ERK/p38MAPK pathways and further induced ER stress in macrophage
i) The up-regulated expression of aP2is required for IL-17A-induced ER stress in macrophages
Recent study has suggested that aP2mediates saturated fatty acid-induced ER stress and apoptosis in macrophage, to study the mechanism of IL-17A induced ER stress in macrophage. Results from RT-PCR and Western Blotting both showed that IL-17A up-regulated directly aP2transcription and translation in macrophages in vitro. Importantly, IL-17A also led to a significant increase in the expression of aP2in atherosclerotic lesions in vivo. To clarify the role of aP2in IL-17A-induced ER stress, we treated macrophages with aP2inhibitor (BMS309403) in presence of IL-17A and found that accompanying inhibition of aP2, ER stress indicators p-eIF2α and the splice of XBP-1in macrophages diminished obviously correspondingly. This data clearly demonstrate that IL-17A for up-regulated aP2expression is required, at least partially, for IL-17A-induced ER stress in macrophages.
ii) IL-17A up-regulated expression of aP2by activating NF-κB and ERK/p38MAPK pathways
Previous works have shown that IL-17A can induce multiple genes via activating the nuclear factor (NF)-κB pathway and MAPK pathway. To explore the mechanism of IL-17A up-regulated the expression of aP2, macrophages were stimulated by IL-17A. And Western Blotting analysis showed that IL-17A could rapidly up-regulate the expression of p-IκB, p-ERK and p-p38, p-JNK. Subsequently, to clarify causative role of these signal pathways in IL-17A-upregulated aP2, we treated mouse peritoneal macrophages with the inhibitor of NF-κB, ERK, JNK and p38and then detected the aP2expression by Western Blotting. Results revealed that the inhibitors of NF-κB, ERK and p38diminish significantly the expression of aP2. However, JNK inhibitor did not suppress the aP2expression. Collectively, IL-17A up-regulates the expression of aP2through activating NF-κB and ERK/p38MAPK signal pathways in macrophages.
Conclusions
1. Th17cells and effector IL-17A could promote the development of atherosclerosis
The expression of Th17cells and IL-17A in atherosclerotic plaque increased and the proportion of Th17cells in spleen increased by the development of atherosclerosis and correlated with the size of atherosclerotic plaque and the level of TCH. In addition, in vivo, we confirm that Th17cells and effector IL-17A promote the formation of atherosclerotic plaque.
2. IL-17A induced ER stress in macrophage and secondary apoptosis is one of mechanisms that IL-17A accelerates the formation of atherosclerotic plaque IL-17A induced ER stress in macrophage in vitro culture and in lesions. Prolongation of ER stress promotes macrophage apoptosis and accelerates the formation of atherosclerotic plaque through activating ER stress-CHOP-caspase3pathway.
3. IL-17A up-regulating the expression of aP2is one of mechanisms of IL-17A induced ER stress in macrophage
Further study verify that IL-17A induces ER stress in macrophage through activating NF-κB and p38、ERK pathway and up-regulating the expression of aP2. And aP2inhibitor could alleviate ER stress that IL-17A induced in macrophage.
Originality and Significance
In this research, we systematicaly study the effect and mechanism of Th17cells and its effector IL-17A in the development of atherosclerosis with experiment in vivo and in vitro. These have important theoretical and potential application value for revealing immunological mechanism of atherosclerosis and preventing atherosclerotic related cardiovascular disease. The originality and significance as follows:
1. We confirm that Th17cells and IL-17A participated in the development of atherosclerosis. And exogenous IL-17A treatment obviously promotes the formation of atherosclerotic plaque. However, neutralizing IL-17A antibody could reduce the size of plaque. These foundings provide strong theoretical and experimental basis for treatment of atherosclerosis with IL-17A as clinical therapy target.
2. We demonstrate, for the first time, that IL-17A by oneself can induce ER stress in macrophage. Secondary apoptosis of prolongation ER stress is one of mechanisms that IL-17A promotes the development of atherosclerosis. Furthermore, the inhibiting of ER stress with chemical chaperon PBA effectively control IL-17A-aggravated atherosclerosis.
3. For the first time, we demonstrate that IL-17A up-regulate the expression of aP2and then induce ER stress in macrophage. And firstly, we establish a previously unrecognized link between cytokines and lipid metabolism through cytosolic lipid chaperone aP2in macrophage. These provide new ideas for studying the relationship and mechanism between inflammation and lipid metabolism diseases.
4. We confirm that that IL-17A antibody and PBA could inhibit atherogenesis, aP2inhibitor could alleviate ER stress in macrophage. All these provide potent theoretical and experimental basis for treatment target as inflammatory cytokines, ER stress and lipid metabolism.
Limitations of this study
1. The mechanism of IL-17A induced ER stress need to be further investigated.
2. The effect of aP2on IL-17A-induced ER stress and promoted atherosclerosis need experiments in vivo to be verified.
动脉粥样硬化是发生于大中动脉的慢性炎症性病变,以大量脂质沉积到血管壁为主要特点,并伴随多种免疫细胞浸润及平滑肌细胞增生,其所诱发的心脑血管疾病是发达国家及发展中国家常见的死亡原因之一。目前研究认为固有免疫应答(巨噬细胞)和适应性免疫应答(CD4+T细胞)均参与动脉粥样硬化的发生和发展,近年来对于CD4+T细胞在该疾病中的作用受到了广泛的关注。CD4+T细胞分为多种亚群,主要包括Th1、Th2和调节性T细胞(regulatory T cells, Treg)以及近年来发现的Thl7细胞。Thl细胞促进动脉粥样硬化的发生,Th2细胞在该疾病中的作用还不是非常明确,尚存在争议。调节性T细胞在动脉粥样硬化中主要发挥了保护性的作用,能够抑制动脉粥样硬化斑块的形成。然而,Th17在动脉粥样硬化中的作用尚不明确。
Th17细胞是以分泌IL-17A为主要特征的CD4+T细胞群体,CD4+初始T细胞受抗原刺激后,在转录因子RORγt和RORα的调控下,IL-6和TGF-β联合作用即可促使初始T细胞分化成为Th17细胞。目前的研究认为Th17细胞及IL-17A参与多种自身免疫性疾病和炎症性疾病的发生,例如：自身反应性脑脊髓炎(EAE)、类风湿性关节炎(RA)和结肠炎等,动脉粥样硬化是发生在动脉血管壁的慢性炎症性疾病,前期有研究显示急性冠状动脉综合症患者外周血中Th17细胞的比例及IL-17A的水平较健康人明显升高,而且在人的动脉粥样硬化斑块局部检测到IL-17A的存在,这就提示Th17细胞及IL-17A很有可能参与该疾病的发生和发展。但是,Th17细胞及其效应分子IL-17A在动脉粥样硬化模型小鼠的发生和发展中变化如何?对动脉粥样硬化斑块的形成究竟发挥什么样的作用,尚存在争议。IL-17A能否作为动脉粥样硬化的治疗靶点之一?IL-17A在该疾病中的作用机制是什么?目前这些问题尚不清楚,都非常值得研究和探索。
为了明确Th17细胞及IL-17A在动脉粥样硬化发生发展中的作用,阐明其发挥作用的机制,为IL-17A的临床应用提供理论依据和实验基础,本论文拟从以下两部分进行研究：
一、通过体内外实验确定Th17细胞及其主要效应分子IL-17A在动脉粥样硬化发展中的促进作用：
(一)标准动脉粥样硬化小鼠模型和快速颈动脉损伤小鼠模型的建立：通过高脂饮食喂养建立标准动脉粥样硬化小鼠模型；高脂喂养联合颈动脉缩窄手术模拟血管狭窄,建立快速颈动脉粥样硬化小鼠模型,便于进行下一步研究。
(二)Th17细胞及IL-17A在动脉粥样硬化斑块局部及外周免疫器官的表达：通过多种方法检测动脉粥样硬化斑块局部存在IL-17A及转录因子RORγt的表达以及Th17细胞的存在；流式细胞术检测动脉粥样硬化发生过程中,Th17细胞的比例及IL-17A在脾脏中呈现明显上升的趋势,而且Th17细胞的比例与动脉粥样硬化的发病程度以及血脂水平(TCH)具有明显的正相关。
(三)Th17细胞及IL-17A对动脉粥样硬化斑块形成的影响：采用IL-17A中和性抗体或小鼠重组IL-17A对小鼠进行体内干预,从正反两个方面证实IL-17A能够促进动脉粥样硬化斑块的形成,并且发现IL-17A能够减少斑块局部平滑肌细胞的含量,提示IL-17A还能够促进斑块向不稳定方向发展。
二、从巨噬细胞内质网应激、细胞凋亡入手探讨IL-17A促进动脉粥样硬化发展的分子机制
(一)IL-17A对巨噬细胞凋亡的影响及作用机制：IL-17A在体外能够促进巨噬细胞的凋亡,进一步研究发现IL-17A在体外能够诱导小鼠及人来源的巨噬细胞发生内质网应激,而且在体内IL-17A也能够诱导斑块局部巨噬细胞发生内质网应激。体内用化学伴侣PBA干预小鼠能够缓解IL-17A诱导的巨噬细胞内质网应激,斑块局部凋亡的巨噬细胞亦明显减少,说明IL-17A能够通过诱导巨噬细胞内质网应激从而促进巨噬细胞凋亡。
(二)IL-17A加剧动脉粥样硬化的发生机制：IL-17A单独作用及IL-17A联合PBA体内干预,发现IL-17A能够通过促进斑块局部巨噬细胞发生内质网应激进而导致巨噬细胞凋亡发挥加速动脉粥样硬化斑块形成的作用。
(三)IL-17A诱导巨噬细胞内质网应激的机制：IL-17A能够上调巨噬细胞内脂质伴侣aP2的表达,而且IL-17A能够激活巨噬细胞NF-κB和MAPK通路(ERK, p38和JNK),进一步研究发现IL-17A是通过激活NF-κB通路和p38.ERK通路上调aP2的表达。最后我们采用aP2抑制剂证实IL-17A是通过aP2的上调诱导巨噬细胞内质网应激的发生。
方法
一、通过体内外实验确定Th17和其主要效应分子IL-17A在动脉粥样硬化发展中的促进作用
(一)标准动脉粥样硬化小鼠模型和快速颈动脉损伤小鼠模型的建立：
1.标准模型的建立：ApoE-/-雄性8周龄小鼠给予高脂饮食到小鼠16周和24周,同品系的C57BL/6野生型小鼠同时给予高脂喂养作为对照组。
2.快速颈动脉损伤模型的建立：ApoE-/-雄性8周龄小鼠给予高脂饮食两周,即小鼠10周时行左侧颈动脉缩窄手术模拟血管狭窄,小鼠继续给予高脂饮食分别至16周和24周,同品系的C57BL/6野生型小鼠作为对照组。
(二)Th17细胞及IL-17A在动脉粥样硬化斑块局部及外周免疫器官中的表达
1.取小鼠主动脉根部和左侧颈动脉制备连续冰冻切片,HE染色和油红O染色检测斑块的形成和脂质沉积情况；免疫组织化学染色检测斑块局部IL-17A和IFN-γ表达,并用免疫荧光双染的方法检测IL-17A的细胞来源。
2.取小鼠主动脉弓部抽提RNA,RT-PCR方法检测IL-17A,RORγt和IFN-γ,T-bet在斑块局部的表达。
3.取小鼠脾脏制备脾细胞悬液,采用流式细胞术检测Th1和Th17细胞的比例。
4.分析Th1和Th17细胞比例与动脉粥样硬化斑块大小的相关性以及与小鼠血
脂水平的相关性。
(三)Th17细胞及IL-17A对动脉粥样硬化斑块形成的影响
1.中和性IL-17A抗体干预
(1)ApoE-/-雄性8周龄小鼠20只给予高脂饮食两周,小鼠10周行左侧颈动脉缩窄手术,术后一周小鼠随机分为两组,实验组小鼠腹腔注射小鼠中和性IL-17A抗体(50μg/只),对照组小鼠腹腔注射同型对照。
(2)干预结束后一周微型超声观察斑块形成情况,取小鼠左侧颈动脉和主动脉根部制备连续冰冻切片,油红O染色检测斑块大小。
(3)免疫组化检测斑块局部巨噬细胞和平滑肌细胞的表达。
2.外源性小鼠IL-17A干预
(1) ApoE-/-雄性8周龄小鼠10只随机分为两组给予高脂饮食,小鼠11周时,实验组小鼠腹腔注射外源性小鼠重组IL-17A (2μg/只/次),对照组小鼠腹腔注射生理盐水。
(2)干预结束后一周,取小鼠主动脉根部制备连续冰冻切片,油红O染色检测斑块大小。
(3)免疫组化检测斑块局部巨噬细胞和平滑肌细胞的表达。
二、从巨噬细胞内质网应激、细胞凋亡入手探讨IL-17A促进动脉粥样硬化发展的分子机制
(一)IL-17A对巨噬细胞凋亡的影响及作用机制以及促进动脉粥样硬化斑块形成的机制
1.体外实验
(1)取小鼠腹腔巨噬细胞,加入IL-17A刺激后,Hoechst染色从细胞形态学上观察细胞凋亡,流式细胞术(Annexin V/PI染色)检测细胞凋亡的数量。
(2)小鼠巨噬细胞系RAW264.7及小鼠腹腔巨噬细胞,IL-17A刺激后收取细胞蛋白,Western Blotting检测内质网应激标志分子p-PERK, p-eIF2a和XBP1s的表达。
(3)人单核细胞来源细胞系Thp-1和人外周血单个核细胞,体外诱导成巨噬细胞,IL-17A刺激后收取细胞蛋白,Western Blotting检测内质网应激标志分子p-PERK, p-eIF2a和XBP1s的表达。
(4)小鼠腹腔巨噬细胞,加入IL-17A刺激后,Western Blotting检测(?)CHOP, caspase12和caspase3剪切体的表达。
2.体内试验
(1) ApoE-/-雄性8周龄小鼠30只给予高脂饮食,小鼠11周时随机分为三组,实验组其中一组给予腹腔注射小鼠重组IL-17A (2μg/只/次,每周一次),另外一组腹腔注射IL-17A(2μg/只/次,每周一次)和化学伴侣PBA(100mg/kg,每周两次),对照组小鼠腹腔注射生理盐水。
(2)干预结束后一周,取小鼠主动脉根部制备连续冰冻切片,免疫组织化学方法检测斑块局部Moma-2(巨噬细胞)、p-PERK和p-eIF2a的表达以及CHOP和caspasel2的表达；Tunnel染色检测斑块局部巨噬细胞凋亡。取小鼠主动脉弓部抽提蛋白,Western Blotting检测内质网应激标志p-PERK, p-eIF2α和XBP1s的表达以及CHOP, caspase12和caspase3剪切体的表达。
(3)油红O染色检测斑块大小。
(二)IL-17A诱导巨噬细胞内质网应激的机制
1.小鼠腹腔巨噬细胞,IL-17A刺激后RT-PCR和Western Blotting检测aP2的表达。
2.小鼠腹腔巨噬细胞在IL-17A作用前加入aP2抑制剂(BMS309403)刺激3小时,收取细胞蛋白,Western Blotting检测(?)p-eIF2α和XBPls的表达。
3.小鼠腹腔巨噬细胞,IL-17A刺激后Western Blotting检测(?)p-IκB, p-ERK, p-p38和p-JNK的表达；IL-17A刺激前分别加入NF-κB (PDTC), ERK (PD98059), JNK (SP600125)和p38(SB203580)抑制剂作用1小时,然后用Western Blotting方法检测aP2的表达。
结果一、通过体内外实验证实Th17及效应分子IL-17A能够促进动脉粥样硬化的发生(一)Th17细胞及IL-17A在动脉粥样硬化斑块局部及外周免疫器官中的表达
1.动脉粥样硬化斑块局部IL-17A和IFN-γ的表达
为了研究Th17细胞和IL-17A在动脉粥样硬化发生中的作用,我们利用ApoE-/-雄性小鼠建立了快速颈动脉粥样硬化模型和标准动脉粥样硬化小鼠模型,取小鼠左侧颈动脉和主动脉根部制备连续冰冻切片并进行HE染色和免疫组化染色。结果发现,ApoE-/-小鼠8周时,血管壁仅出现内膜略微增厚没有明显斑块的形成,此时在斑块局部几乎检测不到IL-17A和IFN-γ。高脂喂养到小鼠16周时,血管内膜有明显的增厚,平滑肌大量增生,形成较厚的纤维帽,大量有核细胞浸润到斑块局部,其斑块结构和组成属于早期动脉粥样硬化斑块,此时斑块局部能够检测到IL-17A和IFN-γ的表达,而对照组小鼠没有明显的斑块形成也检测不到IL-17A和IFN-γ的表达。小鼠继续高脂喂养到24周时,斑块大小较16周增加,纤维帽明显变薄,斑块表面仅有很薄的内皮细胞覆盖,平滑肌细胞减少,斑块内呈现多个空泡样的结构即坏死核,此时的斑块属于动脉粥样硬化晚期斑块,同龄的野生型C57BL/6小鼠此时也没有明显的斑块形成,免疫组化在斑块局部仅能检测到IL-17A,几乎检测不到IFN-γ的存在。
此外,我们还采用RT-PCR检测了主动脉弓部IL-17A和IFN-γ及转录因子RORγt和T-bet的表达,与免疫组化结果一致,在早期斑块和晚期斑块局部均能够检测到IL-17A的表达,且较对照组小鼠明显升高；但是IFN-γ仅在早期斑块局部能够检测到而在晚期则几乎检测不到。RORγt和T-bet分别调控Th17和Th1细胞的分化,在ApoE-/-小鼠8周时表达即明显高于对照组小鼠,而且其表达持续升高能够维持到动脉粥样硬化斑块晚期。为了确定IL-17A的细胞来源,我们采用免疫荧光双染的方法证实斑块局部的IL-17A由CD4+T细胞和巨噬细胞分泌。上述结果提示Th17和Th1细胞均参与动脉粥样硬化的发生,而且Th17细胞在动脉粥样硬化晚期发挥更重要的作用。
2.Th17和Th1细胞随动脉粥样硬化的发生在外周免疫器官中的变化
为了观察Th17和Th1细胞在外周免疫器官中的变化,我们采用流式细胞术的方法检测Th17(CD4+IL-17A+)细胞和Th1(CD4+IFN-γ+)细胞在脾脏中的比例,结果显示在小鼠8周时,即无动脉粥样硬化斑块形成的小鼠脾脏中,实验组和对照组Th17和Th1细胞无明显的差异,在斑块形成的早期,Th17和Thl细胞比例在ApoE-/-小鼠脾脏中较对照组小鼠明显升高；随着疾病的进展,Th17和Th1细胞在斑块形成的晚期进一步升高,此外在疾病晚期小鼠脾脏中还发现一群即能够分泌IL-17A,同时也分泌IFN-γ的CD4+T细胞(CD4+IL-17A+IFN-γ+)较对照组小鼠也明显升高。
进一步统计学分析显示Th17、Th1和CD4+IL-17A+IFN-γ+T细胞的变化与动脉粥样硬化斑块的大小均呈明显的正相关,而且与小鼠血清中总胆固醇(TCH)的水平也呈现明显的正相关,这就提示Th17和Th1细胞均可能参与动脉粥样硬化的发生。
(二)Th17细胞及IL-17A对动脉粥样硬化斑块形成的影响
为了证实Th17细胞及IL-17A在动脉粥样硬化发生中的作用,我们用小鼠中和性IL-17A抗体和外源性小鼠IL-17A分别干预ApoE-/-小鼠,微型超声和油红O染色结果显示抗IL-17A抗体能够明显抑制斑块的形成,而外源性小鼠IL-17A则明显促进动脉粥样硬化斑块的形成,免疫组织化学染色结果显示IL-17A干预组小鼠斑块局部巨噬细胞的比例与对照组小鼠无明显差异,而平滑肌细胞的比例较对照组小鼠明显增多。该结果从正反两个方面证实IL-17A能够促进动脉粥样硬化斑块的形成,而且有可能促进斑块向不稳定型斑块发展。
二、IL-17A通过激活NF-κB和p38, ERK通路上调aP2诱导巨噬细胞内质网应激并继发细胞凋亡进而加速动脉粥样硬化的发生和发展
(一)IL-17A通过诱导巨噬细胞内质网应激促进细胞凋亡
1.IL-17A促进巨噬细胞凋亡
大量巨噬细胞凋亡能够促进动脉粥样硬化发生发展,为了研究IL-17A促进动脉粥样硬化发生的机制,在体外巨噬细胞用IL-17A刺激后,Hoechst染色和Annexin V/PI染色结果显示,IL-17A刺激后,随浓度的增高和时间延长,巨噬细胞内凋亡小体明显增多。流式细胞术定量分析显示IL-17A能够明显促进巨噬细胞发生晚期凋亡。
2.IL-17A诱导小鼠及人来源的巨噬细胞发生内质网应激
内质网应激是动脉粥样硬化发生过程中促进巨噬细胞凋亡的重要原因之一,为了研究IL-17A促进巨噬细胞凋亡的机制,小鼠巨噬细胞系RAW264.7和腹腔巨噬细胞在体外用IL-17A刺激后,Western Blotting结果显示内质网应激标志分子p-PERK、p-eIF2α和XBP1s的表达明显升高,由此可见IL-17A能够诱导小鼠巨噬细胞发生内质网应激。为了观察IL-17A是否对人来源巨噬细胞也具有同样的作用,我们将单核细胞系Thp-1和人外周血单个核细胞(PBMC)体外诱导成巨噬细胞后,加入IL-17A刺激,Western Blotting结果显示p-PERK、p-eIF2α和XBP1s的表达同样明显升高。上述结果说明IL-17A在体外能够诱导人和小鼠来源的巨噬细胞发生内质网应激。
3. IL-17A诱导动脉粥样硬化斑块局部巨噬细胞发生内质网应激
为了验证是否在小鼠体内IL-17A也能诱导巨噬细胞发生内质网应激,我们首先用Western Blotting检测主动脉弓部p-PERK、p-eIF2α和XBP1s的表达,结果显示IL-17A干预组小鼠斑块局部内质网应激标志分子的表达均明显高于对照组小鼠。进一步我们采用免疫组织化学染色的方法定位检测斑块局部Moma-2, p-PERK和p-eIF2α的表达,结果显示在巨噬细胞表达部位,p-PERK和p-eIF2α的表达量占斑块面积的比例在IL-17A干预组小鼠明显高于对照组小鼠,该结果说明IL-17A不仅可以在体外,在斑块局部也能够诱导巨噬细胞发生内质网应激。
4.IL-17A通过诱导巨噬细胞内质网应激促进细胞凋亡
长期内质网应激能够导致细胞发生凋亡,内质网应激诱导细胞凋亡主要通过上调CHOP的表达、激活caspas12分子、激活JNK进而活化Bcl-2,最终通过活化caspase3诱导细胞发生凋亡。为了探索IL-17A是否通过诱导内质网应激促进巨噬细胞凋亡,我们首先在体外发现IL-17A能够促进CHOP和caspase3剪切体的表达,caspase12略有上调。为了在小鼠体内验证该结果,我们将ApoE-/-小鼠随机分为三组：对照组,IL-17A干预组,IL-17A联合PBA干预组。Western Blotting和免疫组化染色显示IL-17A干预组小鼠斑块局部CHOP表达明显上调,而caspasel2无明显的差异,caspase3剪切体的表达亦明显升高；而IL-17A联合PBA干预组,p-PERK和p-eIF2α的表达较IL-17A干预组明显减弱,说明PBA能够缓解IL-17A诱导的巨噬细胞内质网应激,同时CHOP和caspase3的表达较IL-17A干预组亦明显减弱,caspasel2无明显的差异。Tunnel染色结果显示IL-17A干预组小鼠斑块局部凋亡的巨噬细胞数量明显高于对照组小鼠,而IL-17A联合PBA干预组凋亡细胞则较IL-17A干预组明显减少。上述结果证实IL-17A通过诱导巨噬细胞内质网应激进而通过激活CHOP-caspase3通路促进巨噬细胞的凋亡。
(二)IL-17A通过内质网应激引起的巨噬细胞凋亡促进动脉粥样硬化的发生
ApoE-/-小鼠随机分为三组(对照组,IL-17A干预组,IL-17A联合PBA干预组)进行干预后,我们采用油红O染色的方法检测斑块的大小及脂质沉积,结果显示IL-17A干预组小鼠主动脉根部斑块的面积明显较对照组增大,而IL-17A联合PBA干预组小鼠的斑块面积则明显较IL-17A单独干预组减小,由此可见,IL-17A确实是通过诱导巨噬细胞内质网应激进而通过激活CHOP-caspase3通路促进细胞凋亡、加速动脉粥样硬化斑块的形成。
(三)IL-17A通过激活NF-κB和p38, ERK通路上调aP2诱导巨噬细胞内质网应激的发生
1.IL-17A上调aP2的表达诱导巨噬细胞内质网应激
有研究表明aP2表达增高能够促进巨噬细胞内质网应激的发生,为了研究IL-17A诱导巨噬细胞内质网应激的发生机制,在体外我们用RT-PCR和Western Blotting检测发现IL-17A能够在RNA和蛋白水平上调巨噬细胞内aP2的表达；此外在小鼠体内我们也发现IL-17A干预组小鼠斑块局部aP2的表达较对照组小鼠明显升高。为了证实IL-17A是通过上调aP2的表达诱导巨噬细胞内质网应激,在体外我们先用aP2抑制剂作用于巨噬细胞,然后再加入IL-17A刺激,Western Blotting检测结果显示p-eIF2a和XBP1s的表达较IL-17A单独刺激明显减弱,该结果证实IL-17A通过上调aP2的表达诱导巨噬细胞内质网应激的发生。
2.IL-17A激活NF-κB和p38、ERK通路上调aP2的表达
IL-17A与其受体结合后能够通过激活NF-κB和MAPK通路传递信号入细胞,诱导多种分子的表达。为了研究IL-17A上调aP2的机制,IL-17A刺激巨噬细胞后,我们采用Western Blotting检测发现P-IκB和p-p38, p-ERK, p-JNK的表达均明显升高。进一步我们分别用NF-κB和p38, ERK, JNK抑制剂作用巨噬细胞,然后用IL-17A刺激后检测aP2的表达,结果显示NF-κB和p38, ERK抑制作用后aP2的水平明显减弱。由此可以证明IL-17A通过激活NF-κB和p38、 ERK通路进而促进aP2的表达。
结论
1.Th17细胞及效应分子IL-17A具有促进动脉粥样硬化的作用动脉粥样斑块局部Th17细胞和IL-17A高表达,外周免疫器官中Th17细胞的数量随着动脉粥样硬化的发生和发展逐渐升高,并与动脉粥样硬化斑块的大小及总胆固醇的水平呈正相关,体内实验证实Th17细胞及效应分子IL-17A能够促进动脉粥样硬化斑块形成。
2.IL-17A诱导巨噬细胞发生内质网应激和继发的细胞凋亡是IL-17A加速动脉粥样硬化斑块形成的机制之一IL-17A在体外以及斑块局部均能够诱导巨噬细胞发生内质网应激,长期内质网应激可以进一步通过激活CHOP-caspase3通路促进巨噬细胞凋亡,进而加速动脉粥样硬化斑块的形成。
3.IL-17A诱导的aP2上调是其导致巨噬细胞发生内质网应激的机制之一IL-17A可通过激活NF-κB和p38、ERK通路上调脂肪酸结合蛋白aP2的表达,抑制剂阻断aP2的表达则明显抑制IL-17A诱导的巨噬细胞内质网应激。
创新性和意义
本研究通过体内外实验,系统研究了新的T细胞亚群Th17细胞及其主要效应分子IL-17A在动脉粥样硬化斑块形成中的作用及其机制,对揭示动脉粥样硬化的免疫机制、防治动脉粥样硬化相关的心血管疾病具有重要的理论价值和潜在的应用价值。本研究的主要创新点和意义如下：
1.证明Th17和IL-17A参与促进动脉粥样硬化的发生和发展,外源性IL-17A治疗明显促进动脉粥样硬化斑块的形成,而中和性抗IL-17A抗体的应用可有效缓解斑块的发展,为临床以IL-17A为靶点治疗动脉粥样硬化提供了有力的理论和实验依据。
2.在国内外首先发现IL-17A能诱导巨噬细胞发生内质网应激,长期内质网应激继发的细胞凋亡是IL-17A促进动脉粥样硬化发生的重要机制之一,用化学伴侣分子PBA阻断内质网应激可有效控制IL-17A介导的动脉粥样硬化病变的进展。
3.在国内外首先发现IL-17A可通过NF-κB和p38/ERK MAKP信号通路上调脂肪酸结合蛋白aP2,进而诱导内质网应激的发生；首次通过aP2将细胞因子和脂质代谢联系在一起,为研究炎症反应与脂质代谢疾病的相互关系及机制提供了新的思路。
4.本研究证实IL-17A中和性抗体、内质网应激抑制剂PBA能够抑制斑块的形成,aP2抑制剂能够缓解巨噬细胞内质网应激,为临床以炎性因子、细胞内质网应激及脂质代谢为靶点联合治疗动脉粥样硬化提供了有力的理论和实验依据。
研究的局限性
1.IL-17A诱导内质网应激发生的机制还有待于更深入的研究；
2.aP2影响IL-17A诱导的内质网应激和促进斑块作用的体内试验尚有待进一步的确定。
Objective
Atherosclerosis is the most common diseases of heart cerebrovascular system and the leading cause of mortablity in advanced and developing countries. Atherosclerosis is a slowly progressing chronic inflammatory disease occurring in large and medium-sized arteries and is characterized by an accumulation of lipids in the artery wall, together with infiltration of immunocytes, such as macrophages, T cells and mast cells, and the proliferation of vascular smooth muscle cells. Accumulate research showed that innate immune respons (macrophages) and adaptive immune response (CD4+T cells) participated in the development of atherosclerosis. Recently, the effect of CD4+T cells in this disease received widespread concerns. CD4+T cells contain different subsets and compose of conventional Th1and Th2cells and regulatory T cells (Treg) and new subsets Th17cells. Th1cells promoted the development of atherosclerosis. And the effect of Th2cells on the diseases remains controversial. Regulatory T cells play a protective role in atherogenesis and could inhibit the formation of atherosclerotic plaque. However, the function of Th17cells in the development of atherosclerosis remains undefined.
Th17cell is characterized by secreting IL-17A. Naive CD4+T cells stimulated by antigens and differentiated into Th17cells with the regulation of transcription factors RORγt and RORoα and in the presence of IL-6and TGF-β.It has been shown that Th17cells and effector IL-17A play a part in many kinds of chronic inflammation and organ-specific autoimmune disease in animals such as experimental autoimmune encephalomyelitis (EAE), rheumatoid arthritis (RA) and colitis. Atherosclerosis is a chronic inflammatory disease occurring in artery vascular walls, previous studies have shown that Th17cells and its major effector cytokine IL-17A are increased in the peripheral blood of patients with coronary atherosclerosis. And T cells infiltrated in the atherosclerotic plaque express IL-17A in human or in mouse models. These suggest that Th17cells and IL-17A probably participate in the development of atherosclerosis. However, whether Th17cells and IL-17A increased with the development of atherosclerosis? What roles Th17cells and effector IL-17A play in the formation and development of atherosclerosis remains debate. Whether could IL-17A as one of potential therapeutic targets for atherosclerosis? What is the mechanism of IL-17A in this disease? All these problems remain to be investigated.
To confirm the effect of Th17cells and IL-17A on the development of atherosclerosis, illustrate the mechanism, and provide theoretical basis and experimental data for clinical applications, this paper intends to study from the following two parts:
1. To confirm the aggravated role of Th17cells and effector IL-17A in the development of atherosclerosis.
1) The establishment of standard atherosclerotic mice model and rapid carotid lesions mice model:ApoE-/-mice fed with high cholesterol diet to establish standard atherosclerotic mice model. And high cholesterol diet combined with placed perivascular constrictive silica collars on the left common carotid artery to establish rapid carotid lesions mice model.
2) The expression of Th17cells and IL-17A in atherosclerotic plaque and peripheral immune organ:Results for RT-PCR and immunohistochemistry showed that Th17cells and IL-17A could be detected in atherosclerotic lesions. And flow cytometry analysis found that the percentage of Th17cells and IL-17A in spleen increased by the formation and development of atherosclerosis. Furthermore, the proportion of Th17cells was positive correlated with the area of the plaque and the level of serum lipids (TCH).
3) The effect of Thl7cells and IL-17A on the formation of atherosclerotic plaque:We treated ApoE-/-mice with anti-IL-17A neutralizing antibody or exogenous recombinant mouse IL-17A, and from both the positive and negative aspects, we confirmed that IL-17A could promote the formation of atherosclerotic plaque. Furthermore, IL-17A reduces the content of smooth muscle cells in lesions, suggesting increased instability of the plaques.
2. To explore the mechanism of IL-17A promote the development of atherosclerosis from macrophage ER stress and cell apoptosis
1) The effect and mechanism of IL-17A on macrophage apoptosis:IL-17A could promote macrophage apoptosis in vitro. Further mechanism study found that IL-17A induced ER stress in murine and human-derived macrophages in vitro. In addition, IL-17A could also induce ER stress in macrophage-dense areas of the atherosclerotic lesions in ApoE-/-mice. However, in vivo, blocking of ER stress by chemical chaperone (PBA) alleviated IL-17A-induced ER stress and reduced the numbers of apoptotic macrophages in lesions, suggesting IL-17A could promote macrophage apoptosis through inducing ER stress.
2) The mechanism of IL-17A aggravated the development of atherosclerosis:We treated ApoE-/-mice with IL-17A or IL-17A combined with PBA, and confirmed that IL-17A could promote macrophages apoptosis by activating ER stress response and accelerate the formation of atherosclerotic plaque.
3) The mechanism of IL-17A induced ER stress in macrophage:In vitro, IL-17A could up-regulate the expression of aP2and activate NF-κB and MAPK signal pathway (ERK, p38and JNK). Furthermore, we found that IL-17A up-regulated aP2through activating NF-κB and p38、ERK pathway. Finally, we confirmed that aP2mediated IL-17A-induced ER stress in macrophages.
Methods
1. To confirm the aggravated role of Th17cells and effector IL-17A in the development of atherosclerosis.
1) The establishment of standard atherosclerotic mice model and rapid carotid lesions mice model:
i) Standard mice model:8weeks old male ApoE-/-mice fed with high cholesterol diet to16weeks or24weeks old. And same strain C57BL/6mice also give high cholesterol diet as control.
ii) Rapid carotid lesions mice model:8weeks old male ApoE-/-mice fed with high cholesterol diet for2weeks, and then carotid atherosclerotic lesions were induced by perivascular constrictive silica collars placed on the left common carotid artery. Mice were going on high cholesterol diet to16weeks or24weeks old. And same strain C57BL/6mice as control.
2) The expression of Th17cells and IL-17A in atherosclerotic plaque and peripheral immune organ:
i) Separate the left common carotid arteries and the aortic root vessels and prepare serial cryosections, HE staining and Oil red O staining detected the formation of atherosclerotic lesions and the content of lipids. And immunohistochemistry detected the expression of IL-17A and IFN-y in plaque, and confirmed the resource of IL-17A with immunofluorescence double staining.
ii) Separate the aortic arch to extract RNA and detect the expression of IL-17A, RORyt and IFN-γ, T-bet in atherosclerotic plaque with RT-PCR.
iii) Splenocytes were prepared from different age mice and flow cytometry detected the percentage of Thl and Th17cells.
iv) To analyze the correlation of the proportion of Thl and Th17cells with the size of atherosclerotic plaque and the level of serum lipids.
3) The effect of Th17cells and IL-17A on the formation of atherosclerotic plaque:
i) Anti-IL-17A neutralizing antibody treatment:
a) Twenty8weeks old male ApoE-/-mice fed with high cholesterol diet for2weeks, and then carotid atherosclerotic lesions were induced by perivascular constrictive silica collars placed on the left common carotid artery. Five days later, mice were randomly divided into two groups and treated with anti-IL-17A neutralization monoclonal antibody (50μg/mouse/time) or isotype antibody (rat IgG2A), once a week for4weeks.
b) During this interventional period, plaque sizes were evaluated by Micro-ultrasound imaging, and then plaque morphology was detected by histopathology after mice were sacrificed at one week following the last challenge. Oil red O staining detected the size of lesions.
c) Immunohistochemistry detected the expression of macrophage and smooth muscle cells in lesions.
ii) Application of exogenous IL-17A:
a) Ten8weeks old male ApoE-/-mice were randomly divided into two groups and fed with high cholesterol diet, and11weeks old mice were given intraperitoneal injection with recombinant mice IL-17A (2μg/mice/time) once a week for5weeks and normal saline as control.
b) At one week following the last challenge, separate the aortic root vessels and prepare serial cryosections. Oil red O staining observed the size of plaque.
c) Immunohistochemistry detected the expression of macrophage and smooth muscle cells in lesions.
2. To explore the mechanism of IL-17A promote the development of atherosclerosis from macrophage ER stress and cell apoptosis
1) The effect and mechanism of IL-17A on macrophage apoptosis and the formation of atherosclerotic plaque:
i) Experiment in vitro
a) Isolating of mice peritoneal macrophages and stimulated with IL-17A, Hoechst staining observed cell apoptosis from cytomorphology and flow cytometry (Annexin V/PI) detected the numbers of apoptotic cells.
b) Mice macrophage cell line RAW264.7and peritoneal macrophages stimulated by IL-17A, then Western Blotting detected the expression of ER stress markers p-PERK, p-eIF2a and XBP1s.
c) Human monocyte cell line Thp-1and peripheral blood mononuclear cells were induced into macrophages and stimulated with human IL-17A, then Western Blotting detected the expression of ER stress markers p-PERK, p-eIF2α and XBP1s.
d) Mice peritoneal macrophages stimulated by IL-17A, then Western Blotting detected the expression of CHOP, caspase12and caspase3cleavage.
ii) Experiment in vivo
a) Thirty8weeks old male ApoE-/-mice fed with high cholesterol diet, and11weeks old mice were randomly divided into3group:Control (i.p. normal saline), IL-17A-treated group (i.p. mice IL-17A,2μg/mice/time, once a week), IL-17A combined with PBA treated group (i.p. IL-17A,2μg/mice/time, once a week and PBA,100mg/kg, twice a week).
b) At one week following the last challenge, separate the aortic root vessels and prepare serial cryosections. Immunohistochemistry detected the expression of Moma-2(for macrophage), p-PERK, p-eIF2α and CHOP, caspase12in atherosclerotic lesions. And Tunnel staining for apoptotic macrophages in lesions. Separate the aortic arch to extract proteins, Western Blotting detected the expression of ER stress markers (p-PERK, p-eIF2α and XBP1s) and CHOP, caspase12, and caspase3cleavage.
c) Oil red O observed the size of atherosclerotic plaque.
2) The mechanism of IL-17A induced ER stress in macrophage:
i) Mice peritoneal macrophages stimulated by IL-17A, RT-PCR and Western Blotting detected the expression of aP2.
ii) Mice peritoneal macrophages with aP2inhibitor (BMS309403)(50uM) pre-treated three hours and then stimulated by IL-17A, Western Blotting detected the expression of p-eIF2α and XBPls.
iii) Mice peritoneal macrophages stimulated by IL-17A, Western Blotting detected the expression of p-IκB, p-ERK, p-p38and p-JNK. Furthermore, mice peritoneal macrophages with NF-κB (PDTC), ERK (PD98059), JNK (SP600125) and p38(SB203580) inhibitor pre-treated one hour and then stimulated by IL-17A, Western Blotting detected the expression of aP2.
Results
1. We confirmed that Th17cells and effector IL-17A aggravated the development of atherosclerosis with experiment in vivo and in vitro
1) The expression of Th17cells and IL-17A in atherosclerotic plaque and peripheral immune organ
i) The expression of IL-17A and IFN-y in atherosclerotic plaque
To study the effect of Th17cells and IL-17A on atherogenesis, we established rapid carotid lesions mice model and standard atherosclerotic model with ApoE-/-mice. And we separated the left common carotid arteries and the aortic root vessels and prepare serial cryosections, HE staining, Oil red O staining and immunohisto-chemistry detected the formation of atherosclerotic lesions and the expression of IL-17A and IFN-y. Results showed that8week-old ApoE-/-mice had no significant plaque formation in carotids and vascular intima only appeared slightly thickening. At this time, no IL-17A and IFN-y was detected in lesions. When mice fed with high cholesterol diet for additional eight weeks,16weeks old ApoE-/-mice showed that vascular intima significantly thicken and proliferation of smooth muscle cells. Atherosclerotic plaque characterized by thicker fibrous cap and much of nucleated cells infiltrating and was defined as the early stage of atherosclerosis. At16weeks old ApoE-/-mice, substantial expression of IL-17A and IFN-y was detected. The plaque was more severe when ApoE-/-mice were continuously fed with high cholesterol diet for16weeks,24weeks old ApoE-/-mice showed that the size increased obviously, fibrous cap became thinner, the number of smooth muscle cells reduced and many vacuoles present in lesions, that was the late stage of atherosclerosis. This stage, we could detect IL-17A in lesions and almost no IFN-y was found. In contrast, the age-matched wild-type C57BL/6mice (fed with the same regimen as ApoE-/-mice) had no detectable plaque in carotids throughout the whole experimental periods.
In addition, we detected the expression of IL-17A, RORyt and IFN-γ, T-bet with RT-PCR. Consistent with the results from immunohistochemistry or immune-fluorescence staining, the expression of IL-17A in ApoE-/-mice with the early and late stage of plaque was markedly higher than that of wild-type control mice. However, IFN-y only was detected in early stage of plaque and could not be detected in late stage of plaque. Notably, the expression of RORyt and T-bet, the transcription factors controlling Th17and Thl cell differentiation respectively, was significantly higher in the vascular wall of ApoE-/-mice than in that of control mice at as early as8weeks. The levels of RORyt and T-bet continued to increase and were maintained at higher levels in ApoE-/-mice at the early and late stages of atherosclerotic plaque (16-24weeks). To confirm the cellular sources of IL-17A, we co-stained IL-17A with CD4+T cells and macrophages with immunofluorescence staining. IL-17A was mainly expressed in both CD4+T cells and macrophages infiltrated into the atherosclerotic plaque. The data suggest that both Th17and Thl cells were involved in the development of the atherosclerotic inflammation, and Th17play more important roles in late stage of atherosclerosis.
ii) Increased Th17and Thl cells in the spleen of ApoE-/-mice with atherosclerotic plaque
To observe the change of Th17and Thl cells in peripheral immune organ, we detected the number of Th17(CD4+IL-17A+) and Thl (CD4+IFN-γ+) in spleen with flow cytometry. Results showed that the pre-symptomatic ApoE-/-mice (8weeks-old) had minimal numbers of Th17cells with no statistical difference compared with control mice. Although the number of Thl cells was higher than Th17, there was no statistical difference between ApoE-/-and control mice. However, the frequencies of Th17and Thl cells in16week-old ApoE-/-mice with plaque was significantly higher than that in control mice, which were further increased in the ApoE-/-mice with advanced atherosclerotic plaque (24weeks-old). In addition, we observed a population of CD4+T cells expressing both IL-17+and IFN-γ+. The frequency of CD4+IL-17+IFN-γ+T cells significantly increased in the late stage atherosclerotic ApoE-/-mice compared with age-matched control mice.
Furthermore, simple linear regression analysis, we observed that the area of the plaque and the level of TCH was not only correlated with the proportion of Thl cells, but also closely associated with Th17cell subset and CD4+IL17+IFN-γ+cells. These results suggested that Th17and Thl cells both probably participated in atherogenesis.
2) The influence of Th17cells and IL-17A on the development of atherosclerosis
To confirm the role of Th17cells and IL-17A on the development of atherosclerosis, we treated ApoE-/-mice with anti-IL-17A neutralizing antibody and exogenous IL-17A. Results from micro-ultrasound imaging and Oil red O staining showed that neutralizing IL-17A antibody significantly inhibited the formation of atherosclerotic plaque, however, exogenous IL-17A obviously accelerated atherogenesis. Immunohistochemistry show that IL-17A treatment had no significant effect on the number of macrophage and it decreased the numbers of smooth muscle cells in plaques. These results confirmed from positive and negative aspects that IL-17A could promote the development of atherosclerosis and increased instability of the plaques.
2. IL-17A-induced ER stress in macrophage promote cell apoptosis and aggravate atherogenesis by up-regulating NF-κB and p38, ERK pathway mediated the expression of aP2
1) IL-17A promote macrophage apoptosis by inducing ER stress
i) IL-17A promote macrophage apoptosis
Macrophage apoptosis is an important cause for the development of atherosclerosis. To explore the mechanism of IL-17A accelerate the formation of atherosclerotic plaque, we stimulated macrophage with IL-17A in vitro. Results from Hoechst staining showed that macrophages treated by IL-17A revealed increase of apoptotic bodies. Next, results from flow cytometry showed that IL-17A promote the number of late apoptotic macrophages increasing markedly.
ii) IL-17A induced endoplasmic reticulum stress in murine and human-derived
macrophage in vitro
Since increasing evidence supports that ER stress is one important mechanism for macrophage apoptosis, to explore the mechanism of IL-17A promote macrophage apoptosis. Mice macrophage cell line RAW264.7and mice peritoneal macrophage were stimulated by IL-17A, Western Blotting analysis showed that the expression of ER stress markers p-PERK、p-eIF2α and XBP1s increased significantly. This suggested that IL-17A could induce ER stress in murine-derived macrophages. To observe whether IL-17A play the same roles on human-derived macrophage, we induced Thp-1and PBMC into macrophages and then stimulated with IL-17A, Western Blotting analysis showed that the expression of p-PERK、p-eIF2α and XBP1s increased significantly. These results indicated that IL-17A could induce ER stress in human and murine-derived macrophages in vitro.
iii) Exogenous IL-17A induced ER stress in macrophage-dense areas of the atherosclerotic lesions in ApoE-/-mice
To confirm the impact of IL-17A on ER stress in vivo, we treated ApoE-/-mice fed with high cholesterol diet utilizing exogenous IL-17A, Western blotting analysis showed that p-PERK, p-eIF2α and XBP-1s were significantly up-regulated in aortic vessels with atherosclerotic plaque in IL-17A-treated mice compared with control. Consist with results from Western Blotting, immunohistochemistry staining revealed that increased expression of p-eIF2a and p-PERK mainly located in macrophage-dense areas of the atherosclerotic lesions. These indicate that IL-17A is able to induce directly ER stress response in macrophage both in vitro and in vivo.
iv) IL-17A promote macrophage apoptosis by inducing ER stress
It has been shown that prolonged activation of ER-associated CHOP and caspase12pathways are involved in the ER stress-mediated apoptosis. To explore whether IL-17A promote macrophage apoptosis by inducing ER stress, we found that IL-17A significantly promote the expression of CHOP and caspase3cleavage, and slightly up-regulate caspasel2in vitro. To confirm the effect in vivo, we divided ApoE-/-mice into3groups:Control, IL-17A-treated group and IL-17A combined with PBA treated group. Western Blotting and immunohistochemistry showed that the expression of CHOP and caspase3cleavage up-regulated markedly in lesions of IL-17A-treated mice, and caspase12had no alteration. However, the expression of p-PERK and p-eIF2α diminished obviously in lesions of IL-17A combined PBA-treated mice, suggesting PBA could alleviate ER stress in lesions. At the same time, CHOP and caspase3cleavage reduced significantly and caspase12had no obvious change. Tunnel staining showed that IL-17A leads to a marked increase in apoptotic cells in atherosclerotic lesions and IL-17A comined with PBA could reduce apoptotic cells. Those indicated that IL-17A could induce apoptosis in macrophages mainly via ER stress-CHOP-caspase3axis.
2) IL-17A accelerated the progression of atherosclerosis via ER stress-induced apoptosis in macrophage
ApoE-/-mice divided into three groups:control, IL-17A-treated group and IL-17A combined with PBA treated group. Oil red O detected the size of the aortic root for lesions, results showed that mice receiving IL-17A and PBA treatment had an obvious reduction in plaque size compared with IL-17A-treated mice. It follows that IL-17A could induce apoptosis in macrophages mainly via ER stress-CHOP-caspase3axis by which it accelerated progression of atherosclerosis.
3) IL-17A up-regulated expression of aP2by activating NF-κB and ERK/p38MAPK pathways and further induced ER stress in macrophage
i) The up-regulated expression of aP2is required for IL-17A-induced ER stress in macrophages
Recent study has suggested that aP2mediates saturated fatty acid-induced ER stress and apoptosis in macrophage, to study the mechanism of IL-17A induced ER stress in macrophage. Results from RT-PCR and Western Blotting both showed that IL-17A up-regulated directly aP2transcription and translation in macrophages in vitro. Importantly, IL-17A also led to a significant increase in the expression of aP2in atherosclerotic lesions in vivo. To clarify the role of aP2in IL-17A-induced ER stress, we treated macrophages with aP2inhibitor (BMS309403) in presence of IL-17A and found that accompanying inhibition of aP2, ER stress indicators p-eIF2α and the splice of XBP-1in macrophages diminished obviously correspondingly. This data clearly demonstrate that IL-17A for up-regulated aP2expression is required, at least partially, for IL-17A-induced ER stress in macrophages.
ii) IL-17A up-regulated expression of aP2by activating NF-κB and ERK/p38MAPK pathways
Previous works have shown that IL-17A can induce multiple genes via activating the nuclear factor (NF)-κB pathway and MAPK pathway. To explore the mechanism of IL-17A up-regulated the expression of aP2, macrophages were stimulated by IL-17A. And Western Blotting analysis showed that IL-17A could rapidly up-regulate the expression of p-IκB, p-ERK and p-p38, p-JNK. Subsequently, to clarify causative role of these signal pathways in IL-17A-upregulated aP2, we treated mouse peritoneal macrophages with the inhibitor of NF-κB, ERK, JNK and p38and then detected the aP2expression by Western Blotting. Results revealed that the inhibitors of NF-κB, ERK and p38diminish significantly the expression of aP2. However, JNK inhibitor did not suppress the aP2expression. Collectively, IL-17A up-regulates the expression of aP2through activating NF-κB and ERK/p38MAPK signal pathways in macrophages.
Conclusions
1. Th17cells and effector IL-17A could promote the development of atherosclerosis
The expression of Th17cells and IL-17A in atherosclerotic plaque increased and the proportion of Th17cells in spleen increased by the development of atherosclerosis and correlated with the size of atherosclerotic plaque and the level of TCH. In addition, in vivo, we confirm that Th17cells and effector IL-17A promote the formation of atherosclerotic plaque.
2. IL-17A induced ER stress in macrophage and secondary apoptosis is one of mechanisms that IL-17A accelerates the formation of atherosclerotic plaque IL-17A induced ER stress in macrophage in vitro culture and in lesions. Prolongation of ER stress promotes macrophage apoptosis and accelerates the formation of atherosclerotic plaque through activating ER stress-CHOP-caspase3pathway.
3. IL-17A up-regulating the expression of aP2is one of mechanisms of IL-17A induced ER stress in macrophage
Further study verify that IL-17A induces ER stress in macrophage through activating NF-κB and p38、ERK pathway and up-regulating the expression of aP2. And aP2inhibitor could alleviate ER stress that IL-17A induced in macrophage.
Originality and Significance
In this research, we systematicaly study the effect and mechanism of Th17cells and its effector IL-17A in the development of atherosclerosis with experiment in vivo and in vitro. These have important theoretical and potential application value for revealing immunological mechanism of atherosclerosis and preventing atherosclerotic related cardiovascular disease. The originality and significance as follows:
1. We confirm that Th17cells and IL-17A participated in the development of atherosclerosis. And exogenous IL-17A treatment obviously promotes the formation of atherosclerotic plaque. However, neutralizing IL-17A antibody could reduce the size of plaque. These foundings provide strong theoretical and experimental basis for treatment of atherosclerosis with IL-17A as clinical therapy target.
2. We demonstrate, for the first time, that IL-17A by oneself can induce ER stress in macrophage. Secondary apoptosis of prolongation ER stress is one of mechanisms that IL-17A promotes the development of atherosclerosis. Furthermore, the inhibiting of ER stress with chemical chaperon PBA effectively control IL-17A-aggravated atherosclerosis.
3. For the first time, we demonstrate that IL-17A up-regulate the expression of aP2and then induce ER stress in macrophage. And firstly, we establish a previously unrecognized link between cytokines and lipid metabolism through cytosolic lipid chaperone aP2in macrophage. These provide new ideas for studying the relationship and mechanism between inflammation and lipid metabolism diseases.
4. We confirm that that IL-17A antibody and PBA could inhibit atherogenesis, aP2inhibitor could alleviate ER stress in macrophage. All these provide potent theoretical and experimental basis for treatment target as inflammatory cytokines, ER stress and lipid metabolism.
Limitations of this study
1. The mechanism of IL-17A induced ER stress need to be further investigated.
2. The effect of aP2on IL-17A-induced ER stress and promoted atherosclerosis need experiments in vivo to be verified.
引文
[1]Hansson G.K., Robertson A.K., So"derberg-Naucle'r C. Inflammation and atherosclerosis. Annu Rev Pathol.2006; 1:297-329.
[2]Lloyd-Jones, D.M. Cardiovascular risk prediction:basic concepts, current status, and future directions. Circulation 2010; 121:1768-1777.
[3]Hansson, G.K. Inflammation, atherosclerosis, and coronary artery disease. N. Engl. J. Med.2005; 352:1685-1695.
[4]Williams, K.J., Tabas, I. The response to retention hypothesis of early atherogenesis. Arterioscler. Thromb. Vasc. Biol.1995;15:551-561.
[5]Glass, C.K., Witztum, J.L. Atherosclerosis the road ahead. Cell 2001; 104: 503-516.
[6]Mestas, J., Ley, K. Monocyte-endothelial cell interactions in the development of atherosclerosis. Trends Cardiovasc. Med.2008; 18:228-232.
[7]Johnson, J.L., Newby, A.C. Macrophage heterogeneity in atherosclerotic plaques. Curr. Opin. Lipidol.2009; 20:370-378
[8]Paulson, K.E., Zhu, S.N., Chen, M., Nurmohamed, S., Jongstra-Bilen, J., Cybulsky, M.I. Resident intimal dendritic cells accumulate lipid and contribute to the initiation of atherosclerosis. Circ. Res.2010; 106:383-390.
[9]Kunjathoor, V.V., Febbraio, M., Podrez, E.A., Moore, K.J., Andersson, L., Koehn, S., Rhee, J.S., Silverstein, R., Hoff, H.F., Freeman, M.W. Scavenger receptors class A-I/I I and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J. Biol. Chem.2002; 277:49982-49988.
[10]Moore, K.J., Kunjathoor, V.V., Koehn, S.L., Manning, J.J., Tseng, A.A., Silver, J.M., McKee, M., Freeman, M.W. Loss of receptor mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyper-lipidemic mice. J. Clin. Invest.2005; 115:2192-2201.
[11]Manning-Tobin, J.J., Moore, K.J., Seimon, T.A., Bell, S.A., Sharuk, M., Alvarez-Leite, J.I., de Winther, M.P., Tabas, I., Freeman, M.W. Loss of SR-A and CD36 activity reduces atherosclerotic lesion complexity without abrogating foam cell formation in hyperlipidemic mice. Arterioscler. Thromb. Vasc. Biol.2009; 29:19-26.
[12]Mosmann T.R., Cherwinski C., Bond M.W., Giedlin M.A., Coffman R.L. Two types of murine helper T cell clones. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986; 136:2348-2357.
[13]Del Prete G.F., De Carli M., Mastromauro C., Biagiotti R., Macchia D., Falagiani Ricci M., Romagnani S. Purified protein derivative of Mycobacterium tuberculosis and excretory/secretori antigen(s) of Toxocaracanis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. J Clin Invest 1991; 88:346-350.
[14]Asano, M., Toda, M., Sakaguchi, N., Sakaguchi,S. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J.Exp.Med. 1996; 184:387-396.
[15]Rengarajan J., Szabo S.J., Glimcher L.H. Transcriptional regulation of Thl/Th2 polarization. Immunol Today 2000; 21:479-483.
[16]Khattri, R., Cox, T., Yasayko, S.A., Ramsdell, F. An essential role for Scurfin in CD4+CD25+T regulatory cells. Nat. Immunol.2003; 4:337-342.
[17]Fontenot, J.D., Gavin, M.A., Rudensky, A.Y. Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells. Nat. Immunol.2003; 4:330-336.
[18]Fontenot, J.D., Rasmussen, J.P., Williams, L.M., Dooley, J.L., Farr, A.G., Rudensky, A.Y. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity.2005; 22:329-341.
[19]Cabarrocas, J., Cassan, C., Magnusson, F., Piaggio, E., Mars, L., Derbinski, J., Kyewski, B., Gross, D.A., Salomon, B.L., Khazaie, K. Foxp3+CD25+ regulatory T cells specific for a neo-self-antigen develop at the doublepositive thymic stage. Proc. Natl. Acad. Sci.2006; 103:8453-8458.
[20]Campbell, D. J., Ziegler, S.F. Foxp3 modifies the phenotypic and functional properties of regulatory T cells. Nat. Rev. Immunol.2007; 7:305-310.
[21]Tedgui, A., Mallat, Z. Cytokines in atherosclerosis:pathogenic and regulatory pathways. Physiol. Rev.2006; 86:515-581.
[22]Zhou, X., Nicoletti, A., Elhage, R., Hansson, G.K. Transfer of CD4(+) T cells aggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice. Circulation 2000; 102:2919-2922.
[23]Zhou, X., Robertson, A.K., Hjerpe, C., Hansson,G.K. Adoptive transfer of CD4+ T cells reactive to modified low-density lipoprotein aggravates atherosclerosis. Arterioscler. Thromb. Vasc. Biol.2006; 26:864-870.
[24]Buono, C., Come, C.E., Stavrakis, G., Maguire, G.F., Connelly, P.W., Lichtman, A.H. Influence of interferon-gamma on the extent and phenotype of diet-induced atherosclerosis in the LDLR-deficient mouse. Arterioscler. Thromb. Vasc. Biol.2003; 23:454-460.
[25]Gupta, S., Pablo, A.M., Jiang, X., Wang, N., Tall, A.R., Schindler, C. IFN-gamma potentiates atherosclerosis in ApoE knock-out mice. J. Clin. Invest.1997; 99:2752-2761.
[26]Whitman, S.C., Ravisankar, P., Elam, H., Daugherty, A. Exogenous interferon-gamma enhances atherosclerosis in apolipoprotein E-/- mice. Am. J. Pathol. 2000; 157:1819-1824.
[27]Lee, T. S., Yen, H.C., Pan, C.C., Chau, L.Y. The role of interleukin 12 in the development of atherosclerosis in ApoE-deficient mice. Arterioscler. Thromb. Vasc. Biol.1999; 19:734-742.
[28]Davenport, P., Tipping, P.G. The role of interleukin-4 and interleukin-12 in the progression of atherosclerosis in apolipoprotein E-deficient mice. Am. J. Pathol.2003; 163:1117-1125.
[29]Paigen, B., Morrow, A., Brandon, C., Mitchell, D., Holmes, P. Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis 1985; 57:65-73.
[30]Huber, S.A., Sakkinen, P., David, C., Newell, M.K., Tracy, R.P.. T helper-cell phenotype regulates atherosclerosis in mice under conditions of mild hypercholesterolemia. Circulation 2001; 103:2610-2616.
[31]King, V.L., Szilvassy, S.J., Daugherty, A. Interleukin-4 deficiency decreases atherosclerotic lesion formation in a site-specific manner in female LDL receptor-/-mice. Arterioscler. Thromb. Vasc. Biol.2002; 22:456-461.
[32]Sakaguchi S. Regulatory T cells:key controllers of immunologic self-tolerance. Cell 2000; 101:455-458.
[33]Mills, K.H. Regulatory T cells:friend or foe in immunity to infection? Nat. Rev. 2004; 4:841-855.
[34]Aarvak, T., Chabaud, M., Miossec, P., Natvig, J.B. IL-17 is produced by some proinflammatory Th1/Th0 cells but not by Th2 cells. J. Immunol.1999; 162: 1246-1251.
[35]Harrington, L.E., Hatton, R.D., Mangan, P.R., Turner, H., Murphy, T.L., Murphy, K.M., Weaver, C.T. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol.2005; 6: 1123-1132.
[36]Park, H., Li, Z., Yang, X.O., Chang, S.H., Nurieva, R., Wang, Y. H., Wang, Y, Hood, L., Zhu, Z., Tian, Q., Dong, C. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol.2005; 6:1133-1141.
[37]Ivanov,I.I., McKenzie, B.S., Zhou, L., Tadokoro, C.E., Lepelley, A., Lafaille, J.J., Cua, D.J., Littman, D.R. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 2006; 126: 1121-1133.
[38]Veldhoen, M., Hocking, R.J., Atkins, C.J., Locksley, R.M., Stockinger, B. TGF-beta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006; 24:179-189.
[39]Bettelli, E., Carrier, Y, Gao, W., Korn, T., Strom, T. B., Oukka, M., Weiner, H.L. Kuchroo,V.K. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006; 441:235-238.
[40]Mangan, P.R., Harrington, L.E., O'Quinn, D.B., Helms, W.S., Bullard, D.C., Elson, C.O., Hatton, R.D., Wahl, S.M., Schoeb, T.R., Weaver,C.T. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 2006; 441: 231-234.
[41]Ouyang, W., Kolls, J. K., Zheng, Y. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity.2008; 28:454-467.
[42]Langrish, C.L., Chen, Y., Blumenschein, W.M., Mattson, J., Basham, B., Sedgwick, J.D., McClanahan, T., et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med.2005; 201:233-240.
[43]Ferretti, S., Bonneau, O., Dubois, G.R., Jones, C.E., Trifilieff, A. IL-17, produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia:IL-15 as a possible trigger. J. Immunol.2003; 170: 2106-2112.
[44]Hymowitz, S. G., Filvaroff, E. H., Yin, J. P., Lee, J., Cai, L., Risser, P., Maruoka, M., Mao, W., Foster, J., Kelley R. F., Pan, G., Gurney, A. L., de Vos A. M., Starovasnik, M. A.. IL-17s adopt a cystine knot fold:structure and activity of a novel cytokine, IL-17F, and implications for receptor binding. EMBo J.2001; 20: 5332-5341.
[45]Wright, J. F., Guo, Y., Quazi, A., Luxenberg, D. P., Bennett, F., Ross, J. F., Qiu, Y., Whitters, M. J., Tomkinson, K. N., Dunussi-Joannopoulos K, Carreno, B. M., Collins, M., Wolfman, N. M.. Identification of an interleukin 17F/17A heterodimer in activated human CD4+ T cells. J. Biol. Chem.2007; 282:13447-13455.
[46]Chang, S. H.& Dong, C. A novel heterodimeric cytokine consisting of IL-17 and IL-17F regulates inflammatory responses. Cell Res.2007; 17:435-440.
[47]Wright, J. F., Whitters, M. J., Tomkinson, K. N., Fitz, L. J., Wolfman, N. M., Collins, M., Dunussi-Joannopoulos K., Chatterjee-Kishore M., Carreno, B. M.. The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL 17RC receptor complex. J. Immunol.2008;181:2799-2805.
[48]Kuestner, R. et al. Identification of the IL-17 receptor related molecule, IL-17RC, as the receptor for IL-17F. J. Immunol.2007; 179:5462-5473.
[49]Yang, X. O., Chang, S. H., Park, H., Nurieva, R., Shah, B., Acero, L., Wang, Y. H., Schluns, K. S., Broaddus, R. R., Zhu, Z., Dong C. Regulation of inflammatory responses by IL-17F. J. Exp. Med.2008; 205:1063-1075.
[50]Ishigame, H., Kakuta, S., Nagai, T., Kadoki, M., Nambu, A., Komiyama, Y., Fujikado, N., Tanahashi, Y., Akitsu, A., Kotaki, H., Sudo, K., Nakae, S., Sasakawa, C., Iwakura, Y. Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses. Immunity 2009; 30: 108-119.
[51]Gaffen, S. L., Kramer, J. M., Yu, J. J.& Shen, F. in Vitamins and Hormones Vol.74. (ed. G. Litwack) 255-282 (Academic, London,2006).
[52]McAllister, F., Henry, A., Kreindler, J. L., Dubin, P. J., Ulrich, L., Steele, C., Finder, J. D., Pilewski, J. M., Carreno, B. M., Goldman, S. J., Pirhonen, J., Kolls, J. K. Role of IL-17A, IL-17F, and the IL-17 receptor in regulating growth -related oncogene-α and granulocyte colony -stimulating factor in bronchial epithelium: implications for airway inflammation in cystic fibrosis. J. Immunol.2005; 175: 404-412.
[53]Dong, C. Regulation and pro-inflammatory function of interleukin-17 family cytokines. Immunol. Rev.2008; 226:80-86.
[54]Aggarwal, S.& Gurney, A. L. IL-17:a prototype member of an emerging family. J. Leukoc. Biol.2002; 71:1-8.
[55]Yao, Z., Fanslow, W. C., Seldin, M. F., Rousseau, A. M., Painter, S. L., Comeau, M. R., Cohen, J. I., Spriggs, M. K. Herpesvirus Saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor. Immunity 1995; 3:811-821.
[56]Yao, Z., Painter, S. L., Fanslow, W. C., Ulrich, D., Macduff, B. M., Spriggs, M. K., Armitage, R. J. Cutting edge:human IL-17:a novel cytokine derived from T cells. J. Immunol.1995; 155:5483-5486.
[57]Toy, D., Kugler, D., Wolfson, M., Vanden Bos, T., Gurgel, J., Derry, J., Tocker, J., Peschon, J. Cutting edge:interleukin-17 signals through a heteromeric receptor complex. J. Immunol.2006; 177:36-39.
[58]Lee, J., Ho, W. H., Maruoka, M., Corpuz, R. T., Baldwin, D.T., Foster, J. S., Goddard, A. D., Yansura, D.G., Vandlen, R. L., Wood, W. I., Gurney, A. L. IL-17E, a novel proinflammatory ligand for the IL-17 receptor homolog IL-17Rh1. J. Biol. Chem.2001; 276:1660-1664.
[59]Langrish, C.L., Chen, Y, Blumenschein, W.M., Mattson, J., Basham, B., Sedgwick, J.D., McClanahan, T., Kastelein, R.A., Cua, D.J. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med.2005; 201: 233-240.
[60]Koenders, M. I., Lubberts, E., Oppers-Walgreen, B., van den Bersselaar, L., Helsen, M.M., Kolls, J.K., Joosten, L.A., van den Berg, W. B. Induction of cartilage damage by overexpression of T cell interleukin-17A in experimental arthritis in mice deficient in interleukin-1. Arthritis.rheum.2005; 52:975-983.
[61]Eid, R.E, Rao, D.A., Zhou, J., Lo, S.F., Ranjbaran, H., Gallo, A., Sokol, S.I., Pfau, S., Pober, J.S., Tellides, G. Interleukin-17 and Interferon-gamma are produced concomitantly by human coronary artery-infiltrating T cells and act synergistically on vascular smooth muscle cells. Circulation 2009; 119:1424-1432.
[62]Cheng, X, Yu, X., Ding, Y. J., Fu, Q.Q., Xie, J.J., Tang, T.T., Yao, R., Chen, Y, Liao, Y. H. The Th17/Treg imbalance in patients with acute coronary syndrome. Clin. Immunol.2008; 127:89-97.
[63]Xie, J.J., Wang, J., Tang, T.T., Chen, J., Gao, X.L., Yuan, J., Zhou, Z.H., Liao, M.Y., Yao, R., Yu, X., Wang, D., Cheng, Y, Liao, Y.H., Cheng, X. The Th17/Treg functional imbalance during atherogenesis in ApoE-/- mice. Cytokine 2009; 9:7-15.
[64]Erbel C., Chen, L., Bea, F., Wangler, S., Celik, S., Lasitschka, F., Wang, Y, Bockler, D., Katus, H. A., Dengler, T. J. Inhibition of IL-17A attenuates atherosclerotic lesion development in apoE-deficient mice. J. Immunol.2009; 183: 8167-8175.
[65]Taleb S., Romain, M., Ramkhelawon, B., Uyttenhove, C., Pasterkamp, G., Herbin, O., Esposito, B., Perez, N., Yasukawa, H., Van, S. J., Yoshimura, A., Tedgui, A., Mallat, Z. Loss of SOCS3 expression in T cells reveals a regulatory role for interleukin-17 in atherosclerosis. J. Exp. Med.2009; 206:2067-2077.
[66]Reardon, C.A., Blachowicz, L., White, T., Cabana, V., Wang, Y, Lukens, J., Bluestone, J., Getz, G.S. Effect of immune deficiency on lipoproteins and atherosclerosis in male apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol.2001; 21:1011-1016.
[67]Niwa, T., Wada, H., Ohashi, H., Iwamoto, N., Ohta, H., Kirii, H., Fujii, H., Saito, K., Seishima, M. Interferon-gamma produced by bone marrow-derived cells attenuates atherosclerotic lesion formation in LDLR-deficient mice. J. Atheroscler. Thromb.2004; 11:79-87.
[68]Jain, R., Tartar, D.M., Gregg, R.K., Divekar, R.D., Bell, J.J., Lee, H.H., Yu, P., Ellis, J.S., Hoeman, C.M., Franklin, C.L., Zaghouani, H. Innocuous IFNgamma induced by adjuvant-free antigen restores normoglycemia in NOD mice through inhibition of IL-17 production. J. Exp. Med.2008; 205:207-218.
[69]Irmler, I.M., Gajda, M., Brauer, R. Exacerbation of antigen-induced arthritis in IFN-gamma-deficient mice as a result of unrestricted IL-17 response. J. Immunol. 2007; 179:6228-6236.
[70]Zelante, T., De Luca, A., Bonifazi, P., Montagnoli, C., Bozza, S., Moretti, S., Belladonna, M.L., Vacca, C., Conte, C., Mosci, P., Bistoni, F., Puccetti, P., Kastelein, R.A., Kopf, M., Romani, L. IL-23 and the Th17 pathway promote inflammation and impair antifungal immune resistance. Eur. J. Immunol.2007; 37:2695-2706.
[71]Galkina, E., Ley, K. Immune and inflammatory mechanisms of atherosclerosis (*). Annu Rev Immunol.2009; 27:165-197.
72] Kolls, J.K. and Linden, A. Interleukin-17 family members and inflammation. Immunity 2004; 21:67-476.
[73]Aukrust, P., Otterdal, K., Yndestad, A., Sandberg, W.J., Smith, C., Ueland, T. Oie, E., Damas, J.K., Gullestad, L., Halvorsen, B. The complex role of T-cell-based immunity in atherosclerosis. Curr Atheroscler Rep.2008; 10:236-243.
[74]Taleb, S., Tedgui, A., Mallat, Z. Interleukin-17:friend or foe in atherosclerosis? Curr Opin Lipidol.2010; 21:404-408.
[75]Butcher, M. and Galkina, E. Current views on the functions of interleukin-17A-producing cells in atherosclerosis. Thromb Haemost.2011; 106: 787-795.
[76]Hashmi, S. and Zeng, Q.T. Role of interleukin-17 and interleukin-17- induced cytokines interleukin-6 and interleukin-8 in unstable coronary artery disease. Coron. Artery. Dis.2006; 17:699-706.
[77]van Es, T., van Puijvelde, G.H., Ramos, O.H., Segers, F.M., Joosten, L.A., van den Berg, W.B., Michon, I.M., de Vos, P., van Berkel, T.J., Kuiper, J. Attenuated atherosclerosis upon IL-17R signaling disruption in LDLr deficient mice. Biochem. Biophys. Res. Commun.2009; 388:261-265.
[78]Erbel, C., Dengler, T.J., Wangler, S., Lasitschka, F., Bea, F., Wambsganss, N., Hakimi, M., Bockler, D., Katus, H.A., Gleissner, C.A. Expression of IL-17A in human atherosclerotic lesions is associated with increased inflammation and plaque vulnerability. Basic Res. Cardiol. 2011; 106:125-134.
[79]Chen, S., Shimada, K., Zhang, W., Huang, G., Crother, T.R., Arditi, M. IL-17A is proatherogenic in high-fat diet-induced and Chlamydia pneumoniae infection-accelerated atherosclerosis in mice. J. Immunol.2010; 185:5619-5627.
[80]Gao, Q., Jiang, Y., Ma, T., Zhu, F., Gao, F., Zhang, P., Guo, C., Wang, Q., Wang, X., Ma, C., Zhang, Y., Chen, W., Zhang, L. A critical function of Th17 proinflammatory cells in the development of atherosclerotic plaque in mice. J. Immunol.2010; 185:5820-5827.
[81]Smith, E., Prasad, K.M.R., Butcher, M., Dobrian, A., Kolls, J.K., Ley, K., Galkina, E. Blockade of Interleukin-17A Results in Reduced Atherosclerosis in Apolipoprotein E-Deficient Mice. Circulation 2010; 121:1746-1755.
[82]Madhur, M.S., Funt, S.A., Li, L., Vinh, A., Chen, W., Lob, H.E., Iwakura, Y., Blinder, Y., Rahman, A., Quyyumi, A.A., Harrison, D.G. Role of interleukin-17 in inflammation, atherosclerosis, and vascular function in apolipoprotein e-deficient mice. Arterioscler. Thromb. Vasc. Biol.2011; 31:1565-1572.
[83]Shen, F., Ruddy, M. J., Plamondon, P.& Gaffen, S. L. Cytokines link osteoblasts and inflammation:microarray analysis of interleukin-17- and TNF-α-induced genes in bone cells. J. Leukoc. Biol.2005; 77:388-399.
[84]Ruddy, M. J., Wong, G. C., Liu, X. K., Yamamoto, H., Kasayama, S., Kirkwood, K. L., Gaffen, S. L. Functional cooperation between interleukin-17 and tumor necrosis factor-α is mediated by CCAAT/enhancer binding protein family members. J. Biol. Chem.2004; 279:2559-2567.
[85]Gaffen, S. L. Structure and signalling in the IL-17 receptor family. Nat Rev Immunol.2009; 9:556-567.
[86]Chen, S., Crother, T. R., Arditi, M. Emerging role of IL-17 in atherosclerosis. J. Innate Immun.2010; 2:325-333.
[87]Schroder, M. and Kaufman, R.J. The mammalian unfolded protein response. Annu Rev Biochem.2005; 74:739-789.
[88]Schroder, M. and Kaufman, R.J. ER stress and the unfolded protein response. Mutat. Res.2005; 569:29-63.
[89]Ron, D. and Walter, P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell Biol.2007; 8:519-529.
[90]Todd, D.J., Lee, A.H., Glimcher, L.H. The endoplasmic reticulum stress response in immunity and autoimmunity. Nat. Rev. Immunol.2008; 8:663-674.
[91]Harding, H.P., Zhang, Y., Ron, D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 1999; 397:271-274.
[92]Shi, Y., Vattem, K.M., Sood, R., An, J., Liang, J., Stramm, L., Wek, R.C. Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol. Cell Biol.1998; 18: 7499-7509.
[93]Haze, K., Yoshida, H., Yanagi, H., Yura, T., Mori, K. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell 1999; 10:3787-3799.
[94]Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., Mori, K.. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 2001; 107:881-891.
[95]Lee, K., Tirasophon, W., Shen, X., Michalak, M., Prywes, R., Okada, T. Yoshida, H., Mori, K., Kaufman, R. J. IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. Genes Dev.2002; 16:452-466.
[96]Calfon, M., Zeng, H., Urano, F., Till, J.H., Hubbard, S.R., Harding, H.P., Clark, S.G., Ron, D. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 2002; 415:92-96.
[97]Tabas, I. The role of endoplasmic reticulum stress in the progression of atherosclerosis. Circ. Res.2010; 107:839-850.
[98]Feng, B., Yao, P.M., Li, Y., Devlin, C.M., Zhang, D., Harding, H.P., Sweeney, M., Rong, J.X., Kuriakose, G., Fisher, E.A., Marks, A.R., Ron, D., Tabas, I. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages. Nat. Cell Biol.2003; 5:781-792.
[99]Minamino, T., Komuro, I., Kitakaze, M. Endoplasmic Reticulum Stress As a Therapeutic Target in Cardiovascular Disease. Circ. Res.2010; 107:1071-1082.
[100]Thorp, E., Li, G., Seimon, T.A., Kuriakose, G., Ron, D., Tabas, I. Reduced apoptosis and plaque necrosis in advanced atherosclerotic lesions of Apoe-/- and Ldlr-/- mice lacking CHOP. Cell Metab.2009; 9:474-481.
[101]Erbay, E., Babaev, V.R., Mayers, J.R., Makowski, L., Charles, K.N., Snitow, M.E., Fazio, S., Wiest, M.M., Watkins, S.M., Linton, M.F., Hotamisligil, G.S. Reducing endoplasmic reticulum stress through a macrophage lipid chaperone alleviates atherosclerosis. Nat. Med.2009; 15:1383-1391.
[102]Zhu, F., Wang, Q., Guo, C., Wang, X., Cao, X., Shi, Y., Gao, F., Ma, C., Zhang, L. IL-17 induces apoptosis of vascular endothelial cells:a potential mechanism for human acute coronary syndrome. Clin. Immunol.2011; 141:152-160.
[103]Barin, J.G., Baldeviano, G.C., Talor, M.V., Wu, L., Ong, S., Quader, F., Chen, P., Zheng, D., Caturegli, P., Rose, N.R., Eihakova, D. Macrophages participate in IL-17-mediated inflammation. Eur J Immunol.2011; 12, (Epub ahead of print).
[104]Tsukano, H., Gotoh, T., Endo, M., Miyata, K., Tazume, H., Kadomatsu, T. Yano, M., Iwawaki, T., Kohno, K., Araki, K., Mizuta, H., Oike, Y. The Endoplasmic Reticulum Stress-C/EBP Homologous Protein Pathway- Mediated Apoptosis in Macrophages Contributes to the Instability of Atherosclerotic Plaques. Arterioscler. Thromb. Vasc. Biol.2010; 30:1925-1932.
[105]Furuhashi, M., Hotamisligil, G.S. Fatty acid-binding proteins:role in metabolic diseases and potential as drug targets. Nat. Rev. Drug Disc.2008; 7:489-503.
[106]Makowski, L., Boord, J.B., Maeda, K., Babaev, V.R., Uysal, K.T., Morgan, M. A., Parker, R.A., Suttles, J., Fazio, S., Hotamisligil, G.S., Linton, M.F. Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis. Nat. Med.2001; 7:699-705. [107] Tuncman, G., Erbay, E., Hom, X., De Vivo, I., Campos, H., Rimm, E.B., Hotamisligil, G.S. A genetic variant at the fatty acid-binding protein aP2 locus reduces the risk for hypertriglyceridemia, type 2 diabetes, and cardiovascular disease. Proc. Natl. Acad. Sci.2006; 103:6970-6975.
[108]Furuhashi, M., Tuncman, G., Gorgiin, C.Z., Makowski, L., Atsumi, G., Vaillancourt, E., Kono, K., Babaev, V.R., Fazio, S., Linton, M.F., Sulsky, R., Robl, J.A., Parker, R.A., Hotamisligil, G.S. Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2. Nature 2007; 447:959-965.
[109]Ozcan, U., Yilmaz, E., Ozcan, L., Furuhashi, M., Vaillancourt, E., Smith, R.O., Gorgun, C.Z., Hotamisligil, G.S. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 2006; 313: 1137-1140.
[110]Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., Yuan, J. (2000). Caspase-12 mediates endoplasmic-reticulum- specific apoptosis and cytotoxicity by amyloid-beta. Nature 403,98-103.
[111]Xue, X., Piao, J.H., Nakajima, A., Sakon-Komazawa, S., Kojima, Y., Mori, K., Yagita, H., Okumura, K., Harding, H., Nakano, H. Tumor necrosis factor alpha (TNFalpha) induces the unfolded protein response (UPR) in a reactive oxygen species (ROS)-dependent fashion, and the UPR counteracts ROS accumulation by TNFalpha. J. Biol. Chem.2005; 280:33917-33925.
[112]Gotoh, T., Oyadomari, S., Mori, K., Mori, M. Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP. J. Biol. Chem.2002; 277:12343-12350.
[113]Fu, Y., Luo, N., Lopes-Virella, M.F. Oxidized LDL induces the expression of ALBP/aP2 mRNA and protein in human THP-1 macrophages. J Lipid Res.2000; 41: 2017-2023.
[114]Kazemi, M.R., McDonald, C.M., Shigenaga, J.K., Grunfeld, C., Feingold, K.R. Adipocyte fatty acid-binding protein expression and lipid accumulation are increased during activation of murine macrophages by toll-like receptor agonists. Arterioscler. Thromb. Vasc. Biol.2005; 25:1220-1224.
[115]Szegezdi, E., Fitzgerald, U., Samali, A. (2003). Caspase-12 and ER-stress-mediated apoptosis:the story so far. Ann N Y Acad Sci 1010,186-194.
[2]Lloyd-Jones, D.M. Cardiovascular risk prediction:basic concepts, current status, and future directions. Circulation 2010; 121:1768-1777.
[3]Hansson, G.K. Inflammation, atherosclerosis, and coronary artery disease. N. Engl. J. Med.2005; 352:1685-1695.
[4]Williams, K.J., Tabas, I. The response to retention hypothesis of early atherogenesis. Arterioscler. Thromb. Vasc. Biol.1995;15:551-561.
[5]Glass, C.K., Witztum, J.L. Atherosclerosis the road ahead. Cell 2001; 104: 503-516.
[6]Mestas, J., Ley, K. Monocyte-endothelial cell interactions in the development of atherosclerosis. Trends Cardiovasc. Med.2008; 18:228-232.
[7]Johnson, J.L., Newby, A.C. Macrophage heterogeneity in atherosclerotic plaques. Curr. Opin. Lipidol.2009; 20:370-378
[8]Paulson, K.E., Zhu, S.N., Chen, M., Nurmohamed, S., Jongstra-Bilen, J., Cybulsky, M.I. Resident intimal dendritic cells accumulate lipid and contribute to the initiation of atherosclerosis. Circ. Res.2010; 106:383-390.
[9]Kunjathoor, V.V., Febbraio, M., Podrez, E.A., Moore, K.J., Andersson, L., Koehn, S., Rhee, J.S., Silverstein, R., Hoff, H.F., Freeman, M.W. Scavenger receptors class A-I/I I and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J. Biol. Chem.2002; 277:49982-49988.
[10]Moore, K.J., Kunjathoor, V.V., Koehn, S.L., Manning, J.J., Tseng, A.A., Silver, J.M., McKee, M., Freeman, M.W. Loss of receptor mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyper-lipidemic mice. J. Clin. Invest.2005; 115:2192-2201.
[11]Manning-Tobin, J.J., Moore, K.J., Seimon, T.A., Bell, S.A., Sharuk, M., Alvarez-Leite, J.I., de Winther, M.P., Tabas, I., Freeman, M.W. Loss of SR-A and CD36 activity reduces atherosclerotic lesion complexity without abrogating foam cell formation in hyperlipidemic mice. Arterioscler. Thromb. Vasc. Biol.2009; 29:19-26.
[12]Mosmann T.R., Cherwinski C., Bond M.W., Giedlin M.A., Coffman R.L. Two types of murine helper T cell clones. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986; 136:2348-2357.
[13]Del Prete G.F., De Carli M., Mastromauro C., Biagiotti R., Macchia D., Falagiani Ricci M., Romagnani S. Purified protein derivative of Mycobacterium tuberculosis and excretory/secretori antigen(s) of Toxocaracanis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. J Clin Invest 1991; 88:346-350.
[14]Asano, M., Toda, M., Sakaguchi, N., Sakaguchi,S. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J.Exp.Med. 1996; 184:387-396.
[15]Rengarajan J., Szabo S.J., Glimcher L.H. Transcriptional regulation of Thl/Th2 polarization. Immunol Today 2000; 21:479-483.
[16]Khattri, R., Cox, T., Yasayko, S.A., Ramsdell, F. An essential role for Scurfin in CD4+CD25+T regulatory cells. Nat. Immunol.2003; 4:337-342.
[17]Fontenot, J.D., Gavin, M.A., Rudensky, A.Y. Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells. Nat. Immunol.2003; 4:330-336.
[18]Fontenot, J.D., Rasmussen, J.P., Williams, L.M., Dooley, J.L., Farr, A.G., Rudensky, A.Y. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity.2005; 22:329-341.
[19]Cabarrocas, J., Cassan, C., Magnusson, F., Piaggio, E., Mars, L., Derbinski, J., Kyewski, B., Gross, D.A., Salomon, B.L., Khazaie, K. Foxp3+CD25+ regulatory T cells specific for a neo-self-antigen develop at the doublepositive thymic stage. Proc. Natl. Acad. Sci.2006; 103:8453-8458.
[20]Campbell, D. J., Ziegler, S.F. Foxp3 modifies the phenotypic and functional properties of regulatory T cells. Nat. Rev. Immunol.2007; 7:305-310.
[21]Tedgui, A., Mallat, Z. Cytokines in atherosclerosis:pathogenic and regulatory pathways. Physiol. Rev.2006; 86:515-581.
[22]Zhou, X., Nicoletti, A., Elhage, R., Hansson, G.K. Transfer of CD4(+) T cells aggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice. Circulation 2000; 102:2919-2922.
[23]Zhou, X., Robertson, A.K., Hjerpe, C., Hansson,G.K. Adoptive transfer of CD4+ T cells reactive to modified low-density lipoprotein aggravates atherosclerosis. Arterioscler. Thromb. Vasc. Biol.2006; 26:864-870.
[24]Buono, C., Come, C.E., Stavrakis, G., Maguire, G.F., Connelly, P.W., Lichtman, A.H. Influence of interferon-gamma on the extent and phenotype of diet-induced atherosclerosis in the LDLR-deficient mouse. Arterioscler. Thromb. Vasc. Biol.2003; 23:454-460.
[25]Gupta, S., Pablo, A.M., Jiang, X., Wang, N., Tall, A.R., Schindler, C. IFN-gamma potentiates atherosclerosis in ApoE knock-out mice. J. Clin. Invest.1997; 99:2752-2761.
[26]Whitman, S.C., Ravisankar, P., Elam, H., Daugherty, A. Exogenous interferon-gamma enhances atherosclerosis in apolipoprotein E-/- mice. Am. J. Pathol. 2000; 157:1819-1824.
[27]Lee, T. S., Yen, H.C., Pan, C.C., Chau, L.Y. The role of interleukin 12 in the development of atherosclerosis in ApoE-deficient mice. Arterioscler. Thromb. Vasc. Biol.1999; 19:734-742.
[28]Davenport, P., Tipping, P.G. The role of interleukin-4 and interleukin-12 in the progression of atherosclerosis in apolipoprotein E-deficient mice. Am. J. Pathol.2003; 163:1117-1125.
[29]Paigen, B., Morrow, A., Brandon, C., Mitchell, D., Holmes, P. Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis 1985; 57:65-73.
[30]Huber, S.A., Sakkinen, P., David, C., Newell, M.K., Tracy, R.P.. T helper-cell phenotype regulates atherosclerosis in mice under conditions of mild hypercholesterolemia. Circulation 2001; 103:2610-2616.
[31]King, V.L., Szilvassy, S.J., Daugherty, A. Interleukin-4 deficiency decreases atherosclerotic lesion formation in a site-specific manner in female LDL receptor-/-mice. Arterioscler. Thromb. Vasc. Biol.2002; 22:456-461.
[32]Sakaguchi S. Regulatory T cells:key controllers of immunologic self-tolerance. Cell 2000; 101:455-458.
[33]Mills, K.H. Regulatory T cells:friend or foe in immunity to infection? Nat. Rev. 2004; 4:841-855.
[34]Aarvak, T., Chabaud, M., Miossec, P., Natvig, J.B. IL-17 is produced by some proinflammatory Th1/Th0 cells but not by Th2 cells. J. Immunol.1999; 162: 1246-1251.
[35]Harrington, L.E., Hatton, R.D., Mangan, P.R., Turner, H., Murphy, T.L., Murphy, K.M., Weaver, C.T. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol.2005; 6: 1123-1132.
[36]Park, H., Li, Z., Yang, X.O., Chang, S.H., Nurieva, R., Wang, Y. H., Wang, Y, Hood, L., Zhu, Z., Tian, Q., Dong, C. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol.2005; 6:1133-1141.
[37]Ivanov,I.I., McKenzie, B.S., Zhou, L., Tadokoro, C.E., Lepelley, A., Lafaille, J.J., Cua, D.J., Littman, D.R. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 2006; 126: 1121-1133.
[38]Veldhoen, M., Hocking, R.J., Atkins, C.J., Locksley, R.M., Stockinger, B. TGF-beta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006; 24:179-189.
[39]Bettelli, E., Carrier, Y, Gao, W., Korn, T., Strom, T. B., Oukka, M., Weiner, H.L. Kuchroo,V.K. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006; 441:235-238.
[40]Mangan, P.R., Harrington, L.E., O'Quinn, D.B., Helms, W.S., Bullard, D.C., Elson, C.O., Hatton, R.D., Wahl, S.M., Schoeb, T.R., Weaver,C.T. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 2006; 441: 231-234.
[41]Ouyang, W., Kolls, J. K., Zheng, Y. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity.2008; 28:454-467.
[42]Langrish, C.L., Chen, Y., Blumenschein, W.M., Mattson, J., Basham, B., Sedgwick, J.D., McClanahan, T., et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med.2005; 201:233-240.
[43]Ferretti, S., Bonneau, O., Dubois, G.R., Jones, C.E., Trifilieff, A. IL-17, produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia:IL-15 as a possible trigger. J. Immunol.2003; 170: 2106-2112.
[44]Hymowitz, S. G., Filvaroff, E. H., Yin, J. P., Lee, J., Cai, L., Risser, P., Maruoka, M., Mao, W., Foster, J., Kelley R. F., Pan, G., Gurney, A. L., de Vos A. M., Starovasnik, M. A.. IL-17s adopt a cystine knot fold:structure and activity of a novel cytokine, IL-17F, and implications for receptor binding. EMBo J.2001; 20: 5332-5341.
[45]Wright, J. F., Guo, Y., Quazi, A., Luxenberg, D. P., Bennett, F., Ross, J. F., Qiu, Y., Whitters, M. J., Tomkinson, K. N., Dunussi-Joannopoulos K, Carreno, B. M., Collins, M., Wolfman, N. M.. Identification of an interleukin 17F/17A heterodimer in activated human CD4+ T cells. J. Biol. Chem.2007; 282:13447-13455.
[46]Chang, S. H.& Dong, C. A novel heterodimeric cytokine consisting of IL-17 and IL-17F regulates inflammatory responses. Cell Res.2007; 17:435-440.
[47]Wright, J. F., Whitters, M. J., Tomkinson, K. N., Fitz, L. J., Wolfman, N. M., Collins, M., Dunussi-Joannopoulos K., Chatterjee-Kishore M., Carreno, B. M.. The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL 17RC receptor complex. J. Immunol.2008;181:2799-2805.
[48]Kuestner, R. et al. Identification of the IL-17 receptor related molecule, IL-17RC, as the receptor for IL-17F. J. Immunol.2007; 179:5462-5473.
[49]Yang, X. O., Chang, S. H., Park, H., Nurieva, R., Shah, B., Acero, L., Wang, Y. H., Schluns, K. S., Broaddus, R. R., Zhu, Z., Dong C. Regulation of inflammatory responses by IL-17F. J. Exp. Med.2008; 205:1063-1075.
[50]Ishigame, H., Kakuta, S., Nagai, T., Kadoki, M., Nambu, A., Komiyama, Y., Fujikado, N., Tanahashi, Y., Akitsu, A., Kotaki, H., Sudo, K., Nakae, S., Sasakawa, C., Iwakura, Y. Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses. Immunity 2009; 30: 108-119.
[51]Gaffen, S. L., Kramer, J. M., Yu, J. J.& Shen, F. in Vitamins and Hormones Vol.74. (ed. G. Litwack) 255-282 (Academic, London,2006).
[52]McAllister, F., Henry, A., Kreindler, J. L., Dubin, P. J., Ulrich, L., Steele, C., Finder, J. D., Pilewski, J. M., Carreno, B. M., Goldman, S. J., Pirhonen, J., Kolls, J. K. Role of IL-17A, IL-17F, and the IL-17 receptor in regulating growth -related oncogene-α and granulocyte colony -stimulating factor in bronchial epithelium: implications for airway inflammation in cystic fibrosis. J. Immunol.2005; 175: 404-412.
[53]Dong, C. Regulation and pro-inflammatory function of interleukin-17 family cytokines. Immunol. Rev.2008; 226:80-86.
[54]Aggarwal, S.& Gurney, A. L. IL-17:a prototype member of an emerging family. J. Leukoc. Biol.2002; 71:1-8.
[55]Yao, Z., Fanslow, W. C., Seldin, M. F., Rousseau, A. M., Painter, S. L., Comeau, M. R., Cohen, J. I., Spriggs, M. K. Herpesvirus Saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor. Immunity 1995; 3:811-821.
[56]Yao, Z., Painter, S. L., Fanslow, W. C., Ulrich, D., Macduff, B. M., Spriggs, M. K., Armitage, R. J. Cutting edge:human IL-17:a novel cytokine derived from T cells. J. Immunol.1995; 155:5483-5486.
[57]Toy, D., Kugler, D., Wolfson, M., Vanden Bos, T., Gurgel, J., Derry, J., Tocker, J., Peschon, J. Cutting edge:interleukin-17 signals through a heteromeric receptor complex. J. Immunol.2006; 177:36-39.
[58]Lee, J., Ho, W. H., Maruoka, M., Corpuz, R. T., Baldwin, D.T., Foster, J. S., Goddard, A. D., Yansura, D.G., Vandlen, R. L., Wood, W. I., Gurney, A. L. IL-17E, a novel proinflammatory ligand for the IL-17 receptor homolog IL-17Rh1. J. Biol. Chem.2001; 276:1660-1664.
[59]Langrish, C.L., Chen, Y, Blumenschein, W.M., Mattson, J., Basham, B., Sedgwick, J.D., McClanahan, T., Kastelein, R.A., Cua, D.J. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med.2005; 201: 233-240.
[60]Koenders, M. I., Lubberts, E., Oppers-Walgreen, B., van den Bersselaar, L., Helsen, M.M., Kolls, J.K., Joosten, L.A., van den Berg, W. B. Induction of cartilage damage by overexpression of T cell interleukin-17A in experimental arthritis in mice deficient in interleukin-1. Arthritis.rheum.2005; 52:975-983.
[61]Eid, R.E, Rao, D.A., Zhou, J., Lo, S.F., Ranjbaran, H., Gallo, A., Sokol, S.I., Pfau, S., Pober, J.S., Tellides, G. Interleukin-17 and Interferon-gamma are produced concomitantly by human coronary artery-infiltrating T cells and act synergistically on vascular smooth muscle cells. Circulation 2009; 119:1424-1432.
[62]Cheng, X, Yu, X., Ding, Y. J., Fu, Q.Q., Xie, J.J., Tang, T.T., Yao, R., Chen, Y, Liao, Y. H. The Th17/Treg imbalance in patients with acute coronary syndrome. Clin. Immunol.2008; 127:89-97.
[63]Xie, J.J., Wang, J., Tang, T.T., Chen, J., Gao, X.L., Yuan, J., Zhou, Z.H., Liao, M.Y., Yao, R., Yu, X., Wang, D., Cheng, Y, Liao, Y.H., Cheng, X. The Th17/Treg functional imbalance during atherogenesis in ApoE-/- mice. Cytokine 2009; 9:7-15.
[64]Erbel C., Chen, L., Bea, F., Wangler, S., Celik, S., Lasitschka, F., Wang, Y, Bockler, D., Katus, H. A., Dengler, T. J. Inhibition of IL-17A attenuates atherosclerotic lesion development in apoE-deficient mice. J. Immunol.2009; 183: 8167-8175.
[65]Taleb S., Romain, M., Ramkhelawon, B., Uyttenhove, C., Pasterkamp, G., Herbin, O., Esposito, B., Perez, N., Yasukawa, H., Van, S. J., Yoshimura, A., Tedgui, A., Mallat, Z. Loss of SOCS3 expression in T cells reveals a regulatory role for interleukin-17 in atherosclerosis. J. Exp. Med.2009; 206:2067-2077.
[66]Reardon, C.A., Blachowicz, L., White, T., Cabana, V., Wang, Y, Lukens, J., Bluestone, J., Getz, G.S. Effect of immune deficiency on lipoproteins and atherosclerosis in male apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol.2001; 21:1011-1016.
[67]Niwa, T., Wada, H., Ohashi, H., Iwamoto, N., Ohta, H., Kirii, H., Fujii, H., Saito, K., Seishima, M. Interferon-gamma produced by bone marrow-derived cells attenuates atherosclerotic lesion formation in LDLR-deficient mice. J. Atheroscler. Thromb.2004; 11:79-87.
[68]Jain, R., Tartar, D.M., Gregg, R.K., Divekar, R.D., Bell, J.J., Lee, H.H., Yu, P., Ellis, J.S., Hoeman, C.M., Franklin, C.L., Zaghouani, H. Innocuous IFNgamma induced by adjuvant-free antigen restores normoglycemia in NOD mice through inhibition of IL-17 production. J. Exp. Med.2008; 205:207-218.
[69]Irmler, I.M., Gajda, M., Brauer, R. Exacerbation of antigen-induced arthritis in IFN-gamma-deficient mice as a result of unrestricted IL-17 response. J. Immunol. 2007; 179:6228-6236.
[70]Zelante, T., De Luca, A., Bonifazi, P., Montagnoli, C., Bozza, S., Moretti, S., Belladonna, M.L., Vacca, C., Conte, C., Mosci, P., Bistoni, F., Puccetti, P., Kastelein, R.A., Kopf, M., Romani, L. IL-23 and the Th17 pathway promote inflammation and impair antifungal immune resistance. Eur. J. Immunol.2007; 37:2695-2706.
[71]Galkina, E., Ley, K. Immune and inflammatory mechanisms of atherosclerosis (*). Annu Rev Immunol.2009; 27:165-197.
72] Kolls, J.K. and Linden, A. Interleukin-17 family members and inflammation. Immunity 2004; 21:67-476.
[73]Aukrust, P., Otterdal, K., Yndestad, A., Sandberg, W.J., Smith, C., Ueland, T. Oie, E., Damas, J.K., Gullestad, L., Halvorsen, B. The complex role of T-cell-based immunity in atherosclerosis. Curr Atheroscler Rep.2008; 10:236-243.
[74]Taleb, S., Tedgui, A., Mallat, Z. Interleukin-17:friend or foe in atherosclerosis? Curr Opin Lipidol.2010; 21:404-408.
[75]Butcher, M. and Galkina, E. Current views on the functions of interleukin-17A-producing cells in atherosclerosis. Thromb Haemost.2011; 106: 787-795.
[76]Hashmi, S. and Zeng, Q.T. Role of interleukin-17 and interleukin-17- induced cytokines interleukin-6 and interleukin-8 in unstable coronary artery disease. Coron. Artery. Dis.2006; 17:699-706.
[77]van Es, T., van Puijvelde, G.H., Ramos, O.H., Segers, F.M., Joosten, L.A., van den Berg, W.B., Michon, I.M., de Vos, P., van Berkel, T.J., Kuiper, J. Attenuated atherosclerosis upon IL-17R signaling disruption in LDLr deficient mice. Biochem. Biophys. Res. Commun.2009; 388:261-265.
[78]Erbel, C., Dengler, T.J., Wangler, S., Lasitschka, F., Bea, F., Wambsganss, N., Hakimi, M., Bockler, D., Katus, H.A., Gleissner, C.A. Expression of IL-17A in human atherosclerotic lesions is associated with increased inflammation and plaque vulnerability. Basic Res. Cardiol. 2011; 106:125-134.
[79]Chen, S., Shimada, K., Zhang, W., Huang, G., Crother, T.R., Arditi, M. IL-17A is proatherogenic in high-fat diet-induced and Chlamydia pneumoniae infection-accelerated atherosclerosis in mice. J. Immunol.2010; 185:5619-5627.
[80]Gao, Q., Jiang, Y., Ma, T., Zhu, F., Gao, F., Zhang, P., Guo, C., Wang, Q., Wang, X., Ma, C., Zhang, Y., Chen, W., Zhang, L. A critical function of Th17 proinflammatory cells in the development of atherosclerotic plaque in mice. J. Immunol.2010; 185:5820-5827.
[81]Smith, E., Prasad, K.M.R., Butcher, M., Dobrian, A., Kolls, J.K., Ley, K., Galkina, E. Blockade of Interleukin-17A Results in Reduced Atherosclerosis in Apolipoprotein E-Deficient Mice. Circulation 2010; 121:1746-1755.
[82]Madhur, M.S., Funt, S.A., Li, L., Vinh, A., Chen, W., Lob, H.E., Iwakura, Y., Blinder, Y., Rahman, A., Quyyumi, A.A., Harrison, D.G. Role of interleukin-17 in inflammation, atherosclerosis, and vascular function in apolipoprotein e-deficient mice. Arterioscler. Thromb. Vasc. Biol.2011; 31:1565-1572.
[83]Shen, F., Ruddy, M. J., Plamondon, P.& Gaffen, S. L. Cytokines link osteoblasts and inflammation:microarray analysis of interleukin-17- and TNF-α-induced genes in bone cells. J. Leukoc. Biol.2005; 77:388-399.
[84]Ruddy, M. J., Wong, G. C., Liu, X. K., Yamamoto, H., Kasayama, S., Kirkwood, K. L., Gaffen, S. L. Functional cooperation between interleukin-17 and tumor necrosis factor-α is mediated by CCAAT/enhancer binding protein family members. J. Biol. Chem.2004; 279:2559-2567.
[85]Gaffen, S. L. Structure and signalling in the IL-17 receptor family. Nat Rev Immunol.2009; 9:556-567.
[86]Chen, S., Crother, T. R., Arditi, M. Emerging role of IL-17 in atherosclerosis. J. Innate Immun.2010; 2:325-333.
[87]Schroder, M. and Kaufman, R.J. The mammalian unfolded protein response. Annu Rev Biochem.2005; 74:739-789.
[88]Schroder, M. and Kaufman, R.J. ER stress and the unfolded protein response. Mutat. Res.2005; 569:29-63.
[89]Ron, D. and Walter, P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell Biol.2007; 8:519-529.
[90]Todd, D.J., Lee, A.H., Glimcher, L.H. The endoplasmic reticulum stress response in immunity and autoimmunity. Nat. Rev. Immunol.2008; 8:663-674.
[91]Harding, H.P., Zhang, Y., Ron, D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 1999; 397:271-274.
[92]Shi, Y., Vattem, K.M., Sood, R., An, J., Liang, J., Stramm, L., Wek, R.C. Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol. Cell Biol.1998; 18: 7499-7509.
[93]Haze, K., Yoshida, H., Yanagi, H., Yura, T., Mori, K. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell 1999; 10:3787-3799.
[94]Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., Mori, K.. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 2001; 107:881-891.
[95]Lee, K., Tirasophon, W., Shen, X., Michalak, M., Prywes, R., Okada, T. Yoshida, H., Mori, K., Kaufman, R. J. IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. Genes Dev.2002; 16:452-466.
[96]Calfon, M., Zeng, H., Urano, F., Till, J.H., Hubbard, S.R., Harding, H.P., Clark, S.G., Ron, D. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 2002; 415:92-96.
[97]Tabas, I. The role of endoplasmic reticulum stress in the progression of atherosclerosis. Circ. Res.2010; 107:839-850.
[98]Feng, B., Yao, P.M., Li, Y., Devlin, C.M., Zhang, D., Harding, H.P., Sweeney, M., Rong, J.X., Kuriakose, G., Fisher, E.A., Marks, A.R., Ron, D., Tabas, I. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages. Nat. Cell Biol.2003; 5:781-792.
[99]Minamino, T., Komuro, I., Kitakaze, M. Endoplasmic Reticulum Stress As a Therapeutic Target in Cardiovascular Disease. Circ. Res.2010; 107:1071-1082.
[100]Thorp, E., Li, G., Seimon, T.A., Kuriakose, G., Ron, D., Tabas, I. Reduced apoptosis and plaque necrosis in advanced atherosclerotic lesions of Apoe-/- and Ldlr-/- mice lacking CHOP. Cell Metab.2009; 9:474-481.
[101]Erbay, E., Babaev, V.R., Mayers, J.R., Makowski, L., Charles, K.N., Snitow, M.E., Fazio, S., Wiest, M.M., Watkins, S.M., Linton, M.F., Hotamisligil, G.S. Reducing endoplasmic reticulum stress through a macrophage lipid chaperone alleviates atherosclerosis. Nat. Med.2009; 15:1383-1391.
[102]Zhu, F., Wang, Q., Guo, C., Wang, X., Cao, X., Shi, Y., Gao, F., Ma, C., Zhang, L. IL-17 induces apoptosis of vascular endothelial cells:a potential mechanism for human acute coronary syndrome. Clin. Immunol.2011; 141:152-160.
[103]Barin, J.G., Baldeviano, G.C., Talor, M.V., Wu, L., Ong, S., Quader, F., Chen, P., Zheng, D., Caturegli, P., Rose, N.R., Eihakova, D. Macrophages participate in IL-17-mediated inflammation. Eur J Immunol.2011; 12, (Epub ahead of print).
[104]Tsukano, H., Gotoh, T., Endo, M., Miyata, K., Tazume, H., Kadomatsu, T. Yano, M., Iwawaki, T., Kohno, K., Araki, K., Mizuta, H., Oike, Y. The Endoplasmic Reticulum Stress-C/EBP Homologous Protein Pathway- Mediated Apoptosis in Macrophages Contributes to the Instability of Atherosclerotic Plaques. Arterioscler. Thromb. Vasc. Biol.2010; 30:1925-1932.
[105]Furuhashi, M., Hotamisligil, G.S. Fatty acid-binding proteins:role in metabolic diseases and potential as drug targets. Nat. Rev. Drug Disc.2008; 7:489-503.
[106]Makowski, L., Boord, J.B., Maeda, K., Babaev, V.R., Uysal, K.T., Morgan, M. A., Parker, R.A., Suttles, J., Fazio, S., Hotamisligil, G.S., Linton, M.F. Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis. Nat. Med.2001; 7:699-705. [107] Tuncman, G., Erbay, E., Hom, X., De Vivo, I., Campos, H., Rimm, E.B., Hotamisligil, G.S. A genetic variant at the fatty acid-binding protein aP2 locus reduces the risk for hypertriglyceridemia, type 2 diabetes, and cardiovascular disease. Proc. Natl. Acad. Sci.2006; 103:6970-6975.
[108]Furuhashi, M., Tuncman, G., Gorgiin, C.Z., Makowski, L., Atsumi, G., Vaillancourt, E., Kono, K., Babaev, V.R., Fazio, S., Linton, M.F., Sulsky, R., Robl, J.A., Parker, R.A., Hotamisligil, G.S. Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2. Nature 2007; 447:959-965.
[109]Ozcan, U., Yilmaz, E., Ozcan, L., Furuhashi, M., Vaillancourt, E., Smith, R.O., Gorgun, C.Z., Hotamisligil, G.S. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 2006; 313: 1137-1140.
[110]Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., Yuan, J. (2000). Caspase-12 mediates endoplasmic-reticulum- specific apoptosis and cytotoxicity by amyloid-beta. Nature 403,98-103.
[111]Xue, X., Piao, J.H., Nakajima, A., Sakon-Komazawa, S., Kojima, Y., Mori, K., Yagita, H., Okumura, K., Harding, H., Nakano, H. Tumor necrosis factor alpha (TNFalpha) induces the unfolded protein response (UPR) in a reactive oxygen species (ROS)-dependent fashion, and the UPR counteracts ROS accumulation by TNFalpha. J. Biol. Chem.2005; 280:33917-33925.
[112]Gotoh, T., Oyadomari, S., Mori, K., Mori, M. Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP. J. Biol. Chem.2002; 277:12343-12350.
[113]Fu, Y., Luo, N., Lopes-Virella, M.F. Oxidized LDL induces the expression of ALBP/aP2 mRNA and protein in human THP-1 macrophages. J Lipid Res.2000; 41: 2017-2023.
[114]Kazemi, M.R., McDonald, C.M., Shigenaga, J.K., Grunfeld, C., Feingold, K.R. Adipocyte fatty acid-binding protein expression and lipid accumulation are increased during activation of murine macrophages by toll-like receptor agonists. Arterioscler. Thromb. Vasc. Biol.2005; 25:1220-1224.
[115]Szegezdi, E., Fitzgerald, U., Samali, A. (2003). Caspase-12 and ER-stress-mediated apoptosis:the story so far. Ann N Y Acad Sci 1010,186-194.