线粒体DNA-LL-37复合物诱导动脉粥样硬化的发病机理研究
文献类型：学位论文
| 作者 | 张治业 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 赖仞 |
| 关键词 | 动脉粥样硬化 炎症反应 中性粒细胞激活 自噬 |
| 其他题名 | Research on the mechanism of mitochondrial DNA-LL-37 complex promoting atherosclerosis |
| 中文摘要 | 动脉粥样硬化是发生在动脉血管内壁的慢性炎症疾病，研究表明脂代谢异常及免疫反应异常的激活和该疾病的发生密切相关。最近几年的研究发现人阳离子抗菌肽hCAP18/LL-37(小鼠中称为Cramp)和动脉粥样硬化发病相关，在粥样斑病变组织浸润的中性粒细胞内及细胞外trap结构(neutrophil extracellular traps，NETs)中均发现了该多肽的存在。在自身免疫性疾病银屑病中，LL-37能与DNA或RNA结合形成复合物结构，这两种复合物结构分别通过Toll样受体9(TLR9)和TLR7来激活树突状细胞产生干扰素-α(INF-α)，因此而加重了银屑病的发病。另外，有研究表明LL-37和死亡的细胞或NETs结构释放的DNA结合形成复合物后同样可以刺激树突状细胞产生INF-α，因此而促进了动脉粥样硬化疾病的发病。但是，死亡的细胞或NETs结构释放的DNA中既有基因组DNA(nDNA)也包含线粒体DNA(mtDNA)，因此我们推测上述形成的复合物中包括LL-37-nDNA和LL-37-mtDNA两种复合物。由于线粒体从进化上来自于细胞内的共生菌，因而mtDNA保留了和细菌DNA类似的非甲基化的CpG(胞嘧啶鸟嘌呤二核苷酸)结构，而这种结构具有很强的炎症刺激作用。因此，我们假设在炎症细胞激活和促动脉粥样硬化疾病发病过程中是LL-37-mtDNA复合物而非LL-37-nDNA复合物发挥了主要的作用。 为了验证我们的假设，我们首先检测了LL-37和mtDNA是否可以形成复合物。结果表明LL-37和mtDNA可以形成非常稳定的LL-37-mtDNA复合物，且形成的复合物可以抵抗DNase II的降解。同时，LL-37-mtDNA复合物相对于LL-37-nDNA复合物能够激活树突状细胞表达分泌更多的INF-α。因此，在明确了LL-37和mtDNA可以结合形成复合物，且该复合物比LL-37-nDNA复合物具有更强的炎症刺激作用后，我们收集了动脉粥样硬化病人的血液及粥样斑样本进行了LL-37-mtDNA复合物与动脉粥样硬化疾病发病的相关性研究。研究发现，动脉粥样硬化病人血浆中的LL-37、mtDNA或LL-37-mtDNA复合物浓度比正常人偏高，且通过免疫组织化学染色的方法，我们在动脉粥样斑的新内膜中发现了LL-37与LL-37-mtDNA复合物的存在。 为了更好的研究LL-37-mtDNA复合物与动脉粥样硬化疾病发病的关系，我们构建了小鼠动脉粥样硬化模型。在动脉粥样硬化小鼠的粥样斑位置我们发现了高水平的Cramp-mtDNA复合物沉积。当将Cramp-mtDNA复合物通过尾静脉注射的方式连续四周注射模型小鼠后，小鼠胸主动脉根部位的动脉粥样硬化发病更加明显。而将Cramp-nDNA复合物通过同样的方式注射进小鼠体内却对小鼠的发病没有太大的影响。另外，如果以Cramp-mtDNA复合物为治疗靶点，通过使用抗Cramp-mtDNA复合物抗体治疗后，小鼠动脉粥样硬化的发病得到了明显的抑制。以上结果明确的证明了LL-37/Cramp-mtDNA复合物是导致动脉粥样硬化的主要原因。 我们重点研究了LL-37-mtDNA复合物诱导动脉粥样硬化的发病机制，通过实验我们发现该复合物可以诱导中性粒细胞脱颗粒并释放基质金属蛋白酶8(MMP-8)，同时该复合物还可诱导中性粒细胞表达分泌白细胞介素6(IL-6)、 IL-8和肿瘤坏死因子α(TNF-α)，而LL-37-nDNA复合物激活中性粒细胞分泌上述细胞因子的能力却很弱。LL-37-mtDNA复合物作为促炎介质，正常情况下该复合物进入细胞后应该会被细胞内的自噬体-溶酶体系统清除掉。因此我们选取内皮细胞作为靶细胞，用LL-37-mtDNA复合物刺激该细胞后发现细胞内的自噬体数量明显增加。接着我们通过免疫细胞共定位的方法证明了进入细胞内的LL-37和LL-37-nDNA复合物均被细胞内增加的自噬体吞噬掉，但是我们惊奇的发现LL-37-mtDNA复合物却可以逃避细胞的自噬识别。另外，LL-37-mtDNA复合物刺激内皮细胞后该细胞内TLR9阳性染色的内体数量有了明显的增加，推测LL-37-mtDNA复合物激活了TLR9的表达或促进了TLR9从内质网向内体的转运。LL-37-mtDNA复合物作用于人内皮细胞后可诱导该细胞表达分泌单核细胞趋化因子1(MCP-1)、细胞间粘附因子1(ICAM-1)和IL-8。通过以上结果我们推测是LL-37-mtDNA复合物激活了TLR9信号通路，但由于LL-37-mtDNA不容易被DNase II降解同时也可以逃避细胞的自噬识别，导致该复合物在内皮细胞内一直存在，因而导致了TLR9信号通路一直处于激活的状态，最终导致了内皮细胞连续分泌上述趋化因子和炎症因子。这些结果说明了LL-37-mtDNA复合物通过激活中性粒细胞和内皮细胞分泌趋化因子和促炎因子来趋化更多炎症细胞浸润粥样斑或直接损伤内皮细胞，最终导致粥样斑内的炎症反应进一步的级联放大而变的不可控制，加速了粥样斑的病变。 通过以上实验结果我们可以总结如下：通过和人抗菌肽LL-37形成复合物，来自死亡细胞或NET结构中的mtDNA不仅不能被DNase II顺利降解清除，同时形成的LL-37-mtDNA复合物进入细胞后通过逃避细胞的自噬识别而变得不容易被清除，导致了细胞内TLR9信号通路处于连续激活的状态。而处于激活状态的细胞通过分泌趋化因子和炎症因子来招募更多的炎症细胞浸润粥样斑，导致了粥样斑内的炎症反应进一步被放大，最终导致或加剧了动脉粥样硬化的病变。 |
| 英文摘要 | Atherosclerosis is a chronic inflammatory disease of the arterial wall, and an imbalanced lipid metabolism and a maladaptive immune response are closely linked to the pathogenesis of it. Human cationic antimicrobial protein 18 (hCAP18)/LL-37 (known as Cramp in mice) was identified residing in neutrophil and neutrophil extracellular traps (NETs) in atherosclerotic lesions. LL-37 can form complexes with self-DNA and self-RNA, which gain access to endosomal TLR9 and TLR7 respectively, leading to abnormal activation of plasmacytoid dendritic cells (pDCs) to produce interferon-α (IFN-α) in psoriasis. Additionally, mice Cramp forms complex with self-DNA released from dying cells or in NETs, breaking innate tolerance to self-DNA and triggering direct activation of pDCs to produce IFN-α, and thus aggravates atherosclerotic lesion formation. However, there are nuclear DNA (nDNA) and mitochondrial DNA(mtDNA) in NETs and dying cells, accordingly, LL-37-nDNA complex and LL-37-mtDNA complex could be formed. In addition, mitochondria are evolutionary endosymbionts derived from bacteria and contain similar unmethylated CpG DNA repeats to bacterial DNA. Multiple CpG nucleotides display stronger inflammatogenic properties than eukaryotic nDNA, which contains fewer such motifs and most of them are masked by methylation. We hypothesized that LL-37-mtDNA complex (not LL-37-nDNA complex) plays a major role in the activation of leukocytes and promoting atherosclerosis. To prove our hypothesis, firstly, we tested whether LL-37 binds mtDNA to form complex, and we found that LL-37 binds mtDNA to form complex that is protected from DNase II degradation and induce pDCs activation. The blood samples of atherosclerotic patients and volunteers, and plaque samples and normal control arteries were all collected to investigate wether LL37-mtDNA complex is associated with atherosclerosis. Elevated levels of LL-37, mtDNA, and LL-37-mtDNA complex were found in atherosclerotic plasma. High concentration of LL-37-mtDNA was also observed in neointima of human atherosclerotic lesion, suggesting that LL-37-mtDNA complex is associated with atherosclerotic plaque formation. To investigate whether Cramp-mtDNA complex is present in atherosclerotic lesions in mice, Apoe-/- mice fed a high-fat diet (HFD) for 4 weeks to develop atherosclerosis in aortic roots were studied. High level of Cramp-mtDNA complex was also observed in atherosclerotic arteries of Apoe-/- mice. Injection of Cramp-mtDNA complex into HFD-fed Apoe-/- mice significantly promoted atherosclerotic plaque growth while administration of anti-Cramp-mtDNA antibody led to a substantial reduction of atherosclerotic plaque size in Apoe-/- mice. All the data indicated that mtDNA in complex with LL-37/Cramp promotes atherosclerosis, and therapeutic depletion of the complex might inhibit atherosclerosis progression. Our study demonstrated that LL-37-mtDNA exacerbates inflammation by stimulating IFN-α production in pDCs and inducing IL-6, IL-8, TNF-α, and MMP-8 release in neutrophil. By triggering pDCs and PMN activation to release these chemokines or cytokines, LL-37-mtDNA serves as inflammatory amplifier to attract more neutrophils to infiltrate atherosclerotic lesions. Our study also showed that LL-37-mtDNA stimulated endothelial cell HUVECs to produce MCP-1, ICAM-1, and IL-8, suggesting that LL-37-mtDNA serves as a key mediator to attract monocytes to atherosclerotic plaque by inducing expression of chemokines. Given that LL-37-mtDNA acts as an inflammatory stimulator, it might be distinguished by autophagosome or endosome, and subsequently delivered to lysosome for degradation. Unexpectedly, LL-37-mtDNA could escape from autophagic recognition, and it strengthened the formation of autophagosome and TLR9-positive endosomes in HUVECs. The increasing formation of autophagosome might be resulted from TLR9 activation induced by LL-37-mtDNA, as TLRs agonists have been demonstrated to induce high level of autophagosome formation. Together, these data indicate that by forming complex with LL-37, the endogenous mtDNA from NETs and dying cell in atherosclerotic plaque is protected from DNase II degradation which leads to persistent activation of TLR9. The indelible autoimmune activation and generation of chemokines or cytokines eventually aggravate atherosclerosis lesion. These data provide new perspectives on the mechanism of genesis of chronic inflammation in atherosclerosis and a promising therapeutic target. |
| 语种 | 中文 |
| 源URL | [http://159.226.149.26:8080/handle/152453/10137]  |
| 专题 | 昆明动物研究所_动物毒素室 |
| 推荐引用方式 GB/T 7714 | 张治业. 线粒体DNA-LL-37复合物诱导动脉粥样硬化的发病机理研究[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：昆明动物研究所
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