模拟人微卫星DNA脱氧寡核苷酸对TLR9活化诱导全身炎症应答的负调控机制

Recent researches indicate that Toll like receptor 9 (TLR9) is naturally expressed on the surface of neutrophils, and that the surface TLR9 (sTLR9）can be activated by self mitochondrial DNA, inducing systemic inflammatory response syndrome (SIRS). Mitochondiral DNA originates from bacterial DNA and is able to activate TLR9. In addition to mitochondiral DNA, self DNA can also derive from nucleus. The nuclear DNA may act on TLR9. In previous study, we found that a human microsatellite DNA mimicking oligodeoxyribonucleotide (CCT ODN）with CCT repeats could inhibit TLR9 activation and rescue mice from TLR9 agonist CpG ODN-induced lethal inflammatory shock，implying that the nuclear DNA and its mimics are TLR9 ligands and display negative regulatory effect by targeting sTLR9. In this project, by focusing on sTLR9,we plan to study the negatively regulatory effect of CCT ODN on systemic inflammatory response in SIRS using TLR9 activation induced systemic inflammatory response-mouse model and TLR9 agonist activated immune cells, and the influence on the level and activation of sTLR9 in various immune cells. The achivement of the project will shed light on the negative regulation of systemic inflammatory response and provide experimental basis for the prevention and treatment of SIRS.

新近研究表明，TLR9天然地表达在中性粒细胞表面，这种表面TLR9（sTLR9）能通过感受因损失释放的自身线粒体DNA而诱发全身性炎症应答综合征（SIRS）。线粒体DNA源于细菌DNA，可以激活TLR9。除线粒体DNA外，自身DNA也可来自细胞核，细胞核DNA也可作用于TLR9。我们的前期工作表明，模拟人细胞核基因组微卫星DNA的、由CCT重复序列组成的脱氧寡核苷酸（CCT ODN）能抑制TLR9活化，抑制TLR9激动剂CpG ODN诱导小鼠发生致死性炎症休克，提示细胞核DNA及其模拟物也可能是TLR9配体，通过靶向sTLR9发挥免疫负调节作用。在本课题中，我们拟以sTLR9为切入点，采用TLR9活化诱导的全身炎症反应小鼠模型和细胞模型，研究CCT ODN对全身炎症反应的负调节作用及对不同免疫细胞sTLR9水平和激活的影响，从而揭示其对全身炎症应答的负调控机制，为防治SIRS提供实验依据。

在前期发现的CCT ODN能挽救TLR9激动剂CpG ODN诱导的致死性全身炎症应答小鼠的生命的基础上，本项目围绕TLR9研究CCT ODN免疫负调节的机制。为了完成这一目标，我们采用三种小鼠全身炎症模型，包括TLR9活化诱导全身炎症应答综合征（SIRS）、脓毒症腹膜炎及灼伤诱导SIRS模型，并采用小鼠炎症局部免疫细胞确定TLR9移行定位和TLR9活化抑制。结果发现：CCT ODN对TLR9活化诱导全身炎症应答有显著的抑制作用，这种作用是通过上调炎症部位中性粒细胞表面TLR9（sTLR9）实现的；CCT ODN能使TLR9从细胞内向细胞膜表面移行，使炎症部位的sTLR9+中性粒细胞数量及比例明显增加；炎症早期募集到炎症部位的sTLR9+中性粒细胞对致死性全身炎症小鼠起保护作用；CCT ODN主要以阻碍TLR9活化来抑制过度炎症应答，因此对于脓毒症及灼伤诱导SIRS效果不明显。这些结果提示，CCT ODN是通过上调sTLR9对TLR9介导炎症应答发挥负调节作用，具有潜在的临床应用价值，但需要对炎症类型进行选择。研究中意外发现：AAAG ODN抑制全身炎症应答是通过阻碍干扰素调节因子5（IRF5）核转位实现的。由于IRF5是多条信号通路介导炎症因子产生的转录因子，因此，靶向IRF5的AAAG ODN不仅对TLR9活化诱导的SIRS有负调节作用，也对脓毒症性炎症和灼伤诱导SIRS表现出很好抑制作用。这个结果提示AAAG ODN可能成为过度炎症应答的广谱负调节剂，具有潜在的临床应用价值。