mtDNA介导的cGAS-STING信号通路通过调控自噬参与自身免疫性甲状腺炎的机制研究

Autoimmune thyroiditis (AIT) is an organ-specific autoimmune disease caused by the combination of heredity and environment. However, the pathogenesis of AIT is still unclear. Thyrocyte damage induced by iodine or inflammatory factors and the imbalanced of T lymphocytes cell homeostasis play a vital role in the pathogenesis of AIT. It has been reported that mitochondrial DNA (mtDNA) can release into cytoplasm, which activates the stimulator of interferon genes (STING) pathway on dendritic cells and induces autoantigen presentation thus causes immune response. The cGAS-STING pathway has been demonstrated in multiple autoimmune diseases and function to restore homeostasis. Whether the mechanism is involved in AIT has not been reported. Our preliminary study studies found that the mtDNA level was significantly upregulated in the plasma of AIT patients and the thyroid gland of AIT model , and the levels of mtDNA copy numbers were positively correlated with the severity of the disease. Meanwhile, STING expression was upregulated in the thyroid gland of iodine-induced AIT model, which suggested that mtDNA released by thyrocyte injury are involved in the pathogenesis of AIT. Therefore, we hypothesize that “mtDNA released from damaged thyrocyte may further extend the inflammatory response through the cGAS-STING pathway on DC. The mechanism of injury is related to the reduction of autophagy”. To verify this hypothesis, this study is aimed at exploring the molecular mechanism of mtDNA-cGAS-STING pathway through in vivo experiments in AIT mice model and in vitro culture of thyroid follicular cells. This study provides a strong theoretical basis for the development and treatment of AIT.

自身免疫性甲状腺炎（AIT）是常见的器官特异性自身免疫性疾病，具体发病机制尚未明确。高碘或炎性因子可以引起甲状腺组织损伤，T细胞分化失衡是AIT的主要发病机制。文献报道线粒体DNA（mtDNA）释放进入细胞质，激活树突状细胞（DC）的免疫应答分子STING，引起抗原递呈，导致自身免疫反应。申请人前期的研究发现AIT患者血浆及小鼠甲状腺mtDNA明显上调，与疾病严重程度高度相关，AIT小鼠甲状腺STING表达上调，提示甲状腺细胞损伤通过释放mtDNA，促进AIT的发病。因此，我们提出“AIT中释放mtDNA通过结合DC上的cGAS-STING通路，抑制自噬作用，造成甲状腺细胞进一步损伤”的假设。为验证这一假设，本研究将通过AIT小鼠体内实验及体外培养甲状腺细胞，探讨mtDNA-cGAS-STING通路参与AIT的分子机制。本研究为开发和治疗自身免疫性甲状腺炎的新切入点，提供有力的理论依据。

有研究表明，cGAS-STING通路在调控机体自身免疫反应中发挥重要作用。mtDNA可以结合cGAS-STING信号通路，促进抗原呈递细胞尤其是树突状细胞（Dendritic cells，DC）的成熟和活化。但具体机制未明确。.本研究通过AIT小鼠模型体内实验以及体外培养甲状腺细胞，研究对自噬、mtDNA或STING信号通路任一关键点进行干预，来探讨其参与AIT的分子机制。明确AIT动物模型中甲状腺组织及血浆的mtDNA存在质和量的差异，甲状腺细胞来源的mtDNA能够通过结合甲状腺自身抗原，引起不同亚型T细胞相关细胞因子及转录因子的分泌，造成T细胞功能失衡。然后进一步在小鼠体内实验干预，4周龄NOD.H-2h4小鼠被随机分为六组：对照组、SAT组(高碘水喂养)、H-151组（高碘水给予8周后 STING抑制剂H-151）、EGCG组（高碘水给予8周后给予cGAS抑制剂 EGCG）、DMXAA（高碘水给予STING激动剂DMXAA）、mtDNA（高碘水给予mtDNA）后，测定甲状腺局部炎症，血清TgAb水平，树突状细胞成熟，炎性因子，cGAS/STING信号通路蛋白及凋亡的表达。然后以原代培养的小鼠甲状腺细胞为研究对象，进行体外实验。分别给予高碘诱导或STING激动剂或抑制剂干预，揭示微环境的改变（高碘或炎性因子），引起甲状腺细胞mtDNA的变化，通过树突状细胞的cGAS-STING识别，引起抗原递呈，激活下游干扰素因子及炎性因子。结果显示：STING抑制剂能明显抑制SAT小鼠的炎症反应，激动剂同样可以激活其炎症反应。而mtDNA激活以及cGAS抑制剂抑制效果均不显著，研究表明cGAS/STING通路在AIT中的作用，主要是通过STING蛋白的直接调节。mtDNA刺激下，甲状腺细胞膜电位升高，甲状腺细胞中树突状细胞比例升高并受cGAS/STING通路调节。同时mtDNA刺激下，甲状腺细胞中cGAS/STING通路激活。NOD.H-2h4小鼠巨噬细胞中的体外研究，发现STING激动剂可以活化STAT3信号通路来调节巨噬细胞极化作用。.以上结果提示，mtDNA与树突状细胞，cGAS-STING通路及线粒体自噬结合起来，可以更好的阐述mtDNA调控AIT的发病机制，力求找到研究切入点对疾病进行干预。.