肠道菌群失调在Graves病患者调节性T细胞异常中的作用及其机制研究

Gut Microbiota plays important roles in the regulation of regulatory T cells (Treg) and is involved in the pathogenesis of many extraintestinal autoimmune diseases. The diarrhea caused by gut microbiota dysbiosis (GMD) and Treg abnormalities are the significant features of Graves' disease (GD). However, the study of the effects and mechanism of GMD on the regulatory T cell abnormalities in the patients with GD has not yet to be reported. Our preliminary studies have shown that compared to normal and non-diarrhea GD patients, the gut microbiota of GD patients with diarrhea was markedly abnormal, the number and function of Treg cells decreased significantly and the ability to correct abnormalities of Treg cells in mice which gut microbiota was removed using antibiotic cocktails was also significantly lower. Therefore, we hypothesized that the GMD of GD patients might result in abnormalities of some intestinal bacteria and their metabolites and then lead to the dysfunction of Treg cells in intestine and blood directly or indirectly through cytokines. To verify this hypothesis, the technology of metabolomics and metagenomics will be used for identification of the aberrant intestinal bacteria and related metabolites in patients with GD. Then, the aberrant intestinal bacteria and related metabolites that can really regulate Treg cells will be selected using mice which gut microbiota was removed by antibiotic cocktails treatment. Furthermore, the regulatory mechanism will be revealed by flow cytometry and protein chip. This project would help to clarify the role and mechanism of GMD in Treg abnormalities in patients with GD, to understand the regulation of intestinal flora and other environmental factors to Treg cells and the immune system as well as to guide the design of biological immunotherapy.

肠道菌群对调节性T细胞（Treg）有重要调控作用，与肠外自身免疫疾病关系密切；而Treg异常和腹泻所致肠道菌群失调（GMD）是Graves病(GD)的重要特征，但GD患者GMD对Treg异常的作用及其机制目前仍未见研究报道。我们前期研究表明与正常和非腹泻GD相比，腹泻GD患者肠道菌群明显异常，Treg数量和功能显著下降；其肠道菌群对肠道菌清除小鼠Treg异常的纠正能力明显偏低。我们推测GD患者GMD可能使某些肠道菌及其代谢物异常，直接或间接通过细胞因子等在肠道或入血使Treg异常。为验证这一假说，本申请将利用代谢组和宏基因组等技术，揭示GD患者失调的肠道菌及其代谢物，并用小鼠筛选确能调控Treg的菌种/代谢物，进而在体外用蛋白芯片和流式等揭示其分子机制。本项目有助于阐明GMD在GD患者Treg异常中的作用和机制，理解肠道菌群等环境因素对Treg和免疫系统的调控作用，并指导设计生物免疫疗法。

在本项目的支持下，我们按照项目研究计划，综合利用16s rRNA测序、靶向和非靶向代谢组学、Graves病小鼠模型以及体内和体外功能实验技术，深入研究了肠道菌群失调在Graves病患者包括外周Treg细胞减少在内的免疫炎症异常中的作用及其机制，首次揭示了关键肠道菌和代谢物在这些异常中的作用及其与GD发病之间的因果关系，并且进一步揭示了关键肠道菌Bacteroides fragilis YCH46对Treg / Th17动态平衡的影响及其机制。我们发现GD患者外周血多种炎症因子、Treg细胞和Th17细胞含量及Treg / Th17比例显著异常，同时肠道菌群和整体代谢特征也发生显著异常，而且许多肠道细菌和代谢产物的异常变化与GD的临床特征、炎症免疫异常密切相关，如显著减少的短链脂肪酸（SCFA）及生产SCFA的肠道菌（如脆弱拟杆菌）。我们通过选择性培养，进一步发现Bacteroides fragilis YCH46菌株作为肠道中最主要的脆弱拟杆菌，其含量在GD患者肠道中显著下降。该菌能产生丙酸，通过上调游离脂肪酸受体2（FFAR2）并下调组蛋白脱乙酰酶6和组蛋白脱乙酰酶9的表达而增加（减少）Treg（Th17）细胞数量。我们还进一步在动物水平发现移植GD患者的肠道菌群会显著改变GD模型小鼠血清中总甲状腺素（TT4）和免疫炎症标记物（IL-10，IL-17A和TRAb）的水平，使GD模型小鼠的成模率显著增加。另外，我们还发现三个肠道细菌属（Bacteroides, Alistipes, Prevotella）能以85％的准确性区分GD患者和健康人，具有作为一种价廉、便捷的非侵入式GD患者筛查标注物的潜力。我们的研究也为B. fragilis改善GD患者的免疫功能障碍提供了有价值的线索，并阐明了GD的微生态疗法的前景。