早期肠道菌群变化经TLRs通路调控树突状细胞在1型糖尿病发病机制中的研究

Type 1 diabetes (T1DM) is a T cell–mediated autoimmune disease that involves the slow, progressive destruction of islet ß cells and loss of insulin production, as a result of interaction with environmental factors, in genetically susceptible individuals. The gut microbiota is established very early in life. Increasing evidence suggests that the gut microbiota is an important environmental element in the development of type 1 diabetes, but the possible mechanism remains unknown. .Our previous study showed that Neomycin treatment during infancy protected the NOD mice from diabetes development through alterations in the gut microbiota, as well as influence the immunophenotype of the dendritic cells. We speculate that alterations in the gut microbiota may affect the function of the dendritic cells, which led to reduced activation of diabetogenic T cells. .We will determine the change in composition of gut microbiota induced by Neomycin treatment using 454 pyrosequencing of the 16S ribosomal RNA and validate the different gut microbiota by Real-time quantitative (q-PCR). The functional diversity, composition, structure, and metabolic potential or activity of a variety of the microbial communities were analyze by functional gene array GeoChip. Next, we will explore if alterations in the gut microbiota regulate the dendritic cells through TLRs signaling pathway in vitro and in vivo and further influence the intestinal permeability by increasing the expression levels of genes involved in tight junction formation. The finding will provide a rationale for novel therapeutic strategies targeting the gut microbiota to prevent and treat type 1 diabetes.

本项目从前期研究基础出发，以体内外研究为视角，沿着早期干预肠道菌群延缓1型糖尿病（T1DM）发生的思路，朝着系统阐述早期肠道菌群变化经Toll样受体信（TLRs）通路调控树突状细胞（DCs）延缓T1DM发生机制的目标开展工作。首先联合应用16S rRNA高通量基因测序和基因功能芯片（GeoChip）技术，针对早期干预肠道菌群的结构和功能进行系统分析,在高通量水平上甄别出与T1DM发生相关的菌群结构及功能基因，并用体内、外实验验证通量数据，筛选出相关菌群。随后以细胞、NOD小鼠和转基因小鼠为研究对象，从体内、外实验二个层面证实筛选出的菌群不仅通过增加紧密连接蛋白的表达降低肠黏膜通透性，而且借助TLRs通路调控DCs活性及抗原递呈功能，使致病性T淋巴细胞活化增殖减少延缓T1DM发生。本项目的实施有助于深刻理解早期干预肠道菌群参与T1DM发病的机制，为预防和治疗T1DM提供理论依据及新的思路。

肠道菌群与T1DM发展密切相关，而肠道菌群在婴幼儿期对机体免疫系统发育也发挥重要作用。本研究发现在NOD小鼠出生至3周内经Neomycin干预，改变其肠道菌群结构可降低T1DM发病率。我们应用16S rRNA高通量基因测序技术，针对Neomycin干预组肠道菌群结构和功能进行分析，发现与对照组相比，Neomycin组干预3周后肠道菌群结构发生显著变化，在门水平上观察到Bacteroidetes和 Cyanobacteria升高，而 Actinobacteria 降低，且两组间肠道菌群结构变化在干预结束后的12周、T1DM发病时仍长期存在。Neomycin干预后也降低了NOD小鼠肠黏膜通透性。与对照组相比，Neomycin组NOD小鼠脾脏、MLN和PLN中树突状细胞（dendritic cell，DC）协同刺激分子及炎性细胞因子分泌减少。在体外不同TLR激动剂刺激下，Neomycin组NOD小鼠树突状细胞协同刺激分子CD80,CD86及炎性细胞因子IFN-γ分泌减少。而在体外实验中，从Neomycin组以及对照NOD小鼠脾脏中分离纯化DC体外培养，加入正常NOD小鼠脾脏T淋巴细胞及抗CD3和抗CD28单克隆抗体进行3H增殖实验，发现Neomycin组NOD小鼠DC活性功能减弱，对外源性/内源性致病性T细胞的活化减弱。再将Neomycin组以及对照NOD小鼠脾脏纯化DC体外培养，分别加入BDC2.5NOD小鼠中脾CD4+T细胞及NY 8.3NOD小鼠脾CD8+T细胞及刺激物minotope 或IGRP共孵育进行3H 增殖试验后发现Neomycin组导致NOD小鼠DC对CD4+及CD8+T细胞的抗原递呈能力减弱。在体内实验部分，我们将分离纯化好的糖尿病发病的NOD鼠脾脏T淋巴细胞过继转移给接受过放射线照射的Neomycin组或对照组NOD鼠，发现过继转移了致病性T淋巴细胞的Neomycin组其T1DM发病率低于对照组。本研究表明，Neomycin早期干预NOD鼠导致肠道菌群结构发生改变，通过降低肠粘膜通透性，调控DC活化表型及抗原递呈功能，使致病性T淋巴细胞在体内的增殖和活化均减少，从而减轻胰岛自身免疫反应延缓T1DM发病。本项目的实施有助于从免疫及分子水平揭示早期肠道菌群变化在T1DM发病中机制，为将来针对肠道菌群治疗T1DM提供新的靶点。