精准挖掘牙周炎微生物组对糖尿病的免疫调控机制

The study of the immunomodulatory mechanism of microbiome and its association with the organ dysfunction is the frontier of academic research (Cell, 2017). A bidirectional relationship has been purported between diabetes mellitus and periodontitis induced by periodontitis microbiome, but the precise modulatory mechanism is yet unclear. We have already screened the suspected microbial taxa and functional gene associated with periodontitis using Miseq sequencing and HumiChip functional gene array in our previous study, but their functional effect on the host has not been investigated. In the current study, we will inoculate the monoclonal periodontal microorganisms in the subgingival tissue of germ-free diabetic mice. For the first time, we plan to identify the dynamic changes of the body parts colonized by 3 phyla, 8 genera and 15 species associated with periodontitis based on the phylogenetic tree in a broad spectrum. And then, we take the lead in precisely mining the comprehensive activation and modulatory mechanism of periodontal microbiome on 18 innate and adaptive immune cell types and 27 circulating inflammatory factors. Finally, we will assess the epigenetic modulatory effect of periodontal microbiome on the islet genome and LcnRNA using transcriptome array, and further try to explore the interaction mechanism between immune system and genetic transcription through bioinformatics techniques. Our research will establish an effective and sensitive readout system. Numerous data will be generated to illuminate how precisely the periodonttitis microbiome modulate the immune response and islet gene expression. These research findings will provide new thought and precision medicine for the treatment of diabetes mellitus.

人体微生物组与全身免疫系统的调控机制和器官功能疾病的关系是学科研究前沿（Cell, 2017）。牙周炎微生物组所致牙周炎与糖尿病的双向促进关系是公认的学说，但其精细调控机制尚不清楚。我们前期通过Miseq测序和HumiChip芯片筛选出牙周炎核心菌属和功能基因，尚未探索其对宿主的功能体现。本课题拟采用单克隆牙周菌接种于糖尿病无菌小鼠口腔的方法，首次广谱鉴别系统发育树上3门8属15种牙周菌在宿主定植部位的动力学改变，并创新性精准挖掘其对18种固有和适应性免疫细胞、27种循环炎症因子的全面激活作用及调控机制；最后用转录组芯片评价对胰岛基因组和lcnRNA的表观遗传调控作用、生物信息学手段探索免疫系统和基因转录的互作机制。我们将绘制基线，建立一个有效的“敏感”读出系统，生成的大量数据集将阐明牙周炎微生物组是如何精确指挥免疫系统响应和胰岛基因的表达，为糖尿病防治提供新的思路及精准治疗靶点。

本课题开展牙周炎微生物组在宿主定植部位的动力学改变，精准挖掘其对18种固有和适应性免疫细胞以及循环炎症因子的全面激活作用及调控机制。首先我们发现三种牙周炎关键致病菌能显著上调αβT细胞，对脾脏免疫影响大于牙龈和结肠。对结肠免疫细胞谱而言，具核梭杆菌的影响最大，主要以ILC3和Tregs的增加、B细胞减少为主；牙龈卟啉单胞菌对牙龈固有免疫谱的影响最大，以巨噬细胞显著上调为主；齿垢密螺旋体显著激活脾脏的免疫应答，以Tregs下调为主。然后将牙周炎病人的集合微生物组接种T1DM无菌鼠口腔后，可在多种器官中异位定植，以肺炎克雷伯菌为主。与SH组（健康人唾液接种T1DM无菌鼠）相比，SP组（牙周炎唾液接种T1DM无菌鼠）对脾脏的免疫调控作用强于小肠，以脾脏固有免疫细胞MF、pDCs、Mono和ILC3细胞显著上调，适应性免疫细胞DNT、Th17细胞显著上调和αβT细胞显著下调为主，变化幅度均超过了2倍；与P组（牙周炎患者唾液接种无菌鼠）相比，SP组小肠、脾脏的固有免疫和适应性免疫应答显著改变，以激活为主。小肠、脾脏的pDCs、B和CD8+T细胞均显著上调，但DNT细胞相反。RDA分析结果显示，部分菌群与免疫细胞呈现相关关系。同时我们发现牙周炎微生物组抑制2型糖尿病SPF小鼠脾脏固有免疫应答和炎症循环因子的生成，使单核细胞、巨噬细胞和ILC3细胞数量下降、IL-17数量减少，促进适应性免疫细胞Tregs和Th17细胞数量增加。动物实验显示M2巨噬细胞外泌体（M2-exo）可被小鼠骨髓间充质干细胞(BMSCs)摄取内化，促进成骨、抑制破骨；qPCR结果显示，高糖组成骨分化相关基因Alp、Runx2、Ocn及Hedgehog通路相关基因Gli1、Smo、Ptch1 mRNA水平与Control组相比均有不同程度的降低，而M2-exo可促进这些基因的上调；WB结果类似。最后我们发现牙周炎微生物群处理能促进小鼠炎癌转化，并改变免疫微环境，IL-17+γδ T细胞发挥着关键作用。我们筛选出的特定肺炎克雷伯菌对高活性的γδ T-TAM免疫轴或ILC3的调控机制值得深入研究。总之，我们生成的大量数据集阐明了牙周炎微生物组是如何精确指挥免疫系统响应和相关基因表达，为糖尿病防治提供新的思路及精准治疗靶点。