Type 1 diabetes: the role of commensal microbiota

描述（由申请人提供）：1型糖尿病（T1D）是一种衰弱性自身免疫性疾病，在发达国家发病率呈上升趋势。许多事实表明微生物参与了T1D的调节。在初步研究中，我们已经确定：a.缺乏MyD88接头的非肥胖糖尿病（NOD）小鼠在常规的无特异性病原体（SPF）条件下对自发性T1D具有抗性；b. MyD88 KO NOD小鼠未表现出对胰岛抗原的全身耐受，但在局部胰腺淋巴结中确实表现出T细胞对这些抗原的耐受；c.当同样的动物无菌（GF）时，它们患糖尿病的频率约为100%；d.用确定的菌群重建GF MyD88 KO动物后，糖尿病发病率降低。综上所述，这些数据表明肠道中的共生（非致病）微生物影响了T1D的发展。MyD88可能的作用是控制共生微生物。当MyD88缺失时，肠道微生物群的变化随之而来。为了支持这一观点，我们对正常NOD和MyD88 KO动物的肠道微生物群进行了宏基因组分析（16S rRNA基因测序），发现它们之间存在显著且有意义的差异。利用基因敲除和条件敲除小鼠，无菌技术和宏基因组测序方法，我们将追求以下目标：1。揭示myd88依赖的机制，控制共生微生物，从而促进T1D。我们将测试TLR2以及B细胞和肠上皮细胞在控制肠道微生物群中的可能作用，并研究MyD88 KO小鼠通过条件消除MyD88对T1D产生耐药性的细胞基础。2. 阐明共生体引起T细胞耐受的myd88独立机制。我们将测试不同的模式识别受体（PRRs）在预防T1D中的作用。我们将使用基因表达分析的结果：测试PLN细胞亚群中已鉴定基因的表达，以及CCR5在微生物群预防T1D中的重要性。我们将讨论抗原呈递细胞（APC）和调节性T细胞（Treg）在微生物群诱导的局部耐受性中的作用。3. 研究肠道菌群在预防T1D中的作用。我们将验证一种假设，即特定的细菌可以预防T1D，并测试选定的微生物群落单独或联合免疫抑制逆转GF小鼠临床糖尿病的能力。

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) is a debilitating autoimmune disease with the incidence on the increase in developed countries. Many facts point at microbial involvement in the regulation of T1D. In preliminary studies we have established that: a. non-obese diabetic (NOD) mice lacking MyD88 adaptor were resistant to spontaneous T1D when housed in conventional specific-pathogen-free (SPF) conditions; b. MyD88 KO NOD mice did not show systemic tolerance to islet antigens, but did show tolerance of T cells to such antigens in the local pancreatic lymph nodes; c. when the same animals were made germ-free (GF), they developed diabetes with the frequency of about 100%; d. when GF MyD88 KO animals were reconstituted with the defined microflora, the diabetes incidence was reduced. Taken together the data suggested that commensal (not pathogenic) microbes in the gut influenced the development of T1D. The likely role of MyD88 is to control commensal microbes. When MyD88 is missing, the changes in gut microbiota ensue. To support that idea, we have performed the metagenomic analysis of the gut microbiota (16S rRNA genes sequencing) from normal NOD and MyD88 KO animals, and discovered significant and meaningful differences between them. Taking advantage of knock-out and conditional knock- out mice, the germ-free technology and metagenomic sequencing approach, we will pursue the following aims 1. Uncover MyD88-dependent mechanisms that control commensal microbes, thus promoting T1D. We will test a possible role of TLR2 as well as the role of B cells and intestinal epithelial cells in control of intestinal microbiota and study the cellular basis for resistance of MyD88 KO mice to T1D by conditional elimination of MyD88. 2. Elucidate MyD88-independent mechanisms by which commensals cause T cell tolerance. We will test different Pattern Recognition receptors (PRRs) for their role in prevention of T1D. We will use the results of a gene expression analysis: test expression of identified genes in cellular subsets in PLN and an importance of CCR5 in prevention of T1D by microbiota. We will address the role of antigen presenting cells (APC) and regulatory T cells (Treg) in local tolerance induced by microbiota. 3. Study the role of intestinal microbiota in prevention of T1D. We will test a hypothesis that particular bacteria protect against T1D and test the ability of selected microbial communities to reverse clinical diabetes in GF mice by themselves or in combination with immunosuppression. PUBLIC HEALTH RELEVANCE: The proposal is devoted to the studies of animals (NOD, non-obese diabetic strain of mice) prone to Type 1 diabetes (T1D) and also lacking control over intestinal microbes due to the disruption of their innate immune system. These mice are protected from T1D by their intestinal microbes, whereas same animals without microbes (germ-free, GF) are not protected. Using these animals and their GF counterparts, we will dissect the signaling pathways involved in protection from T1D, characterize microbial lineages capable of protection from T1D, and perform microbial transplants to test the possibility of translation of our prevention tactics to humans.