Type 1 diabetes: the role of commensal microbiota

描述(申请人提供)：1型糖尿病(T1D)是一种衰弱的自身免疫性疾病，在发达国家的发病率呈上升趋势。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)小鼠在传统的无特定病原体(SPF)条件下对自发的T1D具有抵抗力；b.MyD88 KO NOD小鼠对胰岛抗原没有系统耐受性，但对局部胰腺淋巴结中的此类抗原表现出耐受性；c.当相同动物被使无菌(GF)时，它们患糖尿病的频率约为100%；D.当GF MyD88KO动物与定义的微生物菌群重组时，糖尿病的发病率降低。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的发育。MyD88可能的作用是控制共生微生物。当MyD88缺失时，肠道微生物区系随之发生变化。为了支持这一观点，我们对正常NOD和MyD88 KO动物的肠道微生物区系(16S rRNA基因测序)进行了元基因组分析，发现它们之间存在显著而有意义的差异。利用基因敲除和条件基因敲除小鼠的优势，无菌技术和元基因组测序方法，我们将追求以下目标：1.揭示MyD88依赖的控制共生微生物的机制，从而促进T1D。我们将测试TLR2以及B细胞和肠道上皮细胞在肠道微生物区系控制中的可能作用，并通过有条件地消除MyD88来研究MyD88 KO小鼠对T1D耐药的细胞学基础。2.阐明共生体导致T细胞耐受的MyD88非依赖性机制。我们将测试不同的模式识别受体(PRR)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细胞亚群中已识别基因的表达，以及CCR5在微生物群预防T1D中的重要性。我们将讨论抗原提呈细胞(APC)和调节性T细胞(Treg)在微生物区系诱导局部耐受中的作用。3.研究肠道微生物区系在预防T1D中的作用。我们将测试一种假说，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与免疫抑制一起逆转GF小鼠临床糖尿病的能力。 公共卫生相关性：该提案致力于对易患1型糖尿病(T1D)的动物(NOD，非肥胖型糖尿病小鼠品系)以及由于其固有免疫系统的破坏而缺乏对肠道微生物的控制的研究。这些小鼠受到肠道微生物的保护，免受T1D的侵袭，而没有微生物的动物(无菌动物，GF)则不受保护。利用这些动物和它们的GF对应物，我们将剖析参与保护T1D的信号通路，表征能够保护T1D的微生物谱系，并进行微生物移植，以测试我们的预防策略适用于人类的可能性。

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.