Functional Analysis of MicroRNAs and Target Genes in Immune Tolerance

项目摘要 自身反应性B细胞在许多自身免疫性疾病中发挥重要作用。多重免疫耐受 存在检查点，以清除自身反应的B细胞或将其保持在控制之下。这些检查点中的缺陷 构成了自身免疫性疾病发展的基础。尽管进行了密集的研究，我们的 对这些检查站的了解仍然不完整和支离破碎。微RNA(MiRNAs)是一种新的 一类小的非编码RNA，调节多种生物过程。数百个miRNAs 在免疫系统中表达。虽然一些miRNAs已被证明在 淋巴细胞的发育和功能，miRNAs在控制免疫耐受中的作用仍然很差 明白了。我们对最近建立的数百个miRNAs进行了体内功能分析 免疫球蛋白-巨噬细胞自体小鼠模型的建立及鉴定miR-148A是B细胞中枢耐受的重要调节因子 和自身免疫(自然免疫学17：433-40,2016)。进一步的分子分析确定了119个靶点 未成熟B细胞中受miR-148A调控的基因我们检查了其中4个目标基因，并证明了 其中3个，Gadd45a，Bim和Pten，调节B细胞中枢耐受。在这项提案中，我们将1)进一步 研究miR-148A调节免疫耐受的细胞和分子机制 和自身免疫，重点是它在控制各种B细胞耐受检查点和浆细胞中的作用 2)评价miR-148A消融治疗系统性自身免疫的可能性。 分别通过遗传和化学方法进行抑制，以及3)进行体外功能筛选 其他115个miR-148A靶基因用于识别B细胞耐受的新调节因子。我们的飞行员屏幕上有 确定B4galt5是一个积极的打击。由于B4galt5是糖脂生物合成途径中的主要酶，我们 推测该途径在免疫耐受中起重要作用。因此，我们将澄清 B4galt5和糖脂生物合成途径在控制B细胞耐受中的作用和机制 和自身免疫力。

Project Summary Autoreactive B cells play critical roles in many autoimmune diseases. Multiple immune tolerance checkpoints exist to remove autoreactive B cells or keep them under control. Defects in these checkpoints constitute the basis for the development of autoimmune diseases. Despite intensive study, our understanding of these checkpoints remains incomplete and fragmentary. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate a large diversity of biological processes. Hundreds of miRNAs are expressed in the immune system. While some miRNAs have been shown to play important roles in lymphocyte development and function, the roles of miRNAs in controlling immune tolerance remain poorly understood. We performed in vivo functional analysis of hundreds of miRNAs in the recently established IgMb-macroself mouse model and identified miR-148a as an important regulator of B cell central tolerance and autoimmunity (Nature Immunology 17:433-40, 2016). Further molecular analysis identified 119 target genes regulated by miR-148a in immature B cells. We examined 4 of these target genes and demonstrated that 3 of them, Gadd45a, Bim and Pten, regulate B cell central tolerance. In this proposal, we will 1) further investigate the cellular and molecular mechanisms underlying miR-148a regulation of immune tolerance and autoimmunity, focusing on its role in controlling various B cell tolerance checkpoints and plasma cell differentiation; 2) evaluate the possibility of treating systemic autoimmunity through miR-148a ablation and inhibition by genetic and chemical approaches, respectively, and 3) perform an in vitro functional screen of the other 115 miR-148a target genes to identify novel regulators of B cell tolerance. Our pilot screen has identified B4galt5 as a positive hit. As B4galt5 is a major enzyme in the glycolipid biosynthesis pathway, we speculate that this pathway plays important roles in immune tolerance. Therefore, we will elucidate the function and mechanism of B4galt5 and the glycolipid biosynthesis pathway in controlling B cell tolerance and autoimmunity.