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起关键作用。多重免疫耐受 存在检查点以除去自身反应性B细胞或使它们处于控制之下。这些检查点的缺陷 构成自身免疫性疾病发展的基础。尽管深入研究，我们 对这些检查站的了解仍然是不完整和不完整的。microRNAs（miRNAs）是一种新的 一类小的非编码RNA，调节多种多样的生物过程。数百种miRNAs 在免疫系统中表达。虽然一些miRNAs已被证明在 尽管miRNAs在淋巴细胞发育和功能中的作用很小，但miRNAs在控制免疫耐受中的作用仍然很差 明白我们在最近建立的细胞系中对数百种miRNAs进行了体内功能分析。 IgM-巨自身小鼠模型，并鉴定了miR-148 a作为B细胞中枢耐受的重要调节因子 和自身免疫（Nature Immunology 17：433-40，2016）。进一步的分子分析确定了119个目标 未成熟B细胞中miR-148 a调控的基因。我们检测了其中4个靶基因，并证明了 其中Gadd 45 a、Bim和Pten 3个基因调控B细胞的中枢耐受。在本建议中，我们将：（1）进一步 研究miR-148 a调节免疫耐受的细胞和分子机制 和自身免疫，重点介绍其在控制各种B细胞耐受检查点和浆细胞免疫中的作用。 2）评估通过miR-148 a消融治疗全身性自身免疫的可能性， 分别通过遗传和化学方法抑制，和3）进行体外功能筛选， 其他115个miR-148 a靶基因，以鉴定B细胞耐受性的新调节因子。我们的试点屏幕有 发现B4 galt 5是阳性的由于B4 galt 5是糖脂生物合成途径中的主要酶， 推测该通路在免疫耐受中起重要作用。因此，我们将阐明 B4 galt 5和糖脂合成途径在控制B细胞耐受中的作用和机制 和自身免疫

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.