Deaf1 isoforms control changes in PTA expression in the NOD PLN during T1D pathog

Deaf1亚型控制T1D病理过程中NOD PLN中PTA表达的变化

• 批准号: 8097962
• 负责人: CHARLES GARRISON FATHMAN
• 金额: $ 39.63万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8097962
• 关键词: Address; Age; Age of Onset; Alternative Splicing; Antigens; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmune Process; Beta Cell; Binding; Biological Assay; Biological Process; Birth; Blood Glucose; Cells; Cervical lymph node group; Collaborations; Cytoplasm; Data; Defect; Development; Disease; Disease Progression; Dominant-Negative Mutation; Down-Regulation; Ectopic Expression; Effector Cell; Gene Expression; Genes; Genetic; Genetic Transcription; Histones; Hormones; Human; Immunohistochemistry; Immunophenotyping; In Situ Hybridization; Inbred NOD Mice; Incidence; Infiltration; Inflammation; Inflammatory; Insulin; Insulin-Dependent Diabetes Mellitus; Islet Cell; Islets of Langerhans; Knock-out; Knockout Mice; Laboratories; Lead; Length; Letters; Lymph Node Tissue; Lymphocyte; Lymphoid Tissue; Measures; Mediating; Microarray Analysis; Modeling; Molecular; Mus; Mutation; Non obese; Onset of illness; Pancreas; Pathogenesis; Pathway interactions; Patients; Peptides; Peripheral; Phenotype; Play; Production; Promoter Regions; Protein Binding; Protein Isoforms; RNA Splicing; Regulation; Regulatory T-Lymphocyte; Reporter; Reporting; Research Personnel; Role; Self Tolerance; Severities; Severity of illness; Sorting - Cell Movement; Spliced Genes; Stromal Cells; Structure of beta Cell of islet; Syndrome; T-Cell Development; T-Lymphocyte; Testing; Thymus Gland; Time; Tissues; Transcriptional Regulation; Uveitis; Variant; age related; autoreactive T cell; base; cell type; congenic; cytokine; design; diabetic; diabetic patient; lymph nodes; mouse model; nuclear factor 1; peripheral tolerance; promoter; public health relevance; type I diabetic; uveoretinitis

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by self-reactive T cells. Potentially self-reactive T cells escaping elimination in the thymus may subsequently be deleted or tolerized in the periphery. Recently, stromal cells of lymph nodes were reported to control self-tolerance by inducing the ectopic expression of various peripheral tissue antigens (PTAs) and presenting them to T cells in a manner that induces tolerance (deletion or regulation). Currently, both the transcriptional control of PTA expression in lymph nodes and the dysregulation of peripheral tolerance in T1D are unclear. Our laboratory recently identified Deaf1 as a transcriptional regulator of PTA gene expression in peripheral lymph nodes. Our findings show that the gene expression of PTAs, including insulin and other beta cell antigens, are down- regulated in the pancreatic lymph nodes (PLN) of non-obese diabetic (NOD) mice at 12 weeks of age. This change in expression coincides in time with the onset of beta cell destruction by autoreactive T cells. Preliminary studies show that Deaf1 regulates the expression of multiple PTA genes in the PLN. Thus far, two forms of Deaf1, a full-length functional form and a non-functional variant (Deaf1-VAR) have been identified in the PLN of 12 week-old NOD mice. At this age, Deaf1-VAR expression is significantly higher and Deaf1 expression is significantly lower in the PLN of NOD compared to NOD.B10 mice. Deaf1-VAR is expressed in the cytoplasm, has little transcriptional activity of its own, and inhibits the transcriptional activity of Deaf1 by heterodimerizing with it and retaining it in the cytoplasm. Remarkably, we identified an equivalent alternatively spliced Deaf1 isoform in the PLN of T1D patients. This human variant which was ~20-fold more abundant in the PLN of T1D patients than controls, behaves like the mouse Deaf1-VAR. The high expression of this variant also correlated with the absence of insulin gene expression in the PLN of T1D patients. Based on these data, it is hypothesized that Deaf1 controls the expression of and maintains peripheral tolerance to certain pancreatic beta-cell antigens, including insulin, in the PLN, and that a change in Deaf1 splicing contributes to the pathogenesis of T1D. To examine this hypothesis, proposed studies address the following: 1) What cell type in the PLN expresses Deaf1, Deaf1-VAR and PTAs? 2) How does Deaf1 mediate PTA gene transcription on a molecular level? 3) Does inflammation of the PLN or various "genetic factors" regulate the splicing of Deaf1? 4) Will knock-out of Deaf1 expression in PTA-expressing cells exacerbate NOD disease, and if so, is this due to a lack of autoreactive T cell deletion or a lack of regulatory T cell development? Finally, do either mouse models of autoimmune uveoretinitis or human APS 1 patients have changes in Deaf1 that can be correlated with disease? The results of these studies will provide an overall understanding of how Deaf1 and its isoforms maintain peripheral tolerance and thus, how a change in the splicing of Deaf1 or a mutation in DEAF1 may contribute to the development of autoimmune disease. PUBLIC HEALTH RELEVANCE: Type 1 diabetes can result from a break in peripheral tolerance that is, in part, mediated by the expression of peripheral tissue antigens (PTAs) on lymph node cells. We have identified a transcriptional regulator, Deaf1, that regulates the expression of PTAs in the pancreatic lymph node (PLN), and have data to suggest that the alternative splicing of this gene leads to reduced PTA expression in the non-obese diabetic mouse model and in human type 1 diabetic patients. The studies proposed here will examine the role of Deaf1 in maintaining peripheral tolerance and help us understand how a change in Deaf1 splicing may contribute to the pathogenesis of T1D.

描述(由申请人提供)：1型糖尿病(T1D)由自身反应性T细胞对胰岛β细胞的自身免疫破坏而发展。在胸腺中逃避消除的潜在的自身反应性T细胞随后可能在外周被删除或耐受。最近，有报道称，淋巴结基质细胞通过诱导各种外周组织抗原(PTA)的异位表达，并以诱导耐受(缺失或调节)的方式将其呈递给T细胞来控制自身耐受。目前，T1D中PTA表达的转录调控和外周耐受的异常都不清楚。我们的实验室最近发现Deaf1是外周淋巴结中PTA基因表达的转录调节因子。我们的研究结果表明，12周龄非肥胖糖尿病(NOD)小鼠胰腺淋巴结(PLN)中PTA的基因表达下调，包括胰岛素和其他β细胞抗原。这种表达的变化在时间上与自身反应性T细胞开始破坏β细胞的时间一致。初步研究表明，Deaf1调控多个PTA基因在PLN中的表达。到目前为止，在12周龄NOD小鼠的PLN中已经发现了两种形式的Deaf1，一种是全长功能形式，另一种是非功能变体(Deaf1-VAR)。在这个年龄段，NOD小鼠PLN中Deaf1-VAR的表达显著高于NOD.B10小鼠，而Deaf1的表达显著低于NOD.B10小鼠。Deaf1-VAR在细胞质中表达，自身几乎没有转录活性，并通过与Deaf1异源二聚并将其保留在细胞质中来抑制Deaf1的转录活性。值得注意的是，我们在T1D患者的PLN中发现了一个等效的选择性剪接的Deaf1亚型。这种人类变异体在T1D患者的PLN中的含量是对照组的20倍，其行为类似于小鼠Deaf1-VAR。该变异的高表达还与T1D患者PLN中胰岛素基因表达缺失有关。根据这些数据，推测Deaf1控制PLN中包括胰岛素在内的某些胰岛β细胞抗原的表达并维持其外周耐受性，Deaf1剪接的变化参与了T1D的发病。为了验证这一假设，拟议的研究涉及以下问题：1)PLN中什么类型的细胞表达Deaf1、Deaf1-VAR和PTA？2)Deaf1如何在分子水平上调节PTA基因的转录？3)PLN的炎症或各种“遗传因素”是否调节Deaf1的剪接？4)表达PTA的细胞中Deaf1的表达缺失会加剧NOD疾病，如果是这样，这是由于缺乏自身反应性T细胞缺失还是缺乏调节性T细胞发育？最后，无论是自身免疫性葡萄膜视网膜炎的小鼠模型还是人类APS 1患者，Deaf1的变化是否与疾病相关？这些研究的结果将有助于全面了解Deaf1及其亚型如何维持外周耐受，从而了解Deaf1剪接的变化或DEAF1的突变如何有助于自身免疫性疾病的发展。 公共卫生相关性：1型糖尿病可以由外周耐受性的破坏引起，这在一定程度上是由淋巴细胞上外周组织抗原(PTA)的表达介导的。我们已经确定了一个转录调控因子Deaf1，它调控胰腺淋巴结(PLN)中PTA的表达，并有数据表明，该基因的选择性剪接导致非肥胖糖尿病小鼠模型和人类1型糖尿病患者中PTA表达减少。本文提出的研究将检验Deaf1在维持外周耐受中的作用，并帮助我们了解Deaf1剪接的变化如何在T1D的发病机制中起作用。