PTPN22 R619W in NOD mice

NOD 小鼠中的 PTPN22 R619W

• 批准号: 10183146
• 负责人: LINDA A SHERMAN
• 金额: $ 74.52万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10183146
• 关键词: Adoptive Transfer; Affect; Alleles; Amino Acids; Antigens; Appearance; Autoantibodies; Autoimmune; Autoimmune Diseases; Autoimmunity; Automobile Driving; B-Lymphocytes; Behavior; Birth; Bone Marrow; C57BL/6 Mouse; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Candidate Disease Gene; Cell physiology; Cells; Child; Data; Defect; Development; Diabetes Mellitus; Discrimination; Disease; Disease susceptibility; Drug Targeting; Environmental Risk Factor; Enzymes; European; Exhibits; Frequencies; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic Polymorphism; Goals; Graves' Disease; Haplotypes; Human; Immune; Immune Tolerance; Immune system; Inbred NOD Mice; Incidence; Insulin; Insulin Antibodies; Insulin-Dependent Diabetes Mellitus; Islets of Langerhans; Label; Lymphocyte; Modeling; Molecular; Monitor; Mus; Mutate; Mutation; Myelogenous; Myeloid Cells; Non obese; Orthologous Gene; Pancreas; Pathway interactions; Penetrance; Peptides; Phenotype; Population; Predisposition; Preventive therapy; Production; Property; Protein Tyrosine Phosphatase; Proteins; Retroviral Vector; Rheumatoid Arthritis; Risk; Risk Factors; Role; Single Nucleotide Polymorphism; Structure of beta Cell of islet; Structure of germinal center of lymph node; Susceptibility Gene; System; Systemic Lupus Erythematosus; T-Lymphocyte; Techniques; Testing; Transgenic Mice; Transgenic Organisms; Vitiligo; anergy; antigen-specific T cells; base; cell type; differential expression; disorder risk; experimental study; genetic risk factor; genome editing; high risk; human disease; insulin tolerance; interest; islet; islet cell antibody; knock-down; lymph nodes; mouse model; prevent; reconstitution; seroconversion; small hairpin RNA; therapy development; transcriptome sequencing

Project Summary: Type 1 diabetes (T1D) results from a breakdown in immune tolerance that progresses to the destruction of the insulin producing beta cells of the pancreatic islets. A large number of genetic and environmental factors contribute to the incidence of disease. One of the strongest non-MHC genetic risk factors is a single nucleotide polymorphism in the gene that encodes the tyrosine phosphatase non-receptor type 22, PTPN22, that results in a single amino acid change in the protein, R620W. PTPN22 is expressed in many bone marrow derived cells, including lymphocytes and myeloid derived cells. Although R620W increases the risk of many autoimmune diseases associated with production of autoantibodies, including T1D, rheumatoid arthritis, systemic lupus erythematosus, Graves disease, vitiligo, and others, little is know about how it enhances disease. In part, this is because it functions in many different types of cells that affect the immune system. In order to produce a mouse model that can be used to identify its mechanism of action, we used Crispr/Cas9 genome editing to introduce the mouse ortholog of the pro-autoimmune allele of PTPN22 (619W) into the non- obese diabetes (NOD) mouse that spontaneously develops T1D. NOD mice harboring this mutation demonstrate accelerated production of anti-insulin antibodies and accelerated T1D of higher penetrance, which is the same phenotype exhibited by humans expressing R620W. This proposal will use the 619W NOD model to reveal the mechanism responsible for enhanced disease by pursuing the following specific aims: Aim1 will identify immune cells in which endogenous expression of 619W enhances IAA production and T1D. This entails adoptive transfer experiments to produce mice expressing the 619W mutation of PTPN22 in key immune cells. Aim 2 will explore the mechanistic basis for enhanced IAA and T1D in 619W mice. The types of immune cells identified under Aim 1 will be probed to identify genes affected in their level of expression by allelic difference at 619. Genes and pathways will be identified by either RNAseq or by analyzing gene expression at the single cell level. Candidate genes will be tested for function by altering level of expression with shRNAs delivered by retroviral vectors. Aim 3 will examine tolerance of insulin specific B lymphocytes in R619W mice. Aim 4 will use TCR transgenic mice and single cell expression analysis to examine the fate of islet antigen specific T cells as they become activated in either 619W or 619R expressing mice. Successful completion of these studies will have relevance to development of treatments that may prevent the many different autoimmune disease associated with PTPN22.

项目概要： 1型糖尿病（T1 D）是由于免疫耐受性的破坏， 胰岛的产生胰岛素的β细胞。大量的遗传和环境因素 导致疾病的发生。最强的非MHC遗传风险因素之一是单核苷酸 编码酪氨酸磷酸酶非受体22型（PTPN 22）的基因中存在多态性， 在蛋白质R620 W中的单个氨基酸变化。PTPN 22在许多骨髓来源的细胞中表达。 细胞，包括淋巴细胞和髓源性细胞。虽然R620 W增加了许多风险， 与自身抗体产生相关的自身免疫性疾病，包括T1 D，类风湿性关节炎， 系统性红斑狼疮，格雷夫斯病，白癜风和其他疾病，很少有人知道它如何增强 疾病在某种程度上，这是因为它在影响免疫系统的许多不同类型的细胞中发挥作用。在 为了产生可用于鉴定其作用机制的小鼠模型，我们使用Crispr/Cas9 基因组编辑以将PTPN 22（619 W）的促自身免疫等位基因的小鼠直系同源物引入非自身免疫细胞中。 肥胖糖尿病（NOD）小鼠自发地发展T1 D。携带这种突变的NOD小鼠 证明抗胰岛素抗体的加速产生和更高的胰岛素抵抗率的加速T1 D， 是与表达R620 W的人表现出的表型相同的表型。本提案将使用619 W NOD型号 通过追求以下具体目标，揭示疾病加剧的机制： Aim 1将鉴定其中内源性表达619 W增强IAA产生和T1 D的免疫细胞。 这就需要进行过继转移实验，以产生表达PTPN 22的619 W突变的小鼠，这些突变在小鼠中的关键表达是PTPN 22的619 W突变。 免疫细胞。 目的2探讨619 W小鼠IAA和T1 D增加的机制。免疫细胞的类型 目的1下鉴定的基因将被探索以鉴定受等位基因差异影响其表达水平的基因 在619。基因和途径将通过RNAseq或通过分析单个细胞的基因表达来鉴定。 细胞水平。候选基因将通过改变由载体递送的shRNA的表达水平来测试其功能 逆转录病毒载体。 目的3检测R619 W小鼠胰岛素特异性B淋巴细胞的耐受性。 目的4利用TCR转基因小鼠和单细胞表达分析检测胰岛抗原的命运 特异性T细胞，因为它们在表达619 W或619 R的小鼠中变得活化。 这些研究的成功完成将有助于开发可能预防糖尿病的治疗方法。 与PTPN 22相关的许多不同的自身免疫性疾病。