The role of Fli1 in T cell function and the pathogenesis of lupus

Fli1在T细胞功能和狼疮发病机制中的作用

• 批准号: 7685574
• 负责人: TAMMY (TAMARA) NOWLING
• 金额: --
• 依托单位: RALPH H JOHNSON VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7685574
• 关键词: Affect; Affinity; African American; Apoptosis; Autoimmune Diseases; B-Lymphocytes; Binding; Biological Assay; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; Caring; Caucasians; Caucasoid Race; Cell Count; Cell Proliferation; Cell physiology; Co-Immunoprecipitations; Coculture Techniques; Cytotoxic T-Lymphocytes; DNA; Data; Development; Disease; Disease Progression; Down-Regulation; Exhibits; Exposure to; Female; Flow Cytometry; Functional disorder; Genetic Transcription; Goals; Health; Health Care Costs; Healthcare; Hispanics; Human; Immune; Immune system; In Vitro; Individual; Inflammatory; Intervention; Knowledge; Laboratories; Lead; Lupus; Lymphocyte; Lymphocyte Activation; Lymphocyte Function; Methods; Military Personnel; Minority; Modeling; Modification; Morbidity - disease rate; Mouse Strains; Mus; Pathogenesis; Patients; Phenotype; Play; Population; Post-Translational Protein Processing; Protein Binding; Proteins; Quality of life; Regulation; Regulatory Pathway; Resources; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; Screening procedure; Severities; Stimulus; System; T-Cell Activation; T-Cell Proliferation; T-Lymphocyte; Testing; Time; Transcription factor genes; Transgenic Mice; Translating; Translations; Veterans; Western Blotting; Woman; base; chromatin immunoprecipitation; human disease; improved; in vivo; insight; lupus prone mice; lupus-like; men; mortality; mouse model; neoplastic; novel therapeutic intervention; overexpression; promoter; public health relevance; systemic autoimmune disease; tool; transcription factor

DESCRIPTION (provided by applicant): The Ets transcription factor Fli1 clearly has effects on the immune system and the pathogenesis of lupus as evidenced by the immune phenotype of Fli1 transgenic mice and the profound effect of Fli1 heterozygosity on a mouse model of lupus. Based on these studies and our preliminary data, we hypothesize that Fli1 fails to be down-regulated following activation of T cells from lupus prone mice, due mechanistically to the loss of binding of negative regulators to the Fli1 promoter, contributing to the over-expression of Fli1 in lupus T cells with specific impact on cell number and regulatory function. We propose to study cell proliferation, apoptosis and suppressor and effector functions of T cells in mice expressing reduced Fli1 activity and to identify the mechanisms involved in the down-regulation of Fli1 expression upon T cell activation that are dysfunctional in lupus. Specific Aims include: 1) Demonstrate that reduced Fli1 activity increases CD8+ T cell proliferation and regulatory function and 2) Identify the mechanisms controlling down-regulation of Fli1 in activated T cells and identify in MRL/lpr lupus T cells which of these mechanisms is dysfunctional. The effects of reduced Fli1 expression on T cell number and function will be determined before and after exposure to inflammatory stimuli in lymphocyte co-cultures from lupus prone and control mice expressing a Fli1 protein that exhibits reduced Fli1 activity compared to their wild-type counterparts. Cell proliferation and apoptosis will be analyzed by standard flow cytometry assays. T suppressor and cytotoxic T lymphocyte (CTL) function will be analyzed by standard suppression and CTL assays, respectively. Mechanisms involved in the down-regulation of Fli1 in activated T cells in lupus prone and control mice will be analyzed by identifying proteins binding the endogenous Fli1 promoter using chromatin immunoprecipitation (IP) and analyses of expression levels, post-translational modifications and interactions with co-factors of the proteins that control the Fli1 promoter by western immunoblots, real-time RTPCR, co-IP and DNA affinity assays in T cells. The proposed studies will directly test our hypotheses and add valuable knowledge in the pathogenesis of lupus and immune regulation in general. Most importantly, it will provide the necessary groundwork for our long-term goal of identifying targets in the Fli1 regulatory pathway and/or methods of down-regulating Fli1 transcription for translation into human lupus studies leading to possible treatments for lupus patients. Lupus predominantly afflicts women and African Americans/Hispanics. As the population of females and minorities in the military continues to rise, the number of individuals with lupus in the VA system will significantly increase. The proposed studies will eventually lead to identifying earlier screening methods and interventions for the treatment of this disease; thus relieving the burden of increased health care costs for these patients and improving their quality of life. PUBLIC HEALTH RELEVANCE: The Ets factor Fli1 has been implicated as a key modulator of lupus disease pathogenesis. Increasing the levels of Fli1 in normal mice results in the development of a lupus-like disease. Fli1 is over- expressed in two lupus prone mouse strains and in lupus patients. The expression of Fli1 in lupus patients correlated with disease activity. Importantly, genetically lowering the levels of Fli1 in two lupus prone mouse strains by 50% significantly improved disease and prolonged survival. Preliminary data generated in our laboratory indicates that Fli1 transcription fails to be turned off when T cells are activated due to dysregulation of transcription in a lupus prone mouse model. We hypothesize that Fli1 fails to be down- regulated upon activation of T cells from lupus prone mice, due mechanistically to the loss of binding of negative regulators to the Fli1 promoter, contributing to the over-expression of Fli1 in lupus T cells with specific impact on T cell number and regulatory function. We propose to study the function of T cells in mice expressing reduced Fli1 activity and to identify the mechanisms involved in the down-regulation of Fli1 expression upon T cell activation that are dysfunctional in lupus. Specific Aims include: 1) Demonstrate that reduced Fli1 activity increases T cell proliferation and regulatory function and 2) Identify the mechanisms controlling down-regulation of Fli1 in activated T cells and identify in lupus prone T cells which of these mechanisms is dysfunctional. Fli1 expression clearly influences the pathogenesis of lupus and warrants further study. Little is known about the role of Fli1 in the function of T cells or how its expression is regulated in the immune system. These studies will provide the necessary groundwork for our long-term goal of identifying targets in the Fli1 regulatory pathway for translation into human lupus studies leading to possible treatments for lupus patients. Lupus afflicts women 10 times more frequently than men. It also is 4-5 times more common in African Americans and Hispanics than Caucasians, and morbidity and mortality are greater in African Americans/Hispanics with lupus. As the population of females and minorities in the military continues to rise, the number of individuals with lupus in the VA system will significantly increase. There are already thousands of veterans with lupus being cared for in the VA system. Due to its severity, patients with lupus use a disproportionate amount of health care dollars including VA resources. Our proposed studies on lupus will lead to a better understanding of the pathogenesis of lupus and the factors involved and could lead to the development of better treatments/therapies as well as potential screening tools for individuals at risk for developing lupus. Additionally, insight will be gained into the regulation of T cell function in both health and disease with relevance to an array of immune and neoplastic diseases.

描述（由申请人提供）： ETS转录因子FLI1显然对FLI1转基因小鼠的免疫表型和FLI1杂合性对小鼠狼疮模型的深远影响证明了对免疫系统和狼疮的发病机理的影响。基于这些研究和我们的初步数据，我们假设FLI1在狼pla的小鼠的T细胞激活后无法下调，这是由于负调节剂与FLI1启动子的结合失去了结合，导致FLI1对卢普斯T细胞过表达的卢普斯T细胞的结合丧失，对细胞数量和调节型的特异性影响都导致了FLI1的过表达。我们建议研究表达降低FLI1活性的小鼠中T细胞的细胞增殖，凋亡以及抑制剂以及效应子功能，并确定在狼疮中T细胞激活后FLI1表达下调的机制。具体目的包括：1）证明FLI1活性的降低会增加CD8+ T细胞的增殖和调节功能，2）确定控制活化T细胞中FLI1下调的机制，并在MRL/LPR狼疮T细胞中识别这些机制中的其中一种功能障碍。 FLI1表达降低对T细胞数量和功能的影响将在暴露于容易发生狼疮的淋巴细胞共培养和表达FLI1蛋白的对照小鼠的淋巴细胞培养物中和之后确定，与其野生型相比，该蛋白表现出降低的FLI1蛋白。细胞增殖和凋亡将通过标准流式细胞仪测定法分析。 T抑制和细胞毒性T淋巴细胞（CTL）功能将分别通过标准抑制和CTL分析分析。 Mechanisms involved in the down-regulation of Fli1 in activated T cells in lupus prone and control mice will be analyzed by identifying proteins binding the endogenous Fli1 promoter using chromatin immunoprecipitation (IP) and analyses of expression levels, post-translational modifications and interactions with co-factors of the proteins that control the Fli1 promoter by western immunoblots, real-time RTPCR, T细胞中的co-IP和DNA亲和力测定。 拟议的研究将直接检验我们的假设，并在狼疮和免疫调节的发病机理中增加有价值的知识。最重要的是，它将为我们的长期目标提供必要的基础，即在FLI1调节途径和/或下调节FLI1转录的方法中识别靶标，以转化为人类狼疮研究，从而为狼疮患者提供治疗。狼疮主要困扰着妇女和非裔美国人/西班牙裔。随着军人中女性和少数民族的人口继续增加，VA系统中狼疮的人数将大大增加。拟议的研究最终将导致确定早期的筛查方法和治疗该疾病的干预措施。从而减轻了这些患者增加医疗保健成本的负担并改善了他们的生活质量。 公共卫生相关性： ETS因子FLI1已被认为是狼疮疾病发病机理的关键调节剂。正常小鼠的FLI1水平增加导致狼疮样疾病的发展。 FLI1在两只容易发生的小鼠菌株和狼疮患者中过度表达。狼疮患者中FLI1的表达与疾病活性相关。重要的是，从遗传上降低两只狼疮小鼠菌株中的FLI1水平可显着改善疾病并长期生存。我们实验室中产生的初步数据表明，由于狼疮小鼠模型中转录的失调，由于转录失调而激活T细胞时，FLI1转录无法关闭。我们假设FLI1因狼疮俯卧小鼠的T细胞的激活而无法受到调节，这是由于负调节剂与FLI1启动子的结合丧失，导致狼疮T细胞中FLI1在T细胞中的过表达对T细胞数和调节功能的特异性影响。我们建议研究表达降低FLI1活性的小鼠中T细胞的功能，并确定狼疮中T细胞激活后FLI1表达下调的机制。具体目的包括：1）证明FLI1活性的降低会增加T细胞增殖和调节功能，2）确定控制激活的T细胞中FLI1下调的机制，并在狼疮俯卧的T细胞中识别这些机制功能障碍。 FLI1表达清楚地影响了狼疮的发病机理，并保证进一步研究。关于FLI1在T细胞功能或其在免疫系统中如何调节其表达的作用知之甚少。这些研究将为我们的长期目标提供必要的基础，即确定FLI1调节途径中的靶标，以转化为人类狼疮研究，从而为狼疮患者提供可能的治疗方法。狼疮比男性遭受的频率高10倍。在非洲裔美国人和西班牙裔人中，这也比高加索人高4-5倍，而在非洲裔美国人/西班牙裔狼疮中，发病率和死亡率更高。随着军人中女性和少数民族的人口继续增加，VA系统中狼疮的人数将大大增加。在VA系统中，已经有成千上万的退伍军人受到狼疮的照顾。由于其严重程度，狼疮患者使用了包括VA资源在内的不成比例的医疗保健资金。我们提出的关于狼疮的研究将使人们更好地了解狼疮的发病机理以及所涉及的因素，并可能导致更好的治疗方法/疗法的发展以及为有狼疮风险的个体的潜在筛查工具。此外，与一系列免疫和肿瘤疾病有关的健康和疾病中T细胞功能的调节将获得洞察力。