Stat transcription factors in immunoregulation and autoimmune disease

免疫调节和自身免疫性疾病中的 Stat 转录因子

• 批准号: 8157151
• 负责人: John O'Shea
• 金额: $ 184.96万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157151
• 关键词: Autoimmune Diseases; immunoregulation; transcription factor

Cytokines are secreted proteins that regulate cellular growth and differentiation. These factors are especially important in regulating immune and inflammatory responses, regulating lymphoid development and differentiation. Cytokines also have critical functions in regulating immune homeostasis, tolerance, and memory. Not surprisingly, cytokines are critical in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease and psoriasis. Understanding the molecular basis of cytokine action provides important insights into the pathogenesis of immune-mediated disease and offers new therapeutic targets. The first step in cytokine signaling is activation of receptor associated Janus kinases or Jaks. The next step in signaling is the activation of a family of transcription factors called Stats (signal transducers and activators of transcription). Stat5A and Stat5B are important for development and homeostasis of T cells, B cells and neutrophils. Other STATs, STAT3, STAT4 and STAT6 are important for the differentiation of CD4 helper T cells. T cells orchestrate immune responses through their production of cytokines, shaping the character of the response during host defense and in autoimmune settings. Subsets of helper T (Th) cells include Th1 Th2, Th17 and regulatory T (Treg) cells We found that deficiency of Stat5A/B in CD4+ T cells abrogated Treg development in vivo and blocked conversion of peripheral cells to become iTregs. IL-17 is a major inflammatory cytokine, which appears to contribute to the pathogenesis of many autoimmune and autoinflammatory disorders including rheumatoid arthritis, spondyloarthropathies, multiple sclerosis and inflammatory bowel diseases (IBD). We showed that Stat3 is essential for Th17 differentiation and that Stat3 binds to the Il17 gene. Additionally, through analysis of patients with hyperimmunoglobulin E or Job's syndrome, who have dominant negative mutations of STAT3, we showed that Stat3 function is also essential for human Th17 differentiation. We extended these studies by further investigating the role of Stat3 in a model of inflammatory bowel disease and found that absence of Stat3 prevents the development of IBD, as well as proliferation and survival of CD4+ T cells. Using chromatin immunoprecipitation and massive parallel sequencing, we found that STAT3 bound many genes involved in Th17 differentiation, ranging from cytokines and cytokine receptors to transcription factors and genes that regulate cell proliferation and survival. Interleukin 23 (IL-23) is required for autoimmune inflammation mediated by IL-17-producing helper T cells (Th-17 cells) and has been linked to many human immune disorders including spondyloarthopathy and inflammatory bowel disease. We restricted deficiency in the IL-23 receptor to defined cell populations in vivo to investigate the requirement for IL-23 signaling in the development and function of T(H)-17 cells in autoimmunity, inflammation and infection. In the absence of IL-23, Th-17 development was stalled at the early activation stage. This was associated with less proliferation; consequently, fewer effector Th-17 cells were produced in the lymph nodes and hence available to emigrate to the bloodstream and tissues Behet's disease is a disease of unknown etiology characterized by recurrent inflammatory attacks affecting the orogenital mucosa, eyes and skin. We collaborated to perform a genome-wide association study on individuals with Behet's disease. We identified an association with polymorphisms of the IL10 and the IL23R genes. The disease-associated IL10 variant was associated with diminished expression. Another factor that regulates IL-17 production is IL-1, which is regulated by the inflammasome. Mutations of one component of the inflammasome, NLRP3, underlie the diseases Neonatal Onset Multisystem Disease, Muckle-Wells and Familial Cold Autoinflammatory syndrome. (cryopyrinopathies). In effort to generate a mouse model of these cryopyrinopathy disorders, mice with mutations of NLRP3 were created. Like patients with mice with NLRP3 mutations have severe, overwhelming inflammatory disease. This disease is characterized by exagerated Th17 differentiation; however, the disease also occurs in the absence of adaptive immune cells. In a separate study, we also searched for sources of IL-17 and IL-22 in innate immune cells and found that lymphoid tissue inducer cells are important producers of these cytokines. To better understand the process of helper T cell differentiation, we have also initiated studies mapping genomewide chromatin modifications, examining the role of Stats in the process. Specifically, we analyzed histone modifications in Th1, Th2, Th17, and Treg cells by Chip-Seq technology. We have also assessed the importance of Stat4 and STAT6 in chromatin modifications in Th1 and Th2 cells Using chromatin immunoprecipitation and massive parallel sequencing, we quantitated the full complement of STAT-bound genes, concurrently assessing global STAT-dependent epigenetic modifications and gene transcription by using cells from cognate STAT-deficient mice. STAT4 and STAT6 each bound over 4000 genes with distinct binding motifs. Both played critical roles in maintaining chromatin configuration and transcription of a core subset of genes through the combination of different epigenetic patterns. Globally, STAT4 had a more dominant role in promoting active epigenetic marks, whereas STAT6 had a more prominent role in antagonizing repressive marks. Clusters of genes negatively regulated by STATs were also identified, highlighting previously unappreciated repressive roles of STATs. Therefore, STAT4 and STAT6 play wide regulatory roles in T helper cell specification. In related work, we applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. In another study, we collaborated to show that deletion of the kinase Lyn in myeloid cells resulted in enhanced Th2 differentiation that was driven by increased IL-4 production by basophils. Neutrophils play a vital role in the immune defense, as evidenced by the severity of neutropenia causing life-threatening infections. Granulocyte macrophage-colony stimulating factor (GM-CSF) controls homeostatic and emergency development of granulocytes. However, little is known about the contribution of the downstream mediating transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A/B). To elucidate the function of this pathway, we generated mice with complete deletion of both Stat5a/b genes in hematopoietic cells. In homeostasis, peripheral neutrophils were markedly decreased in these animals. Moreover, during emergency situations, such as myelosuppression, Stat5a/b-mutant mice failed to produce enhanced levels of neutrophils and were unable to respond to GM-CSF. To investigate the target genes of STAT5A/B activated by cytokines in HSCs and progenitors, we performed microarray analyses using Lineage- Sca-1+ c-Kit+ (KSL) cells in the presence and absence of STAT5A/B. CCN3/NOV is a positive regulator of human HSC self-renewal and development of committed blood cells. Without stimulation, the Ccn3/Nov signal level was low in control KSL cells similar to Stat5a/b-null KSL cells. To determine which cytokine activates the Ccn3/Nov gene we analyzed Lineage- c-Kit+ (KL) and 32D cells using qPCR and ChIP assays. While stimulation with a cocktail lacking IL-3 prevented the induction of Ccn3/Nov in control KL cells, IL-3 alone could induce Ccn3/Nov mRNA in control KL and 32D cells. ChIP assays using 32D cells revealed IL-3-induced binding of STAT5A/B to a GAS site in the Ccn3/Nov gene promoter.

细胞因子是调节细胞生长和分化的分泌蛋白。这些因子对于调节免疫和炎症反应、调节淋巴系统的发育和分化尤其重要。细胞因子在调节免疫稳态、耐受性和记忆方面也具有重要功能。毫不奇怪，细胞因子在类风湿性关节炎、系统性红斑狼疮、炎症性肠病和牛皮癣等自身免疫性疾病的发病机制中至关重要。了解细胞因子作用的分子基础为了解免疫介导疾病的发病机制提供了重要的见解，并提供了新的治疗靶点。 细胞因子信号传导的第一步是激活受体相关的 Janus 激酶或 Jaks。信号转导的下一步是激活称为 Stats（信号转导子和转录激活子）的转录因子家族。 Stat5A 和 Stat5B 对于 T 细胞、B 细胞和中性粒细胞的发育和稳态很重要。 其他 STAT、STAT3、STAT4 和 STAT6 对于 CD4 辅助 T 细胞的分化很重要。 T 细胞通过产生细胞因子来协调免疫反应，从而塑造宿主防御和自身免疫环境中反应的特征。辅助性 T (Th) 细胞亚群包括 Th1、Th2、Th17 和调节性 T (Treg) 细胞。我们发现 CD4+ T 细胞中 Stat5A/B 的缺乏会破坏体内 Treg 的发育，并阻止外周细胞转化为 iTreg。 IL-17 是一种主要的炎症细胞因子，它似乎与许多自身免疫和自身炎症性疾病的发病机制有关，包括类风湿性关节炎、脊柱关节病、多发性硬化症和炎症性肠病 (IBD)。我们表明 Stat3 对于 Th17 分化至关重要，并且 Stat3 与 Il17 基因结合。此外，通过对具有 STAT3 显性失活突变的高免疫球蛋白 E 或乔布氏综合症患者的分析，我们发现 Stat3 功能对于人类 Th17 分化也至关重要。我们通过进一步研究 Stat3 在炎症性肠病模型中的作用来扩展这些研究，发现 Stat3 的缺失可以阻止 IBD 的发展，以及 CD4+ T 细胞的增殖和存活。使用染色质免疫沉淀和大规模并行测序，我们发现 STAT3 结合了许多参与 Th17 分化的基因，从细胞因子和细胞因子受体到转录因子和调节细胞增殖和存活的基因。 白细胞介素 23 (IL-23) 是由产生 IL-17 的辅助 T 细胞（Th-17 细胞）介导的自身免疫炎症所必需的，并且与许多人类免疫疾病有关，包括脊柱关节病和炎症性肠病。 我们将 IL-23 受体的缺陷限制在体内特定的细胞群中，以研究自身免疫、炎症和感染中 T(H)-17 细胞的发育和功能对 IL-23 信号传导的需求。在缺乏 IL-23 的情况下，Th-17 的发育在早期激活阶段停滞。这与较少的增殖有关；因此，淋巴结中产生的效应 Th-17 细胞较少，因此可迁移到血流和组织中 白塞氏病是一种病因不明的疾病，其特征是影响口生殖粘膜、眼睛和皮肤的反复炎症发作。我们合作对患有白塞氏病的个体进行了全基因组关联研究。我们确定了与 IL10 和 IL23R 基因多态性的关联。与疾病相关的 IL10 变异与表达减少有关。 调节 IL-17 产生的另一个因素是 IL-1，它受炎症小体调节。 炎症小体的一种成分 NLRP3 的突变是新生儿发病多系统疾病、Muckle-Wells 疾病和家族性感冒自身炎症综合征的基础。 （冷吡啶病）。 为了建立这些冷热蛋白病的小鼠模型，我们创建了带有 NLRP3 突变的小鼠。就像 NLRP3 突变小鼠的患者患有严重的、难以忍受的炎症性疾病一样。该疾病的特点是 Th17 过度分化；然而，这种疾病也会在缺乏适应性免疫细胞的情况下发生。在另一项研究中，我们还在先天免疫细胞中寻找 IL-17 和 IL-22 的来源，发现淋巴组织诱导细胞是这些细胞因子的重要产生者。 为了更好地了解辅助 T 细胞分化的过程，我们还启动了绘制全基因组染色质修饰图谱的研究，研究 Stats 在该过程中的作用。具体来说，我们通过芯片测序技术分析了 Th1、Th2、Th17 和 Treg 细胞中的组蛋白修饰。我们还评估了 Stat4 和 STAT6 在 Th1 和 Th2 细胞染色质修饰中的重要性。使用染色质免疫沉淀和大规模并行测序，我们定量了 STAT 结合基因的全部互补体，同时通过使用以下方法评估了全局 STAT 依赖性表观遗传修饰和基因转录：来自同源 STAT 缺陷小鼠的细胞。 STAT4 和 STAT6 各自结合超过 4000 个具有不同结合基序的基因。两者通过不同表观遗传模式的组合，在维持染色质构型和核心基因子集转录方面发挥着关键作用。在全球范围内，STAT4在促进活跃的表观遗传标记方面具有更显着的作用，而STAT6在拮抗抑制标记方面具有更突出的作用。还鉴定了 STAT 负调控的基因簇，强调了 STAT 先前未被认识到的抑制作用。因此，STAT4和STAT6在T辅助细胞规范中发挥广泛的调节作用。 在相关工作中，我们应用 miRNA、mRNA 和 ChIP-Seq 在转录组和表观基因组背景下表征淋巴细胞生成过程中的 microRNome。在另一项研究中，我们合作证明，骨髓细胞中激酶 Lyn 的缺失会导致 Th2 分化增强，这是由嗜碱性粒细胞产生的 IL-4 增加驱动的。 中性粒细胞在免疫防御中发挥着至关重要的作用，中性粒细胞减少症的严重性导致危及生命的感染就证明了这一点。粒细胞巨噬细胞集落刺激因子（GM-CSF）控制粒细胞的稳态和紧急发育。然而，人们对下游介导转录因子信号转导子和转录激活子 5A 和 5B (STAT5A/B) 的贡献知之甚少。为了阐明该途径的功能，我们培育了造血细胞中 Stat5a/b 基因完全缺失的小鼠。在体内平衡中，这些动物的外周中性粒细胞显着减少。此外，在紧急情况下，例如骨髓抑制，Stat5a/b 突变小鼠无法产生较高水平的中性粒细胞，并且无法对 GM-CSF 做出反应。为了研究 HSC 和祖细胞中细胞因子激活的 STAT5A/B 靶基因，我们在存在和不存在 STAT5A/B 的情况下使用 Lineage-Sca-1+ c-Kit+ (KSL) 细胞进行微阵列分析。 CCN3/NOV 是人类 HSC 自我更新和定向血细胞发育的正调节因子。在没有刺激的情况下，对照 KSL 细胞中的 Ccn3/Nov 信号水平与 Stat5a/b 缺失的 KSL 细胞相似。为了确定哪种细胞因子激活 Ccn3/Nov 基因，我们使用 qPCR 和 ChIP 检测分析了 Lineage-c-Kit+ (KL) 和 32D 细胞。虽然用缺乏 IL-3 的混合物刺激可阻止对照 KL 细胞中 Ccn3/Nov 的诱导，但单独的 IL-3 可以在对照 KL 和 32D 细胞中诱导 Ccn3/Nov mRNA。使用 32D 细胞的 ChIP 测定揭示了 IL-3 诱导的 STAT5A/B 与 Ccn3/Nov 基因启动子中的 GAS 位点的结合。