A novel splice variant of interleukin-13 and its role in promoting EAE responses

IL-13 的新型剪接变体及其在促进 EAE 反应中的作用

• 批准号: 7660224
• 负责人: MICHAEL K. SHAW
• 金额: $ 19万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-19 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7660224
• 关键词: Adjuvant; American; Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Antigen-Presenting Cells; Autoimmune Diseases; B-Lymphocytes; Biological Assay; Cells; Cellular Morphology; Characteristics; Chronic; Clinical; Dendritic Cells; Development; Disease; Employee Strikes; Encephalomyelitis; Exhibits; Exons; Experimental Autoimmune Encephalomyelitis; Freund' s Adjuvant; Gene Expression; Generations; Hand; Histopathology; Human; Immune response; In Vitro; Inflammation Mediators; Inflammatory; Interleukin-12; Interleukin-13; Knowledge; Length; Lesion; Measures; Messenger RNA; Methods; Multiple Sclerosis; Mus; Pathogenesis; Pattern; Phase; Proteins; RNA Splicing; Relapse; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Source; T-Lymphocyte; Therapeutic; Variant; cytokine; immunopathology; in vivo; insight; lymph nodes; macrophage; monocyte; novel; public health relevance; response

DESCRIPTION (provided by applicant): Interleukin 13 (IL-13) is a pleiotropic, anti-inflammatory cytokine produced mainly by activated Th2 T cells. We have recently cloned a naturally occurring mRNA splice variant of IL-13 in which the second exon is omitted (termed IL-13 E2). Our characterization of this splice variant indicates sharp contrasts in the expression and function of IL-13 E2 and IL-13. IL-13 E2 is expressed mainly during inflammatory (Th1 and Th17) responses, while IL-13 is expressed primarily during Th2 immune responses. IL-13 E2 is produced exclusively by activated B cells, while the main cellular source of IL-13 is activated Th2 T cells. Treatment with IL-13 E2, in vitro or in vivo, results in increases in the inflammatory mediators TNF-1 and IL-12. On the other hand, the net effect of IL-13 expression is a decrease in the secretion of these same inflammatory mediators. Lastly, we have also shown that in mice lacking IL-13 E2 expression is highly resistant to induction of experimental autoimmune encephalomyelitis (EAE). Given the expression pattern of IL-13 E2, its ability to modulate the expression of inflammatory cytokines, and the demonstration that lack of IL-13 E2 impairs the development of EAE, we hypothesis that IL-13 E2 is a unique cytokine involved in inflammatory immune responses, including the priming of encephalitogenic T cells. Given the discovery of this novel cytokine, we propose to further demonstrate the indispensable role of IL-13 E2 in the immune responses which cause (EAE). We also propose to investigate the mechanism by which IL-13 E2 promotes EAE in vitro and in vivo, comparing differences in the cytokine milieu and the generation of Th1 and Th17 cells in the presence or absence of IL-13 E2. The specific aims of this study are: Specific Aim 1: Does IL-13 E2 augment the induction of Th1 and Th17 cells in vitro and in vivo? Specific Aim 2: Does IL-13 E2 exert effects during priming and during the effector phase of EAE? Specific Aim 3: Do B cells augment EAE solely by secreting IL-13 E2? Public Health Relevance: Multiple sclerosis is a human autoimmune disease which afflicts more than 400,000 Americans. EAE is commonly accepted as a suitable animal model for studying the immunopathology of multiple sclerosis. These two diseases share many common characteristics such as clinical manifestations and histopathology of CNS lesions. Murine EAE also resembles MS in the development of the chronic relapsing form of the disease. The basic mechanisms of disease initiation in EAE and MS are still unknown. This proposal seeks to characterize a novel cytokine splice variant and its role in the pathogenesis of EAE. Knowledge obtained from this study should provide insight to possible therapeutics for MS. Interleukin 13 is a Th2 type cytokine produced by activated T cells which exhibits anti-inflammatory function on B cells, monocytes, macrophages and dendritic cells. We have recently cloned a naturally occurring mRNA splice variant of IL-13 in which the second exon is omitted (termed IL-13 E2 or simply E2). We have developed specific RT-PCR assays for quantitation of IL-13 and E2 gene expression. We have also cloned and purified each of the molecules as HIS-tagged proteins for use in in vivo and in vitro studies. We find that unlike its full length counterpart, E2 is expressed predominantly under inflammatory conditions. E2 is the predominant form of IL-13 expressed in the lymph nodes of mice immunized using complete Freund's adjuvant (CFA), an adjuvant which induced Th1 responses. We find that addition of exogenous E2 to cultured antigen presenting cells (either macrophages or dendritic cells) induces striking phenotypic changes. These changes include transformation in cell morphology and increases in inflammatory cytokine secretion after activation. Most notably we have shown that mice lacking E2 expression are highly resistant to induction of experimental autoimmune encephalomyelitis (EAE). Given the expression pattern of E2, its ability to modulate the expression of inflammatory cytokines, and the demonstration that lack of E2 impairs the development of EAE, we hypothesis that E2 is a unique cytokine involved in inflammatory immune responses, including the priming of encephalitogenic T cells. We suggest that analysis of cytokine expression by Th1 T cells in other studies may mistakenly identify E2 as IL- 13, since most methods commonly used to measure IL-13 do not discriminate between the splice variants. Given the discovery of this novel cytokine, we propose to further demonstrate the indispensable role of E2 in the immune responses which cause (EAE). We also propose to investigate the mechanism by which E2 promotes EAE in vitro and in vivo, comparing differences in the cytokine milieu and the generation of Th1, Th2 and Th17 cells in the presence or absence of E2. Furthermore, we propose to determine if the development of EAE can be inhibited by selectively blocking the action of E2 in vivo.

描述（由申请人提供）：白细胞介素13（IL-13）是一种多效性抗炎细胞因子，主要由活化的Th 2 T细胞产生。我们最近克隆了IL-13的天然mRNA剪接变体，其中第二个外显子被省略（称为IL-13 E2）。我们对这种剪接变体的表征表明IL-13 E2和IL-13的表达和功能存在鲜明的对比。IL-13 E2主要在炎症（Th 1和Th 17）应答期间表达，而IL-13主要在Th 2免疫应答期间表达。IL-13 E2仅由活化的B细胞产生，而IL-13的主要细胞来源是活化的Th 2 T细胞。在体外或体内用IL-13 E2治疗导致炎性介质TNF-1和IL-12增加。另一方面，IL-13表达的净效应是这些相同炎症介质的分泌减少。最后，我们还表明，在缺乏IL-13表达的小鼠中，E2对诱导实验性自身免疫性脑脊髓炎（EAE）具有高度抗性。鉴于IL-13 E2的表达模式，其调节炎性细胞因子表达的能力，以及缺乏IL-13 E2会损害EAE发展的证据，我们假设IL-13 E2是参与炎性免疫应答的独特细胞因子，包括致脑炎性T细胞的引发。鉴于这种新型细胞因子的发现，我们建议进一步证明IL-13 E2在引起（EAE）的免疫应答中不可或缺的作用。我们还建议研究IL-13 E2在体外和体内促进EAE的机制，比较IL-13 E2存在或不存在时细胞因子环境和Th 1和Th 17细胞生成的差异。本研究的具体目的是：具体目的1：IL-13 E2是否在体外和体内增强Th 1和Th 17细胞的诱导？具体目标2：IL-13 E2是否在EAE的引发期和效应期发挥作用？特异性目的3：B细胞是否仅通过分泌IL-13 E2而增强EAE？ 公共卫生相关性：多发性硬化症是一种人类自身免疫性疾病，困扰着40多万美国人。EAE通常被认为是研究多发性硬化免疫病理学的合适动物模型。这两种疾病有许多共同的特点，如临床表现和中枢神经系统病变的组织病理学。鼠EAE在疾病的慢性复发形式的发展方面也类似于MS。EAE和MS发病的基本机制尚不清楚。该建议旨在描述一种新的细胞因子剪接变体及其在EAE发病机制中的作用。从这项研究中获得的知识应该为MS的可能治疗提供见解。白细胞介素13是由活化的T细胞产生的Th 2型细胞因子，其对B细胞、单核细胞、巨噬细胞和树突状细胞表现出抗炎功能。我们最近克隆了一种天然存在的IL-13 mRNA剪接变体，其中第二个外显子被省略（称为IL-13 E2或简称E2）。我们已经开发了特异性RT-PCR检测IL-13和E2基因表达的定量。我们还克隆和纯化了每个分子作为HIS标记的蛋白质用于体内和体外研究。我们发现，与其全长对应物不同，E2主要在炎症条件下表达。 E2是使用完全弗氏佐剂（CFA）免疫的小鼠的淋巴结中表达的IL-13的主要形式，CFA是诱导Th 1应答的佐剂。我们发现，除了外源性E2培养的抗原呈递细胞（无论是巨噬细胞或树突状细胞）诱导显着的表型变化。这些变化包括细胞形态的转化和活化后炎性细胞因子分泌的增加。最值得注意的是，我们已经表明，缺乏E2表达的小鼠对实验性自身免疫性脑脊髓炎（EAE）的诱导具有高度抗性。鉴于E2的表达模式，其调节炎性细胞因子表达的能力，以及缺乏E2会损害EAE发展的证据，我们假设E2是一种独特的细胞因子，参与炎症免疫反应，包括致脑炎性T细胞的启动。我们认为，在其他研究中，Th 1 T细胞的细胞因子表达分析可能会错误地将E2识别为IL- 13，因为大多数通常用于测量IL-13的方法不能区分剪接变体。鉴于这种新型细胞因子的发现，我们建议进一步证明E2在引起（EAE）的免疫反应中不可或缺的作用。我们还建议调查E2促进EAE在体外和体内的机制，比较差异的细胞因子环境和产生的Th 1，Th 2和Th 17细胞在E2的存在或不存在。此外，我们建议确定是否可以通过选择性阻断E2在体内的作用来抑制EAE的发展。