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

说明(申请人提供)：白介素13(IL-13)是一种多效性抗炎细胞因子，主要由活化的Th2T细胞产生。我们最近克隆了一种自然产生的IL-13的mRNA剪接变异体，其中第二外显子被省略(称为IL-13E2)。我们对这种剪接变异体的特征表明，IL-13E2和IL-13在表达和功能上形成了鲜明的对比。IL-13E2主要在炎症(Th1和Th17)反应中表达，而IL-13主要在Th2免疫反应中表达。IL-13E2仅由激活的B细胞产生，而IL-13的主要细胞来源是激活的Th2T细胞。无论是在体外还是在体内，使用IL-13E2治疗都会导致炎症介质TNF-1和IL-12的增加。另一方面，IL-13表达的净影响是这些相同的炎症介质的分泌减少。最后，我们还表明，在缺乏IL-13E2表达的小鼠中，对实验性自身免疫性脑脊髓炎(EAE)的诱导具有高度的抵抗力。鉴于IL-13E2的表达模式，其对炎性细胞因子表达的调控能力，以及缺乏IL-13E2会损害EAE的发展，我们假设IL-13E2是一种参与炎性免疫反应的独特细胞因子，包括脑源性T细胞的启动。鉴于这种新的细胞因子的发现，我们建议进一步证明IL-13E2在导致EAE的免疫反应中所起的不可或缺的作用。我们还建议研究IL-13E2在体外和体内促进EAE的机制，比较在有或没有IL-13E2的情况下细胞因子环境以及Th1和Th17细胞生成的差异。本研究的具体目的是：特异性目标1：IL-13E2在体外和体内是否增强Th1和Th17细胞的诱导作用？具体目标2：IL-13E2是否在EAE的始发阶段和效应阶段发挥作用？具体目标3：B细胞是否仅通过分泌IL-13E2来增强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被普遍认为是研究多发性硬化症免疫病理的合适动物模型。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。在慢性复发性疾病的发展过程中，小鼠EAE也与MS相似。EAE和MS发病的基本机制尚不清楚。本研究旨在研究一种新的细胞因子剪接变异体及其在EAE发病机制中的作用。从这项研究中获得的知识应该为MS提供可能的治疗方法。IL-13是一种Th2型细胞因子，由激活的T细胞产生，对B细胞、单核细胞、巨噬细胞和树突状细胞具有抗炎功能。我们最近克隆了一种自然产生的IL-13的mRNA剪接变异体，其中省略了第二外显子(称为IL-13 E2或简称E2)。我们已经建立了特异的RT-PCR方法来定量检测IL-13和E2基因的表达。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，与其全长对应蛋白不同，E2主要在炎症条件下表达。在用完全弗氏佐剂(CFA)免疫的小鼠的淋巴结中，表达的IL-13的主要形式是E2。完全弗氏佐剂是一种诱导Th1反应的佐剂。我们发现，在培养的抗原提呈细胞(巨噬细胞或树突状细胞)中加入外源性E2会引起显著的表型变化。这些变化包括细胞形态的改变和激活后炎性细胞因子的分泌增加。最值得注意的是，我们已经证明，缺乏E2表达的小鼠对实验性自身免疫性脑脊髓炎(EAE)的诱导具有高度的抵抗力。鉴于E2的表达模式，其对炎性细胞因子表达的调控能力，以及缺乏E2会损害EAE的发展，我们假设E2是一种参与炎性免疫反应的独特细胞因子，包括脑源性T细胞的启动。我们认为，在其他研究中，对Th1T细胞细胞因子表达的分析可能会错误地将E2识别为IL-13，因为大多数常用的测量IL-13的方法不区分剪接变体。鉴于这种新的细胞因子的发现，我们建议进一步证明E2在导致EAE的免疫反应中所起的不可或缺的作用。我们还建议在体外和体内研究E2促进EAE的机制，比较在有或没有E2的情况下细胞因子环境和Th1、Th2和Th17细胞的产生的差异。此外，我们建议在体内通过选择性地阻断E2的作用来确定是否可以抑制EAE的发展。

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.