Suppression of pathogenic autoantibodies in lupus by inhibition of AID

描述（由申请人提供）：在拟定的研究中，我们将检验以下假设：IL-17通过上调G蛋白信号传导（RGS）蛋白的调节剂增强自身反应性生发中心（GC）形成，增强的GC T细胞和B细胞相互作用导致激活诱导的胞苷脱氨酶（AID）上调和高致病性自身抗体的产生。这些研究的意义在于确定IL-17和自身抗体产生之间的机制联系，以及区分自身抗体和炎症反应在类风湿性关节炎发展中的作用的能力。BXD 2小鼠发生自发性糜烂性关节炎和全身性自身免疫病。这些小鼠的一个独特的免疫特征是大量的Th 17 CD 4 + T细胞，它们在脾脏中自发活化。我们的数据表明，IL-17促进脾脏中B细胞和CD 4 + T细胞的相互作用以及自发性GC的形成。IL-17的这种作用是由RGS 13和RGS 16的上调介导的。这些基因的上调降低而不是增加了GC B和T细胞上表达的CXCR 4和CXCR 5趋化因子受体的信号传导。在本申请中，我们将解决以下问题。(1)IL-17是否诱导自身反应性GC的形成和功能？IL- 17的作用将根据其对边缘和滤泡B细胞区之间B细胞迁移、AID的B细胞表达和致病性自身抗体的产生的调节来确定。将通过共聚焦显微镜和FACS分析来分析GC形成。(2)IL-17是否通过上调RGS蛋白来调节GC的形成？IL-17调节趋化性的能力将在体外使用Transwell室、流动室实时成像分析来确定，并且在体内通过GFP+ B细胞或从靶向敲除小鼠获得的B细胞在BXD 2小鼠中的归巢分析来确定，其中在GFP+ B细胞中IL-17 R或Rgs基因已经使用shRNA技术来调节。(3)IL-17驱动的自身反应性GC的形成是否独立于IL-17驱动的炎症反应而促进CII关节炎的发展？将鉴定B细胞中的IL-17信号传导机制，并确定与NF-：B促炎途径的相似性。平行地，将使用BXD 2小鼠中新建立的CII关节炎模型来确定IL-17调节B细胞中的自身抗体产生相对于外周慢性炎症在糜烂性关节炎发展中的作用。这些研究的可行性由已经组建的专家小组以及即将使用的试剂和小鼠的即时可用性来确保。在关节炎小鼠模型中驱动致病性自身抗体形成的事件的潜在鉴定以及IL-17驱动的自身免疫和炎症的相对作用的定义都将为关节炎和其他自身免疫性疾病提供新的治疗靶点，并表明目前正在开发的治疗方法的未识别效果。 关节炎或肾炎诱导的自身抗体可以引发或加剧类风湿性关节炎或系统性红斑狼疮患者的炎症。这些自身抗体一旦形成就很难消除。因此，阻断生发中心的形成，这是这些自身抗体产生的生物工厂，可能是这些患者的重要和新的治疗方法。这项提议将分析在淋巴细胞中相互作用导致生发中心形成的特定分子途径。了解这些淋巴细胞产生的导致致病性自身抗体产生的特定因素将为干预这一过程提供新的靶点。

DESCRIPTION (provided by applicant): In the proposed studies, we will test the hypothesis that IL-17 enhances autoreactive germinal center (GC) formation via upregulation of regulators of G-protein signaling (RGS) proteins, the enhanced GC T- and B-cell interactions lead to upregulation of activation-induced cytidine deaminase (AID) and the production of highly pathogenic autoantibodies. The significance of these studies lies in the identification of a mechanistic link between IL-17 and autoantibody production and the ability to distinguish the roles of autoantibodies and inflammatory responses in the development of rheumatoid arthritis. BXD2 mice develop spontaneous erosive arthritis and generalized autoimmune disease. A unique immune feature of these mice is the high numbers of Th17 CD4+ T cells, which are spontaneously activated in the spleen. Our data suggest that IL-17 promotes the interaction of B cells and CD4+ T cells in the spleen and the formation of spontaneous GCs. This action of IL-17 is mediated by upregulation of RGS13 and RGS16. Upregulation of these genes decrease, rather than increase, the signaling to the CXCR4 and CXCR5 chemokine receptors expressed on GC B and T cells. In this application, we will address the following questions. (1) Does IL-17 induce the formation and function of autoreactive GCs? The effects of IL- 17 will be determined in terms of its regulation of B-cell migration between the marginal and follicular B- cell zones, B-cell expression of AID, and the production of pathogenic autoantibodies. GC formation will be analyzed by confocal microscopy and FACS analysis. (2) Does IL-17 regulate GC formation by up- regulation of RGS proteins? The ability of IL-17 to regulate chemotaxis will be determined in vitro using a Transwell chamber, Flow chamber live imaging analysis, and in vivo by analysis of homing in BXD2 mice of GFP+ B cells in which the IL-17R or Rgs genes have been modulated using shRNA technology, or B cells obtained from targeted knockout mice. (3) Does IL-17-driven formation of autoreactive GCs contribute to the development of CII arthritis independently of IL-17-driven inflammatory responses? The IL-17 signaling mechanisms in B cells will be identified and similarities to the NF-:B pro-inflammatory pathways will be determined. In parallel, the effects of IL-17 modulation of autoantibody production in B cells vs. peripheral chronic inflammation in the development of erosive arthritis will be determined using a newly established CII arthritis model in BXD2 mice. The feasibility of these studies is ensured by the team of experts that has been assembled and the immediate availability of the reagents and mice that will be used. The potential identification of the events that drive pathogenic autoantibody formation in mouse models of arthritis and the definition of the relative roles of IL-17-driven autoimmunity and inflammation would both suggest novel therapeutic targets for arthritis and other autoimmune diseases and indicate unrecognized effects of therapies currently under development.Short Narrative Arthritis or nephritis-inducing autoantibodies can either initiate or worsen inflammation in patients with rheumatoid arthritis or systemic lupus erythematosus. It is difficult to eliminate these autoantibodies once they are formed. Thus, blocking the formation of germinal centers, which is the biologic factory that these autoantibodies are generated, can be an important and novel therapy for these patients. This proposal will analyze specific molecular pathways that interact in lymphocytes leading to the formation of germinal centers. Understanding of the specific factors produced by these lymphocytes that lead to pathogenic autoantibodies production will provide new targets for intervention of this process.