Suppression of pathogenic autoantibodies in lupus by inhibition of AID

描述（由申请人提供）：在提议的研究中，我们将测试IL-17通过上调g蛋白信号（RGS）蛋白的调节因子来增强自身反应性生发中心（GC）形成的假设，增强的GC - T和b细胞相互作用导致激活诱导的胞苷脱氨酶（AID）的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了IL-17与自身抗体产生之间的机制联系，并能够区分自身抗体和炎症反应在类风湿关节炎发展中的作用。BXD2小鼠发生自发性糜烂性关节炎和全身性自身免疫性疾病。这些小鼠的一个独特的免疫特征是大量的Th17 CD4+ T细胞，这些细胞在脾脏中自发激活。我们的数据表明，IL-17促进脾脏中B细胞和CD4+ T细胞的相互作用和自发性GCs的形成。IL-17的这种作用是通过上调RGS13和RGS16介导的。这些基因的上调会减少而不是增加GC B和T细胞上表达的CXCR4和CXCR5趋化因子受体的信号。在这个应用程序中，我们将解决以下问题。(1) IL-17是否诱导自反应性GCs的形成和功能？IL- 17的作用将根据其对B细胞在边缘和滤泡B细胞区之间迁移的调节、B细胞AID的表达和致病性自身抗体的产生来确定。GC的形成将通过共聚焦显微镜和FACS分析进行分析。(2) IL-17是否通过上调RGS蛋白调控GC的形成？IL-17调节趋化性的能力将在体外通过Transwell室、Flow室实时成像分析来确定，在体内通过分析BXD2小鼠中使用shRNA技术调节IL-17R或Rgs基因的GFP+ B细胞或从靶向敲除小鼠中获得的B细胞的归巢来确定。(3) il -17驱动的自身反应性GCs的形成是否独立于il -17驱动的炎症反应促进了CII关节炎的发展？IL-17在B细胞中的信号传导机制将被确定，并与NF-:B促炎途径的相似性将被确定。同时，IL-17调节B细胞自身抗体产生对周围慢性炎症在糜糜性关节炎发展中的作用将通过新建立的BXD2小鼠CII关节炎模型来确定。这些研究的可行性由已组建的专家小组和即将使用的试剂和小鼠的立即可用性来保证。在关节炎小鼠模型中，潜在地发现驱动致病性自身抗体形成的事件，以及定义il -17驱动的自身免疫和炎症的相对作用，都将为关节炎和其他自身免疫性疾病提供新的治疗靶点，并指出目前正在开发的治疗方法的未被认识的效果。简短的叙述

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.