Interplay Between Pathogenic and Regulatory T Cells in EAE

描述(由申请人提供)摘要T辅助(H)17细胞是一种新描述的CD4+T细胞亚群，与Th1细胞一起与多发性硬化症(MS)及其动物模型实验性自身免疫性脑脊髓炎(EAE)等自身免疫性疾病的发展有关。另一方面，调节性T细胞保护自身免疫的发展。Th17细胞及其分化相关因子促进自身免疫的机制尚未完全阐明。我们先前已经确定，Th17细胞的分化需要转化生长因子-2和IL-6以及后来的IL-21的共同作用。另一方面，IL-23p19对于维持Th17反应和EAE的发展至关重要，因为缺乏IL-23p19的小鼠对EAE具有抵抗力。我们发现IL-23受体(IL-23R)除了在Th17细胞上表达外，还在巨噬细胞和小胶质细胞等天然免疫系统的细胞上表达。然而，到目前为止，IL-23在非T细胞上的作用尚不清楚。我们创造了一种新型的IL-23R绿色荧光蛋白(GFP)敲打蛋白(KI)报告小鼠，在该小鼠中，所有表达IL-23R的细胞都会同时表达GFP，并对IL-23失去反应。IL-23R GFP Ki小鼠将有助于我们确定IL-23在EAE的发生发展中对Th17细胞和巨噬细胞/小胶质细胞的相对贡献。我们进一步确定，Th17细胞在EAE发病时迅速迁移到中枢神经系统(CNS)，其数量与疾病的严重程度相关。然而，Th17细胞数量迅速下降，而Th1细胞数量保持不变，提示Th17细胞可能在发病早期起主导作用，Th1细胞在EAE慢性期参与更多。我们已经产生了一只IL-17F RFP/DTR小鼠，在其中Th17细胞可以通过注射白喉毒素在任何给定的时间被删除，我们将能够确定Th17在疾病不同阶段的作用。最后，在我们进一步确定Th17细胞增强致病活性的机制的过程中，我们分离了一种选择性表达在Th17细胞上的表面分子，名为podoplan in。泊多拉宁在T细胞上的表达以前从未被报道过，但此前已有研究表明泊多拉宁与肿瘤细胞的迁移有关。我们的初步数据显示，在用髓鞘抗原免疫的小鼠体内注射抗泊多拉宁抗体后，与对照动物相比，治疗组动物中枢神经系统中Th17细胞的数量增加，这表明泊多拉宁可能在Th17细胞的迁移中发挥作用。 公共卫生相关性：基于我们的初步数据，并使用我们产生的新试剂，我们设计了实验，以解决对我们理解自身免疫性疾病非常重要的几个悬而未决的问题：1)确定EAE过程中效应器Th17和Th1细胞以及T-reg细胞之间的相互作用；2)确定泊多普宁在Th17细胞发育中的作用；3)确定IL-23如何通过其对Th17细胞和固有免疫系统细胞的作用而加剧EAE的发展。项目简介这些研究将补充正在进行的关于Th17细胞和调节性T细胞的研究，并有助于我们全面了解自身免疫发展及其调节的机制。

DESCRIPTION (provided by applicant): Abstract T helper (h) 17 cells are a newly described subset of CD4+ T cells which has been implicated, along with Th1 cells in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Regulatory T cells, on the other hand, protect from the development of autoimmunity. The mechanisms by which Th17 cells and factors associated with their differentiation promote autoimmunity have not been fully elucidated. We have previously determined that the differentiation of Th17 cells required the combined action of TGF-2 plus IL-6 and later IL-21. On the other hand IL-23 p19 is critical for sustaining Th17 responses and for the development of EAE since mice deficient for IL- 23 p19 are resistant to EAE. We found that IL-23 receptor (IL-23R), besides being expressed on Th17 cells, is also expressed on cells of the innate immune system such as macrophages and microglia. However, the role of IL-23 on non-T cells is to date unknown. We have generated a novel IL-23R green fluorescent protein (GFP) knockin (KI) reporter mouse in which all cells expressing the IL-23R will concomitantly express the GFP and become non responsive to IL-23. The IL-23R GFP KI mice will help us determine the relative contribution of IL- 23 on Th17 cells and macrophages/microglia in the development and progression of EAE. We have further determined that Th17 cells migrate quickly to the central nervous system (CNS) at the onset of EAE and their numbers correlate with the disease severity. However, the number of Th17 cells rapidly decline while the number of Th1 cells is maintained suggesting that Th17 cells may have a dominant role early on at the initiation phase and that Th1 cells are more involved during the chronic phase of EAE. We have generated an IL-17F RFP/DTR mouse in which Th17 cells can be deleted at any given time through the injection of diphtheria toxin and we will be able to determine the role of Th17 at different stages of the disease. Finally, in our efforts to further determine the mechanisms by which Th17 cells have enhanced pathogenic activity, we have isolated a surface molecule selectively expressed on Th17 cells named podoplanin. The expression of podoplanin has never been reported on T cells before but podoplanin has previously been implicated in the migration of tumor cells. Our preliminary data, injecting anti-podoplanin antibody in vivo in mice immunized with myelin antigen show an increase number of Th17 cells present in the CNS of the treated animals compared to control animals suggesting that podoplanin may play a role in the migration of Th17 cells. PUBLIC HEALTH RELEVANCE: Based on our preliminary data and using novel reagents that we have generated, we have designed experiments to address several outstanding questions that are important for our understanding of autoimmune diseases: 1) Determine the interplay between effector Th17 and Th1 cells, and T-reg cells during EAE, 2) Determine the role of podoplanin in the development of Th17 cells 3) Determine how IL-23 exacerbate the development of EAE through its action on Th17 and cells of the innate immune system. Project Narrative These studies will complement ongoing studies on Th17 cells and regulatory T cells and contribute to our overall understanding of the mechanisms involved in the development of autoimmunity and their regulation.