Ligands and cofactors required for RORgt function in the immune system

描述（由申请人提供）：孤儿核受体ROR？T在免疫系统发育、体内平衡和对微生物病原体的反应中起着关键作用。该转录因子仅在淋巴系细胞中表达，包括未成熟胸腺细胞、淋巴组织诱导剂（Lti）细胞和产生炎症细胞因子IL-17 （Th17细胞）的T辅助细胞。ROR吗?T细胞在这些细胞中都有重要的功能。它是CD4+8+胸腺细胞存活所必需的，因此，选择合适的T细胞库；它对Lti细胞的分化至关重要，从而诱导胎儿的淋巴结和Peyer's斑块的形成，以及出生后肠内的隐斑和分离淋巴滤泡的形成；它是Th17细胞从普通CD4+ T淋巴细胞分化所必需的。Th17细胞最近被证明是几种小鼠自身免疫模型中疾病的主要介质，并且越来越多的证据表明它们在人类中具有类似的作用。我们发现没有ROR？t细胞中的t导致IL-17产生减少，以响应相关细胞因子，并导致肠固有层中大多数Th17细胞的损失，而肠道固有层通常是Th17细胞最丰富的地方。此外，缺乏ROR？T细胞对几种自身免疫性疾病的诱导具有难治性。这些发现表明ROR？T可能是多种炎症条件下药理干预的一个有吸引力的靶点。我们对ROR作用机制的理解？目前对T的研究有限，对其转录活性是如何调控的知之甚少。有令人信服的证据表明，ROR？T和其他核受体一样，受配体结合的调节。在初步研究中，我们已经表明，在假定的配体结合区域发生突变，会使ROR？t诱导转录活性或直接细胞因子基因在t细胞中的表达。我们已经找到了ROR？它在果蝇S2细胞中具有转录活性，并利用这一观察结果进行全基因组RNA干扰筛选。通过脂质生物合成途径中涉及的基因过表达或RNAi敲低的研究补充了这一筛选，这些研究为我们指明了候选配体。基于这些发现和其他初步研究，我们提出以下目标：(1)进一步表征生物合成途径，以确定参与ROR生成的酶？T配体，使用哺乳动物和昆虫细胞为基础的系统；(2)使用生化/生物物理（如结合试验和共晶结构）和功能方法（如小鼠和人类Th17细胞极化）验证候选配体；(3)描述影响ROR的其他因素？转录活性，包括在果蝇RNAi筛选中鉴定的小鼠和人类同源基因，以及通过复合物的共免疫沉淀和酵母中适当文库的相互作用筛选鉴定的分子。总之，这些研究将更好地理解ROR的作用机制。T和将促进自身免疫性疾病治疗的新策略。公共卫生相关性：转录因子ROR？在许多疾病中，辅助性t细胞（Th17）的分化是协调炎症反应所必需的。ROR的能力？靶基因如促炎细胞因子IL-17的直接表达依赖于其与一种尚未定义的配体的结合。相关配体的鉴定将加速抑制ROR的新方法的发展。T活性和治疗大量自身免疫性疾病和癌症，其进展取决于炎症过程。

DESCRIPTION (provided by applicant): The orphan nuclear receptor ROR?t has critical roles in immune system development, homeostasis, and responses to microbial pathogens. This transcription factor is expressed only in lymphoid lineage cells, including immature thymocytes, lymphoid tissue inducer (Lti) cells, and T helper cells that produce the inflammatory cytokine IL-17 (Th17 cells). ROR?t has essential functions in each of these cells. It is required for the survival of CD4+8+ thymocytes and, hence, for the selection of an appropriate T cell repertoire; it is essential for the differentiation of Lti cells that induce formation of lymph nodes and Peyer's patches in the fetus and of cryptopatches and isolated lymphoid follicles in the post-natal intestine; and it is required for the differentiation of Th17 cells from na¿ve CD4+ T lymphocytes. Th17 cells have recently been shown to be major mediators of disease in several models for autoimmunity in the mouse, and there is accumulating evidence that they have similar roles in humans. We found the absence of ROR?t in T cells results in reduced IL-17 production in response to relevant cytokines and in the loss of most Th17 cells in the intestinal lamina propria, where they are normally most abundant. In addition, mice lacking ROR?t are refractory to the induction of several autoimmune diseases. These findings suggest that ROR?t may be an attractive target for pharmacological intervention in multiple inflammatory conditions. Our understanding of the mechanism of action of ROR?t is currently limited, and little is known about how its transcriptional activity is regulated. There is compelling evidence that ROR?t, like other nuclear receptors, is regulated by binding of a ligand. In preliminary studies, we have shown that mutations in the putative ligand binding domain abrogate the ability of ROR?t to induce transcriptional activity or to direct cytokine gene expression in T cells. We have found that ROR?t has transcriptional activity in Drosophila S2 cells, and have exploited this observation to perform a genome-wide RNA interference screen. This screen has been complemented by studies with overexpression or RNAi knockdown of genes involved in lipid biosynthetic pathways that have pointed us towards candidate ligands. Based on these findings and on other preliminary studies, we propose the following aims: (1) to further characterize biosynthetic pathways to pinpoint enzymes involved in generation of a ROR?t ligand, using both mammalian and insect cell-based systems; (2) to validate candidate ligands using both biochemical/biophysical (e.g., binding assays and co-crystal structures) and functional approaches (e.g. mouse and human Th17 cell polarization); and (3) to characterize other factors that influence ROR?t transcriptional activity, including mouse and human orthologs of genes identified in the Drosophila RNAi screen and molecules identified by co-immunoprecipitation of complexes and by interaction screening with appropriate libraries in yeast. Together, these studies will provide a better understanding of the mechanism of action of ROR?t and will facilitate new strategies for therapy in autoimmune diseases. Public Health Relevance: The transcription factor ROR?t is required for the differentiation of T helper (Th17) cells that orchestrate inflammatory responses in numerous diseases. The ability of ROR?t to direct expression of target genes, such as the pro-inflammatory cytokine IL-17, is dependent on its binding of a yet undefined ligand. Identification of the relevant ligand will hasten the development of new approaches for inhibiting ROR?t activity and for treating a large number of autoimmune diseases and cancers whose progression depends on inflammatory processes.