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、动态平衡和对微生物病原体的反应中具有关键作用。这种转录因子只在淋巴系细胞中表达，包括未成熟的胸腺细胞、淋巴组织诱导物(LTI)细胞和产生炎症细胞因子IL-17(Th17细胞)的辅助性T细胞。ROR？T在这些细胞中的每一个中都有必要的功能。它是CD4+8+胸腺细胞生存所必需的，因此也是选择合适的T细胞库所必需的；它是诱导胎儿淋巴结和Peyer‘s结形成的LTI细胞分化所必需的，也是出生后肠道中隐窝和孤立淋巴滤泡形成所必需的；它也是从天然的CD4+T淋巴细胞分化出Th17细胞所必需的。最近，在小鼠的几种自身免疫模型中，Th17细胞被证明是疾病的主要媒介，而且越来越多的证据表明，它们在人类中也扮演着类似的角色。我们发现，T细胞中缺乏ROR？T会导致相关细胞因子反应产生的IL-17减少，并导致肠道固有层中大多数Th17细胞的丧失，而肠道固有层通常是Th17细胞最丰富的地方。此外，缺乏ROR？T的小鼠对几种自身免疫性疾病的诱导是困难的。这些发现表明，在多种炎症条件下，ROR？T可能是一个有吸引力的药物干预靶点。目前我们对ROR？T的作用机制了解有限，对其转录活性如何调控知之甚少。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，我们已经证明，假定的配体结合域的突变使ROR？T在T细胞中诱导转录活性或指导细胞因子基因表达的能力丧失。我们已经发现ROR？T在果蝇S2细胞中具有转录活性，并利用这一观察结果进行了全基因组RNA干扰筛选。这一筛选得到了与脂质生物合成途径有关的基因过度表达或RNAi敲除的研究的补充，这些基因将我们引向候选配体。基于这些发现和其他初步研究，我们提出了以下目标：(1)利用哺乳动物和昆虫细胞系统，进一步确定参与ROR？T配体生成的酶的生物合成途径；(2)同时使用生化/生物物理方法(如结合分析和共晶结构)和功能方法(如小鼠和人类Th17细胞极化)来验证候选配体；以及(3)鉴定影响ROR？T转录活性的其他因素，包括果蝇RNAi筛选中确定的基因的小鼠和人类同源基因，以及通过复合体免疫共沉淀和与酵母中适当文库的相互作用筛选确定的分子。总之，这些研究将更好地了解ROR？T的作用机制，并为自身免疫性疾病的治疗提供新的策略。与公共卫生相关：转录因子ROR？T是许多疾病中协调炎症反应的辅助性T细胞(Th17)分化所必需的。ROR？T直接表达靶基因的能力，如促炎细胞因子IL-17，依赖于它与一种尚未定义的配体的结合。相关配体的识别将加速开发抑制ROR？T活性的新方法，并用于治疗大量依赖炎症过程进展的自身免疫性疾病和癌症。

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.