Ligands and Cofactors Required for RORgt Function in the Immune System

孤儿核受体RORyt在免疫系统发育、动态平衡和对微生物病原体的反应中起着关键作用。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。RORyt在这些细胞中都有必要的功能。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；它还是Th17细胞从幼稚的CD4+T淋巴细胞分化所必需的。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RORyt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RORyt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，RORyt可能是多种炎症条件下药物干预的一个有吸引力的靶点。 目前我们对RORyt的作用机制了解有限，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，RORyt和其他核受体一样，受到配体结合的调控。在初步研究中，我们已经证明，假设的配体结合域的突变使RORyt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RORyt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RORyt配体生成的酶的生物合成途径；(2)使用生化/生物物理方法(例如结合分析和共晶结构)和功能方法(例如小鼠和人类Th17细胞极化)来验证候选配体；以及(3)鉴定影响RORyt转录活性的其他因素，包括在果蝇RNAi筛选中确定的基因的小鼠和人类同源基因，以及通过复合体的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RORyt的作用机制，并将促进治疗自身免疫性疾病的新策略。

The orphan nuclear receptor RORyt 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). RORyt 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 naive 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 RORyt 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 RORyt are refractory to the induction of several autoimmune diseases. These findings suggest that RORyt may be an attractive target for pharmacological intervention in multiple inflammatory conditions. Our understanding of the mechanism of action of RORyt is currently limited, and little is known about how its transcriptional activity is regulated. There is compelling evidence that RORyt, 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 RORyt to induce transcriptional activity or to direct cytokine gene expression in T cells. We have found that RORyt 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 RORyt 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 RORyt 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 RORyt and will facilitate new strategies for therapy in autoimmune diseases.