Ligands and Cofactors Required for RORgt Function in the Immune System

孤儿核受体ROR γ t在免疫系统发育、稳态和对微生物病原体的应答中具有关键作用。该转录因子仅在淋巴谱系细胞中表达，包括未成熟胸腺细胞、淋巴组织诱导（Lti）细胞和产生炎性细胞因子IL-17的T辅助细胞（Th 17细胞）。ROR γ t在这些细胞中的每一个中具有重要功能。它是CD 4 +8+胸腺细胞存活所必需的，因此也是选择适当T细胞库所必需的;它是诱导胎儿淋巴结和派尔集合淋巴结形成的Lti细胞分化以及出生后肠道中隐睾和孤立淋巴滤泡形成所必需的;它是从初始CD 4 + T淋巴细胞分化Th 17细胞所必需的。Th 17细胞最近被证明是小鼠自身免疫的几种模型中疾病的主要介质，并且有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T细胞中RORyt的缺乏导致响应于相关细胞因子的IL-17产生减少，并且导致肠固有层中大多数Th 17细胞的损失，其中它们通常是最丰富的。此外，缺乏ROR γ t的小鼠对几种自身免疫性疾病的诱导是难治的。这些发现表明RORyt可能是多种炎症条件下药理学干预的有吸引力的靶标。 目前我们对RORyt的作用机制的理解是有限的，并且对其转录活性是如何调节的知之甚少。有令人信服的证据表明，ROR γ t，像其他核受体一样，是由配体的结合来调节的。在初步研究中，我们已经表明，在假定的配体结合结构域的突变废除的ROR γ t的能力，诱导转录活性或直接细胞因子基因表达的T细胞。我们发现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全基因组RNA干扰筛选。这种筛选已经通过脂质生物合成途径中涉及的基因的过表达或RNAi敲低的研究得到补充，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，我们提出了以下目标：（1）使用基于哺乳动物和昆虫细胞的系统，进一步表征生物合成途径以确定参与ROR γ t配体生成的酶;（2）使用生物化学/生物物理学（例如，结合测定和共晶体结构）和功能方法（例如小鼠和人Th 17细胞极化）;和（3）表征影响ROR γ t转录活性的其它因子，包括在果蝇RNAi筛选中鉴定的基因的小鼠和人直系同源物，以及通过复合物的共免疫沉淀和通过与酵母中的适当文库的相互作用筛选鉴定的分子。总之，这些研究将更好地了解RORyt的作用机制，并将促进自身免疫性疾病治疗的新策略。

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.