Defining the pathways activated by Toll-like Receptors to stimulate immunity

项目摘要 该方案的目标是确定Toll样受体(TLRs)如何刺激不同的细胞反应 巨噬细胞和树突状细胞(DC)，并了解这些反应如何影响基于DC的癌症 免疫疗法。TLRs诱导炎性基因表达的能力一直在研究中 20年来，由MyD88和TRIF适配器介导的不同信号通路解释了所有 转录反应。直到最近人们才意识到，TLRs也会推动 有反应的细胞，如快速诱导有氧糖酵解。在上一个资助期内，我们 发现TLR诱导的Myddosome复合体包含两类蛋白质。必须上一节课 用于myddosome组件(例如MyD88)，并代表该信号结构的核心。第二节课是 不是myddosome组装所必需的(例如TRAF6)，而是用来招募酶来使 虫体的效应器功能。具体地说，我们确定了激酶TBK1是myddosome的一个组成部分 这是由TRAF6招募的，专门用于诱导糖酵解。因此，Myddosome服务于 作为激活不同细胞反应的信号的亚细胞位置。我们对这些活动如何 在体外受到调节，它们对T细胞介导的保护性免疫的影响仍然有限。 除了myddosome外，选择TLRs(例如TLR4和TLR3)与三聚体接合。中央三聚体 调节因子是TRIF，它刺激干扰素(干扰素)反应，NF-kB和MAPK激活，坏死性下垂和 糖酵解。虽然TRIF在免疫中的重要性早已被认识到，但它激活的手段 这些不同的反应尚不清楚。这种知识差距不仅仅是学术上的好奇，就像TRIF那样 对于内毒素受体TLR4刺激适应性免疫的能力是必不可少的。了解监管规定 刺激myddosome和TRIF依赖的反应的事件将使讨论TLRs如何驱动 对感染和癌症的保护性免疫。在本应用程序中，我们建议探索myddosome 在体外和在癌症免疫疗法方面的活动(目标1)。在目标2中，我们提供了一种创新的合成 基于生物学的方法来定义TRIF信号的机制以及这些机制与这些机制的关系 由互补的先天免疫途径诱导。我们对两个主要信号通路的关注被激活 通过TLRs应该可以提供这一重要受体家族的操作视图。

Project Summary The goal of this proposal is to identify how Toll-like Receptors (TLRs) stimulate diverse cellular responses in macrophages and dendritic cells (DCs), and to understand how these responses influence DC-based cancer immunotherapies. The ability of TLRs to induce inflammatory gene expression has been under investigation for twenty years, with distinct signaling pathways mediated by the MyD88 and TRIF adaptors explaining all transcriptional responses. It has only recently become appreciated that TLRs also drive metabolic changes in responding cells, such as the rapid induction of aerobic glycolysis. During the previous funding period, we discovered that the TLR-induced myddosome complex contains two classes of proteins. One class is necessary for myddosome assembly (e.g. MyD88) and represents the core of this signaling structure. The second class is not necessary for myddosome assembly (e.g. TRAF6), but rather operates to recruit enzymes that diversify the effector functions of the myddosome. Specifically, we identified the kinase TBK1 as a myddosome component that is recruited by TRAF6 and is dedicated specifically to induce glycolysis. The myddosome therefore serves as a subcellular site of signals that activate diverse cellular responses. Our understanding of how these activities are regulated in vitro and their impact on T cell mediated protective immunity remains limited. In addition to the myddosome, select TLRs (e.g. TLR4 and TLR3) engage the triffosome. The central triffosome regulator is TRIF, which stimulates interferon (IFN) responses, NF-kB and MAPK activation, necroptosis and glycolysis. While the importance of TRIF in immunity has long-been recognized, the means by which it activates these diverse responses is unclear. This gap in knowledge is not merely an academic curiosity, as TRIF is essential for the ability of the LPS receptor TLR4 to stimulate adaptive immunity. Understanding regulatory events that stimulate myddosome- and TRIF-dependent responses will enable discussions of how TLRs drive protective immunity against infection and cancer. In this application, we propose to explore myddosome activities in vitro and in the context of cancer immunotherapies (Aim 1). In Aim 2, we offer an innovative synthetic biology-based approach to define the mechanisms of TRIF signaling and how these mechanisms relate to those induced by complementary innate immune pathways. Our focus on the two major signaling pathways activated by TLRs should provide an operational view of this important family of receptors.