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

项目摘要 这项提案的目标是确定Toll样受体（TLR）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DC为基础的癌症 免疫疗法TLR诱导炎症基因表达的能力已经在研究中， 20年来，MyD 88和TRIF衔接子介导的不同信号通路解释了所有 转录反应。直到最近才认识到TLR也驱动代谢变化， 反应细胞，如快速诱导有氧糖酵解。在上一个财政年度，我们 发现TLR诱导的myddosome复合物包含两类蛋白质。一节课是必要的 MyD 88是Mydosome组装的核心，代表了这种信号结构的核心。第二类是 不是myddosome装配所必需的（例如TRAF 6），而是用于招募使细胞多样化的酶。 mydosome的功能。具体地说，我们确定了激酶TBK 1作为myddosome组件 由TRAF 6募集，专门用于诱导糖酵解。因此， 作为激活不同细胞反应的信号的亚细胞位点。我们对这些活动的理解 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之外，选择TLR（例如TLR 4和TLR 3）接合triffosome。中央三角体 调节因子是TRIF，其刺激干扰素（IFN）应答、NF-kB和MAPK活化、坏死性凋亡和 糖酵解虽然TRIF在免疫中的重要性早已被认识到，但它激活的方式 这些不同的反应尚不清楚。这种知识上的差距不仅仅是学术上的好奇心，就像TRIF一样。 对于LPS受体TLR 4刺激适应性免疫的能力至关重要。了解监管 刺激myddosome和TRIF依赖性反应的事件将使TLRs如何驱动 对感染和癌症的保护性免疫。在本申请中，我们建议探索myddosome 在体外和癌症免疫疗法的背景下的活性（目的1）。在目标2中，我们提供一种创新的合成 基于生物学的方法来定义TRIF信号传导的机制以及这些机制如何与那些 由互补的先天免疫途径诱导。我们的重点是两个主要的信号通路激活 应该提供一个操作的观点，这个重要的家庭的受体。

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.