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

项目概要 该提案的目标是确定 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基于 DC 的癌症 免疫疗法。 TLR 诱导炎症基因表达的能力已被研究 二十年来，MyD88 和 TRIF 适配器介导的不同信号通路解释了所有 转录反应。直到最近人们才认识到 TLR 也能驱动代谢变化 反应细胞，例如快速诱导有氧糖酵解。在上一个资助期间，我们 发现 TLR 诱导的 myddosome 复合物含有两类蛋白质。需要上一堂课 用于 myddosome 组装（例如 MyD88）并代表该信号传导结构的核心。第二类是 对于 myddosome 组装（例如 TRAF6）来说不是必需的，而是用于招募使细胞多样化的酶 myddosome 的效应器功能。具体来说，我们将激酶 TBK1 鉴定为 myddosome 成分 由 TRAF6 招募并专门用于诱导糖酵解。因此，myddosome 的作用是 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，选择的 TLR（例如 TLR4 和 TLR3）也与三叶体结合。中央三体 TRIF 是一个调节因子，可刺激干扰素 (IFN) 反应、NF-kB 和 MAPK 激活、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。这种知识差距不仅仅是学术上的好奇心，就像 TRIF 一样 对于 LPS 受体 TLR4 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人们能够讨论 TLR 如何驱动 针对感染和癌症的保护性免疫力。在此应用中，我们建议探索 myddosome 体外和癌症免疫治疗背景下的活动（目标 1）。在目标 2 中，我们提供了一种创新的合成材料 基于生物学的方法来定义 TRIF 信号传导机制以及这些机制如何与这些机制相关 由互补性先天免疫途径诱导。我们关注激活的两条主要信号通路 TLRs 的研究应该提供这个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.