Toll-like receptor 9: trafficking and signaling

描述（由申请人提供）：含有CpG基序的DNA（CpG DNA）具有治疗癌症、传染病和过敏性疾病的惊人潜力。尽管有这种潜力，但对我们自己的核酸（包括DNA）的反应会引发自身免疫性疾病，如系统性红斑狼疮。定位和运输的特定受体，TLR 9，可能发挥关键作用，在自我/外来DNA的歧视。揭示TLR 9运输的分子机制是我们操纵TLR 9运输以实现CpG DNA应答调节的长期目标的第一步。我们已经表明，TLR 9是本地化的细胞内，主要是在内质网（ER），之前的CpG DNA刺激。TLR 9从ER运输到内体和溶酶体，在那里它与内吞的CpG DNA共定位。这些数据提出了一个基本问题，即是什么调节TLR 9与CpG DNA的接触，以及接触如何影响对自身和微生物DNA的反应？使用新的TLR 9运输试验，我们积累了TLR 9组成性退出ER、CpG DNA诱导二次TLR 9运输事件以及这两个事件通过传统细胞运输途径发生的证据。因此，我们假设TLR 9组成性交通在一个高度调节的方式通过细胞分泌途径从高尔基复合体的内溶酶体和高尔基转运是一个先决条件，TLR 9对CpG DNA的反应。我们认为，TLR 9的运输可能与其他TLR和自身免疫B细胞受体的协同作用，并通过TLR 9胞质尾的翻译后修饰来调节。这一假设将在三个具体目标中得到检验。首先，转染和内源性TLR 9运输通过高尔基复合体将使用多种体外方法，包括一种新的蛋白酶切割试验进行检查。其次，我们将研究TLR 9-自身免疫B细胞受体协同作用的机制。第三，我们将确定TLR 9翻译后修饰在调节细胞内运输和TLR-自身免疫B细胞受体协同作用中的作用。通过了解TLR 9运输的机制，我们可以揭示如何调节对外源DNA的反应和缺乏对自身DNA的反应。这些知识将为进一步开发CpG DNA作为佐剂和治疗剂以及开发中断自身免疫病理循环的机制提供基础。公共卫生相关性：尽管CpG DNA具有增强癌症和传染病免疫反应的惊人潜力，但对自身DNA的不适当反应会导致自身免疫。该项目旨在确定控制CpG DNA特异性受体Toll样受体9定位的关键因素。通过调控TLR 9的定位，进而调控CpG DNA的活性，我们希望能够增强疫苗的免疫力，阻断自身免疫性疾病的周期。

DESCRIPTION (provided by applicant): DNA containing CpG motifs (CpG DNA) has incredible potential to treat cancer, infectious and allergic diseases. Despite this potential, response to our own nucleic acids, including DNA, triggers autoimmune diseases such as systemic lupus erythematosus. Localization and trafficking of the specific receptor, TLR9, may play a key role in self/foreign DNA discrimination. Uncovering the molecular mechanisms of TLR9 trafficking is the first step towards our long term goal of manipulating TLR9 trafficking to achieve modulation of CpG DNA response. We have shown that TLR9 is localized intracellularly, predominantly in the endoplasmic reticulum (ER), prior to CpG DNA stimulation. TLR9 traffics from the ER to endosomes and lysosomes where it co-localizes with endocytosed CpG DNA. These data raise the fundamental questions of what regulates TLR9 access to CpG DNA and how does access affect response to self and microbial DNA? Using new TLR9 trafficking assays, we have accumulated evidence that TLR9 constitutively exits the ER, that CpG DNA induces a secondary TLR9 trafficking event, and that both events occur through traditional cell trafficking pathways. Therefore, we hypothesize that TLR9 constitutively traffics in a highly regulated fashion through the cell secretory pathway from the Golgi complex to endolysosomes and that Golgi transit is a prerequisite for TLR9 response to CpG DNA. We believe that TLR9 trafficking may account for synergy with other TLRs and the autoimmune B cell receptor, and is regulated by post-translational modification of the TLR9 cytoplasmic tail. This hypothesis will be tested in three Specific Aims. First, transfected and endogenous TLR9 trafficking through the Golgi complex will be examined using multiple in vitro approaches including a novel protease cleavage assay. Second, we will examine the mechanism of TLR9-autoimmune B cell receptor synergism. Third, we will determine the role of TLR9 post-translational modification in regulating intracellular trafficking and in TLR-autoimmune B cell receptor synergy. By understanding the mechanism of TLR9 trafficking we can uncover how regulation of response to foreign DNA and lack of response to self-DNA is achieved. This knowledge will provide the basis for the further development of CpG DNA as an adjuvant and therapeutic as well as develop mechanisms to interrupt the cycle of autoimmune pathology. PUBLIC HEALTH RELEVANCE: Despite incredible potential of CpG DNA to augment immune responses in cancer and infectious disease, inappropriate response to self DNA results in autoimmunity. This project seeks to identify key factors controlling localization of CpG DNA's specific receptor, Toll-like receptor 9. Through manipulating localization of TLR9, and thereby modulating CpG DNA activity, we hope to enhance vaccines and interrupt the cycle of autoimmune disease.