Toll-like receptor 9: trafficking and signaling

描述（由申请人提供）：含有CpG基序的DNA （CpG DNA）在治疗癌症、感染性和过敏性疾病方面具有不可思议的潜力。尽管有这种潜力，但对我们自身核酸（包括DNA）的反应会引发自身免疫性疾病，如系统性红斑狼疮。特异性受体TLR9的定位和运输可能在自我/外源DNA区分中起关键作用。揭示TLR9转运的分子机制是我们实现操纵TLR9转运以实现CpG DNA反应调节的长期目标的第一步。我们已经证明，在CpG DNA刺激之前，TLR9定位于细胞内，主要位于内质网（ER）。TLR9从内质网转运到核内体和溶酶体，在那里它与内吞CpG DNA共定位。这些数据提出了一些基本问题：是什么调节TLR9对CpG DNA的访问，以及访问如何影响对自身和微生物DNA的反应？利用新的TLR9转运试验，我们已经积累了证据，证明TLR9组成性地退出内质网，CpG DNA诱导二次TLR9转运事件，并且这两个事件都通过传统的细胞转运途径发生。因此，我们假设TLR9通过高尔基复合物到内溶酶体的细胞分泌途径以高度调控的方式组成性运输，并且高尔基转运是TLR9对CpG DNA反应的先决条件。我们认为TLR9转运可能与其他tlr和自身免疫B细胞受体协同作用，并受TLR9细胞质尾部翻译后修饰的调节。这一假设将在三个具体目标中进行测试。首先，通过高尔基复合体转染和内源性TLR9运输将使用多种体外方法进行检查，包括一种新的蛋白酶裂解试验。其次，我们将探讨tlr9 -自身免疫B细胞受体协同作用的机制。第三，我们将确定TLR9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TLR9转运的机制，我们可以揭示如何实现对外来DNA的反应调节和对自身DNA的反应缺乏。这些知识将为进一步开发CpG DNA作为辅助和治疗以及开发机制来中断自身免疫病理循环提供基础。公共卫生相关性：尽管CpG DNA在增强癌症和传染病的免疫反应方面具有不可思议的潜力，但对自身DNA的不适当反应会导致自身免疫。本项目旨在确定控制CpG DNA特异性受体toll样受体9定位的关键因素。通过控制TLR9的定位，从而调节CpG DNA的活性，我们希望能够增强疫苗并中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.