TOLLIP and Tuberculosis Immunopathogenesis

描述（由申请人提供）：申请人是传染病专家，长期关注结核病和国际卫生。他在细胞免疫学方面的背景是这项研究的基础。他在美国国立卫生研究院作为霍华德休斯研究学者研究T细胞和树突细胞的细胞免疫学，并在芝加哥大学作为本科生研究蛋白质生物化学。他有一个优秀的出版记录，从他以前的研究经验，在美国国立卫生研究院，在实验室的罗伯特塞德和工作进行的实验室的托马斯霍恩。他的近期目标是研究TOLLIP调节先天免疫反应的机制。他将使用一些专门的资源作为培训计划的一部分来实现这一目标。首先，他的导师（托马斯霍恩博士）有研究大型人类群体中常见遗传变异的经验，这将首次应用于TOLLIP。其次，他的共同导师（Alan Aderem博士）在巨噬细胞生物学方面具有丰富的经验，将允许申请人研究TOLLIP变异对吞噬作用和自噬作用的作用。第三，他将通过研究TOLLIP如何影响树突状细胞成熟，T细胞极化和细胞因子反应来确定TOLLIP对免疫系统影响的广度。申请人的长期目标是在学术中心的传染病部门寻求临床任命，开发独立研究资金来源，并将超过75%的时间用于运行实验室和合作临床研究。结核病刺激强烈的炎症反应，但调节这种反应是控制感染的关键。对结核分枝杆菌（MTb）的最佳炎症反应的调节和性质在人类中仍然知之甚少。深入了解TLR介导的对MTb的先天免疫应答的负调节机制可以在新疫苗和药物的设计中提供重大突破。我们假设TOLLIP及其常见变异体负调节人类单核细胞中的TLR信号传导，并与结核病易感性相关。在外周血人单核细胞中使用TOLLIP的shRNA敲低，我们发现TOLLIP在用TLR 2和TLR 4配体刺激后抑制TNF和IL-6的产生。我们还发现了一个常见的多态性（rs 5743899），这是与TOLLIP缺乏症在高加索人群。此外，在越南的一项病例人群研究中，760份脐带血样本和671例结核病患者，我们发现SNP rs 5743899与结核病易感性相关（p=6.97x10-7）。总之，这些数据表明TOLLIP对人类TLR信号传导具有抗炎作用，并且TOLLIP缺乏与TB风险增加相关。这些数据暗示了TLR信号传导在人TB发病机制中的意外负调节机制。在本申请中，我们将研究TOLLIP的人类变体及其在调节巨噬细胞、树突状细胞和T细胞对MTb感染的反应中的作用。在目标1中，我将确定在人群中常见的影响TOLLIP功能和影响结核病发生风险的特异性多态性。此外，我们将更全面地描述TOLLIP如何影响巨噬细胞和树突状细胞中的吞噬作用、自噬和分枝杆菌复制。我还将联合收割机结合我们的人口研究与Tollip -/-小鼠的机制研究。在目标2中，我将使用多种技术，包括小鼠研究、离体婴儿细胞因子对BCG反应的研究，以及南非儿童结核病的独特病例对照研究，以研究体外和体内效应 TOLLIP变异对树突状细胞生物学的影响以及TOLLIP变异对适应性免疫应答的先天免疫引发调节的影响。这项工作将在西雅图的华盛顿大学医学院进行。申请人和拟议的导师是在过敏和传染病，其中包括73名全职教师，其总赠款支持超过每年1.35亿美元的司。该部门是培养医生科学家的理想环境，因为过去80%以上的奖学金学员获得了学术医学的教职。申请人提出的研究利用了他在细胞免疫学方面的强大背景，但需要在分子生物学，先天免疫学和人类遗传学方面进行额外的培训。他有详细的课程，研讨会和会议，以满足这些需求，并制定了评估他的进展指标。他还组建了一个科学咨询小组，提供一对一的专业知识。最后，拟议的导师有一个证明这些技术的跟踪记录，并成功地指导了其他几个年轻的科学家。 说明：了解免疫和遗传因素，赋予保护免受结核病是至关重要的成功的疫苗和治疗的发展。TOLLIP是最近发现的Toll样受体途径的调节剂，该途径对结核病的有效免疫应答至关重要。我建议把重点放在TOLLIP调节先天免疫的机制，通过确定影响TOLLIP功能的特定常见变异。我还将研究TOLLIP在调节细胞摄取和结核病破坏中的作用。最后，我将研究TOLLIP如何影响婴儿对BCG的长期免疫反应。我将把这项工作与正在进行的Tollip缺陷小鼠的研究联系起来。 提供了关于TOLLIP在影响结核病免疫应答中的作用的重要信息，提供了对感染有效免疫应答的组成部分的深入了解，并可能导致结核病疫苗和疫苗佐剂的改进。

DESCRIPTION (provided by applicant): The applicant is a specialist in infectious diseases with a long-term interest in tuberculosis and international health. His background in cellular immunology is the basis for the proposed research. He studied cellular immunology of T cells and dendritic cells at the NIH as Howard Hughes Research Scholar as well as protein biochemistry as an undergraduate at the University of Chicago. He has an excellent publication record from his previous research experiences at the NIH, in the laboratory of Robert Seder and from work performed in the laboratory of Thomas Hawn. His immediate goals are to study the mechanisms of TOLLIP regulation of the innate immune response. He will accomplish this using a number of specialized resources as part of a training program. First, his mentor (Dr. Thomas Hawn) has experience studying common genetic variation in large human cohorts that will be applied for the first time to TOLLIP. Second, his co-mentor (Dr. Alan Aderem) has extensive experience in macrophage biology and will allow the applicant to study the role of TOLLIP variation on phagocytosis and autophagy. Third, he will determine the breadth of the effects of TOLLIP on the immune system by studying how TOLLIP may affect dendritic cell maturation, T cell polarization, and cytokine responses. The applicant's long term goals are to seek a clinical appointment in a Division of Infectious Diseases at an academic center, develop sources of independent research funding, and devote >75% of his time to running a lab and collaborative clinical studies. Tuberculosis stimulates intense inflammatory response, but regulation of this response is key to control of infection. The regulation and nature of an optimal inflammatory response to Mycobacterium tuberculosis (MTb) remains poorly understood in humans. Insight into mechanisms of negative regulation of the TLR- mediated innate immune response to MTb could provide significant breakthroughs in the design of new vaccines and drugs. We hypothesized that TOLLIP and its common variants negatively regulate TLR signaling in human monocytes and are associated with susceptibility to tuberculosis. Using shRNA knockdown of TOLLIP in peripheral blood human monocytes, we found that TOLLIP suppresses TNF and IL-6 production after stimulation with TLR2 and TLR4 ligands. We also discovered a common polymorphism (rs5743899) that is associated with TOLLIP deficiency in a Caucasian population. Furthermore, in a case-population study in Vietnam with 760 cord-blood samples and 671 TB case patients, we found that the SNP rs5743899 was associated with susceptibility to tuberculosis (p=6.97x10-7). Together, these data demonstrate that TOLLIP has an anti-inflammatory effect on TLR signaling in humans and that TOLLIP deficiency is associated with an increased risk of TB. These data implicate an unexpected mechanism of negative regulation of TLR signaling in human TB pathogenesis. In this application, we will examine human variants of TOLLIP and their role in regulating macrophage, dendritic cell, and T cell responses to MTb infection. In Aim 1, I will determine the specific polymorphisms commonly found in the population that affect TOLLIP function and influence risk for developing tuberculosis. Furthermore, we will more fully characterize how TOLLIP influences phagocytosis, autophagy, and mycobacterial replication in macrophages and dendritic cells. I will also combine our human population studies with mechanistic studies in Tollip -/- mice. In Aim 2, I will use multiple techniques, including mouse studies, study of ex vivo infant cytokine response to BCG, and a unique case-control study of pediatric TB in South Africa to study the in vitro and in vivo effects of TOLLIP variation on dendritic cell biology and the effect of TOLLIP variation on the regulation of innate immune priming of the adaptive immune response. This work will be performed at the University of Washington School of Medicine in Seattle. The applicant and proposed mentor are in the Division of Allergy and Infectious Diseases, which consists of 73 fulltime faculty members whose total grant support exceeds $135 million annually. This Division is an ideal environment for training physician-scientists as more than 80% of past fellowship trainees have obtained faculty positions in academic medicine. The applicant proposed research takes advantage of his strong background in cellular immunology but requires additional training in molecular biology, innate immunology, and human genetics. He has detailed coursework, seminars, and meetings to address these needs and has developed metrics for assessing his progress. He has also assembled a scientific advisory panel to provide one-on-one expertise. Finally, the proposed mentor has a demonstrated track record with these techniques and has successfully mentored several other junior scientists. NARRATIVE: Understanding the immunologic and genetic factors that confer protection from tuberculosis is vital to the development of successful vaccines and therapeutics. TOLLIP is a recently discovered regulator of the Toll-like receptor pathway that is critical for an effective immune response to tuberculosis. I propose to focus on the mechanisms by which TOLLIP regulates innate immunity by determining the specific common variants that affect TOLLIP function. I will also examine the role TOLLIP plays in regulating cellular uptake and destruction of tuberculosis. Lastly, I will study how TOLLIP influences long term immune responses to BCG in infants. I will correlate this work with ongoing studies in Tollip-deficient mice This work will provide important information about the role of TOLLIP in influencing the immune response to tuberculosis, provide insight into the components of an effective immune response to infections, and may lead to improved vaccines and vaccine adjuvants to tuberculosis.