TOLLIP and Tuberculosis Immunopathogenesis

申请人简介（由申请人提供）：申请人是传染病专家，对结核病和国际卫生有长期兴趣。他在细胞免疫学方面的背景是这项拟议研究的基础。他作为霍华德休斯研究学者在美国国立卫生研究院学习T细胞和树突状细胞的细胞免疫学，在芝加哥大学读本科时学习蛋白质生物化学。他之前在NIH， Robert Seder的实验室和Thomas Hawn的实验室进行的研究工作都有很好的发表记录。他的近期目标是研究TOLLIP调控先天免疫反应的机制。作为培训计划的一部分，他将使用一些专门的资源来完成这项工作。首先，他的导师（Dr. Thomas Hawn）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拥有丰富的经验，将允许申请人研究TOLLIP变异在吞噬作用和自噬中的作用。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科寻求临床职位，发展独立研究资金来源，并将其75%的时间用于管理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。人类对结核分枝杆菌（MTb）的最佳炎症反应的调节和性质仍然知之甚少。了解TLR介导的MTb先天免疫应答的负调控机制可能为新疫苗和新药物的设计提供重大突破。我们假设TOLLIP及其常见变异负调控人类单核细胞中的TLR信号，并与结核病易感性相关。通过shRNA敲低人外周血单核细胞TOLLIP，我们发现TLR2和TLR4配体刺激后TOLLIP抑制TNF和IL-6的产生。我们还在高加索人群中发现了与TOLLIP缺乏相关的常见多态性（rs5743899）。此外，在越南的一项包含760份脐带血样本和671例结核病患者的病例群体研究中，我们发现SNP rs5743899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信号具有抗炎作用，TOLLIP缺乏与结核病风险增加有关。这些数据暗示了TLR信号负调控在人类结核病发病中的一个意想不到的机制。在本应用中，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细胞对结核分枝杆菌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功能和影响患结核病风险的特定多态性。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、自噬和分枝杆菌复制。我还将把我们的人口研究与Tollip -/-小鼠的机制研究结合起来。在Aim 2中，我将使用多种技术，包括小鼠研究，体外婴儿细胞因子对卡介苗反应的研究，以及南非儿童结核病的独特病例对照研究，研究体外和体内效应

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.