Palmitic Acid Regulation of Dendritic Cell Toll-Like Receptor 4 Signaling

棕榈酸对树突状细胞 Toll 样受体 4 信号传导的调节

基本信息

批准号：
8741033
负责人：
Dequina Angelina Nicholas
金额：
$ 4.09万
依托单位：
LOMA LINDA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-17 至 2015-08-20
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8741033
关键词：
Achievement Antioxidants Atomic Force Microscopy Binding Biological Assay Blood Cells Calorimetry Cell Culture Techniques Cell model Cell physiology Cells Cellular biology Data Degenerative polyarthritis Dendritic Cells Dendritic cell activation Development Diabetes Mellitus Diet Dietary Intervention Dimerization Disease Dissociation Electrophoretic Mobility Shift Assay Etiology Flow Cytometry Fluorescence Goals Health Benefit Hydrogen Peroxide Immune Immune response Immune system Immunofluorescence Immunologic Immunology In Vitro Inflammation Inflammatory Knowledge Ligands Ligation Lipids Lupus Mediating Modeling Molecular Molecular Target Natural Immunity Nature Palmitic Acids Pathway interactions Phenotype Phosphorylation Play Production Public Health Publishing Reactive Oxygen Species Regulation Reporting Research Research Project Grants Reverse Transcriptase Polymerase Chain Reaction Role Saturated Fatty Acids Signal Transduction Signaling Protein Small Interfering RNA T-Cell Proliferation T-Lymphocyte Testing Therapeutic Therapeutic Intervention Thermodynamics Time Titrations Translating Western Blotting Work adapter protein base cytokine cytotoxic drug discovery improved inhibitor/antagonist innovation new therapeutic target novel pre-doctoral prevent protein tyrosine phosphatase 1B public health relevance response therapeutic target toll-like receptor 4

项目摘要

DESCRIPTION (provided by applicant): Dietary saturated fatty acids (FAs), such as palmitic acid (PA), play a major role in inflammation- associated disease. The mechanisms by which PA produces pro-inflammatory conditions through innate immunity are well-documented, but the pathways through which it acts on the adaptive immune system to produce disease are less clear. PA has been shown to act on dendritic cells (DCs), the immune cells that coordinate adaptive immune responses by regulating T cell activity. However, the molecular mechanisms by which PA activates DCs to influence downstream adaptive immune responses are unknown. This gap in knowledge prevents an understanding of immune cell biology that could provide new molecular targets for therapeutic drug discovery for diseases with inflammation-based etiology such as lupus and osteoarthritis. Thus, our long-term goal is to understand the molecular mechanisms underlying DC function for inflammatory diseases in order to identify therapeutic targets. The overall objective of this pre-doctoral research project is to determine the role of PA in regulating Toll-like receptor 4 (TLR4) signaling in DCs. Our central hypothesis is that PA stimulates DCs by inducing TLR4 signals and regulates these signals through ROS. This hypothesis was formulated based in part on the published work of others and on preliminary data and will be objectively tested through the following specific aims: (1) Determine whether PA binds TLR4 and its adapter protein MD-2; (2) Characterize PA induced DC activation; and (3) Evaluate PA induced ROS regulation of TLR4 signaling in DCs. In Specific Aim 1, we will use isothermal titration calorimetry, fluorescence binding assays, and atomic force microscopy to determine the dissociation constant and thermodynamic parameters of PA interaction with TLR4/MD-2. In Specific Aim 2, flow cytometry, RT-PCR, and siRNA knockdown will be used to characterize PA impact on co-stimulatory factor production, cytokine secretion, and T cell stimulation by DCs. In Specific Aim 3, Immunofluorescence miscroscopy, and novel fluorescent H2O2 probes will be used to assess PA induced H2O2 production in DCs. Electrophoretic mobility shift assay (EMSA), western blot, and siRNA knockdown will be used to assess the roles that ROS play in TLR4 signaling in DCs. The approach is innovative, because it focuses on a new paradigm for the role H2O2 has in normal DC biology, and especially because it will employ application of recently developed fluorescent H2O2 probes and the development of a ternary binding model for TLR/MD-2 and a ligand. The proposed research is significant because, it will, for the first time, define a mechanistic role for H2O2 in the regulation of TLR4 signaling in DCs and define PA as a TLR4 ligand. Once H2O2 mechanisms are defined, targeted therapeutics such as ROS scavengers, anti-oxidants, and signaling inhibitors can be combined with dietary interventions to achieve maximum health benefits.

描述（由申请人提供）：膳食饱和脂肪酸（FA），例如棕榈酸（PA），在炎症相关疾病中发挥重要作用。 PA 通过先天免疫产生促炎症条件的机制已得到充分记录，但其作用于适应性免疫系统以产生疾病的途径尚不清楚。 PA 已被证明可以作用于树突状细胞 (DC)，树突状细胞是通过调节 T 细胞活性来协调适应性免疫反应的免疫细胞。然而，PA 激活 DC 影响下游适应性免疫反应的分子机制尚不清楚。这种知识上的差距阻碍了对免疫细胞生物学的理解，而免疫细胞生物学可以为狼疮和骨关节炎等炎症性疾病的治疗药物发现提供新的分子靶点。因此，我们的长期目标是了解 DC 对炎症性疾病功能的分子机制，以确定治疗靶点。该博士前研究项目的总体目标是确定 PA 的作用调节 DC 中 Toll 样受体 4 (TLR4) 信号传导。我们的中心假设是 PA 通过诱导 TLR4 信号刺激 DC，并通过 ROS 调节这些信号。这一假设的提出部分基于他人已发表的工作和初步数据，并将通过以下具体目标进行客观检验：（1）确定PA是否与TLR4及其衔接蛋白MD-2结合； (2) 表征 PA 诱导的 DC 激活； (3) 评估 DC 中 PA 诱导的 ROS 对 TLR4 信号传导的调节。在具体目标 1 中，我们将使用等温滴定量热法、荧光结合测定和原子力显微镜来确定 PA 与 TLR4/MD-2 相互作用的解离常数和热力学参数。在具体目标 2 中，流式细胞术、RT-PCR 和 siRNA 敲低将用于表征 PA 对共刺激因子产生、细胞因子分泌和 DC 刺激 T 细胞的影响。在具体目标 3 中，免疫荧光显微镜和新型荧光 H2O2 探针将用于评估 PA 诱导 DC 中 H2O2 的产生。电泳迁移率变动分析 (EMSA)、蛋白质印迹和 siRNA 敲低将用于评估 ROS 在 DC 中 TLR4 信号传导中所发挥的作用。该方法具有创新性，因为它专注于 H2O2 在正常 DC 生物学中的作用的新范例，特别是因为它将采用最近开发的荧光 H2O2 探针的应用以及 TLR/MD-2 和 TLR/MD-2 的三元结合模型的开发。配体。这项研究意义重大，因为它将首次定义 H2O2 在 DC 中 TLR4 信号传导调节中的机制作用，并将 PA 定义为 TLR4 配体。一旦确定了 H2O2 机制，ROS 清除剂、抗氧化剂和信号抑制剂等靶向治疗方法就可以与饮食干预相结合，以实现最大的健康益处。