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

描述(申请人提供)：膳食饱和脂肪酸(FA)，如棕榈酸(PA)，在炎症相关疾病中发挥主要作用。PA通过先天免疫产生促炎条件的机制已经得到了很好的证明，但它作用于适应性免疫系统从而产生疾病的途径还不太清楚。PA已被证明作用于树突状细胞(DC)，树突状细胞是一种通过调节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影响下游获得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，免疫细胞生物学可能为狼疮和骨关节炎等基于炎症的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在炎症性疾病中作用的分子机制，以便确定治疗靶点。这个博士前研究项目的总体目标是确定PA的角色 在DC中调节Toll样受体4(TLR4)信号。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。这一假说部分是基于已发表的工作和初步数据提出的，并将通过以下具体目标进行客观检验：(1)确定PA是否与TLR4及其适配蛋白MD-2结合；(2)表征PA诱导的DC激活；(3)评估PA诱导的ROS对DC中TLR4信号的调节。在具体目标1中，我们将使用等温滴定量热法、荧光结合分析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，将使用流式细胞术、RT-PCR和siRNA敲除来表征PA对DC产生共刺激因子、分泌细胞因子和刺激T细胞的影响。在具体目标3中，将使用免疫荧光显微镜和新型荧光双氧水探针来评估PA诱导的DC产生双氧水。凝胶迁移率改变分析(EMSA)、蛋白质印迹和siRNA敲除将被用来评估ROS在DC的TLR4信号转导中所起的作用。这种方法是创新的，因为它关注了过氧化氢在正常DC生物学中所起作用的新范式，特别是因为它将使用最近开发的荧光过氧化氢探针，并开发TLR/MD-2与配体的三元结合模型。这项拟议的研究意义重大，因为它将首次确定过氧化氢在树突状细胞TLR4信号调节中的机械作用，并将PA定义为TLR4配体。一旦确定了过氧化氢的作用机制，靶向治疗药物，如ROS清除剂、抗氧化剂和信号转导抑制剂，就可以与饮食干预相结合，以实现最大的健康益处。

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.