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

描述（由申请人提供）：膳食饱和脂肪酸（FA），如棕榈酸（PA），在炎症相关疾病中起主要作用。PA通过先天免疫产生促炎性疾病的机制已得到充分证实，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已显示作用于树突状细胞（DC），树突状细胞是通过调节T细胞活性来协调适应性免疫应答的免疫细胞。然而，PA激活DC影响下游适应性免疫应答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基于炎症病因的疾病（如狼疮和骨关节炎）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炎症性疾病中DC功能的分子机制，以确定治疗靶点。这个博士前研究项目的总体目标是确定PA的作用 在调节Toll样受体4（TLR 4）信号转导中的作用。我们的中心假设是PA通过诱导TLR 4信号刺激DC，并通过ROS调节这些信号。该假设部分基于其他人的已发表工作和初步数据进行了阐述，并将通过以下具体目标进行客观测试：（1）确定PA是否结合TLR 4及其衔接蛋白MD-2;（2）表征PA诱导的DC活化;和（3）评价PA诱导的ROS对DC中TLR 4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、荧光结合试验和原子力显微镜来确定PA与TLR 4/MD-2相互作用的解离常数和热力学参数。在特定目标2中，流式细胞术、RT-PCR和siRNA敲低将用于表征PA对DC的共刺激因子产生、细胞因子分泌和T细胞刺激的影响。在具体目标3中，免疫荧光显微镜和新型荧光H2 O2探针将用于评估PA诱导的DC中H2 O2产生。将使用电泳迁移率变动分析（EMSA）、蛋白质印迹和siRNA敲低来评估ROS在DC中TLR 4信号传导中的作用。该方法是创新的，因为它侧重于一个新的范例的作用H2 O2在正常的DC生物学，特别是因为它将采用最近开发的荧光H2 O2探针的应用和TLR/MD-2和配体的三元结合模型的发展。这项研究意义重大，因为它将首次确定H2 O2在调节DC中TLR 4信号传导中的机制作用，并将PA定义为TLR 4配体。一旦确定了H2 O2的机制，靶向治疗，如ROS清除剂，抗氧化剂和信号传导抑制剂，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.