Towards a Molecular Signature of Neutrophil Priming

描述（由申请人提供）：中性粒细胞预充是调节良好的炎症反应的一个组成部分，是控制微生物感染和修复损伤的第一步。在该模型中，初始损伤“引发”中性粒细胞，使得在未引发的细胞中原本会缓和的二次损伤现在导致夸大的炎症反应。细胞因子和有毒氧自由基的过度和不受调节的释放导致邻近组织的损伤，导致组织功能障碍、器官衰竭和死亡。对人中性粒细胞引发后启动的精确分子途径知之甚少。目前的建议集中在定量磷酸蛋白质组学技术的应用，以了解独特的蛋白质组范围内的酪氨酸磷酸化事件与人类中性粒细胞引发暴露于低剂量的N-甲酰-蛋氨酸-亮氨酸-苯丙氨酸，脂多糖或血小板活化因子。使用目前可用的定量磷酸化蛋白质组学技术和专业知识，在我们的实验室与原代人类中性粒细胞，我们将在不同的时间点，样本的致敏中性粒细胞和量化的相对变化，在大规模的酪氨酸磷酸化从总细胞裂解物的细胞肽。从该项目中获得的信息包括：中性粒细胞引发后随时间发生的酪氨酸磷酸化事件的全球识别;在引发的中性粒细胞中激活的信号转导途径网络的可视化;随着时间的推移从不同促炎介质引发的中性粒细胞中发生的激活事件的比较。这种类型的定量分析的全局酪氨酸磷酸化位点在中性粒细胞引发的时间过程中沿着与传统的生化分析将允许中性粒细胞引发的信号通路网络模型的构建。这些研究结果将共同检验以下假设：中性粒细胞的启动状态包括促炎介质中常见的信号传导事件，并且可以呈现为这种基本细胞反应的分子特征。鉴于响应的中性粒细胞必须解释炎症部位的许多信号，这些研究的结果可能有助于开发新的诊断或治疗试剂，旨在限制患者因败血症和创伤而遭受的自身炎症组织损伤。公共卫生相关性：神经元引发是调节良好的炎症反应的一个组成部分，是控制微生物感染和损伤修复的第一步。对人中性粒细胞引发后启动的精确分子途径知之甚少。在这个建议中，我们介绍了定量质谱的现代方法，以促进中性粒细胞启动后，由各种代理商的细胞信号转导通路的表征，提供一个全球性的观点正常和引发细胞的磷酸化状态。

DESCRIPTION (provided by applicant): Neutrophil priming is an integral aspect of a well-regulated inflammatory response and is the first step in controlling microbial infection and repair of injury. In this model, an initial injury "primes" neutrophils such that a secondary insult, which would otherwise be tempered in an unprimed cell, now results in an exaggerated inflammatory response. The excessive and unregulated release of cytokines and toxic oxygen radicals causes damage to adjacent tissues leading to tissue dysfunction, organ failure and death. The precise molecular pathways initiated upon priming of human neutrophils are poorly understood. The present proposal focuses on the application of quantitative phosphoproteomic technology to understand the unique proteome-wide tyrosine phosphorylation events associated with human neutrophil priming by exposure to low dose N-formyl-met-leu-phe, lipopolysaccharide or platelet activating factor. Using currently available quantitative phosphoproteomic technology and expertise with primary human neutrophils in our laboratories, we will sample primed neutrophils at various time points and quantify relative changes in wide-scale tyrosine phosphorylation from total cell lysates of cellular peptides. Information to be derived from this project include: global identification of tyrosine phosphorylation events that occur over time after neutrophil priming; visualization of the network of signal transduction pathways that are activated in the primed neutrophils; comparison of activation events that take place over time from neutrophils primed with different proinflammatory mediators. This type of quantitative analysis of global tyrosine phosphorylation sites during a time course of neutrophil priming along with traditional biochemical analysis will permit the construction of a signaling pathway network model for neutrophil priming. Together these findings will test the hypothesis that the primed state of the neutrophil includes signaling events that are common among proinflammatory mediators and can be presented as a molecular signature of this fundamental cellular response. Given that responding neutrophils must interpret a number of signals at a site of inflammation, the results from these studies may assist in the development of novel diagnostic or therapeutic reagents aimed at limiting the auto-inflammatory tissue damage patients suffer as a result of sepsis and trauma. PUBLIC HEALTH RELEVANCE: Neutrophil priming is an integral aspect of a well-regulated inflammatory response and is the first step in controlling microbial infection and repair of injury. The precise molecular pathways initiated upon priming of human neutrophils are poorly understood. In this proposal we introduce modern methods in quantitative mass spectrometry to facilitate the characterization of the cellular signaling pathways initiated upon neutrophil priming by a variety of agents by providing a global view of the phosphorylation state of normal and primed cells.