ENDOTOXIN EXPOSURE IN THE MACROPHAGE: ANALYSIS OF LIPID RAFT PROTEOMICS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 虽然严重创伤后的初始存活率有所改善，但由于多器官功能障碍综合征(MODS)的发展，长期恢复经常受到影响。创伤后，MODS通常是对感染和相关因素的反应，如脂多糖(LPS)，这是脓毒症的特征。创伤后和脓毒症期间，循环中的单核细胞和组织固定的巨噬细胞经历表型分化和重新编程，导致器官损伤。在我们的背景研究中，我们发现，在体外，严重创伤诱导的因素，如血小板活化因子(PAF)、氧化应激和补体5a(C5a)，以及耐受诱导，都可以模拟这种状态。 细胞重编程与炎症介质的显著释放无关。相反，细胞重新编程导致炎症介质产生失调，以应对随后的感染刺激，从而增加器官功能障碍的风险。其机制与神经鞘磷脂酶最初产生神经酰胺有关，导致被称为脂筏的特殊膜成分的流动性改变。由于这种凝胶相流动性，动力学发生改变，导致RAFT蛋白的重组。这种蛋白质含量的变化与Toll样受体(TLR)组分的预组装有关，这种预组装导致TLR复合体的组装和激活，以响应随后的刺激。为了探索相关的分子机制，我们将使用蛋白质组学技术在RAFT和非RAFT蛋白分离株上测试这些复合体的形成发生在细胞重新编程和激活过程中的假设。 该项目的具体目标是： 1)测定脂多糖刺激后单个核细胞的脂筏特征 2)测定严重损伤诱导因子重编程后单个核细胞的脂筏特征

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. While initial survival following severe trauma has improved, long term recovery is frequently impaired by the development of multiple organ dysfunction syndrome (MODS). Following trauma, MODS is usually in response to infection and associated factors, such as lipopolysaccharide (LPS), which is characteristic of sepsis. Following trauma and during sepsis, the circulating monocyte and tissue-fixed macrophage undergo phenotypic differentiation and reprogramming that results in organ injury. In our background studies, we found that this state can be mimicked in vitro by factors induced by severe trauma, such as platelet activating factor (PAF), oxidant stress and complement 5a (C5a) and by tolerance induction. Cellular reprogramming is not associated with significant liberation of inflammatory mediators. Rather, cellular reprogramming leads to dysregulated inflammatory mediator production in response to subsequent infectious stimuli, thus increasing the risk for organ dysfunction. The mechanism responsible is associated with initial ceramide generation by sphingomyelinase, resulting in altered fluidity within specialized membrane components termed lipid rafts. Due to this gel-phase fluidity, altered kinetics occur leading to re-organization of raft proteins. This alteration in protein content is associated with the pre-assembly of Toll-like receptor (TLR) components that results in accelerated TLR complex assembly and activation in response to subsequent stimuli. To explore the molecular mechanisms responsible, we will test the hypothesis that formation of these complexes occurs during cellular reprogramming and activation using proteomic techniques on raft and non-raft protein isolates. The specific aims of this project are to: 1) Determine the lipid raft characteristics of mononuclear cells following LPS stimulation 2) Determine the lipid raft characteristics of mononuclear cells following reprogramming by factors induced by severe injury