TRAUMA-INDUCED REPROGRAMMING: CHANGES IN LIPID RAFT PROTEIN CONTENT

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。 严重创伤后，单个核细胞被重新编程，导致先天免疫功能改变。这些表型导致对入侵生物的易感性增加，从而导致器官衰竭的发生。在体外，通过使单个核细胞受到创伤诱导的因素，包括血小板激活因子(PAF)、氧化应激和补体5a，重新建立了这种状态。 (C5a)。尽管负责这种重编程的机制(S)尚不清楚，但先前的工作已经证明，这一过程可能与特定质膜微域中蛋白质含量的变化有关，这些微域富含胆固醇和被称为脂筏的鞘磷脂。损伤后，我们假设创伤诱导的因素导致脂筏产生脂质介导物神经酰胺。神经酰胺曾经在筏子内产生熔丝，导致大区域的形成，导致膜流动性的变化。由于这些变化，各种蛋白质被招募到脂筏上，导致焦点粘附样复合体的形成，这些复合体包含部分但不是所有的Toll样受体(TLR)成分。实验方法如下：将分化的THP-1细胞置于脂多糖(LPS)刺激下不同的时间段，最长为60分钟。选定的细胞将用PAF、过氧化氢或C5a进行长达30-60分钟的预处理。脂筏蛋白的提取将采用蔗糖梯度离心法。然后，收集的蛋白质将用于LC-ESI-MS系统的分析。我们的假设是，这些复合体的组装和脂筏含量的变化是导致随后的重新编程导致增强激活以应对后续感染的原因。基于这些体外观察，我们希望随后探索严重创伤患者发生的潜在变化，以确定潜在的预后和治疗靶点。

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. Following severe trauma mononuclear cells are reprogrammed leading to alterations in innate immunity. These phenotypes are responsible for increased susceptibility to invading organisms leading to the development of organ failure. This state has been recreated in vitro by subjecting mononuclear cells to factors induced by trauma, including platelet activating factor (PAF), oxidant stress and complement 5a (C5a). Although the mechanism(s) responsible for this reprogramming remain unknown, previous work has demonstrated that this process may be associated with alterations in the protein content within specific plasma membrane microdomains that are rich in cholesterol and sphingolipids termed lipid rafts. Following injury, we hypothesize that factors induced by trauma result in the production of the lipid mediator ceramide from lipid rafts. Ceramide once produced fuses within rafts leading to the formation of macrodomains resulting in changes in membrane fluidity. Due to these changes, various proteins are recruited to the lipid raft resulting in the formation of focal adhesion-like complexes that contain some but not all of the Toll-like receptor (TLR) components. The following experimental approach will be followed: Differentiated THP-1 cells will be subjected to lipopolysaccharide (LPS) stimulation for various periods of time up to 60 min. Selected cells will be pre-treated with PAF, hydrogen peroxide or C5a for periods of time up to 30-60 min. Lipid raft protein extraction will be performed using sucrose gradient centrifugation. Harvested proteins will then be used for analysis using the LC-ESI-MS system. It is our hypothesis that assembly of these complexes and changes in lipid raft content are responsible for subsequent reprogramming that induces enhanced activation in response to subsequent infection. Based on these in vitro observations, it is our hope to then explore potential changes that occur in severely injured trauma patients in order to determine potential prognostic and therapeutic targets.