Free Radicals and Redox Signaling Triggered by Lipopolysccharide in Macrophages

环境和代谢相关的人类疾病中氧化应激诱导的炎症机制仅部分了解。该提案的长期目标是应用本项目中发现/开发的见解和技术，以了解环境-肺-慢性炎症性疾病的分子基础。具体的假设是：以生物大分子为中心的自由基是在巨噬细胞活化期间由脂多糖（LPS）触发的氧化还原信号传导的一部分。该假说基于以下观察结果：1）LPS诱导细胞中的活性氧（ROS）; 2）ROS可以从溶酶体中动员氧化还原活性铁; 3）铁与大分子结合并与ROS原位反应产生羟基自由基; 4）羟基自由基从大分子中提取电子产生自由基。具体的目的是为了测试我们的假设，使用免疫自旋捕获（1ST）与硝酮自旋陷阱5，5，-二甲基-吡咯啉A/-氧化物（DMPO），并改善我们的方法检测和表征这些自由基作为其DMPO硝酮加合物衍生物。具体目标1：确定铁催化氧化在触发氧化还原信号传导中的作用，该氧化还原信号传导产生LPS诱导的巨噬细胞活化。为了实现这一目标，我们将：a）确定触发氧化还原信号传导的ROS的来源和类型，B）确定LPS诱导的ROS是否介导铁从溶酶体释放到细胞质，以及c）研究巨噬细胞活化期间生物大分子的铁催化氧化的LPS诱导的标志物。具体目标2：优化、验证和应用DMPO的1ST来表征LPS诱导的巨噬细胞活化过程中产生的蛋白质自由基。为了实现这一目标，我们将：a）免疫（细胞）化学分析DMPO-蛋白硝酮加合物，B）制备用抗DMPO抗体包被的珠以免疫纯化硝酮加合物，和c）通过质谱分析表征纯化的DMPO硝酮加合物。具体目的#3：优化、验证和应用具有DMPO的1ST来检测LPS诱导的巨噬细胞活化中的总mRNA自由基。为了实现这一目标，我们将：a）检测DMPO-RNA硝酮加合物，B）开发并验证用于检测mRNA自由基的1ST，以及c）将1ST应用于检测LPS激活的巨噬细胞和暴露于LPS的小鼠肺中诱导的总mRNA自由基。相关性（参见说明）：旨在确定新靶点和暴露早期标志物的新研究将帮助我们开发更好的诊断和治疗方法，用于氧化应激诱导的炎症介导的人类疾病。

The mechanism of oxidative stress-induced inflammation in environmental- and metabolism-related human diseases is only partially understood. The long-term goal of this proposal is to apply insights and techniques discovered/developed in the present project to understand the molecular basis of environmental-lung-chronic inflammatory diseases. The specific hypothesis is: biomacromolecule-centered free radicals are part of the redox signaling triggered by lipopolysaccharide (LPS) during macrophage activation. This hypothesis is based on the following observations: 1) LPS induces reactive oxygen species (ROS) in cells; 2) ROS can mobilize redox-active iron from lysosomes; 3) iron binds to macromolecules and reacts in situ with ROS producing hydroxyl radicals; 4) hydroxyl radicals abstract electron(s) from macromolecules producing free radicals. The specific aims are designed to test our hypothesis using immuno-spin trapping (1ST) with the nitrone spin trap 5,5,-dimethyM-pyrroline A/-oxide (DMPO) and to improve our methods of detecting and characterizing those free radicals as their DMPO nitrone adduct-derivatives. SPECIFIC AIM#1: To determine the role of iron-catalyzed oxidations in triggering the redox signaling that produces LPS-induced activation of macrophages. To achieve this aim we will: a) determine the source and the type of ROS that triggers the redox signaling, b) determine whether the ROS induced by LPS mediates the release of iron from lysosomes to the cytoplasm, and c) study LPS-induced markers of iron-catalyzed oxidation of biomacromolecules during macrophage activation. SPECIFIC AIM # 2: To optimize, validate, and apply 1ST with DMPO to characterize protein radicals produced during LPS-induced activation of macrophages. To achieve this aim we will: a) immuno(cyto)chemically analyze DMPO-protein nitrone adducts, b) prepare beads coated with anti-DMPO antibodies to immunopurify nitrone adducts, and c) characterize purified DMPO nitrone adducts by mass spectrometric analyses. SPECIFIC AIM #3: To optimize, validate, and apply 1ST with DMPO to.detect total mRNA radicals in LPS- induced activation of macrophages. To achieve this aim we will: a) detect DMPO-RNA nitrone adducts, b) develop and validate 1ST for detecting mRNA radicals, and c) apply 1ST to the detection of total mRNA radicals induced in LPS-activated macrophages and in the lungs of mice exposed to LPS. RELEVANCE (See instructions): New studies aimed at identifying novel targets and early markers of exposure will help us develop better diagnostic and therapeutic approaches for oxidative stress-induced inflammation-mediated human diseases.