Free Radicals and Redox Signaling Triggered by Lipopolysccharide in Macrophages

在环境和代谢相关的人类疾病中，氧化应激诱导炎症的机制仅部分被理解。本提案的长期目标是应用本项目中发现/开发的见解和技术来了解环境-肺-慢性炎症性疾病的分子基础。具体假设为：巨噬细胞活化过程中，以生物大分子为中心的自由基是脂多糖（LPS）触发的氧化还原信号的一部分。这一假设基于以下观察结果：1)LPS诱导细胞中的活性氧（ROS）；2)活性氧可以调动溶酶体中的氧化还原活性铁；3)铁与大分子结合，与ROS发生原位反应，产生羟基自由基；羟基自由基从产生自由基的大分子中吸取电子。具体目的是利用免疫自旋捕获(1)和硝基自旋捕获5,5，-二甲基吡咯啉A/-氧化物（DMPO）来验证我们的假设，并改进我们检测和表征这些自由基作为其DMPO硝基加合物衍生物的方法。具体目的1：确定铁催化氧化在触发氧化还原信号中的作用，氧化还原信号产生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向细胞质的释放，以及c)研究巨噬细胞活化过程中LPS诱导的铁催化生物大分子氧化标记物。特定目标# 2：优化、验证和应用DMPO来表征lps诱导的巨噬细胞活化过程中产生的蛋白自由基。为了实现这一目标，我们将：a)免疫（细胞）化学分析DMPO-蛋白硝基酮加合物，b)制备涂有抗DMPO抗体的微球来免疫纯化硝基酮加合物，c)通过质谱分析来表征纯化的DMPO硝基酮加合物。特异性目标#3：优化、验证和应用DMPO检测LPS诱导的巨噬细胞激活中的总mRNA自由基。为了实现这一目标，我们将：a)检测DMPO-RNA硝基加合物，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的小鼠肺部诱导的总mRNA自由基。相关性（见说明）：旨在确定新靶点和早期暴露标记的新研究将帮助我们为氧化应激诱导的炎症介导的人类疾病开发更好的诊断和治疗方法。

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.