Ozone, oxysterols, and lung inflammation

臭氧、氧甾醇和肺部炎症

• 批准号: 10132321
• 负责人: ILONA JASPERS
• 金额: $ 55.53万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10132321
• 关键词: 7-dehydrocholesterol; Affect; Air; Alkynes; Antidepressive Agents; Antipsychotic Agents; Area; Biochemical; Biological; Biotin; Biotinylation; Cell Communication; Cell membrane; Cell physiology; Cells; Cholesterol; Cholesterol Homeostasis; Coculture Techniques; Data; Drug Prescriptions; Drug usage; Epithelial; Epithelial Cells; Event; Experimental Models; Exposure to; Fostering; Health; Human; IL8 gene; In Vitro; Individual; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Knowledge; Link; Liquid substance; Lung; Lung Inflammation; Modeling; Modification; Mus; Oxidants; Oxides; Ozone; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phenotype; Play; Population; Predisposition; Proteins; Proteomics; Protocols documentation; Public Health; Reaction; Reagent; Research; Risk; Role; Scientist; Signal Transduction; Signaling Molecule; Signaling Protein; Surface; System; Testing; Translating; Translational Research; Unsaturated Fats; adduct; airway epithelium; airway inflammation; aripiprazole; base; chemical reaction; cholesterol biosynthesis; covalent bond; cycloaddition; design; experimental study; in vivo; innovation; macrophage; novel; oxidation; oxidized lipid; ozone exposure; pollutant interaction; protein function; response; small molecule; translational approach

PROJECT SUMMARY/ABSTRACT Ozone (O3) continues to be of great public health concern with more than 1/3 of the U.S. population, 122 million people, currently living in areas exceeding the National Ambient Air Quality Standard (NAAQS), which are exposure levels known to cause inflammatory responses in humans. O3 is highly reactive and known to oxidize biomolecules, including unsaturated lipids, such as cholesterol. Yet, how O3-induced chemical reactions translate into intracellular effects presents a knowledge gap. O3-derived products of cholesterol include electrophiles, such as oxysterol, which have the ability to form adducts with nucleophilic centers of proteins, thus affecting cellular signaling. The overall objective of this application is to determine how formation of oxysterols and oxysterol-protein adducts link O3-induced chemical reactions with biological effects. We developed experimental protocols in which airway epithelial cells (ECs) are treated with alkyne-modified O3- derived oxysterols followed by reacting the cell lysates with an azido biotin reagent under “click” cycloaddition conditions, resulting in the biotinylation of any protein that forms a covalent bond with alkyne-modified oxysterol. This biotinylated protein mixture can be “pulled down” for proteomic analysis of the “adductome” or individual oxysterol-protein adduct formation. Using a proteomic screen of oxysterol-protein adducts formed in ECs, we identified NLRP2 as a potential target. Specific Aim 1 will expand these initial studies, characterize the overall protein “adductome” generated by O3-derived oxysterol in ECs, and focus on the role of oxysterol- adducted NLRP2 in O3-induced pro-inflammatory responses. Specific Aim 2 will focus on how O3-derived oxysterols affect macrophage function and whether similar to ECs, oxysterols form protein adducts in macrophages, thus affecting cellular function. Using a co-culture system composed of ECs and macrophages, this aim will also determine whether oxysterols formed at or near EC membranes communicate with macrophages. Specific Aim 3 will focus on the relationship between 7-dehydrocholesterol (7-DHC), the last step during cholesterol biosynthesis, and O3-induced inflammation. 7-DHC is more susceptible to O3-induced oxysterol formation and we have evidence that increased lung 7-DHC levels correlate with O3-induced inflammation in humans in vivo. Furthermore, we show that modifying 7-DHC levels by commonly prescribed small molecule antidepressants enhance O3-induced inflammation. Using linked in vitro, mouse in vivo, and human in vivo experiments this aim is designed to determine how pharmacologically modulating pulmonary 7- DHC levels could increase the susceptibility to O3-induced inflammation. The findings developed in this study will uncover novel interactions between oxidized lipids and modification of cellular function in the context of O3 exposure and foster a better understanding of how commonly prescribed drugs could sensitize to O3-induced inflammation.

项目摘要/摘要 臭氧（O3）仍然对超过1/3人口的公共卫生关注，122 目前，有百万人民居住在超过国家环境空气质量标准（NAAQS）的地区 是已知会导致人类炎症反应的暴露水平。 O3具有高度反应性，已知 氧化生物分子，包括不饱和脂质，例如胆固醇。但是，O3诱导的化学品如何 反应转化为细胞内效应，列出了知识差距。 O3衍生的胆固醇产物 包括电泳，例如氧蛋白酶，它们具有与核螺旋中心形成加合物的能力 蛋白质，因此影响细胞信号。该应用程序的总体目的是确定如何形成 氧蛋白酶和氧蛋白酶蛋白质加合物的o3诱导的化学反应与生物学作用联系起来。我们 开发的实验方案，其中气道上皮细胞（EC）用醇修饰的O3-处理 衍生的氧蛋白酶，然后在“喀哒 条件，导致任何形成与醇修饰的共价键的蛋白质的生物素化 氧化酚。可以将这种生物素化蛋白混合物“拉下”以进行“加合体”或 单个氧蛋白质蛋白加合物的形成。使用形成的氧蛋白质蛋白质加合物的蛋白质组学筛选 ECS，我们将NLRP2确定为潜在目标。特定目标1将扩展这些初始研究，表征 O3衍生的氧甲醇在EC中产生的总体蛋白质“加合体”，并专注于氧甲醇的作用 在O3诱导的促炎反应中加入的NLRP2。特定目标2将重点介绍O3衍生的方式 氧蛋白酶会影响巨噬细胞功能，并且是否与EC相似，氧蛋白酶在 巨噬细胞，从而影响细胞功能。使用由EC和巨噬细胞组成的共培养系统， 该目的还将确定在EC机制或附近形成的氧甲甲醇是否与 巨噬细胞。具体目标3将重点放在7-脱氢胆固醇（7-DHC）之间的关系上，最后 在胆固醇生物合成和O3诱导的炎症期间的一步。 7-DHC更容易受到O3诱导的 氧化酚形成，我们有证据表明肺7-DHC水平与O3诱导的 体内人类炎症。此外，我们证明了通过普遍规定的7-DHC级别的修改 小分子抗抑郁药增强了O3诱导的炎症。使用链接的体外鼠标，体内和 人体体内实验该目标旨在确定药物如何调节肺7-- DHC水平可能会增加对O3诱导的注射的敏感性。这项研究的发现 将发现氧化脂质与O3背景下细胞功能的修饰之间的新型相互作用 曝光并促进对常见药物对O3诱导的方式有更好的了解 炎。