Ozone, oxysterols, and lung inflammation

臭氧、氧甾醇和肺部炎症

• 批准号: 9907305
• 负责人: ILONA JASPERS
• 金额: $ 6.83万
• 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 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907305
• 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 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(臭氧)仍然是一个严重的公共卫生问题，超过三分之一的美国人口，122 目前居住在超过国家环境空气质量标准(NAAQS)的地区， 已知暴露水平会在人体内引起炎症反应。O_3是高度活性的，已知 氧化生物分子，包括不饱和脂肪，如胆固醇。然而，臭氧如何诱导化学物质 转化为细胞内效应的反应存在知识鸿沟。胆固醇的臭氧衍生产物 包括具有与亲核中心形成加合物能力的亲电体，例如氧甾醇 蛋白质，从而影响细胞信号。这个应用程序的总体目标是确定如何形成 将臭氧诱导的化学反应与生物效应联系起来。我们 开发了用炔修饰的臭氧处理呼吸道上皮细胞(ECs)的实验方案。 衍生的氧甾醇，然后在“点击”环加成下，细胞裂解物与叠氮生物素试剂反应 条件，导致任何与炔修饰的蛋白质形成共价键的蛋白质的生物素化 氧类固醇。这种生物素化的蛋白质混合物可以“下拉”用于“内收组”的蛋白质组学分析。 个体氧固醇-蛋白质加合物的形成。利用蛋白质组学方法筛选形成于 ECS，我们确定NLRP2是一个潜在的目标。特定目标1将扩展这些初步研究，描述 在内皮细胞中由臭氧衍生的氧合甾醇产生的整体蛋白质“加合物”，并重点关注氧合甾醇- 在臭氧诱导的促炎反应中加成NLRP2。具体目标2将重点介绍臭氧是如何产生的 氧化甾醇影响巨噬细胞的功能以及是否类似于内皮细胞，氧化甾醇在 巨噬细胞，从而影响细胞功能。使用由内皮细胞和巨噬细胞组成的共培养系统， 这一目标还将确定在EC膜上或其附近形成的氧固醇是否与 巨噬细胞。具体目标3将集中在7-脱氢胆固醇(7-DHC)之间的关系，最后 胆固醇生物合成过程中的步骤，以及臭氧诱导的炎症。7-DHC对臭氧诱导更敏感 氧固醇的形成和我们有证据表明肺7-DHC水平升高与臭氧诱导相关 活体内的人体炎症。此外，我们还表明，通过通常规定的方式修改7-DHC水平 小分子抗抑郁剂可增强臭氧诱导的炎症反应。使用链接的体外、小鼠体内和 人体活体实验这个目的是为了确定如何从药物上调节肺7- DHC水平可增加对臭氧致炎的易感性。这项研究中得出的发现 将在臭氧的背景下揭示氧化脂质和细胞功能改变之间的新的相互作用 并促进更好地了解常用处方药是如何使人对臭氧致敏的 发炎。