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.

项目总结/摘要 臭氧（O3）仍然是美国三分之一以上人口的重大公共卫生问题，122 2000万人，目前生活在超过国家环境空气质量标准（NAAQS）的地区， 是已知会引起人类炎症反应的暴露水平。O3具有高活性， 氧化生物分子，包括不饱和脂质，如胆固醇。然而，O3诱导的化学物质 反应转化为细胞内效应存在知识缺口。胆固醇的O3衍生产物 包括亲电体如氧甾醇，其具有与亲核中心形成加合物的能力， 蛋白质，从而影响细胞信号传导。本申请的总体目标是确定 氧化甾醇和氧化甾醇-蛋白质加合物将O3诱导的化学反应与生物效应联系起来。我们 开发了实验方案，其中气道上皮细胞（EC）用炔修饰的O3-处理， 随后使细胞裂解物与叠氮基生物素试剂在“点击”环加成下反应 条件下，导致任何蛋白质的生物素化，该蛋白质与炔修饰的 氧化固醇这种生物素化的蛋白质混合物可以被“拉下”用于“内收体”或“内收体”的蛋白质组学分析。 单独氧固醇-蛋白质加合物形成。使用蛋白质组学筛选形成的氧化甾醇-蛋白质加合物， 在EC中，我们将NLRP 2确定为潜在靶点。具体目标1将扩大这些初步研究， O3-衍生的氧化固醇在EC中产生的总蛋白“内收体”，并重点研究氧化固醇在EC中的作用。 在O3诱导的促炎反应中内收NLRP 2。具体目标2将侧重于如何O3衍生 氧化固醇影响巨噬细胞功能，并且无论是否类似于EC，氧化固醇在细胞中形成蛋白加合物， 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， 这一目的还将确定在EC膜处或附近形成的氧甾醇是否与 巨噬细胞具体目标3将集中在7-脱氢胆固醇（7-DHC），最后一个 胆固醇生物合成过程中的步骤和O3诱导的炎症。7-DHC对O3诱导的 氧固醇形成，我们有证据表明，增加肺7-DHC水平与O3诱导 人体内的炎症。此外，我们表明，修改7-DHC水平，通过通常规定的 小分子抗抑郁药增强O3诱导炎症。使用体外连接、小鼠体内连接和 人体内实验这一目的被设计为确定肺内7-羟色胺是如何调节肺内7-羟色胺的。 DHC水平可增加对O3诱导的炎症的易感性。这项研究的发现 将揭示氧化脂质和O3背景下细胞功能修饰之间的新型相互作用 暴露，并促进更好地了解常用处方药如何对O3诱导的 炎症