Ozone, oxysterols, and lung inflammation

臭氧、氧甾醇和肺部炎症

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要臭氧 (O3) 仍然是超过 1/3 的美国人口的重大公共卫生问题，122 目前居住在超过国家环境空气质量标准（NAAQS）的地区的人口是已知会引起人类炎症反应的暴露水平。O3 具有高度反应性，并且已知会引起炎症反应。氧化生物分子，包括不饱和脂质，例如胆固醇然而，O3- 是如何通过化学方式诱导的。反应转化为细胞内效应存在知识差距。包括亲电子试剂，例如氧甾醇，其能够与亲核中心形成加合物蛋白质，从而影响细胞信号传导，该应用的总体目标是确定如何形成。氧甾醇和氧甾醇蛋白加合物将 O3 诱导的化学反应与生物效应联系起来。开发了实验方案，其中气道上皮细胞 (EC) 用炔烃修饰的 O3- 进行处理衍生的氧甾醇，然后在“点击”环加成下使细胞裂解物与叠氮生物素试剂反应条件下，导致任何与炔烃修饰形成共价键的蛋白质发生生物素化这种生物素化的蛋白质混合物可以被“拉下来”用于“加合物”或的蛋白质组学分析。使用形成的氧甾醇-蛋白质加合物的蛋白质组学筛选。 EC，我们将 NLRP2 确定为潜在目标，具体目标 1 将扩展这些初步研究，描述其特征。 ECs 中 O3 衍生的氧甾醇产生的整体蛋白质“加合物”，并重点关注氧甾醇的作用具体目标 2 将关注 O3 诱导的促炎症反应中的加合 NLRP2。氧甾醇是否影响巨噬细胞功能并且与 EC 类似，氧甾醇在巨噬细胞中形成蛋白质加合物巨噬细胞，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，这一目标还将确定在 EC 膜处或附近形成的氧甾醇是否与具体目标 3 将重点关注最后一个 7-脱氢胆固醇 (7-DHC) 之间的关系。胆固醇生物合成过程中的一个步骤，并且 O3 诱导的炎症更容易受到 O3 诱导的炎症的影响。氧甾醇形成，我们有证据表明肺部 7-DHC 水平升高与 O3 诱导的相关此外，我们发现通过常用处方可以改变 7-DHC 水平。小分子抗抑郁药可增强 O3 诱导的炎症。人体体内实验此目的旨在确定如何药理调节肺 7- DHC 水平可能会增加对 O3 引起的炎症的敏感性。将揭示 O3 背景下氧化脂质与细胞功能改变之间的新相互作用接触并促进更好地了解常用处方药物如何对 O3 诱导敏感炎。