Effect of PPARy Ligands on Alcohol-Induced Alveolar Macrophage Oxidative Stress

PPARy 配体对酒精诱导的肺泡巨噬细胞氧化应激的影响

基本信息

批准号：
9188026
负责人：
Samantha M. Yeligar
金额：
$ 24.9万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2018-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9188026
关键词：
Address Adult Respiratory Distress Syndrome Alcohol abuse Alcohols Alveolar Cell Alveolar Macrophages Animal Model Apoptotic Applications Grants Area Attenuated Award Biology Biomedical Research Biostatistical Methods California Cell Line Chronic Clinical Research Clinical Trials Data Disease Down-Regulation Endothelial Cells Enzymes Ethanol Experimental Models Faculty Functional disorder Future Genetic Transcription Goals Grant Hormones Impairment In Vitro Inflammation Mediators Inflammatory Ingestion Institution Investigation Klebsiella pneumonia bacterium Knock-out Knockout Mice Laboratories Learning Skill Ligands Liver Lung Lung Capacity Maintenance Manuscripts Mediating Mentors Mentorship Messenger RNA Methods MicroRNAs Microbe Molecular Biology Molecular Biology Techniques Mus NADPH Oxidase NADPH Oxidase 1 Nuclear Oxidative Stress PPAR gamma Pathway interactions Patient risk Patients Phagocyte Bactericidal Dysfunction Phagocytes Phagocytosis Phase Physiological Pioglitazone Play Positioning Attribute Postdoctoral Fellow Preparation Production Proteins Publications Reactive Oxygen Species Recording of previous events Regulation Research Research Personnel Resources Respiratory Burst Respiratory Tract Infections Risk Role Source Talents Techniques Therapeutic Thiazolidinediones Training Transgenic Mice Transgenic Organisms Universities Writing alcohol effect alcohol exposure alcohol research career career development chronic alcohol ingestion clinically relevant diabetic patient experience graduate student human disease human subject immune function improved in vivo in vivo Model insulin sensitivity interest killings loss of function mRNA Transcript Degradation macrophage meetings mouse model novel novel therapeutic intervention post-doctoral training pre-doctoral problem drinker programs receptor responsible research conduct rosiglitazone skills transcription factor translational impact

项目摘要

Chronic alcohol abuse increases patients’ risk of developing Acute Respiratory Distress Syndrome (ARDS) and respiratory infections. In alveolar macrophages (AMs), NADPH oxidase (Nox) 1, Nox2, and Nox4 are critical sources of reactive oxygen species (ROS), and Nox2 is essential for the respiratory burst involved in killing microbes after phagocytosis. However, excessive ROS production suppresses phagocytosis. Chronic alcohol ingestion increases Nox enzyme levels, leading to AM oxidative stress and dysfunction. These alcohol-induced derangements can be reversed by treatment with peroxisome proliferator-activated receptor gamma (PPARγ) ligands, such as pioglitazone and rosiglitazone. In these studies, we will determine if the effects of alcohol on AM Nox expression and activity are modulated by microRNAs (miRs): Nox1-related miR- 1264, Nox2-related miR-107, and Nox4-related miRs-363 and -92a/b (Aim 1). Then, we will determine the capacity of PPARγ ligands to modulate these miRs to reverse alcohol-mediated AM Nox1, Nox2, and Nox4 expression, oxidative stress and compromised phagocytosis (Aim 2). These hypotheses will be investigated by using a murine model of chronic alcohol consumption, an in vitro ethanol exposed mouse AM cell line, MHS, and AMs isolated from human subjects. During the K99 phase, the applicant will receive hands-on training in the intra-tracheal delivery of Klebsiella pneumonia to mice to assess the physiological capacity of PPARγ ligands to attenuate alcohol-induced effects on miRs, Nox expression, and AM killing and bacterial clearance. Additional training will include colony maintenance of transgenic and knockout murine models and novel methods of intra-nasal delivery of PPARγ ligands or other therapeutics to mice. The objective of the studies outlined in this proposal is to demonstrate that targeting PPARγ constitutes a novel therapeutic approach to ameliorate alcohol-induced AM dysfunction. If successful, these investigations could have considerable translational impact on the management of patients with a history of alcohol abuse by setting the stage for future clinical studies. The PI’s focus in alcohol research began with during her pre-doctoral dissertation project investigating the detrimental effects of chronic alcohol abuse in the liver, focusing on mechanisms underlying the participation of ROS and inflammatory mediators that alter liver endothelial cell and macrophage function. During post-doctoral training in the laboratories of Drs. Brown and Hart, she acquired additional expertise with numerous molecular biology techniques and with animal models of chronic alcohol ingestion. During the mentored phase of the proposed project she will further expand her repertoire of skills by learning: a) techniques to perform and characterize bacterial challenges in the airways of murine models to assess alveolar macrophage phagocytosis in vivo, b) methods to directly deliver therapeutics to the lungs of mouse models, and c) skills required to develop and manage colonies of knockout and transgenic mice. These skills will be acquired through the execution of the proposed studies with the assistance and training of the mentors’ labs during the first two years of the proposed project period. The focus of these studies will permit a natural extension of the candidate’s interest in the regulation of microRNAs in the context of chronic alcohol ingestion. It is anticipated that her additional expertise in this area will naturally promote her growing independence from her mentors. Further, during the proposed award, in addition to didactic courses in current molecular biology techniques and biostatistical methods, the applicant will gain non-laboratory skills important for her career development as an independent research investigator by participating in seminars and activities related to the responsible conduct of research, laboratory management, and faculty career development. Support from this K99/R00 grant will provide the applicant an outstanding opportunity to expand and consolidate her experimental and laboratory skills and support her career goal to become an independent investigator and obtain a faculty position at an academic institution. The likelihood that she will achieve these goals is supported by planned mentorship from well-established investigators, the abundant opportunities and resources available within the Emory University Alcohol and Lung Biology Center, and a hypothesis-driven proposal exploring novel mechanisms of an important and clinically relevant pathophysiological disorder. The proposed program will permit the applicant to build her publication record, collect critical preliminary data for subsequent grant applications, present research findings at national meetings, and gain experience in manuscript writing and grant preparation. Thus this K99/R00 application provides an excellent opportunity to advance the career of a talented and promising investigator.

长期酗酒会增加患者患急性呼吸窘迫综合征的风险 (ARDS) 和呼吸道感染中，肺泡巨噬细胞 (AM)、NADPH 氧化酶 (Nox) 1、Nox2 和 Nox4。是活性氧 (ROS) 的重要来源，Nox2 对于呼吸爆发至关重要吞噬作用后杀死微生物，但过量的 ROS 产生会抑制慢性吞噬作用。摄入酒精会增加 Nox 酶水平，导致 AM 氧化应激和功能障碍。酒精引起的紊乱可以通过过氧化物酶体增殖物激活受体治疗来逆转 γ (PPARγ) 配体，例如吡格列酮和罗格列酮，在这些研究中，我们将确定是否酒精对 AM Nox 表达和活性的影响由 microRNA (miR) 调节：Nox1 相关 miR- 1264、Nox2 相关 miR-107 和 Nox4 相关 miR-363 和 -92a/b（目标 1）。 PPARγ配体调节这些miR以逆转酒精介导的AM Nox1、Nox2和Nox4的能力表达、氧化应激和吞噬作用受损（目标 2）。通过使用慢性饮酒的小鼠模型，体外乙醇暴露的小鼠 AM 细胞系，MHS，在K99阶段，申请人将接受实践培训。将肺炎克雷伯菌气管内输送至小鼠以评估 PPARγ 的生理能力配体可减弱酒精对 miR、Nox 表达、AM 杀伤和细菌清除的影响。额外的培训将包括转基因和敲除小鼠模型的群体维护以及新颖的向小鼠鼻内递送 PPARγ 配体或其他治疗剂的方法研究的目的。该提案概述的目的是证明靶向 PPARγ 构成了一种新的治疗方法改善酒精引起的 AM 功能障碍如果成功的话，这些研究可能会产生巨大的影响。通过为有酒精滥用史的患者的管理奠定基础，产生转化影响未来的临床研究。 PI 对酒精研究的关注始于她的博士前论文项目调查长期滥用酒精对肝脏的影响，重点关注其背后的机制 ROS 和炎症介质的参与改变了肝内皮细胞和巨噬细胞的功能。在布朗博士和哈特博士实验室的博士后培训期间，她获得了额外的专业知识。许多分子生物学技术和慢性酒精摄入的动物模型。在拟议项目的指导阶段，她将通过学习以下内容进一步扩展她的技能：a) 技术来执行和表征小鼠模型气道中的细菌挑战，以评估肺泡体内巨噬细胞吞噬作用，b) 将治疗药物直接递送至小鼠模型肺部的方法， c) 开发和管理基因敲除小鼠和转基因小鼠群体所需的技能。在导师实验室的协助和培训下通过执行拟议研究而获得在拟议项目期间的前两年，这些研究的重点将是自然的。扩展了候选人对慢性酒精摄入背景下 microRNA 调节的兴趣。预计她在该领域的额外专业知识自然会促进她越来越独立于此外，在拟议的奖励期间，除了当前分子生物学的教学课程外。技术和生物统计方法，申请人将获得对其职业生涯重要的非实验室技能通过参加与以下领域相关的研讨会和活动，发展成为一名独立的研究人员负责任地进行研究、实验室管理和教师职业发展。这项 K99/R00 赠款的支持将为申请人提供一个绝佳的机会来扩展和巩固她的实验和实验室技能并支持她成为独立的职业目标调查员并在学术机构获得教职她实现这些目标的可能性。目标得到了知名研究者有计划的指导、丰富的机会和埃默里大学酒精和肺生物学中心提供的资源，以及假设驱动的提案探索一种重要且临床相关的病理生理疾病的新机制。拟议的计划将允许申请人建立其发表记录，收集关键的初步数据随后的拨款申请，在国家会议上展示研究成果，并获得以下方面的经验：因此，这个 K99/R00 申请提供了一个绝佳的机会。促进有才华和有前途的调查员的职业生涯。