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

描述（由申请人提供）：长期酗酒会增加患者发生急性呼吸窘迫综合征（ARDS）和呼吸道感染的风险。在肺泡巨噬细胞（AM）中，NADPH氧化酶（Nox）1、Nox 2和Nox 4是活性氧（ROS）的关键来源，并且Nox 2对于参与吞噬后杀死微生物的呼吸爆发是必需的。然而，过量的ROS产生抑制吞噬作用。慢性酒精摄入增加Nox酶水平，导致AM氧化应激和功能障碍。这些酒精诱导的紊乱可以通过过氧化物酶体增殖物激活受体γ（PPAR？）配体，如吡格列酮和罗格列酮。在这些研究中，PI将通过研究microRNA（miR）：Nox 1相关miR-1264、Nox 2相关miR-107和Nox 4相关miR-363和-92 a/B阐明调节酒精诱导AM Nox表达和活性的分子机制（Aim 1）。然后，PI将检查如何进行PPAR？配体减弱这些miR以逆转酒精介导的AM Nox 1、Nox 2和Nox 4表达、氧化应激和受损的吞噬作用（Aim 2）。这些假设将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、MH-S和从人类受试者分离的AM来研究。本申请中概述的研究目的是证明靶向PPAR？构成了一种新的治疗方法，以改善酒精诱导的AM功能障碍。如果成功的话，这些研究可以通过为未来的临床研究奠定基础，对有酒精滥用史的患者的管理产生相当大的转化影响。PI对酒精研究的关注始于她的博士前论文项目，研究慢性酒精滥用对肝脏的有害影响，重点是ROS和炎症介质参与改变肝脏内皮细胞和巨噬细胞功能的机制。在布朗博士和哈特博士的实验室进行博士后培训期间，她获得了许多分子生物学技术和慢性酒精摄入动物模型的额外专业知识。在拟议项目的K99指导阶段，申请人将通过接受以下方面的实践培训进一步扩展其技能库：a）在小鼠模型气道中进行和表征肺炎克雷伯菌细菌挑战的技术，以评估体内肺泡巨噬细胞吞噬作用，B）直接递送PPAR？使用鼻内给药的小鼠模型的肺的配体治疗剂，和c）开发和管理敲除和转基因小鼠的集落所需的技能。这些技能将通过在拟议项目期的前两年在导师实验室的协助和培训下执行拟议研究来获得。这些研究的重点将使候选人对慢性酒精摄入背景下miR调节的兴趣自然扩展。预计她在该领域的额外专业知识将自然促进她从导师那里获得越来越多的独立性，进入R 00独立阶段。在拟议的奖励期间，除了当前分子生物学技术和生物统计方法的教学课程外，PI还将通过参加与负责任的研究，实验室管理和教师职业发展有关的研讨会和活动，获得对其职业发展至关重要的非实验室技能。从这个K99/R 00补助金的支持将提供PI一个杰出的机会，扩大和巩固她的实验和实验室技能，并支持她的职业目标，成为一个独立的调查员，并获得在学术机构的教师职位。她将实现这些目标的可能性得到了来自成熟研究者的计划指导，埃默里大学酒精和肺生物学中心内丰富的科学机会以及探索重要和临床相关病理生理学疾病的新机制的假设驱动的应用程序的支持。拟议的计划将允许PI建立她的出版记录，为随后的资助申请收集关键的初步数据，在国家会议上展示研究成果，并获得手稿写作和资助准备方面的经验。因此，这个K99/R 00应用程序提供了一个很好的机会，以推进一个有才华和有前途的研究人员的职业生涯。

DESCRIPTION (provided by applicant): 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 (PPAR?) ligands, such as pioglitazone and rosiglitazone. In these studies, the PI will elucidate the molecular mechanisms that modulate alcohol-induced AM Nox expression and activity by studying microRNAs (miRs): Nox1-related miR-1264, Nox2-related miR-107, and Nox4-related miRs-363 and -92a/b (Aim 1). Then, the PI will examine how PPAR? ligands attenuate 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 mouse model of chronic alcohol consumption, an in vitro ethanol exposed mouse AM cell line, MH-S, and AMs isolated from human subjects. The objective of the studies outlined in this application is to demonstrate that targeting PPAR? 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 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 K99 mentored phase of the proposed project, the applicant will further expand her repertoire of skills by receiving hands-on training in: a) techniques to perform and characterize Klebsiella pneumonia bacterial challenges in the airways of mouse models to assess alveolar macrophage phagocytosis in vivo, b) methods to directly deliver PPAR? ligand therapeutics to the lungs of mouse models using intranasal administration, 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 miRs in the context of chronic alcohol ingestion. t is anticipated that her additional expertise in this area will naturally promote her growing independence from her mentors into the R00 independent phase. During the proposed award, in addition to didactic courses in current molecular biology techniques and biostatistical methods, the PI 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 PI 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 scientific opportunities within the Emory University Alcohol and Lung Biology Center, and a hypothesis-driven application exploring novel mechanisms of an important and clinically relevant pathophysiological disorder. The proposed program will permit the PI 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.