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

• 批准号: 8581531
• 负责人: Samantha M. Yeligar
• 金额: $ 11.72万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8581531
• 关键词: 2,4-thiazolidinedione; Address; Adult Respiratory Distress Syndrome; Alcohol abuse; Alcohols; Alveolar Cell; Alveolar Macrophages; Animal Model; Apoptotic; Applications Grants; Area; Attenuated; Award; Biology; Biostatistical Methods; Cell Line; Chronic; Clinical Research; Clinical Trials; Data; Disease; Endothelial Cells; Enzymes; Ethanol; Experimental Models; Faculty; Functional disorder; Future; Goals; Grant; In Vitro; Infectious Agent; Inflammation Mediators; Ingestion; Institution; Intranasal Administration; Investigation; Klebsiella pneumonia bacterium; Knockout Mice; Laboratories; Ligands; Liver; Lung; Lung Capacity; Lung diseases; Manuscripts; Mediating; Mentors; Mentorship; Methods; MicroRNAs; Microbe; Molecular; Molecular Biology Techniques; Mus; NADPH Oxidase; NADPH Oxidase 1; Nuclear Hormone Receptors; Oxidative Stress; PPAR gamma; Patients; Phagocyte Bactericidal Dysfunction; Phagocytosis; Phase; Pioglitazone; Play; Positioning Attribute; Preparation; Production; Proteins; Publications; Reactive Oxygen Species; Recording of previous events; Regulation; Research; Research Personnel; Respiratory Burst; Respiratory Tract Infections; Risk; Role; Solid; Source; Specificity; Staging; Techniques; Therapeutic; Therapeutic Intervention; Thiazolidinediones; Training; Transgenic Mice; Translations; Universities; Up-Regulation; Writing; alcohol exposure; alcohol research; career; career development; chronic alcohol ingestion; clinically relevant; diabetic patient; experience; human disease; human subject; immune function; improved; in vivo; in vivo Model; insulin sensitivity; interest; killings; mRNA Transcript Degradation; macrophage; meetings; mouse model; novel; novel therapeutic intervention; post-doctoral training; pre-doctoral; problem drinker; programs; public health relevance; receptor; responsible research conduct; rosiglitazone; skills; transcription factor

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.

描述（由申请人提供）：长期酗酒会增加患者患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、Nox2 和 Nox4 是活性氧 (ROS) 的重要来源，Nox2 对于吞噬作用后杀死微生物的呼吸爆发至关重要。然而，过量的 ROS 产生会抑制吞噬作用。慢性酒精摄入会增加 Nox 酶水平，导致 AM 氧化应激和功能障碍。这些酒精引起的紊乱可以通过过氧化物酶体增殖物激活受体γ（PPAR？）配体（例如吡格列酮和罗格列酮）治疗来逆转。在这些研究中，PI 将通过研究 microRNA (miR) 来阐明调节酒精诱导的 AM Nox 表达和活性的分子机制：Nox1 相关的 miR-1264、Nox2 相关的 miR-107 以及 Nox4 相关的 miR-363 和 -92a/b（目标 1）。那么，PI会如何检查PPAR呢？配体减弱这些 miR，以逆转酒精介导的 AM Nox1、Nox2 和 Nox4 表达、氧化应激和吞噬作用受损（目标 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 和从人类受试者中分离的 AM 来进行研究。本申请中概述的研究的目的是证明针对 PPAR？构成了一种改善酒精引起的 AM 功能障碍的新治疗方法。如果成功，这些研究可能会为未来的临床研究奠定基础，对有酗酒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，该项目调查了慢性酒精滥用对肝脏的有害影响，重点关注 ROS 和炎症介质参与改变肝内皮细胞和巨噬细胞功能的机制。在博士实验室进行博士后培训期间。在布朗和哈特的帮助下，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来进一步扩展其技能：a）在小鼠模型气道中执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的吞噬作用，b）直接递送 PPAR 的方法？使用鼻内给药对小鼠模型的肺部进行配体治疗，以及 c) 开发和管理敲除小鼠和转基因小鼠群体所需的技能。这些技能将通过在拟议项目期间的前两年在导师实验室的协助和培训下执行拟议的研究来获得。这些研究的重点将允许候选人对慢性酒精摄入背景下的 miR 调节的兴趣自然延伸。预计她在该领域的额外专业知识自然会促进她逐渐独立于导师，进入 R00 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行为、实验室管理和教师职业发展相关的研讨会和活动，获得对其作为独立研究调查员的职业发展很重要的非实验室技能。这项 K99/R00 赠款的支持将为 PI 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，并支持她成为一名独立研究者并在学术机构获得教职的职业目标。她实现这些目标的可能性得到了知名研究人员的计划指导、埃默里大学酒精和肺生物学中心内丰富的科学机会，以及探索重要且临床相关的病理生理障碍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，收集后续资助申请的关键初步数据，在全国会议上展示研究成果，并获得手稿写作和资助准备的经验。因此，这个 K99/R00 申请提供了一个极好的机会来促进有才华和有前途的研究者的职业生涯。