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: 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
• 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

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.

长期酗酒增加病人患急性呼吸窘迫综合症的风险 （ARDS）和呼吸道感染。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，而Nox 2是呼吸爆发所必需的 在吞噬作用后杀死微生物。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治疗来逆转 γ（PPARγ）配体，如吡格列酮和罗格列酮。在这些研究中，我们将确定 酒精对AM Nox表达和活性的影响受microRNAs（miRs）调节：Nox 1相关的miRs- 1264、Nox 2相关miR-107和Nox 4相关miR-363和-92 a/B（目标1）。然后，我们将确定 PPARγ配体调节这些miR逆转酒精介导的AM Nox 1、Nox 2和Nox 4的能力 表达、氧化应激和受损的吞噬作用（Aim 2）。这些假设将被调查 通过使用慢性酒精消耗的小鼠模型，体外乙醇暴露的小鼠AM细胞系，MHS， 和从人类受试者中分离的AM。在K99阶段，申请人将接受实践培训 在肺炎克雷伯氏菌气管内给药小鼠中，以评估PPARγ的生理能力 配体来减弱酒精诱导的对miR、Nox表达以及AM杀伤和细菌清除的作用。 额外的培训将包括转基因和敲除小鼠模型的集落维持和新的 向小鼠鼻内递送PPARγ配体或其它治疗剂的方法。研究的目的 该提案中概述的是证明靶向PPARγ构成了一种新的治疗方法， 改善酒精诱导的AM功能障碍。如果成功的话，这些调查可能会产生相当大的影响。 翻译的影响，对管理的病人有酗酒史，通过设置阶段， 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 慢性酒精滥用对肝脏的有害影响，重点关注 参与ROS和炎症介质，改变肝内皮细胞和巨噬细胞功能。 在布朗博士和哈特博士的实验室进行博士后培训期间，她获得了更多的专业知识， 许多分子生物学技术和慢性酒精摄入的动物模型。期间 在建议项目的指导阶段，她将通过学习进一步扩大她的技能库：a） 在小鼠模型的气道中进行和表征细菌挑战以评估肺泡 体内巨噬细胞吞噬作用，B）将治疗剂直接递送至小鼠模型肺的方法， 和c）开发和管理基因敲除和转基因小鼠群体所需的技能。这些技能将 在导师实验室的协助和培训下，通过执行拟议的研究获得 在拟议项目期的头两年。这些研究的重点将允许自然的 扩展了候选人对慢性酒精摄入背景下microRNA调控的兴趣。 预计她在这一领域的额外专门知识将自然促进她日益独立于 她的导师们此外，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，申请人将获得对她的职业生涯重要的非实验室技能 作为一名独立的研究调查员，参加有关的研讨会和活动， 负责任地进行研究，实验室管理和教师职业发展。 来自K99/R 00资助的支持将为申请人提供一个出色的机会， 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 并在学术机构获得教职。她实现这些目标的可能性 目标得到了来自知名调查人员的有计划的指导的支持， 埃默里大学酒精和肺生物学中心内可用的资源，以及假设驱动的 一项探索重要的临床相关病理生理学疾病的新机制的提案。的 拟议的计划将允许申请人建立她的出版记录，收集关键的初步数据， 随后的赠款申请，在国家会议上介绍研究成果，并获得经验， 手稿写作和资助准备。因此，K99/R 00应用程序提供了一个绝佳的机会， 推动一个有才华和有前途的调查员的职业生涯。