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.

长期酗酒增加患者患急性呼吸窘迫综合征的风险 (ARDS)和呼吸道感染。在肺泡巨噬细胞(AM)中，NADPH氧化酶(NOx)1、NOX2和NOX4 是活性氧物种(ROS)的关键来源，而NOX2对涉及的呼吸爆发是必不可少的 在吞噬后杀死微生物。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 用过氧化物酶体增殖物激活受体治疗可逆转酒精引起的排列紊乱 伽玛(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表达和活性的影响是由microRNAs(MiRs)调节的：nox1相关miR- 1264、与NOX2相关的miR-107和与NOX4相关的miRs-363和-92a/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 NOX1、NOX2和NOX4的能力 表达、氧化应激和吞噬功能受损(目标2)。我们将对这些假设进行调查 通过建立小鼠慢性饮酒模型，建立了体外乙醇暴露的小鼠AM细胞系MHS， 和从人类受试者身上分离出来的AM。在K99阶段，申请者将接受实践培训 用肺炎克雷伯菌气管内给药评价PPARγ的生理能力 用于减弱酒精诱导的miRs、NOx表达、AM杀伤和细菌清除的配体。 额外的培训将包括转基因和基因敲除小鼠模型和新的 PPARγ配体或其他治疗药物鼻腔给药的方法。研究的目的是 这项提案中概述的是证明靶向PPARγ构成了一种新的治疗方法 改善酒精所致的AM功能障碍。如果成功，这些调查可能会有相当大的 通过为有酒精滥用史的患者的管理设置舞台，对翻译的影响 未来的临床研究。 私家侦探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 ROS和炎症介质参与改变肝脏内皮细胞和巨噬细胞的功能。 在布朗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。在.期间 拟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) 评估肺泡的小鼠模型的呼吸道细菌挑战的实施和表征技术 体内巨噬细胞吞噬作用，b)将治疗药物直接输送到小鼠模型的肺部的方法， 以及c)开发和管理基因敲除和转基因小鼠群体所需的技能。这些技能将是 通过在导师实验室的协助和培训下执行拟议的研究而获得的 在拟议项目期的头两年。这些研究的重点将允许自然的 扩大候选人对慢性酒精摄入背景下的microRNAs调控的兴趣。 预计她在这一领域的额外专业知识将自然促进她日益独立于 她的导师。此外，在拟议的奖项期间，除了当代分子生物学的授课课程外， 技术和生物统计学方法，申请者将获得对她的职业生涯重要的非实验室技能 作为一名独立研究调查员，通过参加与 负责研究、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者提供一个扩大和 巩固她的实验和实验室技能，支持她成为独立人士的职业目标 研究人员，并在学术机构获得教职。她实现这些目标的可能性 Goals得到了来自知名研究人员的有计划的指导，丰富的机会和 埃默里大学酒精和肺部生物学中心提供的资源，以及假设驱动的 建议探索一种重要的和临床相关的病理生理障碍的新机制。这个 建议的计划将允许申请者建立她的出版记录，收集关键的初步数据 随后的赠款申请，在国家会议上提出研究结果，并在以下方面获得经验 稿件的撰写和赠款的准备。因此，此K99/R00应用程序提供了一个极好的机会 促进有才华和有前途的调查员的职业生涯。