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, 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 PPAR? 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 PPAR? 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 PPAR? ligands or other therapeutics to mice. The objective of the studies outlined in this proposal 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 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.
The risk of respiratory infections is increased in alcoholics because the capacity of lung macrophages to clear infectious microbes is impaired. This impaired immune function is primarily due to the over-production of NADPH oxidase (Nox) proteins which generate reactive oxygen species. In this proposal, we will determine if PPAR? receptor ligands such as pioglitazone and rosiglitazone can reverse the effects of alcohol abuse and down-regulate Nox enzymes, decrease oxidative stress, and improve lung macrophage immune functions.
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