There is significant evidence that anesthetics which act at the GABAA receptor alter immune function and that multiple immune cell types express functional GABAA receptors. However, the mechanism of this immune modulation is unknown. In this proposal, we present exciting preliminary data demonstrating that mice lacking expression of the GABAA receptor ?4 subunit (gabra4), a subunit expressed in immune cells including CD4+ lymphocytes, have significantly greater lung inflammation after sensitization with house dust mite antigen, a common allergic asthma model. This heightened inflammation results in greater in vivo airway reactivity. We also demonstrate that gabra4 knockout CD4+ cells express increased inflammatory cytokines compared to wild type CD4+ cells when stimulated via the T cell receptor in vitro, suggesting the hyper-inflammatory phenotype of the gabra4 knockout mouse may be CD4+ cell-mediated. We hypothesize that GABAA receptor currents, which are depolarizing in CD4+ cells, alter calcium signaling processes key in lymphocyte activation and function, specifically calcium oscillations and store-operated calcium entry. We propose to demonstrate that ?4 subunit containing-GABAA receptors mediate currents in CD4+ cells which are augmented by GABAA receptor ligands, including XHE-III-74A, a novel ?4 subunit selective positive allosteric modulator developed by a collaborator. We will also determine if the CD4+ cell is key in producing the gabra4 KO mouse?s hyper-inflammatory phenotype by performing adoptive T cell transfer experiments. Furthermore, we propose to determine if altered GABAAR signaling affects CD4+ cell calcium dynamics (oscillations and store operated calcium entry) using a novel in situ calcium imaging technique that utilizes precision-cut lung slice technology. This offers the key advantage of maintaining the native environment of the inflamed lung. Finally, we will determine if XHE-III-74A, an ?4 subunit- selective GABAA receptor activator that does not cross the blood brain barrier, will limit house dust mite antigen induced lung inflammation and airway hyper-responsiveness when administered chronically during the sensitization process. Although the studies proposed here focus on lung inflammation, the implications of this work may be relevant to inflammatory processes of many types. Given the widespread use of GABAergic medications, this research promises to be of high clinical significance. My department and Columbia University provide an ideal environment to complete these studies, as all necessary support is already in place. In addition, my proven mentoring committee provides a perfect blend of skills to help guide the science, including expertise in ion channel physiology, lung biology, immunology, and calcium signaling. A comprehensive career development plan has been established, including coursework and additional training. Taken as a whole, this K08 proposal outlines a robust pathway to scientific independence and the foundation of a successful and sustained career as a physician-scientist.
Evidence suggests that anesthetics acting at the GABAA receptor alter immune cell function, but the mechanism of this modulation is unknown. We hypothesize that GABAA receptor-mediated currents alter calcium signaling pathways which are key to CD4+ cell activation and function. This work promises to be of significant clinical relevance in a number of inflammatory processes, including allergic lung inflammation.
