GABAA Receptor-Mediated Modulation of Lung Inflammation

项目摘要/摘要 有重要证据表明，作用于GABAA受体的麻醉剂会改变免疫功能，而且 多种免疫细胞类型表达功能性的GABAA受体。然而，这种免疫的机制 调制方式未知。在这项提案中，我们提供了令人兴奋的初步数据，表明老鼠缺乏 GABAA受体α4亚单位(GARR4)的表达 淋巴细胞，在用尘螨抗原致敏后有明显更大的肺部炎症。 常见过敏性哮喘模型。这种高度的炎症会导致体内更大的呼吸道反应性。我们 还表明，与野生型相比，GARR4基因敲除的CD4+细胞表达更多的炎性细胞因子 当在体外通过T细胞受体刺激时，提示高炎性表型 GARR4基因敲除小鼠可能是由CD4+细胞介导的。我们假设GABAA受体电流，它 在CD4+细胞中去极化，改变在淋巴细胞激活和功能中关键的钙信号过程， 具体地说，钙振荡和商店操作的钙进入。我们建议证明α4亚基 包含-GABAA受体的受体介导了被GABAA受体配体增强的CD4+细胞的电流， 包括协作者开发的新型α4亚基选择性正变构调节剂XHE-III-74A。 我们还将确定CD4+细胞是否是产生GRAR4 KO小鼠高炎症表型的关键 通过进行采用的T细胞转移实验。此外，我们建议确定是否改变了GABAAR 使用一种新的原位信号转导机制影响CD4+细胞钙动力学(振荡和存储操作的钙内流) 利用精密切割肺切片技术的钙成像技术。这提供了以下主要优势： 维持发炎肺部的自然环境。最后，我们将确定XHE-III-74A，一个α4亚单位- 选择性GABAA受体激活剂不会穿过血脑屏障，将限制屋尘螨抗原 慢性给药引起的肺部炎症和呼吸道高反应性 敏化过程。尽管这里提出的研究重点是肺部炎症，但这一点的影响 工作可能与许多类型的炎症过程有关。鉴于GABA的广泛使用 药物方面，这项研究有望具有很高的临床意义。我的系和哥伦比亚大学 为完成这些研究提供一个理想的环境，因为所有必要的支持都已经到位。此外, 我的经验丰富的指导委员会提供了完美的技能组合来帮助指导科学，包括专业知识 在离子通道生理学、肺生物学、免疫学和钙信号方面。全面的职业生涯 已经制定了发展计划，包括课程作业和额外培训。作为一个整体，这 K08提案勾勒出了一条通往科学独立的坚实道路，并奠定了成功和 作为一名内科科学家的持久职业生涯。

Project Summary/Abstract 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 α4 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 α4 subunit containing-GABAA receptors mediate currents in CD4+ cells which are augmented by GABAA receptor ligands, including XHE-III-74A, a novel α4 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 α4 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.