CFTR and the Immune Response

描述（由申请人提供）：肺部感染和强烈的炎症反应是囊性纤维化（CF）的主要临床特征，也是CF患者发病和死亡的主要原因。有研究表明，异常的气道上皮细胞和异常的免疫反应共同导致严重的慢性肺部疾病。气道上皮对CF炎症反应的贡献正在被广泛研究，然而，对原代免疫细胞在介导CF肺部疾病中观察到的高反应性中的作用知之甚少。也不知道免疫细胞是否对肺表型有直接的贡献，或者过度的炎症反应是否仅仅是继发于原发性上皮缺陷。最近的报道表明，CFTR可能在巨噬细胞和中性粒细胞的正常功能中起重要作用。此外，我们的初步数据表明，巨噬细胞可能在CF气道的高反应性中发挥重要作用。CFTR-/-巨噬细胞高反应性的机制尚不清楚。众所周知，对LPS的信号转导是由toll样受体4 （TLR4）介导的，TLR4特异性地与LPS结合。我们发现，在LPS刺激下，CF巨噬细胞与WT巨噬细胞相比，在其膜上表达更多的TLR4。TLR4信号与辅助lps结合蛋白（包括MD-2和CD14）和TLR4信号一起，在PA先天免疫应答的激活中起主要作用。这些发现表明免疫细胞直接参与了CF的过度免疫反应。为了验证这一假设，我们提出：i.)研究造血移植CFTR+细胞是否会改善CFTR-/-小鼠的过度炎症免疫反应。我们将使用体外实验和体内研究来确定CFTR无效免疫细胞是否在CFTR-/-小鼠的异常免疫反应中起主要作用，以及WT免疫细胞是否可以纠正这种反应；ii.)我们将利用基因互补策略剖析CFTR+上皮细胞和CFTR+巨噬细胞在慢性炎症肺中对LPS的体内反应中的作用。我们将确定巨噬细胞特异性CFTR表达的CFTR-/-小鼠与上皮细胞特异性CFTR表达的CFTR-/-小鼠是否具有与CFTR-/-、CFTR-/+或WT小鼠相似的炎症反应。iii)最后，我们将通过阻断WT细胞中的CFTR和增强CFTR-/-细胞中的CFTR表达来检查CFTR缺乏是否直接导致巨噬细胞对LPS的过度免疫反应

DESCRIPTION (provided by applicant): Pulmonary infection and a robust inflammatory response are dominant clinical features of cystic fibrosis (CF), and comprise the major cause of morbidity and mortality in CF patients. It has been suggested that abnormal airway epithelial cells and abnormal immune responses collaborate and result in severe chronic lung disease. The contribution of airway epithelia to the inflammatory response in CF is being extensively studied, however much less is known about the role of primary immune cells in mediating the hyper- responsiveness observed in CF lung disease. Neither is it known if immune cells have a direct contribution to the lung phenotype or if the exaggerated inflammatory response is merely secondary to the primary epithelial defect. Recent reports suggest that CFTR may have an important role in the normal function of both macrophages and neutrophils. Additionally, our preliminary data suggest that the macrophage may play an important role in the hyper-responsiveness of the CF airway. The mechanism(s) underlying the hyper-responsiveness of CFTR-/- macrophages is not known. It is known that signal transduction in response to LPS is mediated by Toll-like receptor 4 (TLR4), which binds to LPS specifically. We have found that upon LPS stimulation, CF macrophages and express higher amounts of TLR4 on their membrane when compared to WT macrophages. In concert with accessory LPS-binding proteins including MD-2 and CD14, and TLR4 signaling plays a major role in the activation of the innate immune response to PA. These findings suggest that immune cells directly contribute to the exaggerated immune response in CF. In order to investigate this hypothesis we propose: i.) to investigate if hematopoietic engraftment of CFTR+ cells will ameliorate the hyper-inflammatory immune response in CFTR-/- mice. We will use both in vitro assays, as well as, in vivo studies to determine if CFTR null immune cells play a primary role in the abnormal immune response in CFTR-/- mice and whether WT immune cells can rectify this response; ii.) we will dissect the roles of CFTR+ epithelial cells and CFTR+ macrophages in the in vivo response to LPS in the chronically inflamed lung using a gene complementation strategy. We will determine if CFTR-/- mice with macrophage specific CFTR expression versus epithelial-cell specific CFTR expression has an inflammatory response that is similar to CFTR-/-, CFTR-/+ or WT mice and iii.) lastly, we will examine if the lack of CFTR is directly responsible for the exaggerated immune response to LPS in macrophages by blocking CFTR in WT cells and by enhancing expression of CFTR in CFTR-/- cells PUBLIC HEALTH RELEVANCE: The ultimate goal of this project is to apply insights gained from these studies to improve the clinical management of people with cystic fibrosis and develop new therapies to treat this disease.