CFTR and the Immune Response

描述(申请人提供)：肺部感染和强烈的炎症反应是囊性纤维化的主要临床特征，并构成囊性纤维化患者发病率和死亡率的主要原因。有研究认为，异常的呼吸道上皮细胞和异常的免疫反应协同作用，导致严重的慢性肺部疾病。呼吸道上皮细胞在慢性肺纤维化炎症反应中的作用正在被广泛研究，然而，关于初级免疫细胞在调节慢性肺疾病高反应性中所起的作用却知之甚少。目前也不知道免疫细胞是否对肺部表型有直接贡献，或者夸大的炎症反应是否仅仅是初级上皮缺陷的继发性。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发挥重要作用。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(S)尚不清楚。已知，Toll样受体4(TLR4)是通过Toll样受体4(TLR4)特异性结合内毒素进行信号转导的。我们发现，在内毒素刺激下，与WT巨噬细胞相比，CF巨噬细胞膜上表达更多的TLR4。与包括MD-2和CD14在内的辅助内毒素结合蛋白和TLR4信号通路在激活对PA的先天免疫反应中起主要作用。这些发现表明，免疫细胞直接参与了CF中过度的免疫反应。为了研究这一假说，我们提出：i.)目的：探讨CFTR+细胞移植是否能改善CFTR-/-小鼠的高炎性免疫反应。我们将使用体外分析和体内研究来确定CFTR缺失的免疫细胞是否在CFTR-/-小鼠的异常免疫反应中发挥主要作用，以及WT免疫细胞是否可以纠正这种反应；ii)我们将使用基因互补策略，剖析CFTR+上皮细胞和CFTR+巨噬细胞在慢性炎症肺组织对脂多糖的体内反应中所起的作用。我们将确定巨噬细胞特异性CFTR表达相对于上皮细胞特异性CFTR表达的CFTR-/-小鼠是否有类似于CFTR-/-、CFTR-/+或WT小鼠和III的炎症反应。)最后，我们将通过阻断巨噬细胞中的cftr和增强cftr-/-细胞中cftr的表达，来检验cftr的缺乏是否直接导致巨噬细胞对内毒素的过度免疫反应。

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