Interaction of P. aeruginosa with Alveolar Macrophages

描述(由申请人提供)：感染铜绿假单胞菌(PA)是囊性纤维化(CF)肺部疾病的标志。肺泡巨噬细胞(AM)在肺部炎症反应中起重要作用。对感染PA的AM转录图谱的分析确定了与炎症和凋亡相关的各种预期的和新的因子。虽然AM表达囊性纤维化跨膜调节因子(CFTR)，但其在AM中的作用尚不清楚，有证据表明在CF中AM存在炎症反应的失调。这项建议侧重于在AM中表达的CFTR在他们对PA的反应中的作用。由于难于从CF患者体内获得未受刺激或未感染的AM，利用腺病毒(Ad)载体携带的小干扰RNA(SiRNA)进行基因修饰，可沉默AM中CFTR的表达。总体目标是评估CFTR在AM对PA的反应中的作用。两个具体目标概述了为实现这一目标而进行的研究。目的1.评估在AM中沉默CFTR是否导致促炎表型。将在上皮细胞系中筛选针对CFTRmRNA的siRNA结构。表达最有效的siRNA-CFTR(AdsiRNA-CFTR)的Ad载体将被用来沉默AM中CFTR的表达将监测CFTRmRNA和蛋白的表达，并评估刺激后IL-8的分泌，以评估AM中CFTR的沉默是否会导致已知的上皮细胞的促炎表型。将评估两种交替的对照以实现CFTR功能的降低：(1)表达CFTRR结构域的Ad载体和(2)构建成沉默人AM中CFTR表达的噻唑罗定CFTRinh-172目的2.在体外评价沉默CFTR表达的AM对PA的反应是否发生改变。为了评估CFTR在AM中的表达影响对PA的反应的假设，将AdsiRNA-CFTR基因修饰的人AM感染PA或其他对照细菌，并评估(1)PA的吞噬作用、细胞死亡/凋亡以及趋化因子/细胞因子的释放；以及(2)炎症和凋亡相关基因的表达，这些基因以前通过暴露于PA的人AM的转录谱来鉴定。

DESCRIPTION (provided by applicant): Infection with Pseudomonas aeruginosa (Pa) is a hallmark of lung disease in cystic fibrosis (CF). Alveolar macrophages (AM) play an important part in the pulmonary inflammatory response. The analysis of transcript profiles in AM infected with Pa identified a variety of expected and novel factors related to inflammation and apoptosis. Although AM express the cystic fibrosis transmembrane regulator (CFTR), its function in AM is not known and there is evidence of a dysregulated inflammatory response of AM in CF. This proposal focuses on the role of CFTR expressed in AM in their response to Pa. Based on the difficulty to obtain unstimulated or uninfected AM from individuals with CF, CFTR-expression in AM will be silenced using genetic modification with small interfering RNA (siRNA) delivered by an adenovirus (Ad) vector. The overall goal is to evaluate the role of CFTR in the response of AM to Pa. Two specific aims outline the studies to achieve this goal. Aim 1. To assess if silencing of CFTR in AM results in a proinflammatory phenotype. SiRNA constructs specific for the CFTR mRNA will be screened in epithelial cell lines. An Ad vector expressing the most effective siRNA-CFTR (AdsiRNA-CFTR) construct will be used to silence CFTR expression in AM. The expression of CFTR mRNA and protein will be monitored and the secretion of IL-8 following stimulation will be evaluated to assess if silencing of CFTR in AM results in a proinflammatory phenotype as known for epithelial cells. Two alternate controls will be evaluated to achieve decreased CFTR function: (1) An Ad vector expressing the CFTR R-domain and (2) the thiazilodine CFTR inhibitor CFTRinh-172.constructed to silence CFTR expression in human AM. Aim 2. To evaluate if AM with silenced CFTR expression display an altered response to Pa in vitro. To assess the hypothesis that CFTR expression in AM affects the response to Pa, human AM , genetically modified with AdsiRNA-CFTR will be infected with Pa or other control bacteria and evaluated for (1) Pa phagocytosis, cell death/apoptosis, and chemokine/cytokine release; and (2) the expression of genes related to inflammation and apoptosis, previously identified by transcript profiles of human AM exposed to Pa.