Interaction of P. aeruginosa with Alveolar Macrophages

描述（由申请方提供）：铜绿假单胞菌（Pa）感染是囊性纤维化（CF）肺部疾病的标志。肺泡巨噬细胞（AM）在肺部炎症反应中发挥重要作用。对感染Pa的AM中的转录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中的功能尚不清楚，并且有证据表明CF中AM的炎症反应失调。该建议的重点是在AM中表达的CFTR在其对Pa的反应中的作用。基于难以从患有CF的个体获得未受刺激或未感染的AM，AM中的CFTR表达将使用具有由腺病毒（Ad）载体递送的小干扰RNA（siRNA）的遗传修饰来沉默。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实现这一目标的研究。目标1.评估AM中CFTR沉默是否导致促炎表型。将在上皮细胞系中筛选CFTR mRNA特异性的SiRNA构建体。表达最有效的siRNA-CFTR（AdsiRNA-CFTR）构建体的Ad载体将用于沉默AM中的CFTR表达。将监测CFTR mRNA和蛋白质的表达，并评价刺激后IL-8的分泌，以评估AM中CFTR的沉默是否导致如上皮细胞已知的促炎表型。将评价两种替代对照以实现CFTR功能降低：（1）表达CFTR R结构域的Ad载体和（2）噻唑碘CFTR抑制剂CFTRinh-172，其被构建以沉默人AM中的CFTR表达。目标2.评估CFTR表达沉默的AM是否在体外显示对Pa的反应改变。为了评估AM中的CFTR表达影响对Pa的应答的假设，将用Pa或其他对照细菌感染用AdsiRNA-CFTR遗传修饰的人AM，并评价（1）Pa吞噬作用、细胞死亡/凋亡和趋化因子/细胞因子释放;和（2）先前通过暴露于Pa的人AM的转录谱鉴定的与炎症和凋亡相关的基因的表达。

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.