Regulation of Airway Lactoperoxidase Host Defense

描述（由申请人提供）：乳过氧化物酶（LPO）系统是气道宿主防御细菌、病毒和真菌的重要贡献者，也是气道H2 O2的主要消耗者，而H2 O2又与气道炎症相关。LPO活性的丧失可能导致气道感染增加，而LPO的任何增加都可能产生有害后果。本申请将检验以下假设：LPO系统的所有组分，包括LPO酶表达和分泌、SCN~-跨上皮转运和H2 O2产生，都受到协调调节，并且在囊性纤维化（CF）中LPO底物SCN~-的缺乏可能有助于发病机制。这一假设得到了气道表面液体中LPO系统活性的计算模型的支持，该模型预测组分中的任何不平衡都会使系统不足以进行宿主防御。该假设和模型得到初步数据的支持，初步数据显示：a）CF气道上皮细胞缺乏LPO底物SCN~-向顶端表面的正常转运，这可能导致气道感染和炎症; B）气道上皮细胞通过Duox（一种NADPH氧化酶，可受细胞内[Ca 2 +]调节）产生H2 O2; c）活性氧物质（ROS）增加LPO的表达;和d）Duox和LPO mRNA在CF气道上皮中上调。具体目标1将通过[Ca 2 +]i、cAMP、SCN-或促炎性和感染相关刺激的变化来调节正常和CF气道上皮细胞中Duox H2 O2的产生。具体目标2将检验以下假设：CF气道上皮中有缺陷的SCN-转运导致体内CF气道分泌物中的SCN-减少，这然后可能损害宿主防御。将使用液相色谱法和电喷雾电离质谱法测定呼出气冷凝液中的SCN~和过氧化物酶终产物。具体目标3将通过使用周转研究和核径流测定来检验ROS和/或SCN-增加正常和CF气道上皮中LPO mRNA表达的假设。将通过RACE定位LPO mRNA的5'端，并鉴定上游序列中的启动子元件。具体目标4将检验以下假设：降低的CF气道SCN-水平有助于嗜中性粒细胞介导的氧化损伤和CF气道中观察到的定殖细菌表型。概述：这些实验将提供有关囊性纤维化患者生理缺陷的新信息，可能导致治疗这种疾病的新治疗方法。

DESCRIPTION (provided by applicant): The lactoperoxidase (LPO) system is a significant contributor to airway host defense against bacteria, viruses and fungi and also is a major consumer of airway H2O2 that in turn is associated with airway inflammation. A loss of LPO activity may lead to increased airway infection, while any increase in LPO could have injurious consequences. This application will test the hypothesis that all components of the LPO system, including LPO enzyme expression and secretion, SCN~ transport across the epithelium and H2O2 production, are coordinately regulated and that an absence of the LPO substrate, SCN~, in cystic fibrosis (CF) may contribute to pathogenesis. This hypothesis is supported by a computational model of LPO system activity in airway surface liquid that predicts any unbalance in the components will render the system inadequate for host defense. The hypothesis and model are supported by preliminary data showing: a) CF airway epithelia lack normal transport of LPO's substrate, SCN~, to the apical surface that may contribute to airway infections and inflammation; b) airway epithelia produce H2O2 through Duox, an NADPH oxidase that may be regulated by intracellular [Ca2+]; c) reactive oxygen species (ROS) increase expression of LPO; and d) Duox and LPO mRNAs are up-regulated in CF airway epithelia. Specific Aim 1 will address regulation of Duox H2O2 production in normal and CF airway epithelial cells by changes in [Ca2+]i, cAMP, SCN~ or proinflammatory and infection related stimuli. Specific Aim 2 will test the hypothesis that defective SCN~ transport in CF airway epithelia results in decreased SCN~ in CF airway secretions in vivo that then may compromise host defense. SCN~ and peroxidase endproducts will be determined in exhaled breath condensate using liquid chromatography in tandem with electrospray ionization-mass spectrometry. Specific Aim 3 will test the hypothesis that ROS and/or SCN~ increases LPO mRNA expression in normal and CF airway epithelia by using turnover studies and nuclear runoff assays. The 5' end of LPO mRNA will be mapped by RACE and promoter elements in upstream sequences identified. Specific Aim 4 will test the hypothesis that depressed CF airway SCN~ levels contribute to neutrophil-mediated oxidative damage and to the phenotype of colonizing bacteria observed in CF airways. Lay Summary: These experiments will provide new information about physiological defects in cystic fibrosis patients that may lead to new therapeutic approaches to treating this disease.