Regulation of Airway Lactoperoxidase Host Defense

描述(申请人提供)：乳酸过氧化物酶(LPO)系统是呼吸道宿主抵抗细菌、病毒和真菌的重要贡献者，也是呼吸道过氧化氢的主要消耗者，而过氧化氢又与呼吸道炎症有关。LPO活性的丧失可能会导致呼吸道感染的增加，而LPO的任何增加都可能产生有害的后果。这一应用将验证一种假设，即LPO系统的所有组成部分，包括LPO酶的表达和分泌、SCN~+跨上皮运输和H_2O_2的产生都是协调调节的，囊性纤维化(CF)中LPO底物SCN~+的缺失可能参与了发病机制。这一假说得到了呼吸道表面液体中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将使系统不足以抵抗宿主的防御。初步数据表明：a)Cf气道上皮细胞缺乏LPO底物SCN~-的正常转运，这可能导致气道感染和炎症；b)Cf气道上皮细胞通过DUOX产生H_2O_2，DUOX是一种可能受细胞内[Ca~(2+)]调节的NADPH氧化酶；c)活性氧物种(ROS)增加LPO的表达；d)Cf气道上皮细胞DUOX和LPO mRNAs表达上调。具体目标1将通过改变[Ca~(2+)]i、cAMP、SCN~或促炎和感染相关刺激来调节正常和CF呼吸道上皮细胞DUOX H_2O_2的产生。特异性目标2将验证这样一种假说，即Cf呼吸道上皮细胞SCN~+转运缺陷会导致体内Cf气道分泌物中SCN~+的减少，从而可能损害宿主防御。呼气冷凝液中SCN~+和过氧化物酶终产物的测定采用电喷雾电离-质谱仪联用的方法。具体目标3将通过周转研究和核径流分析来验证ROS和/或SCN~+增加正常和CF气道上皮细胞LPO mRNA表达的假说。LPO mRNA的5‘端将通过RACE和上游序列中的启动子元件进行定位。特指目标4将验证一种假设，即降低的CF气道SCN~+水平有助于中性粒细胞介导的氧化损伤，并与在CF气道观察到的定植细菌的表型有关。这些实验将提供有关囊性纤维化患者的生理缺陷的新信息，可能导致治疗这种疾病的新的治疗方法。

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.