Role of sodium-thiocyanate symporters in the antibacterial airway host defense

描述(由申请人提供)：最近的数据表明，氧化宿主防御机制可以防止呼吸道细菌感染。这种新的杀菌机制是通过在乳过氧化物酶(LPO)催化的反应中产生杀菌的次硫氰酸盐(OSCN-)来杀菌：H_2O_2+SCN-？OSCN-。因此，OSCN的产生需要粘膜下腺分泌LPO，表面上皮细胞的双氧化酶(DUOX)产生H_2O_2，以及SCN通过呼吸道上皮运输。SCN的转运主要是由顶端质膜中的CFTR介导的。因此，CF呼吸道上皮细胞减少了SCN的分泌，从而在OSCN的产生和杀菌方面存在缺陷。基侧SCN转运体的分子同一性仍有待体内系统的确定。当前知识中的这一差距阻碍了增强肺部氧化天然免疫和了解氧化系统的临床重要性的努力。药理学数据表明，至少在培养的呼吸道上皮细胞中，钠碘转运体(NIS)是基底外侧的SCN转运体。然而，我们的初步结果表明，NIS及其近亲同源物SLC5A8可能都介导了呼吸道上皮细胞分泌SCN。此外，SLC5A8表达于表面上皮，而NIS主要定位于粘膜下腺的浆液细胞。我们的中心假设是SLC5A8和NIS的分区表达在呼吸道中创造了两条分泌SCN的途径，DUOX/LPO/SCN-系统对抗菌免疫至关重要。我们还假设，涉及NIS的分泌途径可以被用来将碘(I-)输送到呼吸道表面液体中，其中DUOX/LPO将I-氧化为次碘酸(H_2O_2+I-？HoI)，一种非常有效的抗菌抗病毒分子。因此，我们的目标是确定NIS和SLC5A8与呼吸道分泌SCN的功能相关性，并评价在SCN-和I-存在的情况下DUOX/LPO酶对呼吸道先天免疫的生物学意义。我们提出了以下具体目标：1.明确NIS和SLC5A8的区区化表达对呼吸道SCN分泌的重要性。2.评价DUOX/LPO/SCN-系统对人体呼吸道细菌杀灭的重要性。3.探讨I-在呼吸道中的药代动力学，以及呼吸道液体I-对细菌杀灭的影响。我们将使用1)体内方法，如人类呼吸道异种移植和人体主题研究；2)细胞生物学工具，包括原代呼吸道上皮细胞培养、腺病毒介导的基因传递和细菌杀伤试验；3)分子生物学方法，如RNA干扰；以及4)生化分析，包括离子交换层析和比色氧化还原反应。该项目有望确定OSCN介导的呼吸道细菌杀灭所需的SCN转运体，并深入了解OSCN和HoI介导的宿主防御的体内重要性和治疗潜力，这一点到目前为止还没有被探索过。与公共卫生相关。拟议的项目将探索一种新的呼吸道先天免疫机制，通过产生活性氧类来消除细菌。体外实验表明，这种新机制在囊性纤维化患者的呼吸道上皮细胞中是有缺陷的。因此，建议的研究可能确定在囊性纤维化和其他以反复呼吸道感染为特征的疾病中恢复或增强氧化宿主防御系统的治疗潜力。

DESCRIPTION (provided by applicant): Recent data suggest that an oxidative host defense mechanism may prevent bacterial infections in the airway. This novel mechanism kills bacteria by producing bactericidal hypothiocyanite (OSCN-) in a lactoperoxidase (LPO)-catalyzed reaction: H2O2 + SCN- ? OSCN-. Accordingly, OSCN- production requires LPO secretion by the submucosal glands, H2O2 generation by the dual oxidase (Duox) enzymes of surface epithelia, and SCN transport across the airway epithelium. SCN- transport is mediated predominantly by CFTR in the apical plasma membrane. Thus, CF airway epithelia have reduced SCN- secretion and, consequently, are defective in OSCN- production and bacterial killing. The molecular identity of the basolateral SCN- transporter remains to be determined in an in vivo system. This gap in the current knowledge is hindering efforts to enhance oxidative innate immunity in the lung and to understand the clinical importance of the oxidative system. Pharmacological data suggest that - at least in cultured airway epithelia - the sodium-iodide symporter (NIS) is the basolateral SCN- transporter. However, our preliminary results indicate that both NIS and its close homolog slc5a8 may mediate SCN- secretion by airway epithelial cells. Furthermore, slc5a8 is expressed in the surface epithelium, whereas NIS is localized primarily to the serous cells of the submucosal glands. Our central hypothesis is that compartmentalized expression of slc5a8 and NIS creates two routes of SCN- secretion in the airway, and that the Duox/LPO/SCN- system is critical for antibacterial immunity. We also hypothesize that the secretory route involving NIS can be utilized to deliver iodide (I-) to the airway surface fluid, where Duox/LPO oxidizes I- to hypoiodous acid (H2O2 + I- ? HOI), a very effective antibacterial and antiviral molecule. Thus, our objective is to define the functional relevance of NIS and slc5a8 to SCN- secretion in the airway, and to evaluate the biological importance of the Duox/LPO enzymes to airway innate immunity in the presence of SCN- and I-. We propose the following Specific Aims: 1. Define the importance of compartmentalized NIS and slc5a8 expression to SCN- secretion in the airways. 2. Evaluate the importance of the Duox/LPO/SCN- system to bacterial killing in the human airway. 3. Explore the pharmacokinetics of I- in the airway, and the effect of airway fluid I- on bacterial killing. We will pursue these aims using 1) in vivo approaches such as human airway xenografts and human subject studies; 2) cell biology tools including primary airway epithelial cultures, adenovirus-mediated gene delivery, and bacterial killing assays; 3) molecular biology methods such as RNA interference; and 4) biochemical assays including ion-exchange chromatography and colorimetric redox reactions. This project is expected to identify the SCN- transporters that are required for OSCN- mediated bacterial killing in the airway, as well as to provide insight into the in vivo importance and therapeutic potential of OSCN-- and HOI-mediated host defense, which until now has not been explored. PUBLIC HEALTH RELEVANCE. The proposed project will explore a novel innate immune mechanism of airways that eliminates bacteria via the production of reactive oxygen species. Ex vivo experiments have demonstrated that this novel mechanism is defective in airway epithelia of patients with cystic fibrosis. Therefore, the proposed studies may ascertain the therapeutic potential of restoring or enhancing the oxidative host defense system in cystic fibrosis and other diseases that are characterized by recurrent airway infections.