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预防气道细菌感染。这种新机制通过在乳酸过氧化物酶（LPO）催化反应H2O2 + SCN- ？OSCN -。因此，OSCN-的产生需要粘膜下腺分泌LPO，表面上皮的双氧化酶（Duox）产生H2O2，以及SCN在气道上皮上的运输。SCN-转运主要由CFTR在根尖质膜中介导。因此，CF气道上皮的SCN分泌减少，因此，在OSCN的产生和细菌杀灭方面存在缺陷。基底外侧SCN转运体的分子特性仍有待在体内系统中确定。当前知识的这一差距阻碍了增强肺部氧化先天免疫的努力和了解氧化系统的临床重要性。药理学数据表明，至少在培养的气道上皮中，碘化钠同向转运体（NIS）是基底外侧SCN转运体。然而，我们的初步结果表明，NIS及其同源物slc5a8都可能介导气道上皮细胞分泌SCN-。此外，slc5a8在表面上皮中表达，而NIS主要局限于粘膜下腺的浆液细胞。我们的中心假设是slc5a8和NIS的区隔表达在气道中形成了两条SCN-分泌途径，并且Duox/LPO/SCN-系统对抗菌免疫至关重要。我们还假设涉及NIS的分泌途径可用于将碘化物（I-）递送到气道表面液体，其中Duox/LPO将I-氧化为次碘酸(H2O2 + I- ？HOI)，一种非常有效的抗菌和抗病毒分子。因此，我们的目标是确定NIS和slc5a8与气道中SCN-分泌的功能相关性，并评估在SCN-和I-存在下Duox/LPO酶对气道先天免疫的生物学重要性。我们提出以下具体目标：明确区分NIS和slc5a8表达对气道中SCN分泌的重要性。2. 评估Duox/LPO/SCN-系统对人体气道细菌杀灭的重要性。3. 探讨I-在气道中的药代动力学，以及气道液体I-对细菌杀灭的影响。我们将使用1)体内方法，如人类气道异种移植和人类受试者研究来实现这些目标；2)细胞生物学工具，包括原代气道上皮培养、腺病毒介导的基因传递和细菌杀伤测定；3) RNA干扰等分子生物学方法；4)生化分析包括离子交换色谱法和比色法氧化还原反应。该项目预计将确定OSCN介导的气道细菌杀伤所需的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，这一点迄今尚未被探索。公共卫生相关性。该项目将探索一种新的气道先天免疫机制，通过产生活性氧来消除细菌。体外实验表明，这种新机制在囊性纤维化患者的气道上皮中存在缺陷。因此，提出的研究可能会确定在囊性纤维化和其他以反复气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.