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

硫氰酸钠同向转运体在抗菌气道宿主防御中的作用

• 批准号: 7862596
• 负责人: Botond Banfi
• 金额: $ 37.58万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7862596
• 关键词: Acids; Adenoviruses; Affinity; Anti-Bacterial Agents; Antibiotics; Antiviral Agents; Apical; Bacteria; Bacterial Infections; Biochemical; Biological; Biological Assay; Cell Culture Techniques; Cell membrane; Cells; Cellular biology; Chemicals; Clinical; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Disease; Drug Kinetics; Enzymes; Epithelial; Epithelial Cells; Epithelium; FDA approved; Gene Delivery; Generations; Gland; HOI; Harvest; Homologous Gene; Host Defense; Host Defense Mechanism; Human; Human Activities; Hydrogen Peroxide; Immune; Immunity; In Vitro; Infection; Ingestion; Intake; Iodides; Ion-Exchange Chromatography Procedure; Knowledge; Liquid substance; Lung; Mediating; Methods; Modeling; Molecular; Molecular Biology; Natural Immunity; Nose; OSCN-; Oral; Organ; Oxidases; Oxidation-Reduction; Patients; Pattern; Potassium Iodide; Production; Proteins; Publishing; RNA Interference; Reaction; Reactive Oxygen Species; Recurrence; Role; Route; Serous; Serum; Surface; System; Tablets; Testing; Therapeutic; Xenograft Model; Xenograft procedure; airway epithelium; antimicrobial; bactericide; base; catalase; cell type; cystic fibrosis airway epithelia; cystic fibrosis patients; defined contribution; healthy volunteer; human subject; hypothiocyanite; in vivo; in vivo Model; inhibitor/antagonist; insight; killings; lactoperoxidase; novel; oxidation; pathogen; prevent; public health relevance; research study; sodium thiocyanate; sodium-iodide symporter; symporter; tool; uptake

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.

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中的细菌感染。这种新的机制杀死细菌产生杀菌次硫氰酸盐（OSCN-）在乳过氧化物酶（LPO）催化的反应：H2 O2 + SCN-？OSCN-。因此，OSCN的产生需要粘膜下腺体分泌LPO，表面上皮细胞的双氧化酶（Duox）产生H2 O2，以及SCN穿过气道上皮的转运。SCN-转运主要由顶端质膜中的CFTR介导。因此，CF气道上皮细胞具有减少的SCN-分泌，并因此在OSCN-产生和细菌杀伤方面有缺陷.基底外侧SCN-转运蛋白的分子身份仍有待于在体内系统中确定。目前知识的这一差距阻碍了增强肺中氧化先天免疫和理解氧化系统的临床重要性的努力。药理学数据表明-至少在培养的气道上皮细胞中-钠-碘同向转运体（NIS）是基底外侧SCN-转运体。然而，我们的初步结果表明，NIS和它的同源物slc 5a 8都可以介导气道上皮细胞的SCN-分泌。此外，slc 5a 8在表面上皮中表达，而NIS主要定位于粘膜下腺体的浆液细胞。我们的中心假设是slc 5a 8和NIS的区室化表达在气道中产生两种SCN-分泌途径，并且Duox/LPO/SCN-系统对于抗菌免疫是关键的。我们还假设，涉及NIS的分泌途径可用于将碘（I-）输送到气道表面液体，Duox/LPO将I-氧化为次碘酸（H2 O2 + I-？HOI），一种非常有效的抗菌和抗病毒分子。因此，我们的目标是定义NIS和slc 5a 8与气道中SCN-分泌的功能相关性，并评估Duox/LPO酶在存在SCN-和I-的情况下对气道先天免疫的生物学重要性。我们提出以下具体目标：1。定义区室化NIS和slc 5a 8表达对气道中SCN分泌的重要性。2.评价Duox/LPO/SCN系统对人体气道细菌杀灭的重要性。3.探讨I-在气道内的药代动力学，以及气道液中I-对细菌的杀灭作用.我们将使用以下方法来实现这些目标：1）体内方法，如人气道异种移植物和人体受试者研究; 2）细胞生物学工具，包括原代气道上皮细胞培养、腺病毒介导的基因递送和细菌杀伤试验; 3）分子生物学方法，如RNA干扰; 4）生化试验，包括离子交换色谱法和比色氧化还原反应。该项目预计将确定OSCN介导的气道细菌杀伤所需的SCN转运蛋白，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，迄今为止尚未探索。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。体外实验已经证明，这种新的机制是有缺陷的囊性纤维化患者的气道上皮细胞。因此，拟议的研究可以确定恢复或增强囊性纤维化和其他以反复气道感染为特征的疾病中的氧化宿主防御系统的治疗潜力。