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.
. 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.
|Derscheid, Rachel J; van Geelen, Albert; Berkebile, Abigail R et al. (2014) Increased concentration of iodide in airway secretions is associated with reduced respiratory syncytial virus disease severity. Am J Respir Cell Mol Biol 50:389-97|
|Meitzler, Jennifer L; Hinde, Sara; Bánfi, Botond et al. (2013) Conserved cysteine residues provide a protein-protein interaction surface in dual oxidase (DUOX) proteins. J Biol Chem 288:7147-57|
|Lorentzen, Daniel; Durairaj, Lakshmi; Pezzulo, Alejandro A et al. (2011) Concentration of the antibacterial precursor thiocyanate in cystic fibrosis airway secretions. Free Radic Biol Med 50:1144-50|
|Fischer, Anthony J; Lennemann, Nicholas J; Krishnamurthy, Sateesh et al. (2011) Enhancement of respiratory mucosal antiviral defenses by the oxidation of iodide. Am J Respir Cell Mol Biol 45:874-81|