Glucose Depletion in the Airway Surface Liquid Is Essential for Sterility of the Airways Alejandro A. Pezzulo1, Jeydith Gutie´ rrez1, Kelly S. Duschner1, Kelly S. McConnell1, Peter J. Taft1, Sarah E. Ernst1, Timothy L. Yahr2, Kamal Rahmouni1, Julia Klesney-Tait1, David A. Stoltz1, Joseph Zabner1* 1 Department of Internal Medicine, University of Iowa, Iowa City, Iowa, United States of America, 2 Department of Microbiology, University of Iowa, Iowa City, Iowa, United States of America Abstract Diabetes mellitus predisposes the host to bacterial infections. Moreover, hyperglycemia has been shown to be an independent risk factor for respiratory infections. The luminal surface of airway epithelia is covered by a thin layer of airway surface liquid (ASL) and is normally sterile despite constant exposure to bacteria. The balance between bacterial growth and killing in the airway determines the outcome of exposure to inhaled or aspirated bacteria: infection or sterility. We hypothesized that restriction of carbon sources –including glucose– in the ASL is required for sterility of the lungs. We found that airway epithelia deplete glucose from the ASL via a novel mechanism involving polarized expression of GLUT-1 and GLUT-10, intracellular glucose phosphorylation, and low relative paracellular glucose permeability in well-differentiated cultures of human airway epithelia and in segments of airway epithelia excised from human tracheas. Moreover, we found that increased glucose concentration in the ASL augments growth of P. aeruginosa in vitro and in the lungs of hyperglycemic ob/ob and db/db mice in vivo. In contrast, hyperglycemia had no effect on intrapulmonary bacterial growth of a P. aeruginosa mutant that is unable to utilize glucose as a carbon source. Our data suggest that depletion of glucose in the airway epithelial surface is a novel mechanism for innate immunity. This mechanism is important for sterility of the airways and has implications in hyperglycemia and conditions that result in disruption of the epithelial barrier in the lung. Citation: Pezzulo AA, Gutie´rrez J, Duschner KS, McConnell KS, Taft PJ, et al. (2011) Glucose Depletion in the Airway Surface Liquid Is Essential for Sterility of the Airways. PLoS ONE 6(1): e16166. doi:10.1371/journal.pone.0016166 Editor: Mauricio Rojas, University of Pittsburgh, United States of America Received September 9, 2010; Accepted December 8, 2010; Published January 20, 2011 Copyright: 2011 Pezzulo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a University of Iowa Department of Internal Medicine Translational Research Pilot Grant, and in part by National Heart, Lung and Blood Institute HL51670 and HL091842, National Institutes of Diabetes and Digestive and Kidney Diseases DK54759 and a Cystic Fibrosis Foundation RDP Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: joseph-zabner@uiowa.edu Introduction Hyperglycemia in both diabetic and non-diabetic patients is associated with an increased risk of complications and mortality from community acquired pneumonia [1–3]. Interestingly, in the absence of hyperglycemia, diabetes itself does not increase these risks [2–4]. A number of possible explanations for the association between hyperglycemia and pulmonary infection exist, but the mechanisms are still unknown. The luminal surface of airway epithelia is covered by a thin layer of fluid, termed the airway surface liquid (ASL) [5], and is normally sterile despite frequent exposure to bacteria, fungi and viruses. Inhaled and aspirated bacteria [6] are cleared upon reaching this surface by mechanisms that include ASL antimicro- bials, mucociliary clearance and other components of innate and adaptive immunity. In addition to these antimicrobial compo- nents, the ASL also contains factors that are required for bacterial growth, such as electrolytes, proteins, lipids, amino acids and oligo-/mono-saccharides, including glucose [7,8]. It has been speculated that amino acid, peptide and sugar residue transporters contribute to lung host defense by clearing these compounds from the surface of the airways and alveoli and making them unavailable to bacteria [8,9]. Recently, Baker et al. [10] showed that the concentration of glucose in the ASL is increased in hyperglycemic non-diabetics and diabetics, and in humans with cystic fibrosis (CF). In other studies, the same group demonstrated that the detection of glucose in bronchial aspirates of intubated patients correlated with the risk of MRSA infection [11] and that glucose in the nasal secretions of humans with CF-related diabetes facilitated growth of Staphylococ- cus aureus and Pseudomonas aeruginosa [12]. In this study, we hypothesized that restriction of carbon sources -specifically glucose- in the ASL is an important component of innate immunity. We report a mechanism responsible for a glucose concentration gradient generated by human airway epithelia resulting in low ASL glucose concentration, impaired bacterial growth, and airway sterility. Materials and Methods For detailed materials and methods, see Supporting Methods in Text S1. Ethics Statement Animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments were approved by the Animal Care and Use Committee of the University of Iowa (ACURF # 1004078). PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16166