Primary Cultures of human airway epithelia Primary human airway epithelia were grown at the air-liquid interface as previously described [13]. Transmission Electron Microscopy Primary cultures of human airway epithelia were fixed using a fluorocarbon-based protocol that allows preservation of the ASL and mucus layer. Collection of airway surface liquid (ASL) ASL was collected from primary cultures of human airway epithelia and glucose concentration was determined using a glucose oxidase assay. Measurement of transepithelial electrical properties of intact human tracheal epithelia Epithelia dissected from 3 human tracheas (obtained from the University of Iowa In Vitro Models and Cell Culture Core) were studied in Ussing chambers. Microarray data analysis A MIAME-compliant microarray dataset previously deposited in GEO under accession #GSE20502 was used to determine the mRNA expression profile of GLUT and SGLT transporters in human airway epithelia in vivo and in vitro. For method details, see GEO accession. Immunocytochemistry Primary cultures of HAE were immunostained and visualized with confocal microscopy as described in [14]. For primary and secondary antibodies, see Text S1. As controls, we studied epithelia without primary antibody and isotype-matched control antibody. Bidirectional fluxes and uptake of glucose Bidirectional fluxes and uptake of radiolabeled L-glucose and 2- deoxyglucose were done using glucose-free DMEM supplemented with 5 mM of the corresponding non-radiolabeled and 0.5 mCi of radiolabeled glucose analog. Bacterial strains The PAO1 strain of P. aeruginosa was used where indicated. The edd 2 mutant was obtained from the UW P. aeruginosa Mutant Library [15]. Adequate transposon insertion was confirmed by PCR as indicated by providers of the strain. P. aeruginosa growth in vitro PAO1 was grown overnight in LB medium at 37uC. Bacteria were centrifuged at 4uC for 15 min. at 32006 g. and resuspended in 50 mL M9 medium twice followed by dilution in M9 or ASL pooled from 6 different donors, to an OD 0.05 at 600 nm. Glucose was added as indicated. Samples were kept in a shaking incubator at 300 rpm and 37uC, and OD 600 nm was determined at times shown. P. aeruginosa growth on cultures of human airway epithelia The apical surface of cultures of human airway epithelia was inoculated with PAO1 in a volume of 0.1 mL and bacteria were quantified after 24 hours. P. aeruginosa pulmonary challenge in mice Sex-matched 22 to 24 week-old ob/ob and db/db mice and their littermate controls were used. Intranasal inoculation with the Airway Glucose and Innate Immunity indicated bacterial strain was followed by quantification of bacteria in lung homogenate after 6 hours. Statistical analysis Statistical analyses were performed in Graphpad Prism 5 for MacOSX. Unpaired T-test was performed on every analysis except in Figure 4, C and D, where a Z-test for proportions was used and Figure 5, A and B, where Mann-Whitney U test for nonparametric data was performed. A p-value ,0.05 was considered statistically significant [16]. Results Airway epithelia generate a transepithelial glucose gradient Studies of exhaled breath condensate have shown that the glucose concentration in ASL is approximately one tenth that in serum [10]. We hypothesized that the airway epithelium can maintain a transepithelial glucose gradient. We collected ASL from well-differentiated human airway epithelia cultures grown at the air-liquid interface (Figure 1, A), and compared the glucose concentration to that in the basolateral nutrient media. While the basolateral glucose concentration was 16.660.4 mM, the concen- tration in the ASL was 2.260.5 mM (Figure 1, B). Na+/glucose co-transporter activity is absent from airway epithelia Na+-glucose co-transporters (SGLT-1 and SGLT-2) transport glucose against a concentration gradient in intestinal, renal, and lung alveolar epithelia [17,18]. Transport of glucose follows the Na+ gradient generated by the Na+/K+ ATPase pump. This mechanism is capable of generating a current that can be measured as a short circuit current (Isc) in Ussing chamber studies [19]. We hypothesized that glucose depletion from the ASL occurs through SGLT-mediated Na+-glucose co-transport. Airway epi- thelia dissected from human tracheas were mounted in Ussing chambers for Isc measurements. Apical glucose concentration was sequentially increased (Figure 2, A–B). Apical glucose did not generate a change in Isc, in contrast to tissues that express Na+- Figure 1. Human airway epithelia maintain a transepithelial glucose gradient. (A) Transmission electron microscopy of perfluor- ocarbon-fixed cultures of human airway epithelia. Figure indicates airway surface liquid (ASL), epithelial surface (ES) and filter membrane support (FM). (B) Glucose concentration in ASL collected from cultures of human airway epithelia was compared to glucose concentration in basolateral nutrient media. Data shown as mean 6 s.e.m. n = 6 samples per group. (***: p,0.0001). doi:10.1371/journal.pone.0016166.g001 PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16166