Bacteria constantly challenge the airways, and whether they are eliminated, resulting in a sterile lung, or proliferate to cause infection or colonization depends on host factors and the bacteria. In the airways, multiple innate immune mechanisms have evolved to rapidly kill, expel, or engulf bacteria. These diverse mechanisms act within seconds (antimicrobials, cough) or minutes (mucociliary clearance and phagocytes) and are most effective with small inocula of bacteria. Abnormally high glucose concentration in the ASL could both directly promote bacterial growth and impair antimicrobials [49]. When bacteria are not eliminated, growth can be exponential and virulence factors of pathogenic and non- pathogenic bacteria can play a role. Thus, in the airways, the time- dependent balance between bacterial killing and growth determine the outcome. Our data suggest that carbon source deprivation in airway surface liquid helps tip the balance towards airway sterility. Supporting Information Figure S1 Na+-glucose cotransport activity is absent from well- differentiated cultures of human airway epithelia. Short-circuit current in cultures of human airway epithelia was studied in Ussing chambers. Amiloride and bumetanide were added to Ussing chamber solution, followed by glucose in the apical or basolateral solution in increasing concentrations. Representative tracings of 3 replicates are shown. (TIFF) Figure S2 Basolateral to apical (BL to AP) and apical to basolateral (AP to BL) fluxes of L-[1-14C]glucose (A) or 2-deoxy-d- [1-14C]glucose (B) were measured in intact pig tracheal epithelia over 1 hour. Data shown as mean 6 s.e.m. n = 9 samples per group. (***: p,0.0001, ns: p $ 0.05). (C) Basolateral (BL) and apical (AP) uptake of 2-deoxy-d-[1-14C]glucose (2-DOG) in intact pig tracheal epithelia were measured over 1 hour. Data shown as mean 6 s.e.m. n = 9 samples per group. (***: p,0.0001). (D) Correlation of transepithelial electrical conductance at baseline vs. transepithelial conductance of glucose in intact pig tracheal epithelia. In the intact tracheal epithelia of pigs, bidirectional fluxes of L-glucose were higher than in cultured human epithelia and fluxes of 2-DOG occurred at similar rates in the presence of uptake from both the apical and basolateral membranes. These data indicate that glucose supplied to intact airway epithelia can reach the ASL through the paracellular but not the transcellular pathway. Moreover, the result suggests that intracellular phos- phorylation of glucose resulting in a transepithelial concentration gradient occurs at a higher rate in vivo compared to cultured cells. (TIFF) References 1. Baker EH, Wood DM, Brennan AL, Clark N, Baines DL, et al. (2006) Hyperglycaemia and pulmonary infection. Proc Nutr Soc 65: 227–235. 2. Kornum JB, Thomsen RW, Riis A, Lervang HH, Schønheyder HC, et al. (2007) Type 2 diabetes and pneumonia outcomes: a population-based cohort study. Diabetes Care 30: 2251–2257. 3. McAlister FA, Majumdar SR, Blitz S, Rowe BH, Romney J, et al. (2005) The relation between hyperglycemia and outcomes in 2,471 patients admitted to the hospital with community-acquired pneumonia. Diabetes Care 28: 810–815. 4. Vardakas KZ, Siempos II, Falagas ME (2007) Diabetes mellitus as a risk factor for nosocomial pneumonia and associated mortality. Diabet Med 24: 1168–1171. 5. Widdicombe JH, Widdicombe JG (1995) Regulation of human airway surface liquid. Respir Physiol 99: 3–12. 6. Johanson WG, Pierce AK, Sanford JP (1969) Changing pharyngeal bacterial flora of hospitalized patients. Emergence of gram-negative bacilli. N Engl J Med 281: 1137–1140. 7. Bartlett JA, Fischer AJ, McCray PB (2008) Innate immune functions of the airway epithelium. Contrib Microbiol 15: 147–163. Airway Glucose and Innate Immunity Figure S3 Swimming motility, twitching motility, protease activity and Type III secretion system protein expression are preserved in edd2. (A) A swimming motility assay comparing PAO1 and edd2 was performed as described in [50]. Represen- tative sample of n = 3 is shown. (B) A twitching motility assay comparing PAO1 and edd2 was performed as described in [51], duplicate samples are shown. (C) Protease activity was character- ized as described in [52], triplicate samples shown. (D) Expression of PcrV and ExoS, which are Type III secretion system proteins, was determined by using the method described in [53]. (TIFF) Figure S4 edd2 lethality is preserved in a D. melanogaster infection model. Survival of D. melanogaster was monitored for 72 h after inoculation of the abdominal cavity with PAO1 or edd2 as described in [54] (n = 20). (TIFF) Figure S5 edd2 lethality is preserved in a murine peritoneal infection model. All experiments were reviewed and approved by the Animal Care and Use Committee of the University of Iowa. Male 6 to 8 week old C57B/J6 mice obtained from Harlan Industries, Inc (Indianapolis, IN, USA) were used and were allowed access to food and water ad libitum. Mice were anesthetized with isoflurane and injected intraperitoneally with 36107 CFU of PAO1 or edd2 in a volume of 100 mL H2O. exsA2 strain of P aeruginosa was used as a negative control. Mice were carefully monitored and euthanized when end-point conditions were met (moribund, distressed, and unable to eat or drink). (TIFF) Supporting Information Methods. Text S1 (DOC) Acknowledgments We thank M. Welsh, K. Excoffon, B. Boles, P. McCray, M. Rogan and G. Davis for insightful discussion; N. Gansemer, T. Moninger, N. Rossen, A. Small, D. Hunter, J. Jensen-Arnold, P. Karp, T. Nesselhauf and P. Hughes for excellent assistance. We also thank the University of Iowa In Vitro Models and Cell Culture Core, and the University of Iowa Central Microscopy Research Facility. Author Contributions Conceived and designed the experiments: AAP JG KSD KSM PJT SEE TLY KR JK-T DAS JZ. Performed the experiments: AAP JG KSD KSM PJT SEE TLY. Analyzed the data: AAP JG KSD KSM SEE TLY DAS JZ. Contributed reagents/materials/analysis tools: KR JK-T. Wrote the paper: AAP JZ. 8. Mager S, Sloan J (2003) Possible role of amino acids, peptides, and sugar transporter in protein removal and innate lung defense. Eur J Pharmacol 479: 263–267. 9. Kalsi KK, Baker EH, Medina RA, Rice S, Wood DM, et al. (2008) Apical and basolateral localisation of GLUT2 transporters in human lung epithelial cells. Pflugers Arch 456: 991–1003. 10. Baker EH, Clark N, Brennan AL, Fisher DA, Gyi KM, et al. (2007) Hyperglycemia and cystic fibrosis alter respiratory fluid glucose concentrations estimated by breath condensate analysis. J Appl Physiol 102: 1969–1975. 11. Philips BJ, Redman J, Brennan A, Wood D, Holliman R, et al. (2005) Glucose in bronchial aspirates increases the risk of respiratory MRSA in intubated patients. Thorax 60: 761–764. 12. Brennan A, Gyi K, Wood D, Johnson J, Holliman R, et al. (2007) Airway glucose concentrations and effect on growth of respiratory pathogens in cystic fibrosis. J Cyst Fibros 6: 101–109. 13. Karp PH, Moninger TO, Weber SP, Nesselhauf TS, Launspach JL, et al. (2002) An in vitro model of differentiated human airway epithelia. Methods for establishing primary cultures. Methods Mol Biol 188: 115–137. PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16166