Figure 3. Airway epithelia can deplete airway surface liquid glucose. (A and B) 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 cultures of human airway epithelia over 1 hour. Data shown as mean 6 s.e.m. n = 6 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 cultures of human airway epithelia were measured over 1 hour. Data shown as mean 6 s.e.m. n = 6 samples per group. (***: p,0.0001). (D) Data from Figures 3 A, B and C are integrated into a model of glucose transport in human airway epithelia. Data in nmol/h. Black solid arrow represents paracellular diffusion of glucose. Gray solid arrows represent 2-DOG uptake capacity across the apical (top) and basolateral (bottom) membranes. Dotted arrows represent transcellular bidirectional fluxes of 2-DOG (adjusted by subtracting corresponding L-glucose fluxes). GLUT-10 is present in the apical membrane and GLUT-1 is present in the basolateral membrane. Intracellular glucose (G) is phosphorylated by hexokinase (HK) for subsequent glycolysis. G6P = glucose-6-phosphate. doi:10.1371/journal.pone.0016166.g003 directional transport. To calculate transcellular glucose transport, we repeated the bidirectional flux assays using 14C-2-deoxyglucose (14C-2-DOG), a glucose analog that is a substrate for glucose transporters and can also be phosphorylated by the intracellular hexokinase activity of the airway epithelial cells [24]. Once phosphorylated, glucose or 2-DOG cannot be transported across the cell membrane. We found a basolateral to apical transport rate (8.1560.52 nmoles/cm2/h) and an apical to basolateral transport rate (5.3560.38 nmoles/cm2/h) (Figure 3, B) that were both higher than that observed for paracellular transport. These results indicate that glucose supplied to airway epithelia can reach the ASL through both the paracellular and transcellular pathway. We next hypothesized that the apical glucose uptake capacity would be equal or greater than the net basolateral to apical transepithelial flux of glucose. Airway epithelia absorb glucose from the apical and basolateral compartments We conducted radiolabeled tracer uptake experiments in which we measured the amount of glucose that entered the intracellular compartment after addition to the apical or basolateral side of well-differentiated cultures of human airway epithelia. Uptake of 14C-2-DOG per sample was 12.362.4 nmoles/cm2/h after apical addition and 80.864.8 nmoles/cm2/h after basolateral addition (Figure 3, C). These data show that in human airway epithelia, both the apical and the basolateral membrane are permeable to Airway Glucose and Innate Immunity glucose and basolateral uptake occurs at a higher rate than apical uptake of glucose. Therefore, although glucose can reach the ASL through both the paracellular and transcellular pathways at a specific rate, the epithelial cells have the capacity to absorb it through the apical membrane at an equal or higher rate (Figure 3, D). This would allow the presence of a transepithelial glucose gradient at homeostasis and in the absence of Na+-glucose co- transport activity, as detailed in the discussion section. As a comparison, we investigated glucose fluxes in ex vivo tracheal epithelia from Sus scrofa (domestic pig) in Figure S2. The data indicate that in ex vivo tracheal epithelia, glucose can reach the ASL through the paracellular but not the transcellular pathway and uptake occurs at both the apical and basolateral membranes. These results are concordant with our in vitro data. We next studied the physiological significance of this transepithelial glucose gradient. Low glucose concentration impairs bacterial growth in ASL Airway surface liquid contains multiple antimicrobials known to play a role in innate immunity [7,25]. We hypothesized that the low glucose concentration in ASL may result in an impaired bacterial growth rate, favoring antimicrobial activity over bacterial growth. We chose P. aeruginosa (strain PAO1) as a pathogen because it can use multiple carbon sources for growth, including glucose [26]. The concentration of glucose in minimal growth media (M9) directly determined the growth rate of PAO1 in a dose-dependent manner (Figure 4, A). When we inoculated ASL in vitro (Figure 4, B), PAO1 grew only in the presence of 20 mM glucose (mean OD at 24 h for ASL = 0.23160.008 vs. mean OD at 24 h for ASL + glucose = 0.64360.012. p,0.0001). The growth rate observed without added glucose is in the range seen in minimal growth media with 0.5 to 2 mM glucose. These data suggest that ASL does not contain a sufficient concentration of carbon sources to support growth of P. aeruginosa. Low glucose concentration impairs bacterial growth on the airway surface in situ Airway surface liquid kills bacteria using salt-sensitive peptides such as lysozyme and defensins [7,25] that are efficient only against small inoculum sizes [25,27]. An increased bacterial growth rate in the presence of excess nutrient sources could overwhelm this system by allowing the bacteria to reach the threshold at which innate immune killing becomes ineffective. We investigated the effect of glucose on the ability of PAO1 to grow on the surface of human airway epithelia. We predicted that increasing the glucose concentration in ASL would impair the bactericidal activity against PAO1. We inoculated the apical surface of human airway epithelia cultures from 5 different donors with a PAO1 inoculum of 2.3 or 3.3 log CFU per group. As previously shown [27], human airway epithelia can effectively kill 2.3 log CFU of P. aeruginosa but not 3.3 log CFU. Importantly, when the inoculum and glucose were simultaneously applied to the ASL, airway epithelia failed to kill even 2.3 log CFU of PAO1 (Figure 4, C). We then predicted that lack of apical glucose transport would result in increased bacterial growth in ASL. While human airway epithelia were able to effectively kill an inoculum of up to 2.5 log CFU, preincubation with 5 mM apical glucose and phloretin –an inhibitor of GLUT transporters– resulted in failure to kill inoculums of 1.5 and 2.5 log CFU (Figure 4, D). These data are consistent with our in vitro results and suggest that low glucose concentration in ASL is important in preventing bacterial growth on the airway surface. PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16166