Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC195
The mucosal glucose concentration of polarised human airway epithelial cell monolayers affects luminal Staphylococcus aureus growth
J. P. Garnett1, C. Ozcan1, D. Lonsdale1, C. Da Tan1, E. H. Baker1, D. L. Baines1
1. Division of Biomedical Sciences, St George's, University of London, London, United Kingdom.
Glucose in the airway surface liquid (ASL) is normally maintained at a low concentration compared to glucose levels in the blood (~12.5 times lower). In healthy humans, ASL glucose concentrations rise in response to increased blood glucose (1). Patients on intensive care with elevated ASL glucose are more likely to have respiratory infection, particularly with methicillin-resistant Staphylococcus aureus (MRSA; 2). The aim of this study was to investigate the relationship between mucosal glucose concentration, glucose in the ASL and bacterial growth using an in vitro model of human airway epithelium. H441 epithelial cells were grown on transwell inserts for 10 days, at air-liquid interface, to form polarised monolayers. Cells were washed and bathed in a HCO3--buffered Krebs solution to remove antibiotics and growth supplements which could effect bacterial growth. The glucose concentration of the mucosal Krebs solution was set at 10, 20 or 40mM. For ASL glucose analysis, cells were incubated for 1 hour with the different glucose concentrations. 150μl of solution containing 0.67μg ml-1 FITC-dextran was used to wash the surface of the transwells. Glucose concentration of the washes was quantified using a Annalox glucose analyser. ASL volume was calculated by quantifying dilution of the FITC dextran using a fluorescent spectrophotometer. For co-culture studies, Staph. aureus (8325-4) were grown overnight in RPMI medium to produce a culture of ~108 colony forming units (CFU) ml-1. 50μl of 107 CFU ml-1 in glucose-free RPMI was applied to the luminal surface of H441 monolayers. The H441-Staph. aureus co-culture was incubated for 7 hours at 37°C with 5% CO2. Transepithelial electrical resistance (TEER) of the monolayers was measured using a voltohmmeter (WPI). The co-culture was then scraped from the transwells and CFU’s were determined using the Miles-Misra method. Calculated ASL glucose was less than mucosal glucose concentration but increased from 0.5±0.2mM to 1.5±0.2 and 6.5±1.3mM as basolateral glucose concentration was raised from 10mM to 20mM and 40mM, respectively (P<0.05; n=3-5). The TEER of H441 monolayers was significantly reduced following the addition of Staph. aureus (248±44 Ω.cm2), compared to control monolayers (498±104 Ω.cm2; P<0.05; n=5). Altering mucosal glucose had no effect on H441 TEER. Luminal Staph. aureus growth directly correlated with mucosal glucose concentration, increasing from 8.6±8.1x107 to 1.5±0.8x108 CFU ml-1 and 3.4±1.4x108 CFU ml-1 upon elevation of mucosal glucose from 10mM to 20mM and 40mM, respectively (P<0.05; n=5). These results indicate that raising mucosal glucose increases glucose in ASL and that Staph. aureus utilise this glucose to promote their growth. Moreover, by reducing tight junction integrity bacteria may also increase the paracellular diffusion of glucose into the ASL.
Where applicable, experiments conform with Society ethical requirements