Metformin alters airway epithelial tight junction protein abundance in epithelia-bacteria co-culture model

JP Garnett, W Patkee, A Weekes, EH Baker, DL Baines. St George's, University of London, London, UK

Glucose in the airway surface liquid (ASL) is normally maintained at a low concentration compared to the blood (~12.5 x lower). Raised blood glucose elevates ASL glucose (1), which increases the risk of respiratory infection, particularly with methicillin-resistant Staphylococcus aureus (2) and Pseudomonas aeruginosa (3). Previously, we developed an in vitro co-culture model to investigate the relationship between basolateral/blood glucose concentration and apical/luminal S. aureus growth using H441 human airway epithelial monolayers (4). Using this model we demonstrated that glucose diffuses across the epithelium into the ASL via paracellular pathways where it can be utilised by S. aureus to support its growth and that S. aureus growth can be reduced by limiting the paracellular leak of glucose using metformin. The metformin-induced reduction in paracellular permeability was associated with an increase in transepithelial electrical resistance (TEER), an indicator of tight junction integrity. The aim of this study was to investigate whether metformin alters bacteria-induced changes in airway epithelial tight junction protein abundance, by western blot analysis of co-culture samples.

S. aureus (8325-4) addition to the apical surface of H441 monolayers for 7 hours produced a significant decrease in E-cadherin (p<0.001,n=13) and occludin abundance (p<0.05,n=5), but had no effect on claudin-1 (p>0.05,n=3). Pre-treatment of H441 monolayers with metformin (1 mM; 18 hours) prior to S. aureus addition, had no significant effect on E-cadherin, but did enhance occludin abundance (p<0.05,n=5).

We also investigated the effects of metformin using P. aeruginosa (PA01) - Calu-3 airway epithelia co-cultures. Similar to S.aureus-H441 co-cultures, metformin pre-treatment attenuated the P. aeruginosa induced decrease in Calu-3 monolayer TEER (control= 669±27Ω.cm2; +PA01= 489±20Ω.cm2; +PA01+metformin= 551±16Ω.cm2; p<0.05, n=5) and significantly inhibited glucose-induced bacterial growth (p<0.05, n=4). P. aeruginosa had no effect on E-cadherin or claudin abundance in Calu-3 monolayers, but completely abolished occludin expression (p<0.0001, n=3) which could not be reversed by metformin pre-treatment. Metformin did however produce an increase in Calu-3 claudin abundance in the presence and absence of P. aeruginosa (p<0.01, n=3).

These data provide new evidence that metformin pre-treatment of airway epithelial monolayers can partially reverse the effects of bacteria on paracellular permeability by enhancing the abundance of tight junction proteins and in doing so limits the paracellular flux of glucose into the ASL, restricting bacterial growth. Such mechanisms may have important implications in identifying drug targets for the treatment of respiratory infections associated with raised ASL glucose, including chronic obstructive pulmonary disease and cystic fibrosis.

1. Baker EH et al. (2007) J Appl Physiol 102: 1969-1975.

2. Philips BJ et al. (2005) Thorax 60: 761-764.

3. Brennan AL et al. (2007). J Cyst Fibros 6: 101-109.

4. Garnett JP et al. (2013). Thorax 68(9):835-45.