The effects of tobramycin on pulmonary fibroblast function

Tobramycin (Tob) is widely used to treat gram-negative bacterial infection of the airway, is taken up by fibroblasts, chelates copper and inhibits copper dependent lysyl oxidase (LOX) activity. LOX cross links collagen and elastin into the extracellular matrix (ECM), and Tob is hypothesised to have anti fibrotic properties. The effect of Tob on primary normal human lung fibroblast (NHLF) function was investigated using the induction of spheroid formation from monolayer culture as a model of fibrosis. NHLF were grown in 24 well plates, quiesced, and treated with 1, 10 and 100 µM Tob and CuSO4 in media crowded with 100 µg/ml >500 kDa dextran sulphate and activated with 10 ng/ml TGFβ1. Spheroid formation was measured as size, number and time until initial formation by video microscopy over 48 hours. Elastin and MMP-3 expression by NHLF was measured over 14 days using a colourimetric assay and ELISA respectively. Spheroid formation was dependent on added TGFβ1. 100 µM Tob significantly reduced the number of spheroids/well compared to TGFβ alone from 80±5 to 18.5±1.5, and significantly increased time to formation from 16.5±0.4 to 25.2±2.1 hours. MMP-3 was significantly increased from 19.5±0.8 to 32.1±2.8 ng/ml and soluble elastin was significantly increased from 85.3±6.4 to 169.7±7.4 µg/well. CuSO4 (100 µM) had a pro fibrotic effect and increased spheroid size. In conclusion, Tob inhibits spheroid formation, and increases potential ECM breakdown, whilst exogenous CuSO4 enhances spheroid formation. This suggests that Tob prevents a pro-fibrotic fibroblast phenotype from developing via inhibition of LOX or other copper dependent mechanisms.