Azithromycin (AZM) decreases Glutathione-S-Transferase (GST) -T1 expression and activity in CF airway epithelial cells.

Lung damage in cystic fibrosis (CF) patients is determined by mucus accumulation, Pseudomonas aeruginosa infection and chronic inflammation. Extracellular GSH is a scavenger of free radicals produced by neutrophils in inflamed tissues. Glutathione transferases (GST) are a superfamily of dimeric proteins which conjugate glutathione to a wide range of substrates including oxidants and are involved the synthesis of leukotriens. Clinical beneficial effects have been reported in CF patients following treatment with the macrolide azythromicin (AZM); anti-inflammatory properties have been proposed as possible mechanism. The aim of this study is to investigate the regulation of the GSTT1 and GSTM1 activity and expression by AZM. Reductions of about 25% and 40% on GST enzymatic activity were detected in IB3-1 and 2CFSMEo- cells respectively. GSTs mRNA expression in CF airway epithelial cell lines was analysed by quantitative PCR (qPCR). The level of GSTT1 and GSTM1 basal expression in CF cells IB3- 1 was significantly higher than in isogenic non-CF cells C38. We found statistically significant decreases of GSTT1 and GSTM1 mRNA of about 30% and 25% respectively in IB3-1 cells after treatment with AZM for 24 hours restoring the levels observed in C38 cells. In 2CFSMEo- cells after exposure to AZM we observed 50% and 45% reductions in GSTT1 and GSTM1 mRNA respectively. The macrolide JM, known to lack clinical anti-inflammatory properties, had no significant effects on GSTT1 and GSTM1 mRNA expression in all cell lines. Furthermore, AZM did not alter the mRNA expression levels of GSTP1, a glutathione-S-transferase not differentially expressed in CF and isogenic non-CF cells. Decreased expression of 50% and 85% of GSTT1 protein has been detected by immunoblotting in IB3-1 and 2CFSMEo- cells, respectively, following treatment with AZM. In the same conditions we found a drastic reduction of protein level of GSTM1 in both CF cell lines.Finally, GSTs activity and the expression of GSTT1 and GSTM1 proteins in CF cells, were reduced approximately to the same level detected after treatment with interleukin 10 (IL-10), an anti-inflammatory cytokine, shedding light on a possible correlation between GSTs inhibition and antiinflammatory properties of AZM. The effects of AZM described in this study suggest that downregulation of GSTT1 and GSTM1 expression may result in increased availability of intracellular GSH making CF cells less susceptible to oxidative stressinduced by chronic inflammation. Inhibition of GSTT1 and GSTM1 mightprovide a therapeutic approach for limiting the effects of inflammation criticalfor lung damage in CF patients. This study is supported by Italian CF Research Foundation; Comitato diVicenza-Associazione Veneta per la lotta contro la Fibrosi Cistica; AziendaOspedaliera Verona, Italy.