In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3–1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 3′-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated using three luciferase vectors, including one carrying the complete 3′-UTR region of the IL-8 mRNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3–1, CuFi-1, and NuLi-1 cells by pre–miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-κB–dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93.

IL-8 released from bronchial epithelial cells infected with Pseudomonas aeruginosa plays a crucial role in the chronic lung pathology of patients affected by cystic fibrosis. Novel anti-inflammatory approaches will benefit from a thorough understanding of the regulatory mechanisms involved in the transcription of this chemokine to identify potential pharmacological targets. We addressed this issue by investigating the role of phosphoproteins and transcription factors (TFs) on transcription of IL-8 gene in the human bronchial epithelial IB3-1, CuFi-1, and Calu-3 cells. P. aeruginosa increased the basal phosphorylation of the ERK1/2 pathway components 90-kDa ribosomal S6 kinase (RSK)1/2 and mitogen- and stress-activated kinase-2 and of the p38 MAPK pathway components p38α/δ/γ and heat shock protein 27 (HSP27). The involvement of these kinases in the expression of IL-8 gene was confirmed with pharmacological inhibitors of ERK1/2, RSK, p38, and HSP27 both at transcription and secretion levels. Transfection of TF decoy oligodeoxynucleotides, designed to interfere with the interaction of the TFs NF-κB, NF-IL6, AP-1, CREB, and CHOP with the corresponding consensus sequences identified in the IL-8 promoter, reduced the P. aeruginosa-dependent transcription of IL-8, suggesting their participation in the transcriptional machinery. Stimulation of IB3-1 cells with IL-1β led to a similar pattern of activation, whereas the pattern of phosphoproteins and of TFs modulated by TNF-α differentiated sharply. In conclusion, the results highlight a novel role for RSK1/2 and HSP27 phosphoproteins and of the cooperative role of the TFs NF-κB, NF-IL6, AP-1, CHOP, and CREB in P. aeruginosa-dependent induction of transcription of the IL-8 gene in human bronchial epithelial cells.