Sulforaphane enhances the inhibitory activity of agomiR-93 on the expression of proinflammatory genes induced by Pseudomonas aeruginosa in Cystic Fibrosis IB3-1 cells

Cystic fibrosis (CF) is an inherited genetic disease characterized by the following clinical and biochemical features: (a) dysregulation of the cystic fibrosis transmembrane regulator (CFTR) gene, (b) a chronic hyperinflammatory state and (c) frequent and severe bacterial infections of the lungs. With respect to the molecular basis of the activation of pro-inflammatory genes, an increasing number of reports have been published showing a clear role of microRNAs in chronic Pseudomonas aeruginosa lung infection in cystic fibrosis. This observation was the rationale for proposing “microRNA Therapeutics” based on treatment of target cells with agomiR molecules mimicking the activity of down-regulated microRNAs, obtaining inhibitory effects on the expression of miRNA‐regulated pro‐inflammatory genes. In this study, we aimed to determine whether this “miRNA therapeutics” approach can be combined with treatment with natural compounds, in order to maximize the biological effects on the expression of pro-inflammatory genes. Among the several anti-inflammatory natural compounds, we selected Sulforaphane (SFN), which is highly concentrated in broccoli sprouts (Brassica oleracea) and has been demonstrated to exhibit significant antioxidant, anti-inflammatory, antiviral and anticancer properties. In the present study, human bronchial epithelial IB3-1 cells were used, transfected with the anti-inflammatory agomiR-93. The cystic fibrosis IB3-1 cellular model system has been extensively employed to study the biochemical and molecular effects of P. aeruginosa infection. In our experimental plan, after 48 hours of treatment with 100 nM agomiR-93 or with 100 nM agomiR-93 plus 5 µM SFN (step “1”), the cells were infected with the P. aeruginosa PAO1 strain for 4 hours (step “2”). Changes in gene expression were studied by RT-qPCR and with Bio-Plex analyses (step “3”) as described by Fabbri et al. The results obtained demonstrated that PAO1 infection induced an increase in gene expression of the main pro-inflammatory cytokines involved in the cystic fibrosis inflammation sustained by PAO infection, such as IL-6, IL-8, IL-1β and G-CSF, as demonstrated by RT-qPCR analysis and the absolute quantification of proteins in cell supernatants obtained with Bio-Plex. This increase was inhibited by treatment with both the agomiR-93 and with SFN with different percentages depending on the selected pro-inflammatory protein. Notably, Bio-Plex results demonstrated that the inhibitory effect on the expression of IL-6, IL-8, G-CSF genes increased in the presence of both agomiR-93 and SFN. The same cooperative effect was also observed for IL-1β when analyzed by RT‐qPCR. Since the expression of these genes play an important role in regulating the inflammatory state of cystic fibrosis, this finding suggests that the use of natural products can be combined with the “miRNA Therapeutics” approach to obtain a cooperative effect in inhibiting the expression of pro-inflammatory genes involved in cystic fibrosis.