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The lungs of patients with cystic fibrosis (CF) are characterized by an exaggerated inflammation driven by secretion of IL-8 from bronchial epithelial cells and worsened by Pseudomonas aeruginosa infection. To identify novel antiinflammatory molecular targets, we previously performed a genetic study of 135 genes of the immune response, which identified the c.2534C>T (p.S845L) variant of phospholipase C-β3 (PLCB3) as being significantly associated with mild progression of pulmonary disease. Silencing PLCB3 revealed that it potentiates the Toll-like receptor's inflammatory signaling cascade originating from CF bronchial epithelial cells. In the present study, we investigated the role of the PLCB3-S845L variant together with two synthetic mutants paradigmatic of impaired catalytic activity or lacking functional activation in CF bronchial epithelial cells. In experiments in which cells were exposed to P. aeruginosa, the supernatant of mucopurulent material from the airways of patients with CF or different agonists revealed that PLCB3-S845L has defects of 1) agonist-induced Ca 2+ release from endoplasmic reticulum and rise of Ca 2+ concentration, 2) activation of conventional protein kinase C isoform β, and 3) induction of IL-8 release. These results, besides identifying S845L as a loss-of-function variant, strengthen the importance of targeting PLCB3 to mitigate the CF inflammatory response in bronchial epithelial cells without blunting the immune response.

The angelicin analogue 4,6,4'-trimethylangelicin (TMA) was recently reported as a strong inhibitor of nuclear factor- κ B (NF- κ B) activity and of the expression of the interleukin-8 (IL-8) gene in bronchial epithelial cells in which the inflammatory response has been challenged with P. aeruginosa , the most common bacterium found in the airways of patients affected by cystic fibrosis (CF). These findings encouraged us to analyze new synthetic analogues of TMA in order to evaluate their biological activities on human bronchial epithelial CF IB3-1 cells and to find more potent anti-NF- κ B agents exhibiting only minor antiproliferative effects. Analogues able to inhibit NF- κ B/DNA interaction at lower concentration than TMA were found and selected to investigate their biological activity on IB3-1 cells induced with TNF- α . In this biological system, NF- κ B-mediated IL-8 gene expression was investigated. Some analogues showed similar activity to the lead compound TMA. Other analogues displayed higher activities; in particular, the most interesting compounds showing relevant anti-inflammatory effects were found to cause 56-83% reduction of IL-8 mRNA expression at low concentrations (1-10  μ M), without changes in cell proliferation pattern, demonstrating their potential interest for a possible development of anti-inflammatory therapy of cystic fibrosis.

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.

Cystic fibrosis (CF) is characterized by a deep inflammatory process, with production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against IL-8, with the aim of reducing the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. TFD is based on biomolecules mimicking the target sites of transcription factors (TFs) and able to interfere with TF activity when delivered to target cells. Here, we review the inhibitory effects of decoy oligodeoxyribonucleotides (ODNs) on expression of IL-8 gene and secretion of IL-8 by cystic fibrosis cells infected by Pseudomonas aeruginosa. In addition, the effects of decoy molecules based on peptide nucleic acids (PNAs) are discussed. In this respect PNA-DNA-PNA (PDP) chimeras are interesting: (a) unlike PNAs, they can be complexed with liposomes and microspheres; (b) unlike oligodeoxyribonucleotides (ODNs), they are resistant to DNAses, serum and cytoplasmic extracts; (c) unlike PNA/PNA and PNA/DNA hybrids, they are potent decoy molecules. Interestingly, PDP/PDP NF-kappaB decoy chimeras inhibit accumulation of pro-inflammatory mRNAs (including IL-8 mRNA) in P. aeruginosa infected IB3-1, cells reproducing the effects of decoy oligonucleotides. The effects of PDP/PDP chimeras, unlike ODN-based decoys, are observed even in absence of protection with lipofectamine. Since IL-8 is pivotal in pro-inflammatory processes affecting cystic fibrosis, inhibition of its functions might have a clinical relevance., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Persistent recruitment of neutrophils in the bronchi of cystic fibrosis patients contributes to airway tissue damage, suggesting the importance of intervening on the expression of the neutrophil chemokine IL-8. Extracts from plants have been investigated to select components able to reduce IL-8 expression in bronchial epithelial cells challenged with Pseudomonas aeruginosa. Extracts and purified components have been added to cells 24 h before pro-inflammatory challenge with P. aeruginosa and IL-8 transcription was quantified in the IB3-1 CF cells in vitro. P. aeruginosa-dependent IL-8 mRNA induction was increased by Argemone mexicana and Vernonia anthelmintica whereas no significant modification of transcription was observed with Aphanamixis polystachya, Lagerstroemia speciosa and Hemidesmus indicus. Finally, inhibition of IL-8 was observed with Polyalthia longifolia (IC50=200 microg/ml) and Aegle marmelos (IC50=20 microg/ml). Compounds from A. marmelos were isolated and identified by GC-MS. No significant effect was observed with butyl-p-tolyl sulphate, whereas the inhibition obtained with 6-methyl-4-chromanone concentration was accompanied by an anti-proliferative effect. On the contrary, 5-methoxypsoralen resulted in IL-8 inhibition at 10 microM concentration, without effects on cell proliferation. In synthesis, 5-methoxypsoralen can be taken into consideration to investigate mechanisms of neutrophil chemotactic signalling and for its potential application in modulating the excessive CF lung inflammation.

Chronic pulmonary inflammation in patients affected by cystic fibrosis (CF) is characterized by massive bronchial infiltrates of neutrophils, which is sustained by the interaction of pathogens (e.g., Pseudomonas aeruginosa) with surface bronchial cells. To explore new treatment options focused on the reduction of neutrophil chemotaxis, we applied the transcription factor (TF) decoy approach, based on the intracellular delivery of double-stranded oligodeoxynucleotides (ODNs) causing inhibition of the binding of TF-related proteins to the different consensus sequences in the promoter of specific genes. In CF bronchial IB3-1 cells, P. aeruginosa induced transcription of the neutrophil chemokines IL-8 and GRO-gamma, of the adhesion molecule intercellular adhesion molecule (ICAM)-1, and of the cytokines IL-1beta and IL-6. Since consensus sequences for the TF, NF-kappaB, are contained in the promoters of all these genes, IB3-1, CuFi-1, Beas-2B, and CaLu-3 cells were transfected with double-stranded TF "decoy" ODNs mimicking different NF-kappaB consensus sequences. IL-8 NF-kappaB decoy ODN partially inhibited the P. aeruginosa-dependent transcription of IL-8, GRO-gamma, and IL-6, whereas decoy ODNs to both HIV-1 long terminal repeat and Igk produced a strong, 80 to 85% inhibition of transcription of IL-8, without reducing that of GRO-gamma, ICAM-1, IL-1beta, and IL-6. In conclusion, intracellular delivery of "decoy" molecules aimed to compete with the TF, NF-kappaB, is a promising strategy to obtain inhibition of IL-8 gene transcription.

The angelicin analogue 4,6,4′-trimethylangelicin (TMA) was recently reported as a strong inhibitor of nuclear factor-κB (NF-κB) activity and of the expression of the interleukin-8 (IL-8) gene in bronchial epithelial cells in which the inflammatory response has been challenged with P. aeruginosa, the most common bacterium found in the airways of patients affected by cystic fibrosis (CF). These findings encouraged us to analyze new synthetic analogues of TMA in order to evaluate their biological activities on human bronchial epithelial CF IB3-1 cells and to find more potent anti-NF-κB agents exhibiting only minor antiproliferative effects. Analogues able to inhibit NF-κB/DNA interaction at lower concentration than TMA were found and selected to investigate their biological activity on IB3-1 cells induced with TNF-α. In this biological system, NF-κB-mediated IL-8 gene expression was investigated. Some analogues showed similar activity to the lead compound TMA. Other analogues displayed higher activities; in particular, the most interesting compounds showing relevant anti-inflammatory effects were found to cause 56–83% reduction of IL-8 mRNA expression at low concentrations (1–10 μM), without changes in cell proliferation pattern, demonstrating their potential interest for a possible development of anti-inflammatory therapy of cystic fibrosis. [ABSTRACT FROM AUTHOR]

One of the clinical feature of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NFkappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NFkappaB interferes with the NFkappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3-1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NFkappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NFkappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P. aeruginosa infected IB3-1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis. [ABSTRACT FROM AUTHOR]