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In the frame of a research program aimed to explore the relationship between chirality of iminosugars and their therapeutic potential, herein we report the synthesis of N-akyl l-deoxyiminosugars and the evaluation of the anti-inflammatory properties of selected candidates for the treatment of Pseudomonas aeruginosa infections in Cystic Fibrosis (CF) lung disease. Target glycomimetics were prepared by the shortest and most convenient approach reported to date, relying on the use of the well-known PS-TPP/I 2 reagent system to prepare reactive alkoxyalkyl iodides, acting as key intermediates. Iminosugars ent-1-3 demonstrated to efficiently reduce the inflammatory response induced by P. aeruginosa in CuFi cells, either alone or in synergistic combination with their d-enantiomers, by selectively inhibiting NLGase. Surprisingly, the evaluation in murine models of lung disease showed that the amount of ent-1 required to reduce the recruitment of neutrophils was 40-fold lower than that of the corresponding d-enantiomer. The remarkably low dosage of the l-iminosugar, combined with its inability to act as inhibitor for most glycosidases, is expected to limit the onset of undesired effects, which are typically associated with the administration of its d-counterpart. Biological results herein obtained place ent-1 and congeners among the earliest examples of l-iminosugars acting as anti-inflammatory agents for therapeutic applications in Cystic Fibrosis., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

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.

Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by P. aeruginosa and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by P. aeruginosa. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely N-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by P. aeruginosa. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2.

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.

Cystic fibrosis (CF) is a genetic disease characterized by chronic bacterial lung infection, most commonly sustained by Pseudomonas aeruginosa. Upon infection, elevated concentrations of pro-inflammatory cytokines (i.e. IL-6 and IL1beta) and chemokines (i.e. IL-8 and GROgamma) are found in the bronchoalveolar fluid of CF patients. We report in this paper that: (a) IL-8, IL-6, IL-1beta, ICAM-1, and GRO-gamma genes are upregulated following infection of CF bronchial epithelial IB3-1 cells with P. aeruginosa; (b) Sp1 transcription factor activity is induced following infection of the cystic fibrosis IB3-1 and CuFi-1 cell lines; (c) inhibition of Sp1 activity using transcription factor decoy molecules leads to inhibition of the expression of IL-6 gene. From the theoretical point of view, our results demonstrate that Sp1 transcription factor activity is induced following infection of CF cells with P. aeruginosa, and that this effect is important in the activation of IL-6 gene transcription. From the practical point of view, our data sustain the potential use of decoy molecules targeting the transcription factor Sp1 to control a relevant molecule involved in the inflammatory process associated with the cystic fibrosis airway pathology.

Chronic lung inflammation in cystic fibrosis (CF) is specifically characterized by predominant endobronchial neutrophil infiltrates, colonization by Pseudomonas aeruginosa, and elevated levels of cytokines and chemokines, first of all IL-8. The extensive inflammatory process in CF lungs is the basis of progressive tissue damage and is largely considered detrimental, making antiinflammatory approaches a relevant therapeutic target. This neutrophil-dominated inflammation seems to be related to an excessive proinflammatory signaling, originating from the same surface epithelial cells expressing the defective CF transmembrane conductance regulator (CFTR) protein, although the underlying mechanisms have not been completely elucidated. To investigate the relationship between defective CFTR and the inflammatory response to P. aeruginosa in CF airway cells, we studied the effect of the DeltaF508 CFTR corrector, benzo(c)quinolizinium (MPB)-07 (Dormer et al., J Cell Science 2001;114:4073-4081). CF bronchial epithelial IB3-1 and CuFi-1 cells overproduced the inflammatory molecules, IL-8 and intercellular adhesion molecule (ICAM)-1, in response to P. aeruginosa, compared with the wild-type, CFTR-expressing bronchial cells, S9, and NuLi-1 cells. In both IB3-1 and CuFi-1 cells, the corrector MPB-07 dramatically reduces the IL-8 and ICAM-1 mRNA expression elicited by P. aeruginosa infection. Correction of CFTR-dependent Cl- efflux was confirmed in MPB-07-treated IB3-1 and CuFi-1 cells. In conclusion, the DeltaF508 CFTR corrector MPB-07 produces an antiinflammatory effect in CF bronchial cells exposed to P. aeruginosa in vitro.

Natural peptides with antimicrobial properties are deeply investigated as tools to fight bacteria resistant to common antibiotics. Small peptides, as those belonging to the temporin family, are very attractive because their activity can easily be tuned after small modification to their primary sequence. Structure-activity studies previously reported by us allowed the identification of one peptide, analogue of temporin B, TB_KKG6A, showing, unlike temporin B, antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigated the antimicrobial and anti-inflammatory activity of the peptide TB_KKG6A against Pseudomonas aeruginosa. Interestingly, we found that the peptide exhibits antimicrobial activity at low concentrations, being able to downregulate the pro-inflammatory chemokines and cytokines interleukin (IL)-8, IL-1 β, IL-6 and tumor necrosis factor- α produced downstream infected human bronchial epithelial cells. Experiments were carried out also with temporin B, which was found to show pro-inflammatory activity. Details on the interaction between TB_KKG6A and the P. aeruginosa LPS were obtained by circular dichroism and fluorescence studies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by P. aeruginosa and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by P. aeruginosa. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely N-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by P. aeruginosa. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2. [ABSTRACT FROM AUTHOR]

Abstract: The role of CFTR deficiency in promoting inflammation remains unclear. Perez et al. [A. Perez, A.C. Issler, C.U. Cotton, T.J. Kelley, A.S. Verkman and P.B. Davis, CFTR inhibition mimics the cystic fibrosis inflammatory profile. Am J Physiol Lung Cell Mol Physiol 2007; 292:L383–L395.] recently demonstrated that the inhibition of function of w/t CFTR produces an inflammatory profile that resembles that observed in CF patients, whereas we found that correction of F508del-CFTR function with MPB-07 down-modulates the inflammatory response to P. aeruginosa in CF bronchial cells [M.C. Dechecchi, E. Nicolis, V. Bezzerri, A. Vella, M. Colombatti, B.M. Assael, et al., MPB-07 reduces the inflammatory response to Pseudomonas aeruginosa in cystic fibrosis bronchial cells. Am J Respir Cell Mol Biol 2007; 36, 615–624.]. Since both evidence support a link between CFTR function and inflammation, we extended our investigation to other F508del-CFTR correctors, such as miglustat (Norez, 2006), an approved drug for Gaucher disease, in comparison with the galactose analogue NB-DGJ. We report here that miglustat but not NB-DGJ restores F508del-CFTR function in CF bronchial epithelial IB3-1 and CuFi-1 cells. Miglustat and NB-DGJ reduce the inflammatory response to P. aeruginosa in both CF and non-CF bronchial cells, indicating that the anti-inflammatory effect is independent of the correction of F508del-CFTR function. Miglustat also inhibits the inflammatory response induced by the supernatant of mucopurulent material obtained from the lower airway tract of cystic fibrosis patients with chronic bacterial colonization (Ribeiro, 2005). Both compounds do not interfere with the adherence of P. aeruginosa to the cells and reduce the expression of IL-8 not only after challenge with P. aeruginosa but also after exposure to TNF alpha or IL-1 beta, suggesting an effect on transduction proteins downstream and in common with different receptors for pathogens. Finally, miglustat has no major effects on overall binding activity of transcription factors NF-κBNF-kB and AP-1. Since miglustat is an approved drug, it could be investigated as a novel anti-inflammatory molecule to ameliorate lung inflammation in CF patients. [Copyright &y& Elsevier]

• Despite new CFTR modulators give a great hope to patients, research on anti-inflammatories and antibiotics remains a priority in CF. We previously demonstrated that β-sitosterol (BSS), inhibits the expression of IL-8 in CF bronchial epithelial cells exposed to Pseudomonas aeruginosa. We hypothesized that BSS can be used to reduce the inflammatory response in CF. • In the mouse model of lung chronic infection BSS reduced leukocyte recruitment in the bronchoalveolar lavage fluid. Both neutrophils and alveolar macrophages were decreased. Moreover, decreased bacteria recovered in the airways of mice treated with BSS was found. Treatment with BSS reduced the expression of key cytokines involved in immune response, mainly neutrophil chemotaxis. This activity is accompanied by an improvement of health status. • BSS could become a new drug to target the excessive neutrophil recruitment in lungs chronically infected by P. aeruginosa and encourage future investigations on mechanism of protection driven by BSS. We previously demonstrated that β-sitosterol (BSS) inhibits the expression of the chemokine IL-8 in CF bronchial epithelial cells exposed to P. aeruginosa. In the mouse model of lung chronic infection, herein shown, BSS significantly reduced leukocyte recruitment in the bronchoalveolar lavage fluid and decreased bacteria recovered in the airways. Treatment with BSS decreased the expression of key cytokines involved in immune response, mainly neutrophil chemotaxis, in the lung homogenate. This anti-inflammatory activity is accompanied by a beneficial protecting activity against infection and improvement of health status. Our data suggest that BSS has the potential to become a new drug to target the excessive neutrophil recruitment in lungs chronically infected by P. aeruginosa and encourage future investigations on mechanism of protection driven by BSS. [ABSTRACT FROM AUTHOR]

Abstract: The most relevant cause of morbidity and mortality in cystic fibrosis (CF) patients is the lung pathology characterized by chronic infection and inflammation sustained mainly by Pseudomonas aeruginosa (P. aeruginosa). Innovative pharmacological approaches to control the excessive inflammatory process in the lung of CF patients are thought to be beneficial to reduce the extensive airway tissue damage. Medicinal plants from the so-called traditional Asian medicine are attracting a growing interest because of their potential efficacy and safety. Due to the presence of different active compounds in each plant extract, understanding the effect of each component is important to pursue selective and reproducible applications. Extracts from Emblica officinalis (EO) were tested in IB3-1 CF bronchial epithelial cells exposed to the P. aeruginosa laboratory strain PAO1. EO strongly inhibited the PAO1-dependent expression of the neutrophil chemokines IL-8, GRO-α, GRO-γ, of the adhesion molecule ICAM-1 and of the pro-inflammatory cytokine IL-6. Pyrogallol, one of the compounds extracted from EO, inhibited the P. aeruginosa-dependent expression of these pro-inflammatory genes similarly to the whole EO extract, whereas a second compound purified from EO, namely 5-hydroxy-isoquinoline, had no effect. These results identify Pyrogallol as an active compound responsible for the anti-inflammatory effect of EO and suggest to extend the investigation in pre-clinical studies in airway animal models in vivo, to test the efficacy and safety of this molecule in CF chronic lung inflammatory disease. [Copyright &y& Elsevier]

Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by P. aeruginosa and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by P. aeruginosa. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely N-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by P. aeruginosa. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2. [ABSTRACT FROM AUTHOR]