Anti-inflammatory effect of miglustat in bronchial epithelial cells

AbstractThe 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

INHIBITION OF PRO-INFLAMMATORY GENES IN CF BRONCHIAL EPITHELIAL CELLS BY MEDICINAL PLANT EXTRACTS

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The CAMP/PKA-dependent annexin 2-S100A10 complex with CFTR is functionally important

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

Chemical conjugation of ΔF508-CFTR corrector deoxyspergualin to transporter human serum albumin enhances its ability to rescue Cl- channel functions.

The most common mutation of the cystic fibrosis (CF) gene, the deletion of Phe508, encodes a protein (DeltaF508-CFTR) that fails to fold properly, thus mutated DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) is recognized and degraded via the ubiquitin-proteasome endoplasmic reticulum-associated degradation pathway. Chemical and pharmacological chaperones and ligand-induced transport open options for designing specific drugs to control protein (mis)folding or transport. A class of compounds that has been proposed as having potential utility in DeltaF508-CFTR is that which targets the molecular chaperone and proteasome systems. In this study, we have selected deoxyspergualin (DSG) as a reference molecule for this class of compounds and for ease of cross-linking to human serum albumin (HSA) as a protein transporter. Chemical cross-linking of DSG to HSA via a disulfide-based cross-linker and its administration to cells carrying DeltaF508-CFTR resulted in a greater enhancement of DeltaF508-CFTR function than when free DSG was used. Function of the selenium-dependent oxidoreductase system was required to allow intracellular activation of HSA-DSG conjugates. The principle that carrier proteins can deliver pharmacological chaperones to cells leading to correction of defective CFTR functions is therefore proven and warrants further investigations

ANTI-INFLAMMATORY EFFECT OF MIGLUSTAT: SPHINGOLIPID METABOLISM AS A THERAPEUTIC TARGET FOR CF LUNG INFLAMMATION

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Combined effects of furocoumarin compounds as anti-inflammatory and CFTR potentiator in Calu-3 epithelial cells.

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