Flagellin concentrations in expectorations from cystic fibrosis patients.

International audienceBACKGROUND: The aim was to measure flagellin concentrations in the expectorations of CF patients and to examine whether there are correlations with the level of respiratory insufficiency and inflammation. METHODS: Sputum samples from 31 adult patients chronically colonized with P. aeruginosa were collected and analysed for their content of flagellin and IL-8. Clinical data were extracted from patient files. RESULTS: Regardless of whether patients are colonized with mucoid strains or not, they carry clones of P. aeruginosa that express flagellin. While flagellin was present in airways of all of our CF patients, it is difficult to ascertain its contribution to inflammation (IL-8) and lung function deterioration. CONCLUSIONS: This is the first demonstration that flagellin is present in the sputum of patients. Thus, attempts to down regulate inflammation by the use of TLR5 (flagellin receptor) antagonists remain a possibility. However, this result needs to be extended to a larger number of patients to validate it for future research on this subject

Cytoskeletal involvement in the cellular trafficking of plasmid/PEI derivative complexes

We have studied the cytoskeletal involvement in the cellular trafficking of complexes made with plasmid/PEI or plasmid/lactosylated PEI in cystic fibrosis airway epithelial cells (SigmaCFTE29o- cells). Complexes were incubated in the presence of cytoskeletal inhibitors, and the number of transfected cells was determined by flow cytometry. Complexes were also generated with fluorescein-labeled PEI derivatives and the cell fluorescence intensity was determined by flow cytometry. In the presence of cytochalasin D to depolymerize actin filaments or nocodazole to disrupt microtubules, gene transfer efficiency with both PEI derivatives was decreased by 90%. The uptake of fluoresceinylated complexes studied by flow cytometry was decreased by 50% in the presence of cytochalasin D for both types of complexes (p<0.005) and unchanged in the presence of nocodazole. When cytoskeletal inhibitors were added to the cell culture after the complex uptake had occurred, gene transfer efficiency was decreased by 75% and 50% in the presence of nocodazole and cytochalasin D, respectively. Upon nocodazole-microtubule network disruption, the lysosomal localization of complexes was reduced, as assessed by confocal microscopy. Our results show a major cytoskeletal involvement in the cellular trafficking of complexes made with both PEI derivatives: actin filaments mainly in complex uptake, and microtubules in the trafficking of complexes towards the nucleus, probably through guided transport of complex-containing endosomal vesicles

CFTR dysfunction induces vascular endothelial growth factor synthesis in airway epithelium

International audiencePeribronchial angiogenesis may occur in cystic fibrosis and vascular endothelial growth factor (VEGF)-A regulates angiogenesis in airways. Peribronchial vascularity and VEGF-A expression were examined using immunocytochemistry and morphometric analysis in lung sections obtained in 10 cystic fibrosis patients at transplantation versus 10 control nonsmokers, and in two strains of Cftr-deficient mice versus wild-type littermates. Airway epithelial NCI-H292 cells and primary cultures of noncystic fibrosis human airway epithelial cells were treated with cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors (CFTR-inh(172) or PPQ-102) or transfected with a CFTR small interfering (si)RNA with or without a selective epidermal growth factor receptor tyrosine kinase inhibitor. Concentrations of VEGF-A and phosphorylated epidermal growth factor receptor were measured by ELISA. Peribronchial vascularity was increased in cystic fibrosis patients, but not in Cftr-deficient mice. VEGF-A immunostaining was localised to airway epithelium and was increased in cystic fibrosis patients and in Cftr-deficient mice. In cultured airway epithelial cells, treatment with CFTR inhibitors or transfection with CFTR siRNA induced a twofold increase in VEGF-A production. CFTR inhibitors triggered epidermal growth factor receptor phosphorylation that was required for VEGF-A synthesis. Cystic fibrosis airways at transplantation showed increased peribronchial vascularity and epithelial VEGF-A expression. CFTR dysfunction triggered epithelial synthesis of VEGF-A, which may contribute to vascular remodelling