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The functional role of CFTR in the innate immune response of respiratory epithelia

Dernière mise à jour 25.07.2017 à 10h56

Axe de recherche : Fonction CFTR Délégation territoriale : Île de France

Porteur du projet : Gergely LUKACS
INSERM  U1151 team2 - Canalopathies épithéliales : la mucoviscidose et autres maladies

Contexte :
Cystic fibrosis (CF), most prevalent, lethal genetic diseases in the Caucasian population, is associated with chronic lung inflammation and reoccurring infection, the leading causes of CF mortality. Accumulating evidence suggests that defective function of the CF gene product, which is a chloride channel that ensures ions and water movement into the airways’ surface layer, may have additional functions. It has been proposed that functional expression defect of the CF gene sensitizes the airway cells for launching an immune response even in the absence of infection. Although this phenomenon has been reported in the CF ferret lung and the pig pancreas, its molecular mechanism remains enigmatic. Our experimental models reproduce the augmented immune response in the CF human airway epithelial cells, offer the possibility to understand its molecular basis and identify possible drug targets to attenuate the hyperactive innate immune response in CF.

Objectifs :
Our short-term objective is to identify the underlying molecular mechanism of the augmented inflammation reported in human respiratory epithelial cells. To this end we postulate that documented or other compositional and/or physico-chemical changes in the liquid layer covering the airway epithelia alter the cell surface protein profile and characteristic, which in turn can elicit a complex cascade of signaling that leads to excessive inflammation. We propose to use biochemical methods to isolate the cell surface membrane and cytosol from CF and non-CF cells and examine quantitatively their protein compositional changes and posttranslational modifications by using high-resolution mass spectrometry approaches. Counteracting the compositional changes and modification of signaling molecules at the apical cell surface and in the cytoplasm will offer the possibility to attenuate the inflammation in our model system and ultimately in patients.

Perspectives :
By identifying the composition and posttranslational modifications of membrane and cytosolic proteins, we hope to find new targets that are involved in the regulation of inflammation and ion transport across CF epithelial. These targets will be important at two levels. First, for better understanding of altered signaling that links the primary CFTR transport defect to inflammation in CF, and second, for designing new therapeutics to control the inflammatory response CF patients.

Résultats obtenus :
"The apical PM protein from under air-liquid interface (ALI) condition upon inducible expression of wild-type CFTR have been prepared in differentiated human CF bronchial epithelial (CFBE) cells. Apical PM proteins were obtained by cationic silica beads density shift and cell surface biotinylation of CFBE cells.  After enrichment, the protocols for protein digestion for subsequent MS analysis were optimized.
The main findings are:
- 1700 and 1300 proteins were identified in CFBE in the presence or absence of WT CFTR, using cell surface biotinylation and the silica beads method, respectively. 
- CFTR derived peptides were identified specifically in CFBE expressing WT CFTR, using either MP preparation methods, giving credence for these preparations.  Silica method allowed enriching higher amounts of CFTR.
- More than 100 membrane proteins were identified including EGFR, TNF  and EPHA2A receptors, involved in the inflammatory response of respiratory epithelia.
- 40 proteins are specifically found at the APM depending on the presence of WT CFTR at the apical PM.

Initial immunoblot analysis confirmed that EPH2A receptor signaling is influenced by CFTR expression."