Project name: Project category: Kind of research: Country: Principle investigator:

Proteases and CFTR : in vitro and in vivo regulation in lung epithelial cells and alveolar macrophages

Immunology-Inflammation
France Jean-Michel Sallenave
Status: Ongoing
Abstract:

Cystic fibrosis (CF) is the most common genetically inherited disease in Caucasians (1 in 2500 newborns) and is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). The most prevalent CFTR mutation dF508 (which constitutes 70% of all mutations) results in an incorrect targeting of the CFTR molecule to the membrane.
It is a well accepted concept that mucosal immune responses are dys-regulated in cystic fibrosis through a cycle of infectious (Pseudomonas aeruginosa/P.a) being an important pathogen in that disease)) and inflammatory episodes, and that there is an imbalance between proteases and antiproteases in that pathology.

We have shown that NE has a deleterious effect on CFTR structure and channel function in epithelial cells in vitro and in vivo in a P.a lung infection model (Le Gars et al, AJRCCM, 2013). Interestingly, we showed that NE inactivate CFTR through an intracellular calpain activation pathway, but the potential inflammatory exacerbative effect of proteases (NE and others ) on dF508 in vivo remains to be studied. Importantly, we have shown that other factors than NE may also be important in inducing the degradation of CFTR in the P.a infection model, and among these, we determined in preliminary results that Las B, a zinc metallo-exoprotease secreted by P.a type 2 secretion system can also degrade CFTR in vitro.

We wish here to consolidate these findings by testing in vitro whether NE can
inactivate other CFTR mutants, such as G551D (class III mutation), which, contrary to dF508, is not impaired in its membrane trafficking to the membrane.
In addition, the potential deleterious activity of P.a LasB (and the mechanisms involved) will also be studied in WT, dF508, and G551D CFTR cells.
Because the alveolar macrophage is an important cell type involved in the recognition of P.a and the induction of innate immune responses, we will also test whether the proteases studied here can impair/inactivate the fonction of WT and mutated CFTR on this cell type, and whether other receptors such as TLR-5 may also be linked functionally to CFTR.
It will also be important to further study in vivo the effects of recombinant NE and LasB on CFTR in WT and dF508-CFTR mice, to assess whether in the latter model, proteases may have an exacerbative effect on the CF phenotype. Similarly, to further assess the role of LasB as a virulence factor in the CF context, WT and dF508-CFTR mice will be infected intra-nasally with either WT or LasB KO P.a and the effect on function on CFTR and the inflammatory response will also be studied.

City: PARIS
Project amount (€): 65.004

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