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Amal Kouadri

Copper influence oxidative stress and inflammation associated with cystic fibrosis and regulates bronchic junctional barrier via the cellular prion protein (PrPC)

Published on 15 December 2017


Thesis presented on December 15, 2017

Abstract:
In cystic fibrosis (CF), inflammation is detected early on in the airways, even before the onset of bacterial infection, suggesting that mechanisms other than infection are at the origin of the initial inflammatory processes. Among these processes, there is the oxidative stress. The latter is widely accepted as a critical component of several diseases. My PhD project was focused on the characterization of the role of the oxidative stress in triggering inflammation in the CF disease. I used normal (HBE) and cystic fibrosis (CFBE) human bronchial epithelial cells. I also used two cell models derived from CFBE upon either, a stable expression of the wild-type protein (CFBE-wt), or its mutant form; delF508 (CFBEdeltaF508). The characterization of the inflammatory profile in our cellular models confirmed the presence of an intrinsic inflammation in CFBE cells. This profile was independent of the expression of the CFTR protein and was not modified upon the treatments with different molecules known to correct the trafficking of the mutant CFTR (delF508) protein. This suggest that other parameters might be the cause of the CF intrinsic inflammation. Recent studies from our group showed that cells from bronchial CF patients displayed a decrease in copper (Cu) concentrations, and increase in the production of free radicals, and a decrease in antioxidant enzyme activities, such as Cu/Zn-SOD. These results allowed us to establish a link between Cu levels, oxidative stress and inflammation. While investigating the levels of expression of a number of genes encoding proteins that control Cu homeostasis in HBE and CFBE cells, we found that the expression of the cellular prion protein (PrPC) was altered in CFBE cells. PrPC is a glycosyl phosphatidyl inositol (GPI)-anchored glycoprotein involved in prion infection, propagation and aggregation in the central nervous system that leads to transmissible spongiforme encephalopathies (TSE) diseases. Despite several in vitro and in vivo studies demonstrating the capacity of PrPC to interact with other proteins, to bind copper (Cu) with high affinity and to protect cells against oxidative stress, its physiological functions were still under investigations, particularly in extra-neuronal tissues, such as the bronchial epithelium. We have addressed the role of PrPC in the lung cellular architecture, by determining its impact on the integrity of the lung epithelial junctions. This was performed in relation to Cu homeostasis and oxidative stress in bronchial epithelial cells. To further understand PrPC function, we developed a new HBE PrPC knock-out cell line using the CRISPR/Cas9 strategy. Overall, my project brought new insights into the understanding copper involvement in inflammation, oxidative stress and junctional barriers, and how PrPC protein protect bronchial epithelial cells form copper- associated oxidative stress.


Keywords:
Prion, oxidative stress, inflammation, copper