Abstract
Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult.The results show that endogenous ROS and lipid peroxidation levels are higher in Cftr −/− lung when compared to wild-type (Cftr +/+) in basal conditions, despite a strong enzymatic antioxidant response involving superoxide dismutases, glutathione peroxidases and peroxiredoxin 6 (Prdx6). The latter has the unique capacity to directly reduce membrane phospholipid hydroperoxides (PL-OOH). A dramatic increase in PL-OOH levels in Cftr −/− lung consecutive to in vivo oxidative challenge by paraquat (PQ) unmasks a susceptibility to phospholipid peroxidation. PQ strongly decreases Prdx6 expression in Cftr −/− mice compared to Cftr +/+. Similar results were obtained after P. aeruginosa LPS challenge. Two-dimensional gel analysis of Prdx6 revealed one main molecular form in basal conditions and a PQ-induced form only detected in Cftr +/+ lung. Mass spectrometry experiments suggested that, as opposed to the main basal form, the one induced by PQ is devoid of overoxidized catalytic Cys47 and could correspond to a fully active form that is not induced in Cftr −/− lung. These results highlight a constitutive redox imbalance and a vulnerability to oxidative insult in Cftr −/− lung and present Prdx6 as a key component in CF antioxidant failure. This impaired PL-OOH detoxification mechanism may enhance oxidative damage and stress-related signaling, contributing to an exaggerated inflammatory response in CF lung.
Highlights
Cystic fibrosis (CF), the most prevalent lethal autosomal recessive disorder in the Caucasian population, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, Cftr
The level of DCF fluorescence was 1.5 times greater (P = 0.0005) in Cftr2/2 ciliated cells when compared to Cftr+/+, revealing a higher steady state level of intracellular reactive oxygen species (ROS) in Cftr2/2 airway epithelial cells. To investigate whether this elevated level of intracellular ROS in Cftr2/2 cells was associated with an increased level of oxidationdamaged macromolecules, we evaluated lipid peroxidation in lung homogenates using the Thiobarbituric acid-reactive substances (TBARS) assay (Fig. 1B)
To determine whether decreased antioxidant defences could account for the increased levels of ROS and peroxidized lipids in Cftr2/2 lung, the activities of the main antioxidant enzymes in lung, e.g., superoxide dismutase (SOD), glutathione peroxidase (GPx) and CAT, were evaluated
Summary
Cystic fibrosis (CF), the most prevalent lethal autosomal recessive disorder in the Caucasian population, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, Cftr. Elevated levels of lipid and protein oxidation products found in BALF, exhaled breath condensate and sputum of CF patients [1] [2] [3] [4], provide evidence of oxidative stress in the ELF compartment These oxidative damages are due to an increased oxidant burden, resulting from the release of oxidants by neutrophils and Pseudomonas aeruginosa that chronically infects CF airways [5]. Thereby, CFTR could be considered as an important actor of ELF antioxidant homeostasis and an intrinsic cause of oxidative imbalance in CF airways from human patients as well as Cftr knockout mice. The latter appears as a suitable model to investigate the constitutive redox imbalance in CF
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