Novel antioxidant therapy with the immediate precursor to glutathione, γ-glutamylcysteine (GGC), ameliorates LPS-induced cellular stress in anin vitrocystic fibrosis model

Abstract

IntroductionGlutathione deficiency and chronic bacterial inflammation exacerbates the oxidative stress damage to airways in cystic fibrosis. Improvements to current antioxidant therapeutic strategies are needed. Dietary supplement, γ-glutamylcysteine (GGC), the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against Pseudomonas aeruginosa derived lipopolysaccharide (LPS), a prominent factor in mediating both bacterial virulence and host responses, in CF remains unassessed.MethodsPrimary F508del/F508del mucociliary differentiated bronchial and nasal epithelial cells were created to model LPS-induced oxidative stress and inflammation of CF. The proteomic signature of GGC treated cells was resolved by qLC-MS/MS. Parameters including cell redox state (glutathione, ROS), anti-inflammatory mediators (IL-8, IDO-1) and cellular health (membrane integrity, stress granule formation and cell viability) were assayed.ResultsProteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced ROS and stress granule formation, while significantly increasing intracellular glutathione levels and improving epithelial cell barrier integrity. Moreover, we compared the effect of GGC with thiols NAC and glutathione on cell viability. GGC was the only thiol that increased cell viability; protecting cells against LPS induced cell death. Both therapeutic and prophylactic treatments were successful.ConclusionTogether, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress related damage to airways in Cystic Fibrosis.

Increased susceptibility of cystic fibrosis airway epithelial cells to ferroptosis

“…The presence of oxidative stress in the lung in CF is very well described and plays a pivotal role in the pathogenesis of disease, and there are emerging data that demonstrate cellular lipid metabolism is also disrupted in CF [ 5 , 45 ]. The propensity for ferroptosis in CF is probably directly linked to CFTR dysfunction, which is associated with reduced activity of γ-glutamylcysteine synthetase (GCS) and decreased ability to import cysteine into the cell, which are both required for GSH biosynthesis [ 11 , 46 , 47 ]. Consistent with this, we found that IB3-1 cells had lower intracellular levels of GSH than their cognate C38 cells at baseline and GSH levels were further reduced by FAC and erastin treatment.…”