This study is the first to quantify the relationship between extracellular and intracellular GSH homeostasis and AM function in children with severe asthma. Although extracellular GSH concentrations were not different in the airways of children with moderate and severe asthma, children with severe asthma had significantly higher GSSG concentrations accompanied by increased lipid peroxidation and increased by-products of DNA oxidation in the extracellular space. This shift in the airway GSH to GSSG ratio was also mirrored within the AMs and was further associated with AM functional disturbances, including decreased suppression of histone acetylation, impairment of phagocytosis, and increased apoptosis. Ex vivo supplementation with GSH improved phagocytosis and decreased apoptosis in children with severe asthma while also decreasing intracellular GSSG, suggesting that airway GSH and GSSG are the primary regulators of AM function in these children. Thus, airway GSH homeostasis may represent a novel target for therapeutic intervention in children with severe asthma.
GSH is a tripeptide thiol that is essential for defense against oxidative damage and function of airway cells. Depletion of GSH through either a shift in the ratio of GSH to GSSG or a reduction in the total GSH + GSSG pool results in activation of nuclear factor kappa B (NF-kappaB) and pro-inflammatory cytokine release from HDAC suppression and increased gene transcription (8
). GSH depletion has also been linked to increased AM apoptosis from caspase activation and PARP degradation (21
) and to impaired phagocytosis of infectious particles (22
), perhaps through inhibition of the transcription factor, NF-E2-related factor 2 (Nrf2) (11
). Although molecular mechanisms of altered GSH homeostasis have not been studied in patients with asthma, our findings of increased intracellular GSSG and corresponding AM dysfunction in the form of decreased HDAC activity, increased apoptosis, and impaired phagocytosis suggest that GSH and perhaps NF-kappaB and Nrf2 may regulate many features of the disorder. Indeed, in murine models of allergic airway inflammation, GSH attenuates airway hyperresponsiveness and the airway Th2 imbalance through suppression of chemokine-induced eosinophil chemotaxis and inhibition of NF-kappaB (9
). Similarly, although Nrf2-deficient mice have increased susceptibility to acute lung injury, GSH supplementation prevents lung injury in Nrf2-deficient mice by inhibiting the recruitment of inflammatory cells to the site of airway injury (25
). These data highlight the important role of GSH in the airways and argue for additional study of the mechanisms of GSH-related airway dysfunction in severe asthma.
Although we did not observe differences in ELF GSH concentrations between children with moderate and severe asthma in this study (~30 µM in each group), these levels are significantly lower than those previously reported in healthy adults (500 µM) and in our previous studies of nonasthmatic children with chronic cough (250 µM) (2
). Although it is possible that these decreased GSH concentrations are due to a defect in GSH-related enzymatic activities or polymorphisms in GSH transferases, we recently observed no differences in the activity of GSH peroxidase, GSH-S-transferase, or GSH reductase between children with moderate and severe asthma and healthy controls (2
). Thus, the GSH concentrations that we observed here are likely related to chronic airway oxidation and diffuse airway injury. Indeed, the ELF GSH concentrations that we observed in children with moderate and severe asthma are consistent with those of adults with acute respiratory distress syndrome, a disorder associated with diffuse alveolar injury (26
). Although asthma is traditionally thought to affect the bronchial versus
the alveolar airspaces, there is increasing evidence of distal airway inflammation in both humans (28
) and in experimental models of asthma (30
). Although our BAL samples were pooled and do include a mixture of both bronchial and lower airway constituents, our data may reflect early distal airway damage in moderate to severe asthmatic children that is reflected by low GSH concentrations and altered GSH homeostasis.
This study does have limitations. Because, it is not ethical to perform bronchoscopy for research purposes in healthy children, it is unclear how our findings relate to steroid-naïve children with mild asthma or children with mild asthma that is well controlled with low doses of ICS. Furthermore, it is possible that intracellular and extracellular GSH and GSSG concentrations and AM function could be influenced by asthma treatment. However, we do not believe that corticosteroid use sufficiently explains the discrepancies observed in this study. Given the magnitude of symptoms in children with moderate asthma, all children in this study were treated with ICS. Furthermore, there were no significant correlations between corticosteroid usage and the oxidative and AM functional parameters measured in this study.
In summary, severe asthma in children is a complicated disorder with persistent airway inflammation and ongoing symptoms that are refractory to treatment with high doses of inhaled and oral corticosteroids. This airway inflammation is further accompanied by AM cellular dysfunction, including increased apoptosis and impaired AM phagocytosis of infectious particles. Our findings of altered intracellular and extracellular airway GSH homeostasis provide a possible mechanism for these alterations. Given the vital role of GSH in airway antioxidant defense and cellular function, airway GSH warrants further study in children with severe asthma. Interventions to improve airway GSH homeostasis may ultimately be needed to decrease the morbidity of severe asthma in children.