Thiol redox disturbances in children with severe asthma are associated with posttranslational modification of the transcription factor nuclear factor (erythroid-derived 2)-like 2

Abstract

Background: Airway thiol redox disturbances, including depletion of the antioxidant, glutathione, are differentiating features of severe asthma in children.

Objectives: Given the role of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in maintaining glutathione homeostasis and antioxidant defense, we quantified expression and activity of Nrf2 and its downstream targets in the airways and systemic circulation of children with asthma. We hypothesized that Nrf2 activation and function would be impaired in severe asthma, resulting in depletion of thiol pools and insufficient glutathione synthesis and conjugation.

Methods: PBMCs and airway lavage cells were collected from children 6 to 17 years with severe (n = 51) and mild-to-moderate asthma (n = 38). The thiols glutathione and cysteine were quantified, and expression and activity of Nrf2 and its downstream targets were assessed.

Results: Children with severe asthma had greater oxidation and lower concentrations of glutathione and cysteine in the plasma and airway lavage. Although Nrf2 mRNA and protein increased in severe asthma as a function of increased thiol oxidation, the Nrf2 expressed was highly dysfunctional. Nrf2 activation and downstream targets of Nrf2 binding, including glutathione-dependent enzymes, were not different between groups. The duration of asthma was a key factor associated with Nrf2 dysfunction in severe asthma.

Conclusion: Children with severe asthma have a global disruption of thiol redox signaling and control in both the airways and systemic circulation that is associated with posttranslational modification of Nrf2. We conclude that the Nrf2 pathway is disrupted in severe asthma as a function of chronic oxidative stress, which ultimately inhibits glutathione synthesis and antioxidant defense.

Figure 1

Overview of Nrf2 activation and transcription of selected genes related to glutathione synthesis and conjugation.

Figure 2

Plasma concentrations of (A) Cys, (B) GSH, and the redox potential (E_h) of (C) Cys/Cyss and (D) GSH/GSSG in children with mild-to-moderate asthma (MMA, n = 23) and severe asthma (SA, n = 36). Bar graphs depict the mean ± SEM. Box plots are shown with medians and the upper and lower quartiles in gray.

Figure 3

BAL supernatant concentrations of (A) Cys, (B) GSH, and the redox potential (E_h) of (C) Cys/Cyss and (D) GSH/GSSG in children with mild-to-moderate asthma (MMA, n = 18) and severe asthma (SA, n = 23). Bar graphs depict the mean ± SEM. Box plots are shown with medians and the upper and lower quartiles in gray.

Figure 4

Nrf2 and Keap1 mRNA gene expression in PBMCs (A, B) and airway lavage cells (C, D) from children with mild-to-moderate (MMA) and severe asthma (SA). Data were normalized to 5 housekeeping genes and are shown relative to MMA group as 2^−ΔΔCT values. Horizontal lines represent the mean.

Figure 5

mRNA gene expression of downstream targets of Nrf2 activation responsible for GSH synthesis, including the (A–C) GST isoenzymes mu, pi, and theta, (D) glutathione synthetase, and (E, F) glutamate-cysteine ligase (catalytic and modifier subunits) in PBMCs from children with mild-to-moderate (MMA) and severe asthma (SA). Data were normalized to 5 housekeeping genes and are shown relative to MMA group as 2^−ΔΔCT values. Horizontal lines represent the mean.

Figure 6

Nrf2 (A) activation and (B) protein expression and (C) Keap1 protein expression in PBMCs from children with mild-to-moderate asthma (MMA) and severe asthma (SA). Nrf2 and Keap1 expression were normalized to β-actin and are expressed relative to the MMA group as mean ± SEM values (Nrf2: MMA, n = 8; SA, n = 16; Keap1: MMA, n = 5; SA, n = 5). Representative images of Nrf2 and Keap1 protein expression are shown in panels D and E, respectively.

Figure 7

Scatterplot depicting the relationship between Nrf2 mRNA expression in PBMCs and (A) plasma GSH, (B) the redox potential (E_h) of the GSH:GSSG couple, and (C) asthma duration. Nrf2 mRNA expression is shown as the fold-increase in expression relative to the housekeeping genes. The dashed lines depict the 95% confidence interval.