To our knowledge, this is the first study to describe post-translational modification of Nrf2 in subjects with asthma. Moreover, this is the first study to reveal disruption of the Nrf2 pathway as a mechanism underlying thiol redox disturbances in asthmatic children. Compared to children with mild-to-moderate asthma, children with severe asthma had lower concentrations of the thiols Cys and GSH in the plasma and airway lavage associated with a shift in the redox potential (Eh
) of the Cys/CySS and GSH/GSSG redox couples to the more oxidized state. Remarkably, the magnitude of the Cys/CySS and GSH/GSSG oxidation was similar between the airway and systemic compartments, suggesting a global disruption of redox signaling and control that is not alleviated by endogenous airway defenses. While these thiol redox disturbances were accompanied by an expected upregulation of Nrf2 mRNA and protein, this did not translate to an increase in downstream targets of Nrf2 ARE binding and activation, including key enzymes involved in GSH synthesis and conjugation. Indeed, Nrf2 activation was not different between mild-to-moderate and severe asthmatic children. Given that Nrf2 expression was strongly associated with GSH redox disturbances and the duration of asthma, we conclude that the Nrf2 pathway is disrupted as a function of chronic oxidative stress. Thus in the case of severe asthma, the high expression of Nrf2 at baseline is not beneficial but rather reflects the inability of this pathway to increase GSH synthesis in response to GSH depletion. This Nrf2 “burnout” (i.e., the inability by Nrf2 to counteract even minor, acute redox disturbances) might further explain the increased risk and severity of exacerbations in children with severe asthma,2
particularly in the presence of secondary insults such as respiratory infection.22,23
While our study provides the first evidence of altered Nrf2 expression and activity in subjects with asthma, Nrf2 alterations have been previously described in smokers and adults with chronic obstructive pulmonary disease (COPD)
. In healthy smokers, Nrf2 is activated in the airway epithelium and increases expression of downstream genes containing functional AREs in the promoter region. 24,25
However, in smokers with mild COPD (stage 1–2), GSH-related gene expression is not increased despite airway GSH depletion and oxidative stress.26
As the duration of smoking and severity of COPD increases, airway Nrf2 protein is further depleted in these subjects.26–28
Thus the chronic and sustained oxidative stress conferred by cigarette smoking likely overwhelms the capacity of the Nrf2 pathway to provide antioxidant defense. Because our study was limited to children, it may be that Nrf2 expression increases initially and then progressively declines as severe asthma evolves and worsens from childhood to adulthood. Longitudinal studies of chronic changes in Nrf2 expression in children with severe asthma are needed.
The post-translational modification of Nrf2 that we observed in children with severe asthma is intriguing and suggests that Nrf2, along with thiol redox status, may be a novel biomarker of disease status in asthma. Increased Nrf2 expression in severe asthma may further account for our previous observations of increased IL-8 expression 4,7
and decreased histone deacetylase (HDAC)
in this group. The promoter region of the IL-8 gene contains an ARE element and in vitro
studies have demonstrated increased IL-8 expression and enhanced IL-8 mRNA stability with Nrf2 activation.29
Another recent study noted decreased HDAC expression and activity in lung tissue from Nrf2−/−
mice associated with increased inflammation and steroid resistance.30
While the clinical relevance of these previous studies is unclear, these observations highlight important relationships between Nrf2 and differentiating features of severe asthma in children that warrant further study.
This study does not inform the specific mechanism(s) underlying Nrf2 dysfunction in children with severe asthma. Nrf2 is expressed in nearly all tissues, including PBMCs and airway lavage cells, and is particularly abundant in systems such as the lung that are exposed to the external environment. 31
While the oxidative burden associated with severe asthma may be attributed to environmental exposures, dysfunction of epithelial cells and phagocytes, including granulocytic leukocytes and monocytes/macrophages, may also contribute.32,33
Indeed, we have previously reported persistent activation of airway macrophages in children with severe asthma associated with increased pro-inflammatory cytokine secretion,7
increased lipid and DNA oxidation,4
and impaired phagocytic function34
that may further increase free radical burden. Given the complexities of the innate antioxidant response, coupled with the clinical heterogeneity of severe asthma,35
it is possible that there are several mechanisms responsible for our observations. One such mechanism could be impaired liberation of Nrf2 from Keap1, which results in ubiquitination and proteosomal degradation of Nrf2.36,37
Other mechanisms include acetylation and phosphorylation of Nrf2,38,39
although the functional and clinical relevance of Nrf2 phosphorylation has been questioned.40
Alternatively, Nrf2 dysfunction and redox disturbances may result from single nucleotide polymorphisms in Nrf2, which have been associated with decreased lung function in the general population41
and increased risk of acute lung injury after significant trauma.42
This study has a number of limitations. First, because bronchoscopy can only be performed for clinical indications in children, the inclusion of a healthy, aged-matched control group was not possible. Regardless, the purpose of this study was to provide insight regarding a potential mechanism of severe versus mild-to-moderate asthma in children, and not a mechanism of asthma per se
. Thus comparisons to children with mild-to-moderate asthma help eliminate potential confounding by asthma medication exposure and other key variables in the medical history. However, given the disproportionate representation of black males in our severe asthma group, it is possible that the Nrf2 dysregulation we observed could be due to underlying genetic differences. While enrollment of additional females and non-Hispanic whites would have led to more balanced groups, our group of children with severe asthma likely reflects important genetic differences in asthma severity. Indeed, other genetic-based studies have shown that blacks have the earliest age of asthma onset, the strongest family history of asthma, and the lowest lung function.43
Males with persistent wheezing also have lower lung function compared to females.44
While genetic differences underlying Nrf2 dysfunction in severe asthma were beyond the scope of our study, this is a potentially important finding that should be explored.
Because this study was focused on children, our findings may not be generalized to asthmatic adults. However, given previous observations of Nrf2 dysfunction in adults with COPD,26–28
the inclusion of children provides potentially useful information for the development of future disease-modifying interventions. Furthermore, although we did observe an overall trend of increased Nrf2 expression in children with severe asthma, there was heterogeneity within this group of subjects similar to what has been reported previously.35
Future studies are needed to tease out individualized patterns of Nrf2 responses, including the stability of Nrf2 expression and thiol redox disturbances over time, to understand the clinical relevance of our findings.
In summary, we have demonstrated for the first time significant thiol redox disturbances in the airways and systemic circulation of children with severe asthma associated with dysregulation of the transcription factor, Nrf2. Despite increased Nrf2 expression in children with severe versus mild-to-moderate asthma, Nrf2 activity and expression of downstream Nrf2 genes were not different between groups. Although additional studies are warranted, these findings highlight a potential mechanism for the persistent airway inflammation and oxidative stress that accompany severe asthma. Ultimately, interventions to overcome Nrf2 dysregulation and associated redox disturbances may be warranted in this population.