The contribution of GSTPi to asthma has been supported by epidemiologic, genetic, and animal studies by our group and others14–17, 21
although the mechanism for this has remained elusive. Our findings provide important novel insights into the contribution of GSTPi to asthma in humans and mice. Given that several environmental exposures associated with asthma result in enhanced oxidative stress, one might predict that GST enzymes would be induced in order to increase the capacity to handle the increased electrophilic load and restore redox homeostasis. However, we found that GSTPi transcript levels are markedly down-regulated in mouse models of asthma following allergen challenge via STAT6 dependent and independent pathways. The observed down-regulation was not unique to a specific allergen. The decrease in GSTPi expression was associated with decreased total GST activity in the lungs of mice. Levels of GSTPi mRNA have been shown to correlate to GST activity in other studies (R2
. GSTPi was down-regulated in Balb/c and C57Bl/6 strains of mice supporting that the observation was not restricted to a given strain. Furthermore, GSTPi was similarly down-regulated in children with asthma. Our findings also uncovered a functional role for GSTPi in regulating Cys oxidation, revealing that GSTPi plays an important role in neutralizing oxidative stress in asthma. These data collectively suggest that down-regulation of GSTPi following allergen challenge may result in disruption of redox homeostasis and increased oxidative stress. Interestingly, patients with severe asthma have been found to have lower airway GSH with increased oxidized glutathione (GSSG), consistent with enhanced oxidative stress46
Perturbations in the extracellular thiol/disulfide redox environment have been shown to correlate with the progression and severity of lung injury47
. Cysteine (Cys) and its disulfide Cystine (CySS) constitute the most abundant, low-molecular-weight thiol/disulfide redox couple in the plasma, and Cys homeostasis is adversely affected during the inflammatory response. In models of lung injury, Cys/CySS was selectively oxidized early in inflammation supporting that it is a sensitive marker of redox homeostasis44
. In the absence of GSTPi, the lungs of GSTPi-deficient mice had significantly increased %CySS following allergen challenge supporting that a loss in GSTPi activity significantly altered the ability of the lungs to handle the increased oxidative stress burden following allergen challenge. Key pathways relevant to allergic inflammation have been shown to be sensitive to Cys redox homestasis48
, thus an increase in %CySS may effect on cytokine signaling in addition to the other effects of ROS.
Since GSTPi is an enzyme that plays a critical role in cellular detoxification of endogenous and xenobiotic substrates and protection against oxidative stress, it is not surprising that evidence exists supporting a substantial role for GSTPi in allergic asthma. We had predicted that following allergen exposure there would be an increased necessity for GSTPi-driven detoxification in the lung, marked by an increase in GSTPi expression and total GST activity. Therefore, it was surprising to observe a decrease in GSTPi expression and total GST activity following allergen exposure. One potential explanation for this apparent paradox is that the observed GSTPi down-regulation is transient. Down-regulation of GSTPi expression was evident after sensitization and a single i.t. challenge and remained low for several weeks. However, GSTPi levels and activity normalized. Since asthma is a chronic disease, the impact of the down-regulation may not be as evident in a more chronic model of exposure. The surprising decrease in GSTPi expression and total GST activity could also indicate that GSTPi is maladaptive in response to allergen exposure.
The mechanism by which down-regulation of GSTPi expression and total GST activity occurs may involve Nrf2. HDM and IL-13 treated mice displayed decreased Nrf2 expression in the lungs. IL-13-deficient and STAT6-deficient mice also display decreased Nrf2 expression following HDM exposure. Thus, although IL-13 contributes to HDM-induced Nrf2 down-regulation, neither IL-13 nor Stat6 is necessary for allergen-induced downregulation of Nrf2. There is an alternate pathway downstream of allergen treatment that results in the observed down-regulation in an IL-13 and STAT6 independent manner. Similarly, IL-13- but not HDM-induced GSTPi down-regulation was STAT6 dependent. Thus, HDM can induce down-regulation of GSTPi expression and total GST activity by IL-13 and STAT6 autonomous pathways that involve Nrf2. Furthermore, Nrf2 was found to be an important modulator for mounting an appropriate innate immune response during experimental sepsis49
. Thus, transient down-regulation of Nrf2 and GSTPi following allergen exposure could signal the increase of proinflammatory cytokines and chemokines or the initiation of downstream detoxification pathways.
Our findings are consistent with previous studies that have demonstrated a correlation between GST activity and the level of GSTPi mRNA expression (R2
. We observed a decrease in GSTPi gene expression following sensitization and one i.t. challenge whereas the total GST activity did not decrease until after two i.t. challenges. This suggests that the observed decrease in GSTPi gene expression precedes the decrease in total GST activity. Surprisingly, we did not observe a change in GSTPi protein expression analyzed by western blot (data not shown). One reason for this is may be that the assay used for measuring GSTPi protein expression in the lung may not be sensitive enough to detect modest changes in protein levels. Since the antibody does not distinguish between GSTP1 and GSTP2, it is also possible that one subtype is conserved or even upregulated relative to the other.
The observed decrease in GSTPi expression and total GST activity is not likely due to the dilutional effect of migratory cells infiltrating the lung in response to allergen exposure. STAT6-deficient mice have marked attenuation of lung inflammation, AHR, and mucus production 50, 51
. Since STAT6-deficient mice have diminished airway inflammation, a decrease in GSTPi expression and total GST activity in STAT6-deficient mice treated with HDM indicates that this decrease is likely due to resident cells and not migratory cells.
In summary, GSTPi expression and total GST activity are dysregulated in mouse models of asthma. GSTPi is similarly dysregulated in human asthma. GSTPi plays an important role in neutralizing oxidative stress. The antioxidant and detoxification capacity of GSTPi and its potential role in GSH homeostasis suggest that the GSTPi pathway could be a critical therapeutic target for asthma, especially in response to environmental exposures. In fact, GSH has already been shown to alleviate IL-13 induced asthma in mice 52
. Evaluation of GSTPi expression or GSH homeostasis may prove beneficial in identifying individuals who would benefit from therapy targeting this pathway.