Although HDAC2 has been implicated in increased susceptibility to inflammation and steroid resistance in vitro
], no such studies are available to determine the role of HDAC2 in vivo
in lung in response to inhaled toxicants. In this study, we used mice expressing mutant HDAC2 [14
] to study the steroid resistance in response to CS exposure. HDAC2−/− mice showed increased neutrophil recruitment in the lung in response to CS as compared to WT mice exposed to CS. Neutrophil recruitment was correlated strongly with the release of KC and MCP-1 in lung tissue suggesting the susceptibility of these mice to augmentation of CS-induced inflammation.
Since CS induces degradation of other HDACs (HDAC1 and HDAC3) that may be crucial for inflammation [21
], it was important to determine a specific role for HDAC2 in steroid resistance utilizing an exposure model with no effect on HDAC levels or activity. Hence, we hypothesized that HDAC2−/− mice would exhibit a poor response to budesonide in response to LPS exposure. As expected, budesonide had no significant effect in reducing KC release in HDAC2−/− mice lungs in response to LPS. Furthermore, budesonide partially blocked MCP-1 release in the lungs of HDAC2−/− mice versus complete inhibition of MCP-1 in WT mice. Budesonide was also ineffective in reducing MMP9 activity in the mutant mice. Thus, these data suggest that basal HDAC2 deficiency leads to increased inflammatory response in the lung which is further augmented by LPS exposure. Furthermore, steroids have poor efficacy in HDAC2-ablated mice in response to pro-inflammatory challenge in the lung.
Increased susceptibility to oxidative stress (or deficiency of antioxidants) may exhibit phenotypes similar to HDAC2−/− mice in terms of steroid resistance. We used Nrf2−/− mice, which are susceptible to oxidative stress/cigarette smoke, to study the steroid resistance in controlling the lung inflammatory response. Disruption of Nrf2 enhanced susceptibility to acute CS-induced neutrophilic inflammation which is consistent with an earlier report [13
]. Surprisingly, in air-exposed Nrf2−/− mice, neutrophil infiltration and RelA/p65 phosphorylation on ser276 and ser536 (unpublished data) were markedly enhanced in the lungs. It is possible that loss of Nrf2 leads to activation of NF-κB by increasing the pool of CREB-binding protein (CBP) available to interact with RelA/p65 [26
], and thus causing induction of NF-κB-dependent pro-inflammatory genes. Our data suggest that Nrf2 deficiency led to increased lung neutrophil influx and basal activation of NF-κB, which are augmented by CS exposure.
The finding that loss of Nrf2 induces an oxidative-stress phenotype in the lungs led us to hypothesize that increased oxidative stress status of the lung in Nrf2−/− mice would induce a reduction in HDAC2 activity and level. Lungs from naïve Nrf2−/− mice showed marked reduction in HDAC2 abundance and subsequently reduced HDAC2 deacetylase activity compared to naïve WT mice. Consistent with these data, exposure of Nrf2−/− mice to CS rapidly accelerated the rate of HDAC2 degradation suggesting the oxidant burden in lungs of Nrf2−/− is a key factor in loss of HDAC2 with or without exposure to environmental toxicants. The reduction in levels of HDAC2 in Nrf2−/− mice alternatively suggests that ARE-mediated Nrf2 activation may be required for HDAC2 transcription. However, the molecular regulation of HDAC2 expression or transcription factor(s) binding site is not clearly known [25
]. It may also be possible that nuclear Nrf2 stabilizes the HDAC2 co-repressor complex on pro-inflammatory genes, whereas deficiency of Nrf2 leads to degradation of HDAC2 and activation of NF-κB-CBP binding on pro-inflammatory promoters [26
]. Our preliminary data support this contention showing activation of CBP in lungs of Nrf2 null mice (unpublished data).
We next determined whether the increased inflammatory response and decreased HDAC2 protein levels in Nrf2−/− mice render them resistant to steroids in inhibiting the inflammatory response. WT and Nrf2−/− mice were exposed to aerosolized LPS following intranasal budesonide treatment. LPS induced severe neutrophilic inflammation in mouse lungs (unlike macrophage influx by CS) with no subsequent reduction in HDAC2 protein expression. Pre-treatment of WT mice with a high dose of budesonide significantly blocked LPS-induced lung neutrophilic influx which is consistent with budesonide-mediated inhibition of lung tissue KC levels. However, LPS-exposed Nrf2−/− mice failed to respond to both low and high dose budesonide treatments in blocking neutrophil influx, attenuating KC and MCP-1 release, and inhibiting RelA/p65 nuclear accumulation (unpublished data) in the lung. The observation of steroid resistance in Nrf2−/− and HDAC2−/− mice with the finding that HDAC2 protein levels are diminished in Nrf2−/− mice suggests a strong in vivo linkage between HDAC2 abundance and steroid sensitivity via oxidative stress.
In conclusion, loss of HDAC2 is a critical factor in inhaled toxicant-mediated lung inflammation especially in regulating the anti-inflammatory effects of glucocorticoids in mouse lungs. Oxidative stress-susceptible Nrf2−/− mice showed reduced HDAC2 levels and deacetylase activity in lungs, and are susceptible to CS- and LPS-induced inflammation. Interestingly, the loss of Nrf2 potentially leads to steroid resistance due to HDAC2 reduction. We speculate that the high oxidant status in the lung of Nrf2−/− mice leads to inactivation of endogenous HDAC2 through post-translational modifications, such as nitrosylation of tyrosine residues and carbonylation of cysteine, histidine and lysine residues. This may then block the ability of ligand-bound glucocorticoid receptors to recruit active HDAC2 to promoters of pro-inflammatory genes. Nevertheless, the rapid loss of HDAC2 and NF-κB activation in CS-exposed Nrf2−/− mice suggest steroid resistance is clearly a multifactorial cascade of events that perhaps start with oxidant/antioxidant imbalance and then precipitates at a much more rapid decline in HDAC2. This may have implications for devising better therapies for patients who are refractory/insensitive to steroid treatments, such as patients with COPD, asthma, rheumatoid arthritis, and inflammatory bowel disease [5
- HDAC2 is a critical factor in inhaled toxicant-mediated lung inflammation especially in regulating the anti-inflammatory effects of glucocorticoids.
- Loss of Nrf2 resulted in decreased HDAC2 in lung, and increased inflammatory lung response which was not reversed by steroid.
- Steroid resistance or inability of steroids to control lung inflammatory response is dependent on Nrf2-HDAC2 axis.
- Steroid resistance via Nrf2-HDAC2 axis leads to abnormal inflammatory response which is seen in patients with chronic inflammatory diseases.
- Understanding the redox regulation of Nrf2-HDAC2 may lead to better therapies for patients who are refractory/insensitive to steroid treatments.