Nrf2 deficiency influences susceptibility to steroid resistance via HDAC2 reduction

Abstract

Abnormal lung inflammation and oxidant burden are associated with a significant reduction in histone deacetylase 2 (HDAC2) abundance and steroid resistance. We hypothesized that Nrf2 regulates steroid sensitivity via HDAC2 in response to inflammation in mouse lung. Furthermore, HDAC2 deficiency leads to steroid resistance in attenuating lung inflammatory response, which may be due to oxidant/antioxidant imbalance. Loss of antioxidant transcription factor Nrf2 resulted in decreased HDAC2 level in lung, and increased inflammatory lung response which was not reversed by steroid. Thus, steroid resistance or inability of steroids to control lung inflammatory response is dependent on Nrf2-HDAC2 axis. These findings have implications in steroid resistance, particularly during the conditions of oxidative stress when the lungs are more susceptible to inflammatory response, which is seen in patients with chronic obstructive pulmonary disease, asthma, rheumatoid arthritis, and inflammatory bowel disease.

Fig. 1. HDAC2−/− mice are more susceptible to CS-induced lung inflammation

WT and HDAC2−/− mice were exposed to filtered air or acute CS for 3 days. Mice were sacrificed at 24 h post-last exposure, and lungs were lavaged. Bronchoalveolar lavage cells were prepared on cytospin slides and stained with Diff-Quik. (A) Total number of neutrophils in BALF. (B) MCP-1 levels were measured in mouse lung tissue by ELISA. Data are shown as mean±SEM (n=4 to 6). *P<0.05, **P<0.01, ***P<0.001 significant compared with corresponding air groups. ^#P<0.05, and ^###P<0.001 compared with corresponding WT mice exposed to CS. ^$P<0.05, compared with air-exposed WT mice.

Fig. 2. HDAC2−/− mice are less responsive to budesonide in response to LPS-induced lung inflammation

(A) WT mice were exposed to saline or aerosolized LPS (1 mg/ml). Nuclear extracts from mouse lungs were separated on SDS-PAGE gel, and HDAC2 protein levels were determined by immunoblotting. (B) Band intensity of HDAC2 levels normalized to actin. (C and D) WT and HDAC2−/− mice were pretreated with budesonide (3 mg/kg body weight) for 3 days at 1 h prior to aerosolization of LPS. (C) Neutrophils in BALF were determined following differential staining on cytospin slides. (D) MCP-1 levels were measured from lung homogenates of mouse lung tissue by ELISA. Data are shown as mean±SEM (n=4 to 6). ***P<0.001, significant compared with corresponding controls as denoted in figure. Sal: Saline; Bud: Budesonide.

Fig. 3. Increased lung inflammation is associated with HDAC2 reduction in Nrf2−/− mice exposed to CS

(A) WT and Nrf2−/− mice were exposed to CS for 3 days. Differential counts from BALF were determined by Diff-Quik staining of cytospin slides. (B) Immunoprecipitated HDAC2 from lungs of naïve WT and Nrf2−/− mice were analyzed for deacetylase activity using specific HDAC deacetylase activity kit. (C) WT and Nrf2−/− mice were exposed to filtered air or whole body CS for 3 days. Lung tissue nuclear extracts were analyzed for HDAC2 relative expression. (D) HDAC2 levels normalized to actin expression. Data are shown as mean±SEM (n=4 to 9). *P<0.05, **P<0.01, and ***P<0.001, significant compared with corresponding air-exposed controls. ^#P<0.05 and ^###P<0.001, significant compared with CS-exposed WT controls. ^$$$P<0.001, compared with air-exposed WT mice.

Fig. 4. Nrf2−/− mice are unresponsive to budesonide following LPS exposure

Nrf2−/− mice were treated with budesonide (1 or 3 mg/kg body weight) by intranasal administration followed by LPS exposure. (A) Percentage of neutrophils in BALF was determined following differential staining on cytospin slides. (B) MCP-1 levels were measured in lung tissue homogenates by ELISA. Data are shown as mean±SEM (n=4 to 6). ***P<0.001 significant compared with corresponding controls. Sal: Saline; Bud: Budesonide.