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. 2005 Jul 4;202(1):47-59.
doi: 10.1084/jem.20050538.

Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice

Tirumalai Rangasamy  1 , Jia Guo, Wayne A Mitzner, Jessica Roman, Anju Singh, Allison D Fryer, Masayuki Yamamoto, Thomas W Kensler, Rubin M Tuder, Steve N Georas, Shyam Biswal
Affiliations

Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice

Tirumalai Rangasamy et al. J Exp Med. .

Abstract

Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma.

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Figures

Figure 1.
Figure 1.
Increased allergen-driven asthmatic inflammation in OVA-challenged Nrf2 −/− mice. (A) Total and differential inflammatory cell populations in the BAL fluid of OVA- and saline-challenged Nrf2 + / + and Nrf2 − / − mice (n = 8). (B) First challenge with OVA. (C) Second challenge with OVA. (D and E) Third challenge with OVA. There was a progressive increase in the total number of inflammatory cells in the BAL fluid of both OVA-challenged Nrf2 + / + and Nrf2 − / − mice from the first to third challenges. However, the number of inflammatory cells in the BAL fluid of Nrf2−/ − OVA mice was significantly higher than in the BAL fluid of Nrf2 + / + OVA mice, as well as the respective saline-challenged mice. The number of eosinophils, lymphocytes, neutrophils, and epithelial cells were significantly (*) higher in the BAL fluid of Nrf2 − / − OVA mice compared with Nrf2 + / + OVA mice. Pretreatment with NAC significantly (*) reduced the inflammatory cells (F), predominantly eosinophils (G), in the BAL fluid of Nrf2 − / − OVA mice (n = 6). Data are mean ± SEM. *, P ≤ 0.05.
Figure 2.
Figure 2.
Increased infiltration of inflammatory cells into the lungs of OVA-challenged Nrf2 −/− mice. Lung tissues from the saline- and OVA-challenged (third challenge) Nrf2 + / + and Nrf2 − / − mice (n = 6) were stained with H&E and examined by light microscope (20×). (A) OVA challenge caused a marked infiltration of inflammatory cells into the lungs of Nrf2 − / − OVA compared with Nrf2 + / + OVA mice. Immunohistochemical staining showed the presence of numerous eosinophils around the blood vessels and airways (B) and in the parenchyma (C) of OVA-challenged (third challenge) Nrf2 − / − compared with Nrf2 + / + mice. The figure is representative of three experiments (n = 6). AW, airways; BV, blood vessels. H&E staining of the lung sections from the saline-or NAC-treated (7 d before first OVA challenge) Nrf2-deficient mice. The arrows indicate cells staining positively with anti-MBP antibody. (D) Widespread peribronchial and perivascular inflammatory infiltrates were observed in OVA-sensitized mice after antigen provocation (bottom right). Pretreatment of Nrf2-deficient mice with NAC resulted in substantial reduction in the infiltration of inflammatory cells in the peribronchial and perivascular region (bottom left).
Figure 3.
Figure 3.
Increased oxidative stress markers, eotaxin, and enhanced activation of NF-κB in the lungs of Nrf2 −/− OVA mice. Increased levels of lipid hydroperoxides (A) and protein carbonyls (B) in the lungs of OVA-challenged Nrf2 − / − mice. Values are mean ± SEM. *, Significantly higher than the Nrf2 + / + OVA mice (n = 6). (C) Eotaxin level in the BAL fluid. When compared with OVA-challenged Nrf2 + / + mice, the BAL eotaxin level was markedly higher in OVA-challenged (both first and third challenge) Nrf2 − / − mice (P ≤ 0.05; n = 6). (D–F) Activation of NF-κB in the lungs. Western blot was used to determine the activation of p50 and p65 subunits of NF-κB in the lungs (D). (lanes 1 and 2) Saline-challenged Nrf2 + / + and Nrf2 –/– mice, respectively; (lanes 3 and 4) OVA-challenged Nrf2 + / + and Nrf2 –/– mice, respectively. (E) Quantification of p50 and p65 subunits of NF-κB obtained in Western blots. Values are mean ± SEM of three experiments. (F) ELISA measurement of p65/Rel A subunit of NF-κB using Mercury TransFactor kit. *, P ≤ 0.05 versus OVA-challenged Nrf2 wild-type mice. Data are mean ± SEM of three experiments.
Figure 4.
Figure 4.
Nrf2-deficient mice show increased mucus cell hyperplasia in response to allergen challenge. (A) Lung sections (72 h after the final OVA challenge) stained with PAS. Shown are the purple staining epithelial cells (arrows) in the proximal airways of OVA-challenged mice and pronounced mucus cell hyperplasia in Nrf2 − / − OVA mice (40×). (B) Percentage of airway epithelial cells positive for mucus glycoproteins by PAS staining. Lung sections from the Nrf2 − / − OVA mice showed significantly higher numbers of PAS-positive cells than the lung sections from the Nrf2 + / + OVA mice (*, P ≤ 0.05). Data are mean ± SEM.
Figure 5.
Figure 5.
Nrf2-deficient mice show increased airway responsiveness to acetylcholine challenge. OVA-challenged Nrf2 + / + and Nrf2 − / − mice (third challenge) were challenged with acetylcholine aerosol by nebulization with an Aeroneb Pro-nebulizer (n = 7). Lung resistance and compliance were measured. The percent increase in elastance (E; panel C) and resistance (R; panel D) to acetylecholine challenge were significantly higher (*, P ≤ 0.05) in the Nrf2 − / − OVA mice when compared with Nrf2 + / + OVA mice and the respective saline-challenged mice. However, no significant difference in baseline elastance (A) and resistance (B) were observed in both the saline- and OVA-challenged Nrf2 + / + and Nrf2 − / − mice in the absence of acetylcholine challenge. Data are mean ± SEM.
Figure 6.
Figure 6.
Th2 cytokine levels in the BAL fluid of Nrf2 +/+ and Nrf2 −/− mice challenged with OVA. BAL fluids collected 48 h after the second OVA challenge were used for cytokine assays using ELISA. The amounts of both IL-4 (A) and IL-13 (B) were significantly higher (*, P ≤ 0.05) in the BAL fluid of Nrf2 − / − OVA mice than in Nrf2 + / + OVA mice (n = 8). Data are mean ± SEM.
Figure 7.
Figure 7.
Activation of Nrf2 in the lungs of OVA-challenged Nrf2 +/+ mice. (A) EMSA was used to determine the activation of Nrf2 in the lungs of Nrf2 + / + OVA mice. Equal amounts of nuclear extracts (10 μg) prepared from lungs were incubated with radiolabeled ARE from the NQO1 promoter and analyzed by EMSA. EMSA analysis showed the increased binding of nuclear proteins isolated from the lungs of OVA-challenged Nrf2 + / + mice to ARE sequence. The arrows indicate the supershifted band, the major band, and OCT1, respectively. (B) Level of nuclear Nrf2. Nrf2 level in nuclear extracts were determined by immunoblot analysis with anti-Nrf2 antibody. (lanes 1 and 2) Saline-challenged Nrf2 − / − and Nrf2 + / + mice, respectively; (lanes 3 and 4) OVA-challenged Nrf2 − / − and Nrf2 + / + mice, respectively. The figure is representative of three experiments.
Figure 8.
Figure 8.
Real-time RT-PCR analysis of selected antioxidant genes in the lungs of OVA-challenged Nrf2 +/+ and Nrf2 −/− mice. Real-time RT-PCR analysis showed increased levels of mRNA for genes such as γ GCLm, GCLc, G6PD, GST α3, GST p2, HO-1, SOD2, SOD3, and GSR in the lungs of Nrf2 + / + OVA compared with the lungs of Nrf2 − / − OVA mice and the respective saline-challenged mice. Closed bar, Nrf2 + / + mice; open bar, Nrf2 − / − mice.
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