Nrf2 reduces allergic asthma in mice through enhanced airway epithelial cytoprotective function

Abstract

Asthma development and pathogenesis are influenced by the interactions of airway epithelial cells and innate and adaptive immune cells in response to allergens. Oxidative stress is an important mediator of asthmatic phenotypes in these cell types. Nuclear erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that is the key regulator of the response to oxidative and environmental stress. We previously demonstrated that Nrf2-deficient mice have heightened susceptibility to asthma, including elevated oxidative stress, inflammation, mucus, and airway hyperresponsiveness (AHR) (Rangasamy T, Guo J, Mitzner WA, Roman J, Singh A, Fryer AD, Yamamoto M, Kensler TW, Tuder RM, Georas SN, Biswal S. J Exp Med 202: 47-59, 2005). Here we dissected the role of Nrf2 in lung epithelial cells and tested whether genetic or pharmacological activation of Nrf2 reduces allergic asthma in mice. Cell-specific activation of Nrf2 in club cells of the airway epithelium significantly reduced allergen-induced AHR, inflammation, mucus, Th2 cytokine secretion, oxidative stress, and airway leakiness and increased airway levels of tight junction proteins zonula occludens-1 and E-cadherin. In isolated airway epithelial cells, Nrf2 enhanced epithelial barrier function and increased localization of zonula occludens-1 to the cell surface. Pharmacological activation of Nrf2 by 2-trifluoromethyl-2'-methoxychalone during the allergen challenge was sufficient to reduce allergic inflammation and AHR. New therapeutic options are needed for asthma, and this study demonstrates that activation of Nrf2 in lung epithelial cells is a novel potential therapeutic target to reduce asthma susceptibility.

Keywords: Th2; airway hyperresponsiveness; inflammation; ovalbumin; oxidative stress.

Fig. 1.

Genetic activation of nuclear erythroid 2-related factor 2 (Nrf2) reduces ovalbumin (OVA)-induced asthmatic phenotypes. A: NADPH quinone oxidoreductase 1 (Nqo1) gene expression, as a marker of Nrf2 activity, was measured in lungs of Kelch ECH associating protein 1-floxed (Keap1^fl/fl) and Tam-Keap1^−/− mice that were treated with tamoxifen (Tam) before sensitization. These same mice were used in B–D. B: airway hyperresponsiveness (AHR) was assessed by airway pressure-time index (APTI) (cmH₂O·s) after inhalation of methacholine. Inflammation (C) and cytokines (D) were quantified in the bronchoalveolar lavage (BAL). Values are means ± SE; N = 6–9 mice per group. *P < 0.05 by ANOVA.

Fig. 2.

Pharmacological activation of Nrf2 during the challenge phase reduces OVA-induced asthmatic phenotypes. A: Nqo1 expression was measured by quantitative PCR in lungs at 6 h after treatment with 2-trifluoromethyl-2′-methoxychalone (TMC) or vehicle by gavage (N = 3). AHR (B), inflammation (C), and cytokines (D) were measured in BAL fluid of C57BL/6 mice that were treated with 400 mg/kg TMC or vehicle by gavage 4 h before each challenge. Values are means ± SE; N ≥ 5 mice per group. *P < 0.05 by Student's t-test.

Fig. 3.

Activation of Nrf2 in club cells attenuates OVA-induced asthmatic phenotypes. A: AHR was assessed by APTI (cmH₂O·s) in Keap1^fl/fl and club cell-specific 10-kDa protein (CC10)-Keap1^−/− mice in response to inhalation of methacholine. Inflammation (B) and cytokines (C) were quantified in the BAL. Values are means ± SE; N = 11–12 mice per group. *P < 0.05 by ANOVA. D: mucus secretion was assessed by periodic acid Schiff staining of airways from OVA-exposed Keap1^fl/fl and CC10-Keap1^−/− mice (N = 5 mice per group). Representative images were captured at ×40 (left) and ×200 (right) magnification.

Fig. 4.

Nrf2 regulates club cell function. A: oxidative damage to macromolecules was measured via quantification of protein carbonyls and lipid peroxidation in lung homogenates of CC10-Keap1^−/− and Keap1^fl/fl mice. B: airway leakiness was assessed via quantification of albumin in the cell-free BAL fluid by the Albumin Blue Fluorescent Assay Kit (Active Motif). Values are means ± SE; N = 7–8 mice per group. C: Zonula occludens-1 (Zo-1) and E-cadherin intensity were quantified in the airway epithelium of OVA-sensitized/challenged Keap1^fl/fl and CC10-Keap1^−/− mice, and normalized to background levels using Image J. Quantification was performed on a minimum of 4 airways per mouse. *P < 0.05 by ANOVA. †P < 0.05 by Student's t-test. D: representative ×200 images of airways from OVA-sensitized/challenged Keap1^fl/fl and CC10-Keap1^−/− mice immunostained for Zo-1. Arrows indicate epithelial cells that do not express Zo-1. SM, smooth muscle.

Fig. 5.

Nrf2 enhances barrier function of airway epithelial cells. A: Nqo1 gene expression, as a marker of Nrf2 activity, was measured in epithelial cell monolayers of Tam-Keap1^−/− and Keap1^fl/fl mice that were treated with Tam before harvesting of tracheas. B: after cells formed a confluent monolayer [confirmed by stable transepithelial electrical resistance (TEER) readings], TEER was measured across the monolayer daily for 5 days, and the average of all days was represented as a single replicate. C: FITC-dextran was added to the apical side of the confluent monolayer, and paracellular diffusion was quantified after 20 min. D: Zo-1 localization was measured by immunofluorescence. E: TEER was measured in Keap1^fl/fl and CC10-Keap1^−/− epithelial monolayers. Values are means ± SE; N = 6 mice per group. *P < 0.05 by Student's t-test.