NRF2 is a transcription factor important in the protection against carcinogenesis and oxidative stress through antioxidant response element (ARE)-mediated transcriptional activation of several phase 2 detoxifying and antioxidant enzymes. This study was designed to determine the role of NRF2 in the pathogenesis of hyperoxic lung injury by comparing pulmonary responses to 95–98% oxygen between mice with site-directed mutation of the gene for NRF2 (Nrf2− /−) and wild-type mice (Nrf2+ /+ ). Pulmonary hyperpermeability, macrophage inflammation, and epithelial injury in Nrf2− /− mice were 7.6-fold, 47%, and 43% greater, respectively, compared with Nrf2+ /+ mice after 72 h hyperoxia exposure. Hyperoxia markedly elevated the expression of NRF2 mRNA and DNA-binding activity of NRF2 in the lungs of Nrf2+ /+ mice. mRNA expression for ARE- responsive lung antioxidant and phase 2 enzymes was evaluated in both genotypes of mice to identify potential downstream molecular mechanisms of NRF2 in hyperoxic lung responses. Hyperoxia-induced mRNA levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferase (GST)-Ya and -Yc subunits, UDP glycosyl transferase (UGT), glutathione peroxidase-2 (GPx2), and heme oxygenase-1 (HO-1) were significantly lower in Nrf2− /− mice compared with Nrf2+ /+ mice. Consistent with differential mRNA expression, NQO1 and total GST activities were significantly lower in Nrf2− /− mice compared with Nrf2+ /+ mice after hyperoxia. Results demonstrated that NRF2 has a significant protective role against pulmonary hyperoxic injury in mice, possibly through transcriptional activation of lung antioxidant defense enzymes.

Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated that the expression of about two-thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRA has implications for response to retinoid treatments and adipogenesis. In mouse, 3T3-L1 cells’ SFN treatment affected Rxra expression early in adipogenesis, and knockdown of Nrf2-delayed Rxra expression, both leading to impaired adipogenesis.

Nuclear factor, erythroid 2 related factor 2 (Nrf2) belongs to the Cap'n'collar/basic region leucine zipper (CNC-bZIP) transcription factor family, and is activated by diverse oxidants, pro-oxidants, antioxidants, and chemopreventive agents. After phosphorylation and dissociation from the cytoplasmic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), Nrf2 translocates to the nucleus and binds to an antioxidant response element (ARE). Through transcriptional induction of ARE-bearing genes that encode antioxidant-detoxifying proteins, Nrf2 activates cellular rescue pathways against oxidative injury, inflammation/immunity, apoptosis, and carcinogenesis. ARE-driven genes include direct antioxidants (e.g., GPx), thiol metabolism-associated detoxifying enzymes (e.g., GSTs), stress-response genes (e.g., HO-1), and others (e.g., PSMB5). Application of nrf2 germ-line mutant mice elucidated protective roles for Nrf2 in various models of human disorders in the liver, lung, kidney, brain, and circulation. In the lung, deficiency of nrf2 augmented injury caused by bleomycin and environmental oxidants including hyperoxia, diesel exhaust particles, and cigarette smoke. Microarray analyses of lungs from nrf2-deficient and -sufficient mice identified Nrf2-dependent genes that might be critical in pulmonary protection. Observations from these studies highlight the importance of the Nrf2-antioxidant pathway and may provide new therapeutic strategies for acute respiratory distress syndrome, idiopathic pulmonary fibrosis, cancer, and emphysema in which oxidative stress is implicated.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral pneumonia in infants and young children. Administration of a formalin inactivated vaccine against RSV to children in the 1960s resulted in increased morbidity and mortality in vaccine recipients who subsequently contracted RSV. This incident precluded development of subunit RSV vaccines for infants for over 30 years, because the mechanism of illness was never clarified. An RSV vaccine for infants is still not available. Here, we demonstrate that enhanced RSV disease is mediated by immune complexes and abrogated in complement component C3 and B cell–deficient mice but not in controls. Further, we show correlation with the enhanced disease observed in children by providing evidence of complement activation in postmortem lung sections from children with enhanced RSV disease.

The molecular mechanisms of pulmonary fibrosis are poorly understood, although reactive oxygen species are thought to have an important role. NRF2 is a transcription factor that protects cells and tissues from oxidative stress by activating protective antioxidant and detoxifying enzymes. We hypothesized that NRF2 protects lungs from injury and fibrosis induced by bleomycin, an anti‐neoplastic agent that causes pulmonary fibrosis in susceptible patients. To test this hypothesis, mice with targeted deletion of Nrf2 ( Nrf2 ^‒/‒ ) and wild‐type ( Nrf2 ^+/+ ) mice were treated with bleomycin or vehicle, and pulmonary injury and fibrotic responses were compared. Bleomycin‐induced increases in lung weight, epithelial cell death, and inflammation were significantly greater in Nrf2 ^‒/‒ mice than in Nrf2 ^+/+ mice. Indices of lung fibrosis (hydroxyproline content, collagen accumulation, fibrotic score, cell proliferation) were significantly greater in bleomycin‐treated Nrf2 ^‒/‒ mice, compared with Nrf2 ^+/+ mice. NRF2 expression and activity were elevated in Nrf2 ^+/+ mice by bleomycin. Bleomycin caused greater up‐regulation of several NRF2‐inducible antioxidant enzyme genes and protein products in Nrf2 ^+/+ mice compared with Nrf2 ^‒/‒ mice. Further, bleomycin‐induced transcripts and protein levels of lung injury and fibrosis markers were significantly attenuated in Nrf2 ^+/+ mice compared with Nrf2 ^‒/‒ mice. Results demonstrated that NRF2 has a critical role in protection against pulmonary fibrosis, presumably through enhancement of cellular antioxidant capacity. This study has important implications for the development of intervention strategies against fibrosis.