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.

We recently used positional cloning to identify the transcription factor Nrf2 (NF-E2 related factor 2) as a susceptibility gene in a murine model of oxidant-induced acute lung injury (ALI). NRF2 binds to antioxidant response elements (ARE) and up-regulates protective detoxifying enzymes in response to oxidative stress. This led us to investigate NRF2 as a candidate susceptibility gene for risk of development of ALI in humans. We identified multiple single nucleotide polymorphisms (SNPs) by resequencing NRF2 in ethnically diverse subjects, and one (-617 C/A) significantly (P<0.001) diminished luciferase activity of promoter constructs containing the SNP and significantly decreased the binding affinity (P<0.001) relative to the wild type at this locus (-617 CC). In a nested case-control study, patients with the -617 A SNP had a significantly higher risk for developing ALI after major trauma (OR 6.44; 95% CI 1.34, 30.8; P=0.021) relative to patients with the wild type (-617 CC). This translational investigation provides novel insight into the molecular mechanisms of susceptibility to ALI and may help to identify patients who are predisposed to develop ALI under at risk conditions, such as trauma and sepsis. Furthermore, these findings may have important implications in other oxidative stress related illnesses.

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.

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.

The increasing number of population-based and epidemiological associations between oxidant pollutant exposures and cardiopulmonary disease exacerbation, decrements in pulmonary function, and mortality underscores the important detrimental effects of oxidants on public health. Because inhaled oxidants initiate a number of pathologic processes, including inflammation of the airways which may contribute to the pathogenesis and/or exacerbation of airways disease, it is critical to understand the mechanisms through which exogenous and endogenous oxidants interact with molecules in the cells, tissues, and epithelial lining fluid (ELF) of the lung. Furthermore, it is clear that inter-individual variation in response to a given exposure also exists across an individual lifetime. Because of the potential impact that oxidant exposures may have on reproductive outcomes and infant, child, and adult health, identification of the intrinsic and extrinsic factors that may influence susceptibility to oxidants remains an important issue. In this review, we discuss mechanisms of oxidant stress in the lung, the role of oxidants in lung disease pathogenesis and exacerbation (e.g. asthma, COPD, and ARDS), and the potential risk factors (e.g. age, genetics) for enhanced susceptibility to oxidant-induced 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.

.-This project was designed to determine the genetic (betweenstrain) and environmental (within-strain) variance in daily running wheel activity level in inbred mice. Five male and five female mice, 9.7-15.3 wk old, from each of 13 strains (A/J, AKR/J, BALB/cJ, C3H/HeJ, C57Bl/6J, C57L/J, C3Heb/FeJ, CBA/J, DBA/2J, SWR/J, MRL/MpJ, SPRET/Ei, and CAST/Ei) as well as five female NZB/ BinJ mice were housed individually. A running wheel in each cage was interfaced with a magnetic sensor to measure total daily distance and exercise time for each animal every 24 h for 21 consecutive days (3 wk). Average daily distance (km), duration (min), and velocity (m/min) for each strain was then calculated. Significant interstrain differences in average daily distance (P Ͻ 0.001), average daily exercise duration (P Ͻ 0.0001), and average daily exercise velocity (P Ͻ 0.0001) were found, with C57L/J mice running farther and faster than the other strains. Sex was a significant factor in daily running wheel activity, with female mice running an average of 20% farther (P ϭ 0.01) and 38% faster (P Ͻ 0.0001) than male mice. The male mice ran 15% longer duration on a daily basis (P ϭ 0.0091). Weight was only associated with exercise velocity in the female mice, but this relationship was not significant when subdivided by strain. Broadsense heritability estimates on the physical activity differed by sex (for distance, male 31-48% and female 12-22%; for duration, male 44-61% and female 12-21%; for velocity, male 49-66% and female 44-61%). In conclusion, these data indicate that daily running wheel activity level in mice is significantly affected by genetic background and sex.