Wesley Gladwell

Nrf2-regulated PPARγ Expression Is Critical to Protection against Acute Lung Injury in Mice

RATIONALE The NF-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is essential for protection against oxidative injury and inflammation including hyperoxia-induced acute lung injury. Microarray expression profiling revealed that lung peroxisome proliferator activated receptor gamma (PPARgamma) induction is suppressed in hyperoxia-susceptible Nrf2-deficient (Nrf2(-/-)) mice compared with wild-type (Nrf2(+/+)) mice. PPARgamma has pleiotropic beneficial effects including antiinflammation in multiple tissues. OBJECTIVES We tested the hypothesis that PPARgamma is an important determinant of pulmonary responsivity to hyperoxia regulated by Nrf2. METHODS A computational bioinformatic method was applied to screen potential AREs in the Pparg promoter for Nrf2 binding. The functional role of a potential ARE was investigated by in vitro promoter analysis. A role for PPARgamma in hyperoxia-induced acute lung injury was determined by temporal silencing of PPARgamma via intranasal delivery of PPARgamma-specific interference RNA and by administration of a PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J(2) in mice. MEASUREMENTS AND MAIN RESULTS Deletion or site-directed mutagenesis of a potential ARE spanning -784/-764 sequence significantly attenuated hyperoxia-increased Pparg promoter activity in airway epithelial cells overexpressing Nrf2, indicating that the -784/-764 ARE is critical for Nrf2-regulated PPARgamma expression. Mice with decreased lung PPARgamma by specific interference RNA treatment had significantly augmented hyperoxia-induced pulmonary inflammation and injury. 15 Deoxy-Delta(12,14)-prostaglandin J(2) administration significantly reduced hyperoxia-induced lung inflammation and edema in Nrf2(+/+), but not in Nrf2(-/-) mice. CONCLUSIONS Results indicate for the first time that Nrf2-driven PPARgamma induction has an essential protective role in pulmonary oxidant injury. Our observations provide new insights into the therapeutic potential of PPARgamma in airway oxidative inflammatory disorders.

Differential Expression and Estrogen Response of Lactoferrin Gene in the Female Reproductive Tract of Mouse, Rat, and Hamster

Abstract Lactoferrin, an iron-binding glycoprotein, kills bacteria and modulates inflammatory and immune responses. Presence of lactoferrin in the female reproductive tract suggests that the protein may be part of the mucosal immune system and act as the first line of defense against pathogenic organisms. We have discovered that lactoferrin is a major estrogen-inducible protein in the uterus of immature mice and is up-regulated by physiological levels of estrogen during proestrous in mature mice. In the present study, we examined lactoferrin gene expression and its response to estrogen stimulation in the female reproductive tract of several strains of immature mouse, rat, and hamster. The lactoferrin expression in the cycling adult female rat was also evaluated. Lactoferrin gene polymorphism exists among the different mouse strains. In the three inbred mouse strains studied, lactoferrin gene expression is stimulated by estrogen in the immature uterus, although it is less robust than in the outbred CD-1 mouse. We found that the lactoferrin gene is constitutively expressed in the epithelium of the vagina and the isthmus oviduct; however, it is estrogen inducible in the uterus of immature mice and rats. Furthermore, lactoferrin is elevated in the uterine epithelium of the mature rat during the proestrous and estrous stages of the estrous cycle. Estrogen stimulation of lactoferrin gene expression in the reproductive tract of an immature hamster is limited to the vaginal epithelium. The present study demonstrates differential expression and estrogen responsiveness of the lactoferrin gene in different regions of the female rodent reproductive tract and variation among the rodent species studied.

Targeted deletion of nrf2 impairs lung development and oxidant injury in neonatal mice.

AIMS Nrf2 is an essential transcription factor for protection against oxidant disorders. However, its role in organ development and neonatal disease has received little attention. Therapeutically administered oxygen has been considered to contribute to bronchopulmonary dysplasia (BPD) in prematurity. The current study was performed to determine Nrf2-mediated molecular events during saccular-to-alveolar lung maturation, and the role of Nrf2 in the pathogenesis of hyperoxic lung injury using newborn Nrf2-deficient (Nrf2(-/-)) and wild-type (Nrf2(+/+)) mice. RESULTS Pulmonary basal expression of cell cycle, redox balance, and lipid/carbohydrate metabolism genes was lower while lymphocyte immunity genes were more highly expressed in Nrf2(-/-) neonates than in Nrf2(+/+) neonates. Hyperoxia-induced phenotypes, including mortality, arrest of saccular-to-alveolar transition, and lung edema, and inflammation accompanying DNA damage and tissue oxidation were significantly more severe in Nrf2(-/-) neonates than in Nrf2(+/+) neonates. During lung injury pathogenesis, Nrf2 orchestrated expression of lung genes involved in organ injury and morphology, cellular growth/proliferation, vasculature development, immune response, and cell-cell interaction. Bioinformatic identification of Nrf2 binding motifs and augmented hyperoxia-induced inflammation in genetically deficient neonates supported Gpx2 and Marco as Nrf2 effectors. INNOVATION This investigation used lung transcriptomics and gene targeted mice to identify novel molecular events during saccular-to-alveolar stage transition and to elucidate Nrf2 downstream mechanisms in protection from hyperoxia-induced injury in neonate mouse lungs. CONCLUSION Nrf2 deficiency augmented lung injury and arrest of alveolarization caused by hyperoxia during the newborn period. Results suggest a therapeutic potential of specific Nrf2 activators for oxidative stress-associated neonatal disorders including BPD.

Methylation and expression of the lactoferrin gene in human tissues and cancer cells.

The lactoferrin gene promoter contains GC-rich regions that harbor consensus sequences for a variety of transcription factors. Previous work in our laboratory has demonstrated a link between methylation at the CpG sites of the mouse lactoferrin gene promoter and the level of its expression. The current work investigates the methylation profile in three regions of the human lactoferrin gene by bisulfite genomic sequencing. In addition, the methylation profiles of normal leukocyte DNA, and leukemia cell line and patient DNA were compared. The three regions are located at the −504/−190, which includes the estrogen response element, the −282/+271 which contains the lactoferrin promoter and the +1087/+1476, a region within the first intron that has the alternative delta lactoferrin promoter. Differential methylations were found within all the regions. Increased methylation at the CpG sites and the presence of non-CpG methylation of the lactoferrin promoters were found in the cancer samples.

Exacerbated Airway Toxicity of Environmental Oxidant Ozone in Mice Deficient in Nrf2

Ozone (O3) is a strong oxidant in air pollution that has harmful effects on airways and exacerbates respiratory disorders. The transcription factor Nrf2 protects airways from oxidative stress through antioxidant response element-bearing defense gene induction. The present study was designed to determine the role of Nrf2 in airway toxicity caused by inhaled O3 in mice. For this purpose, Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice received acute and subacute exposures to O3. Lung injury was determined by bronchoalveolar lavage and histopathologic analyses. Oxidation markers and mucus hypersecretion were determined by ELISA, and Nrf2 and its downstream effectors were determined by RT-PCR and/or Western blotting. Acute and sub-acute O3 exposures heightened pulmonary inflammation, edema, and cell death more severely in Nrf2−/− mice than in Nrf2+/+ mice. O3 caused bronchiolar and terminal bronchiolar proliferation in both genotypes of mice, while the intensity of compensatory epithelial proliferation, bronchial mucous cell hyperplasia, and mucus hypersecretion was greater in Nrf2−/− mice than in Nrf2+/+ mice. Relative to Nrf2+/+, O3 augmented lung protein and lipid oxidation more highly in Nrf2−/− mice. Results suggest that Nrf2 deficiency exacerbates oxidative stress and airway injury caused by the environmental pollutant O3.

Identification of novel susceptibility genes in ozone-induced inflammation in mice

Ozone (O3) remains a prevalent air pollutant and public health concern. Inf2 is a significant quantitative trait locus on murine chromosome 17 that contributes to susceptibility to O3-induced infiltration of polymorphonuclear leukocytes (PMNs) into the lung, but the mechanisms of susceptibility remain unclear. The study objectives were to confirm and restrict Inf2, and to identify and test novel candidate susceptibility gene(s). Congenic strains of mice that contained overlapping regions of Inf2 and their controls, and mice deficient in either major histocompatibility complex (MHC) class II genes or the Tnf cluster, were exposed to air or O3. Lung inflammation and gene expression were assessed. Inf2 was restricted from 16.42 Mbp to 0.96 Mbp, and bioinformatic analysis identified MHC class II, the Tnf cluster and other genes in this region that contain potentially informative single nucleotide polymorphisms between the susceptible and resistant mice. Furthermore, O3-induced inflammation was significantly reduced in mice deficient in MHC class II genes or the Tnf cluster genes, compared with wild-type controls. Gene expression differences were also observed in MHC class II and Tnf cluster genes. This integrative genetic analysis of Inf2 led to identification of novel O3 susceptibility genes that may provide important, new therapeutic targets in susceptible individuals.

Association of Nrf2 polymorphism haplotypes with acute lung injury phenotypes in inbred strains of mice.

AIMS Nrf2 is a master transcription factor for antioxidant response element (ARE)-mediated cytoprotective gene induction. A protective role for pulmonary Nrf2 was determined in model oxidative disorders, including hyperoxia-induced acute lung injury (ALI). To obtain additional insights into the function and genetic regulation of Nrf2, we assessed functional single nucleotide polymorphisms (SNPs) of Nrf2 in inbred mouse strains and tested whether sequence variation is associated with hyperoxia susceptibility. RESULTS Nrf2 SNPs were compiled from publicly available databases and by re-sequencing DNA from inbred strains. Hierarchical clustering of Nrf2 SNPs categorized the strains into three major haplotypes. Hyperoxia susceptibility was greater in haplotypes 2 and 3 strains than in haplotype 1 strains. A promoter SNP -103 T/C adding an Sp1 binding site in haplotype 2 diminished promoter activation basally and under hyperoxia. Haplotype 3 mice bearing nonsynonymous coding SNPs located in (1862 A/T, His543Gln) and adjacent to (1417 T/C, Thr395Ile) the Neh1 domain showed suppressed nuclear transactivation of pulmonary Nrf2 relative to other strains, and overexpression of haplotype 3 Nrf2 showed lower ARE responsiveness than overexpression of haplotype 1 Nrf2 in airway cells. Importantly, we found a significant correlation of Nrf2 haplotypes and hyperoxic lung injury phenotypes. INNOVATION AND CONCLUSION The results indicate significant influence of Nrf2 polymorphisms and haplotypes on gene function and hyperoxia susceptibility. Our findings further support Nrf2 as a genetic determinant in ALI pathogenesis and provide useful tools for investigators who use mouse strains classified by Nrf2 haplotypes to elucidate the role for Nrf2 in oxidative disorders.

Genome-wide association mapping of acute lung injury in neonatal inbred mice

Reactive oxygen species (ROS) contribute to the pathogenesis of many acute and chronic pulmonary disorders, including bronchopulmonary dysplasia (BPD), a respiratory condition that affects preterm infants. However, the mechanisms of susceptibility to oxidant stress in neonatal lungs are not completely understood. We evaluated the role of genetic background in response to oxidant stress in the neonatal lung by exposing mice from 36 inbred strains to hyperoxia (95% O2) for 72 h after birth. Hyperoxia‐induced lung injury was evaluated by using bronchoalveolar lavage fluid (BALF) analysis and pathology. Statistically significant interstrain variation was found for BALF inflammatory cells and protein (heritability estimates range: 33.6–55.7%). Genome‐wide association mapping using injury phenotypes identified quantitative trait loci (QTLs) on chromosomes 1, 2, 4, 6, and 7. Comparative mapping of the chromosome 6 QTLs identified Chrm2 (cholinergic receptor, muscarinic 2, cardiac) as a candidate susceptibility gene, and mouse strains with a nonsynonymous coding single‐nucleotide polymorphism (SNP) in Chrm2 that causes an amino acid substitution (P265L) had significantly reduced hyperoxia‐induced inflammation compared to strains without the SNP. Further, hyperoxia‐induced lung injury was significantly reduced in neonatal mice with targeted deletion of Chrm2, relative to wild‐type controls. This study has important implications for understanding the mechanisms of oxidative lung injury in neonates.—Nichols, J. L., Gladwell, W., Verhein, K. C., Cho, H.‐Y., Wess, J., Suzuki, O., Wiltshire, T., Kleeberger, S. R. Genome‐wide association mapping of acute lung injury in neonatal inbred mice. FASEB J. 28, 2538–2550 (2014). www.fasebj.org

Novel Roles for Notch3 and Notch4 Receptors in Gene Expression and Susceptibility to Ozone-Induced Lung Inflammation in Mice

Background Ozone is a highly toxic air pollutant and global health concern. Mechanisms of genetic susceptibility to ozone-induced lung inflammation are not completely understood. We hypothesized that Notch3 and Notch4 are important determinants of susceptibility to ozone-induced lung inflammation. Methods Wild-type (WT), Notch3 (Notch3–/–), and Notch4 (Notch4–/–) knockout mice were exposed to ozone (0.3 ppm) or filtered air for 6–72 hr. Results Relative to air-exposed controls, ozone increased bronchoalveolar lavage fluid (BALF) protein, a marker of lung permeability, in all genotypes, but significantly greater concentrations were found in Notch4–/– compared with WT and Notch3–/– mice. Significantly greater mean numbers of BALF neutrophils were found in Notch3–/– and Notch4–/– mice compared with WT mice after ozone exposure. Expression of whole lung Tnf was significantly increased after ozone in Notch3–/– and Notch4–/– mice, and was significantly greater in Notch3–/– compared with WT mice. Statistical analyses of the transcriptome identified differentially expressed gene networks between WT and knockout mice basally and after ozone, and included Trim30, a member of the inflammasome pathway, and Traf6, an inflammatory signaling member. Conclusions These novel findings are consistent with Notch3 and Notch4 as susceptibility genes for ozone-induced lung injury, and suggest that Notch receptors protect against innate immune inflammation. Citation Verhein KC, McCaw Z, Gladwell W, Trivedi S, Bushel PR, Kleeberger SR. 2015. Novel roles for Notch3 and Notch4 receptors in gene expression and susceptibility to ozone-induced lung inflammation in mice. Environ Health Perspect 123:799–805; http://dx.doi.org/10.1289/ehp.1408852

The Influence of Nrf2 on Cardiac Responses to Environmental Stressors

Nrf2 protects the lung from adverse responses to oxidants, including 100% oxygen (hyperoxia) and airborne pollutants like particulate matter (PM) exposure, but the role of Nrf2 on heart rate (HR) and heart rate variability (HRV) responses is not known. We hypothesized that genetic disruption of Nrf2 would exacerbate murine HR and HRV responses to severe hyperoxia or moderate PM exposures. Nrf2−/− and Nrf2+/+ mice were instrumented for continuous ECG recording to calculate HR and HRV (low frequency (LF), high frequency (HF), and total power (TP)). Mice were then either exposed to hyperoxia for up to 72 hrs or aspirated with ultrafine PM (UF-PM). Compared to respective controls, UF-PM induced significantly greater effects on HR (P < 0.001) and HF HRV (P < 0.001) in Nrf2−/− mice compared to Nrf2+/+ mice. Nrf2−/− mice tolerated hyperoxia significantly less than Nrf2+/+ mice (~22 hrs; P < 0.001). Reductions in HR, LF, HF, and TP HRV were also significantly greater in Nrf2−/− compared to Nrf2+/+ mice (P < 0.01). Results demonstrate that Nrf2 deletion increases susceptibility to change in HR and HRV responses to environmental stressors and suggest potential therapeutic strategies to prevent cardiovascular alterations.