Bronchopulmonary dysplasia (BPD) is a prevalent chronic lung disease of prematurity with limited treatment options. To uncover biomarkers of BPD risk, this study investigated epigenetic and transcriptomic signatures of prematurity at birth and during the neonatal period at day 14 and 28. Peripheral blood DNAs from preterm infants were applied to methylation arrays and cell-type composition was estimated by deconvolution. Covariate-adjusted robust linear regression elucidated BPD- and prolonged oxygen (≥ 14 days) exposure-associated CpGs. RNAs from cord and peripheral blood were sequenced, and differentially expressed genes (DEGs) for BPD or oxygen exposure were determined. Estimated neutrophil–lymphocyte ratios in peripheral blood at day 14 in BPD infants were significantly higher than nonBPD infants, suggesting an heightened inflammatory response in developing BPD. BPD-DEGs in cord blood indicated lymphopoiesis inhibition, altered Th1/Th2 responses, DNA damage, and organ degeneration. On day 14, BPD-associated CpGs were highly enriched in neutrophil activation, infection, and CD4 + T cell quantity, and BPD-DEGs were involved in DNA damage, cellular senescence, T cell homeostasis, and hyper-cytokinesis. On day 28, BPD-associated CpGs along with BPD-DEGs were enriched for phagocytosis, neurological disorder, and nucleotide metabolism. Oxygen supplementation markedly downregulated mitochondrial biogenesis genes and altered CpGs annotated to developmental genes. Prematurity-altered DNA methylation could cause abnormal lymphopoiesis, cellular assembly and cell cycle progression to increase BPD risk. Similar pathways between epigenome and transcriptome networks suggest coordination of the two in dysregulating leukopoiesis, adaptive immunity, and innate immunity. The results provide molecular insights into biomarkers for early detection and prevention of BPD.

Interstitial lung diseases (ILDs) are lethal lung diseases characterized by pulmonary inflammation and progressive lung interstitial scarring. We previously developed a mouse model of ILD using vanadium pentoxide (V2O5) and identified several gene candidates on chromosome 4 associated with pulmonary fibrosis. While these data indicated a significant genetic contribution to ILD susceptibility, they did not include any potential associations and interactions with the mitochondrial genome that might influence disease risk. To conduct this pilot work, we selected the two divergent strains we previously categorized as V2O5-resistant C57BL6J (B6) and -responsive DBA/2J (D2) and compared their mitochondrial genome characteristics, including DNA variants, heteroplasmy, lesions, and copy numbers at 14- and 112-days post-exposure. While we did not find changes in the mitochondrial genome at 14 days post-exposure, at 112 days, we found that the responsive D2 strain exhibited significantly fewer mtDNA copies and more lesions than control animals. Alongside these findings, mtDNA heteroplasmy frequency decreased. These data suggest that mice previously shown to exhibit increased susceptibility to pulmonary fibrosis and inflammation sustain damage to the mitochondrial genome that is evident at 112 days post-V2O5 exposure.

Inbred mouse strains are widely used to identify gene candidates to account for phenotypic variability for various traits, including endurance trainability. While the major mitochondrial DNA (mtDNA) sequence is known for most strains, changes in sequence across mtDNA copies, or heteroplasmy, have yet to be well-defined or completely understood. Therefore, a more thorough understanding of heteroplasmy would augment our understanding of the role of mitochondrial genetics in mediating the individual adaptive response to endurance training. PURPOSE: This preliminary investigation will characterize types of mtDNA variants and heteroplasmy as determined by comparison of the reference strain (C57BL/6 J mouse) to a battery of mouse strains. METHODS: DNA was isolated from the lungs of 32 inbred male mice (n = 2 mice/strain), and mtDNA was amplified by long range PCR, then tagged with Nextera libraries and sequenced on a MiSeq instrument. A site was considered heteroplasmic when the minor allele frequency was ≥1% across the mtDNA copies. We determined the mean heteroplasmy frequency (number of heteroplasmic sites) for each strain, then characterized each heteroplasmic site as either Type 1 or Type 2 heteroplasmy based on the presence of heteroplasmy in either the alternate (Type 1) or reference allele (Type 2). RESULTS: Overall, we found Type 1 heteroplasmies occurred in 30 of the 32 strains, while 7 of the 32 strains had Type 2 heteroplasmy. NZO/HILtJ (198 sites), C3H/HeOuJ (29 sites), and DBA/2 J (19 sites) strains showed the highest frequency Type 1 heteroplasmy, while MOLF/EiJ (356 sites), NZO/HILtJ (81 sites), and PWK/J (71 sites) strains demonstrated the highest frequency Type 2 heteroplasmy. Additionally, analysis of respiratory chain encoding genes revealed a substantial frequency of Type 2 heteroplasmy in the mt-ND5 gene of MOLF/EiJ (6 sites), PWD/PhJ (6 sites), and PWK/J (7 sites) strains, while the NZO strain showed no presence of Type 2 heteroplasmy in the mt-ND5 gene. CONCLUSION: This is the first study in which heteroplasmy has been characterized as occurring in either the reference or alternate allele. Future work in our lab will explore potential relationships between endurance trainability and heteroplasmy type. ACKNOWLEDGEMENTS:

Identifying and understanding genetic factors that influence the propagation of the human respiratory syncytial virus (RSV) can lead to health benefits and possibly augment recent vaccine approaches. We previously identified a p53/immune axis in which the tumor suppressor p53 directly regulates the expression of immune system genes, including the seven members of the APOBEC3 family of DNA cytidine deaminases (A3), which are innate immune sentinels against viral infections. Here, we examined the potential p53 and A3 influence in RSV infection, as well as the overall p53-dependent cellular and p53/immune axis responses to infection. Using a paired p53 model system of p53+ and p53- human lung tumor cells, we found that RSV infection activates p53, leading to the altered p53-dependent expression of A3D, A3F, and A3G, along with p53 site-specific binding. Focusing on A3G because of its 10-fold-greater p53 responsiveness to RSV, the overexpression of A3G can reduce RSV viral replication and syncytial formation. We also observed that RSV-infected cells undergo p53-dependent apoptosis. The study was expanded to globally address at the transcriptional level the p53/immune axis response to RSV. Nearly 100 genes can be directly targeted by the p53/immune axis during RSV infection based on our p53BAER analysis (Binding And Expression Resource). Overall, we identify A3G as a potential p53-responsive restriction factor in RSV infection. These findings have significant implications for RSV clinical and therapeutic studies and other p53-influenced viral infections, including using p53 adjuvants to boost the response of A3 genes.

Background Bronchopulmonary dysplasia (BPD) is a lung disease in premature infants caused by therapeutic oxygen supplemental and characterized by impaired pulmonary development which persists into later life. While advances in neonatal care have improved survival rates of premature infants, cases of BPD have been increasing with limited therapeutic options for prevention and treatment. This study was designed to explore the relationship between gestational age (GA), birth weight, and estimated blood cell-type composition in premature infants and to elucidate early epigenetic biomarkers associated with BPD. Methods Cord blood DNA from preterm neonates that went on to develop BPD ( n = 14) or not (non-BPD, n = 93) was applied to Illumina 450 K methylation arrays. Blood cell-type compositions were estimated using DNA methylation profiles. Multivariable robust regression analysis elucidated CpGs associated with BPD risk. cDNA microarray analysis of cord blood RNA identified differentially expressed genes in neonates who later developed BPD. Results The development of BPD and the need for oxygen supplementation were strongly associated with GA (BPD, p < 1.0E−04; O 2 supplementation, p < 1.0E−09) and birth weight (BPD, p < 1.0E−02; O 2 supplementation, p < 1.0E−07). The estimated nucleated red blood cell (NRBC) percent was negatively associated with birth weight and GA, positively associated with hypomethylation of the tobacco smoke exposure biomarker cg05575921, and high-NRBC blood samples displayed a hypomethylation profile. Epigenome-wide association study (EWAS) identified 38 (Bonferroni) and 275 (false discovery rate 1%) differentially methylated CpGs associated with BPD. BPD-associated CpGs in cord blood were enriched for lung maturation and hematopoiesis pathways. Stochastic epigenetic mutation burden at birth was significantly elevated among those who developed BPD (adjusted p = 0.02). Transcriptome changes in cord blood cells reflected cell cycle, development, and pulmonary disorder events in BPD. Conclusions While results must be interpreted with caution because of the small size of this study, NRBC content strongly impacted DNA methylation profiles in preterm cord blood and EWAS analysis revealed potential insights into biological pathways involved in BPD pathogenesis. Graphical Abstract

Approximately 1 in 10 newborns are born preterm and require supplemental oxygen (O2) in an extrauterine environment following birth. Supplemental O2 can induce oxidative stress that can impair mitochondrial function, resulting in lung injury and increased risk in early life pulmonary diseases. The nuclear factor-erythroid 2 related factor 2 (NRF2) protects the cells from oxidative stress by regulating the expression of genes containing antioxidant response elements and many mitochondrial-associated genes. In this study, we compared Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice to define the role of NRF2 in lung mitochondrial genomic features in late embryonic development in mice (embryonic days, E13.5 and E18.5) versus birth (postnatal day 0, PND0). We also determined whether NRF2 protects lung mitochondrial genome parameters in postnatal mice exposed to a 72 h hyperoxia environment. We found Nrf2−/− embryonic lungs were characterized by decreases in mtDNA copies from E13.5 to E18.5. Interestingly, Nrf2−/− heteroplasmy frequency was significantly higher than Nrf2+/+ at E18.5, though this effect reversed at PND0. In postnatal mice exposed to hyperoxia, we identified three- to four-fold increases in mitochondria-encoded mitochondrial genes, which regulate oxidative phosphorylation. Overall, our findings demonstrate a potentially critical role of NRF2 in mediating long-term effects of hyperoxia on mitochondrial function.

NRF2 protects against oxidant-associated airway disorders via cytoprotective gene induction. To examine if NRF2 is an important determinant of respiratory syncytial virus (RSV) susceptibility after neonate lung injury, Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice neonatally exposed to hyperoxia were infected with RSV. To investigate the prenatal antioxidant effect on neonatal oxidative lung injury, time-pregnant Nrf2−/−and Nrf2+/+mice were given an oral NRF2 agonist (sulforaphane) on embryonic days 11.5–17.5, and offspring were exposed to hyperoxia. Bronchoalveolar lavage and histopathologic analyses determined lung injury. cDNA microarray analyses were performed on placenta and neonatal lungs. RSV-induced pulmonary inflammation, injury, oxidation, and virus load were heightened in hyperoxia-exposed mice, and injury was more severe in hyperoxia-susceptible Nrf2−/− mice than in Nrf2+/+ mice. Maternal sulforaphane significantly alleviated hyperoxic lung injury in both neonate genotypes with more marked attenuation of severe neutrophilia, edema, oxidation, and alveolarization arrest in Nrf2−/− mice. Prenatal sulforaphane altered different genes with similar defensive functions (e.g., inhibition of cell/perinatal death and inflammation, potentiation of angiogenesis/organ development) in both strains, indicating compensatory transcriptome changes in Nrf2−/− mice. Conclusively, oxidative injury in underdeveloped lungs NRF2-dependently predisposed RSV susceptibility. In utero sulforaphane intervention suggested NRF2-dependent and -independent pulmonary protection mechanisms against early-life oxidant injury.

The mitochondrial genome is a small, circular, and highly conserved piece of DNA which encodes only 13 protein subunits yet is vital for electron transport in the mitochondrion and, therefore, vital for the existence of multicellular life on Earth. Despite this importance, mitochondrial DNA (mtDNA) is located in one of the least-protected areas of the cell, exposing it to high concentrations of intracellular reactive oxygen species (ROS) and threat from exogenous substances and pathogens. Until recently, the quality control mechanisms which ensured the stability of the nuclear genome were thought to be minimal or nonexistent in the mitochondria, and the thousands of redundant copies of mtDNA in each cell were believed to be the primary mechanism of protecting these genes. However, a vast network of mechanisms has been discovered that repair mtDNA lesions, replace and recycle mitochondrial chromosomes, and conduct alternate RNA processing for previously undescribed mitochondrial proteins. New mtDNA/RNA-dependent signaling pathways reveal a mostly undiscovered biochemical landscape in which the mitochondria interface with their host cells/organisms. As the myriad ways in which the function of the mitochondrial genome can affect human health have become increasingly apparent, the use of mitogenomic biomarkers (such as copy number and heteroplasmy) as toxicological endpoints has become more widely accepted. In this article, we examine several pathologies of human airway epithelium, including particle exposures, inflammatory diseases, and hyperoxia, and discuss the role of mitochondrial genotoxicity in the pathogenesis and/or exacerbation of these conditions. © 2021 American Physiological Society. Compr Physiol 11:1485-1499, 2021.

Ozone (O3) is the predominant oxidant air pollutant associated with airway inflammation, lung dysfunction, and the worsening of preexisting respiratory diseases. We previously demonstrated the injurious roles of pulmonary immune receptors, tumor necrosis factor receptor (TNFR), and toll-like receptor 4, as well as a transcription factor NF-κB, in response to O3 in mice. In the current study, we profiled time-dependent and TNFR- and NF-κB-regulated lung transcriptome changes by subacute O3 to illuminate the underlying molecular events and downstream targets. Mice lacking Tnfr1/Tnfr2 (Tnfr-/-) or Nfkb1 (Nfkb1-/-) were exposed to air or O3. Lung RNAs were prepared for cDNA microarray analyses, and downstream and upstream mechanisms were predicted by pathway analyses of the enriched genes. O3 significantly altered the genes involved in inflammation and redox (24 h), cholesterol biosynthesis and vaso-occlusion (48 h), and cell cycle and DNA repair (48–72 h). Transforming growth factor-β1 was a predicted upstream regulator. Lack of Tnfr suppressed the immune cell proliferation and lipid-related processes and heightened epithelial cell integrity, and Nfkb1 deficiency markedly suppressed lung cell cycle progress during O3 exposure. Common differentially regulated genes by TNFR and NF-κB1 (e.g., Casp8, Il6, and Edn1) were predicted to protect the lungs from cell death, connective tissue injury, and inflammation. Il6-deficient mice were susceptible to O3-induced protein hyperpermeability, indicating its defensive role, while Tnf-deficient mice were resistant to overall lung injury caused by O3. The results elucidated transcriptome dynamics and provided new insights into the molecular mechanisms regulated by TNFR and NF-κB1 in pulmonary subacute O3 pathogenesis.

Mucin-5AC (MUC5AC) is a major secreted mucin in pathogenic airways. To determine its role in mucus-related airway disorders, Muc5ac-deficient (Muc5ac-/-) and wild-type (Muc5ac+/+) mice were compared in bleomycin-induced pulmonary fibrosis, respiratory syncytial virus (RSV) disease, and ozone toxicity. Significantly greater inflammation and fibrosis by bleomycin were developed in Muc5ac-/- lungs compared to Muc5ac+/+ lungs. More severe mucous cell metaplasia in fibrotic Muc5ac-/- lungs coincided with bronchial Muc2, Muc4, and Muc5b overexpression. Airway RSV replication was higher in Muc5ac-/- than in Muc5ac+/+ during early infection. RSV-caused pulmonary epithelial death, bronchial smooth muscle thickening, and syncytia formation were more severe in Muc5ac-/- compared to Muc5ac+/+. Nasal septal damage and subepithelial mucoserous gland enrichment by RSV were greater in Muc5ac-/- than in Muc5ac+/+. Ozone exposure developed more severe nasal airway injury accompanying submucosal gland hyperplasia and pulmonary proliferation in Muc5ac-/- than in Muc5ac+/+. Ozone caused periodic acid-Schiff-positive secretion only in Muc5ac-/- nasal airways. Lung E-cadherin level was relatively lower in Muc5ac-/- than in Muc5ac+/+ basally and after bleomycin, RSV, and ozone exposure. Results indicate that MUC5AC is an essential mucosal component in acute phase airway injury protection. Subepithelial gland hyperplasia and adaptive increase of other epithelial mucins may compensate airway defense in Muc5ac-/- mice.

PURPOSE: Recently published work by our group identified mitochondrial DNA (mtDNA) variants and heteroplasmy that differ between individuals categorized by high and low aerobic capacity (VO2max) trainability, part of the HERITAGE Family Study Cohort. Our purpose in this investigation was to assess insertions and deletions (indels) in mitochondrial genomes in high (HR) and low responders (LR). METHODS: DNA was isolated from whole blood in HERITAGE Family Study healthy subjects (subjects classified by change in VO2max: HR: n = 15; LR: n = 15). mtDNA was amplified by long-range polymerase chain reaction, then tagged with Nextera libraries, and sequenced on a Miseq instrument. An indel was defined as an insertion or deletion with a frequency of at least 1% at a specific location. For this study, we compared indel sites and the frequency they occurred between HR and LRs. RESULTS: Among subjects, we identified 11 total sites where an indel occurred in at least one individual: mtHVR2 (C285CA: LR = 1 individual; HR = 0 individuals), mtHVR2 (A302AC: LR = 1 individual; HR = 0 individuals), mtHVR3 (G513GCA: LR = 1 individual; HR = 1 individual), mtHVR3 (GCA513G: LR = 3 individuals; HR = 1 individual), mtHVR3 (A567AC: LR = 0 individuals; HR = 1 individual), mtND4 (GA11031G: LR = 0 individuals; HR = 1 individual), mtND5 (CA12417C: LR = 11 individuals; HR = 8 individuals), mtHVR1(CA16179C: LR = 0 individuals; HR = 2 individuals), mtHVR1 (A16182AC: LR = 0 individuals; HR = 2 individuals), mtHVR1 (CT16188C: LR = 0 individuals; HR = 3 individuals), mtHVR1(C16192CT: LR = 0 individual; HR = 1 individual). The average total frequency across the indels in LR was 41.32 % (± 43.55 %) and 31.59 % (± 33.29 %) in HRs. We identified three sites with indels that were present in HR but not LR: mtHVR1 (CA16179C; 21.5% ± 0.71% mean indel frequency), mtHVR1 (A16182AC; 13.4% ± 0.57% mean indel frequency), and mtHVR1 (CT16188C; 17.4% ± 21.45% mean indel frequency). CONCLUSIONS: We found three sites with indels in the HVR1 region of the mitochondrial genome in HR but not LR. While our sample size is small, our findings highlight an area of the mitochondrial genome that is responsible for mtDNA replication that presents indels in individuals classified by low trainability. Our on-going work is to increase the sample size and assess indel implications on VO2max trainability.

Background Bronchopulmonary dysplasia (BPD) affects 20-40% of very low birth weight infants (VLBW) who endure short and long-term limitations throughout their life. Experts have classified the disease based on oxygen and pressure exposure at different time points, on the continuum of disease progression, ignoring the rich respiratory and clinical history of infants up to this point, characterized by temporal fluctuations of oxygen requirements. As a consequence, most prediction models for BPD have shown to be of limited clinical utility missing strategic early opportunities to improve outcomes. Objective Our objective is to study premature lung disease endotypes by a data-driven, hypothesis-generating approach with the use of machine learning (ML) algorithms that can reliably correlate with comorbidities and outcomes. Design/Methods Clusters (endotypes) are constructed employing longitudinal data without any a priori classification such as the canonical labels “severe” or “mild” BPD. We used the fraction of inspired oxygen(FiO2) variable for the construction of trajectories. Then, these were clustered based on the anchored kmedoids algorithm. We leveraged two existing large longitudinal preterm cohorts. We explored our hypothesis using the Discovery-BPD Program (DBPD) and validated our results with a second, independent data set from the PROP cohort. Of all VLBW infants in the DBPD cohort, the ones more than 29 weeks of gestation wereexcluded in the comparison analysis (DBPD-P) to match the inclusion criteria of the PROP Cohort. Results The DBPD and PROP cohorts have comparable demographic characteristics. Unsupervised classification employing ML algorithms yields 3 distinct clusters. The individual trajectories are colored according to cluster membership as assigned by the unsupervised algorithm and then grouped by cluster (Fig 1). Comorbidities such as intra ventricular hemorrhage, sepsis and days of mechanical ventilation segregate better when analyzed by the ML clusters than with standard BPD categories. Additionally, first year of life re-hospitalizations and death (Fig. 2) align better with ML clusters, thus further supporting cluster assignment as a superior categorization algorithm. Conclusion(s) Our preliminary results demonstrate an advantage of our ML model over the conventional BPD definition. We hope that this will enable a better understanding of the molecular basis underlying individual endotypes facilitating the development of better diagnostics and, eventually, personalized treatments for each endotype.

Background An unusual feature of SARS-Cov-2 infection and the COVID-19 pandemic is that children are less severely affected than adults. This is especially paradoxical given the epidemiological links between poor air quality and increased COVID-19 severity in adults and that children are generally more vulnerable than adults to the adverse consequences of air pollution. Objectives To identify gaps in knowledge about the factors that protect children from severe SARS-Cov-2 infection even in the face of air pollution, and to develop a transdisciplinary research strategy to address these gaps. Methods An international group of researchers interested in children’s environmental health was invited to identify knowledge gaps and to develop research questions to close these gaps. Discussion Key research questions identified include: what are the effects of SAR-Cov-2 infection during pregnancy on the developing fetus and child; what is the impact of age at infection and genetic susceptibility on disease severity; why do some children with COVID-19 infection develop toxic shock and Kawasaki-like symptoms; what are the impacts of toxic environmental exposures including poor air quality, chemical and metal exposures on innate immunity, especially in the respiratory epithelium; what is the possible role of a “dirty” environment in conveying protection – an example of the “hygiene hypothesis”; and what are the long term health effects of SARS-Cov-2 infection in early life. Conclusion A concerted research effort by a multidisciplinary team of scientists is needed to understand the links between environmental exposures, especially air pollution and COVID-19. We call for specific research funding to encourage basic and clinical research to understand if/why exposure to environmental factors is associated with more severe disease, why children appear to be protected, and how innate immune responses may be involved. Lessons learned about SARS-Cov-2 infection in our children will help us to understand and reduce disease severity in adults, the opposite of the usual scenario.

In 1967, two toddlers immunized with a formalin-inactivated vaccine against respiratory syncytial virus (FIRSV) in the United States died from enhanced RSV disease (ERD), a severe form of illness resulting from aberrant priming of the antiviral immune response during vaccination. Up to 80% of immunized children subsequently exposed to wild-type virus were hospitalized. These events hampered RSV vaccine development for decades. Here, we provide a characterization of the clinical, immunopathological, and transcriptional signature of fatal human ERD, outlining evidence for safety evaluation of RSV vaccines and a framework for understanding disease enhancement for pathogens in general.

Although it is well accepted that air pollution exposure exacerbates preexisting airway disease, it has not been firmly established that long-term pollution exposure increases the risk of new-onset asthma or chronic obstruction pulmonary disease (COPD). This Workshop brought together experts on mechanistic, epidemiological, and clinical aspects of airway disease to review current knowledge regarding whether air pollution is a causal factor in the development of asthma and/or COPD. Speakers presented recent evidence in their respective areas of expertise related to air pollution and new airway disease incidence, followed by interactive discussions. A writing committee summarized their collective findings. The Epidemiology Group found that long-term exposure to air pollution, especially metrics of traffic-related air pollution such as nitrogen dioxide and black carbon, is associated with onset of childhood asthma. However, the evidence for a causal role in adult-onset asthma or COPD remains insufficient. The Mechanistic Group concluded that air pollution exposure can cause airway remodeling, which can lead to asthma or COPD, as well as asthma-like phenotypes that worsen with long-term exposure to air pollution, especially fine particulate matter and ozone. The Clinical Group concluded that air pollution is a plausible contributor to the onset of both asthma and COPD. Available evidence indicates that long-term exposure to air pollution is a cause of childhood asthma, but the evidence for a similar determination for adult asthma or COPD remains insufficient. Further research is needed to elucidate the exact biological mechanism underlying incident childhood asthma, and the specific air pollutant that causes it.

Cellular antioxidants protect against hyperoxic lung injury. The role of the glutathione (GSH) system in lung development and bronchopulmonary dysplasia (BPD) pathogenesis has not been systematically investigated. The current study utilized GSH reductase-deficient (Gsr-KO) neonatal mice to test the hypothesis that early disruption of the GSH system negatively impacts lung development and hyperoxic responses. Lungs from wild-type (Gsr-WT) and Gsr-KO mice were analyzed for histopathology, developmental markers, redox indices, and transcriptome profiling at different developmental stages following exposure to room air or hyperoxia (85% O2) for up to 14 d. Lungs from Gsr-KO mice exhibited alveolar epithelial dysplasia in the embryonic and neonatal periods with relatively normal lung architecture in adulthood. GSH and its oxidized form (GSSG) were 50–70% lower at E19-PND14 in Gsr-KO lungs than in age-matched Gsr-WT. Differential gene expression between Gsr-WT and Gsr-KO lungs was analyzed at discrete developmental stages. Gsr-KO lungs exhibited downregulated cell cycle and DNA damage checkpoint genes at E19, as well as lung lipid metabolism and surfactant genes at PND5. In addition to abnormal baseline lung morphometry, Gsr-KO mice displayed a blunted response to hyperoxia. Hyperoxia caused a more robust upregulation of the lung thioredoxin system in Gsr-KO compared to Gsr-WT. Gsr-dependent, hyperoxia-responsive genes were highly associated with abnormal cytoskeleton, skeletal-muscular function, and tissue morphology at PND5. Overall, our data in Gsr-KO mice implicate the GSH system as a key regulator of lung development, cellular differentiation, and hyperoxic responses in neonatal mice.

In this pilot work, we selected two inbred strains that respond well to endurance training (ET) (FVB/NJ, and SJL/J strains), and two strains that respond poorly (BALB/cByJ and NZW/LacJ), to determine the effect of a standardized ET treadmill program on mitochondrial and nuclear DNA (nucDNA) integrity, and mitochondrial DNA (mtDNA) copy number. DNA was isolated from plantaris muscles (n = 37) and a gene‐specific quantitative PCR‐based assay was used to measure DNA lesions and mtDNA copy number. Mean mtDNA lesions were not different within strains in the sedentary or exercise‐trained states. However, mtDNA lesions were significantly higher in trained low‐responding NZW/LacJ mice (0.24 ± 0.06 mtDNA lesions/10 Kb) compared to high‐responding strains (mtDNA lesions/10 Kb: FVB/NJ = 0.11 ± 0.01, p = .049; SJL/J = 0.04 ± 0.02; p = .003). ET did not alter mean mtDNA copy numbers for any strain, although both sedentary and trained FVB/NJ mice had significantly higher mtDNA copies (99,890 ± 4,884 mtDNA copies) compared to low‐responding strains (mtDNA copies: BALB/cByJ = 69,744 ± 4,675; NZW/LacJ = 65,687 ± 5,180; p < .001). ET did not change nucDNA lesions for any strain, however, SJL/J had the lowest mean nucDNA lesions (3.5 ± 0.14 nucDNA lesions/6.5 Kb) compared to all other strains (nucDNA lesions/6.5 Kb: FVB/NJ = 4.4 ± 0.11; BALB/cByJ = 4.7 ± 0.09; NZW/LacJ = 4.4 ± 0.11; p < .0001). Our results demonstrate strain differences in plantaris muscle mtDNA lesions in ET mice and, independent of condition, differences in mean mtDNA copy and nucDNA lesions between strains.