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BPD is a chronic lung disease developed after oxygen inhalation therapy or mechanical ventilation usually occurring in certain premature infants or newborn infants with respiratory distress syndrome. Histologically, it is characterized by the unusual abnormalities of the bronchioles, such as metaplasia, decrease in alveolar number, and formation of cysts. The pathophysiology of this condition is complex and may involve a genetic predisposition, perinatal inflammation, or environmental toxin exposure. Frequently, the use of respiratory support, including supplemental oxygen and mechanical ventilation, is required in neonates with BPD (1,2). The animal model of BPD involves exposing newborn mice to hyperoxia. In this model, researchers can specifically target the saccular phase, one of the immature stages of lung development, by exposing the newborn mice to hyperoxia because newborn mouse lungs are equivalent to the lung developmental stage of a human infant born at 26–28 wk of gestation (3). Hyperoxia is one of the known risk factors for the development of BPD. In this mouse model, hyperoxic conditions can be easily reproduced and oxygen concentrations can be manipulated to study dose dependence. Therefore, continuous exposure of newborn rodents to high concentrations of oxygen has often been used to research the effects of hyperoxia on developing immature lungs (4). Hyperoxia causes significant changes to the morphology of lung tissue in newborn mice, including decreased septation of the alveoli, enlarged and simplified terminal air spaces, and a greater degree of pulmonary fibrosis (5,6,7,8).

Hyperoxia-induced lung injury is initiated by reactive oxygen species, and this is followed by the secretion of proinflammatory chemokines and cytokines by resident macrophages and epithelial cells. Neutrophils then migrate into the airspace (9), and macrophages (10) reduce inflammatory responses through the ingestion of apoptotic neutrophils and other dying cells or dead cell debris, thereby mitigating tissue damage (11,12,13). The delayed removal of dying cells may directly affect the natural ability of the injured organism to shut down inflammation and initiate tissue repair (14).

BTB and CNC homology 1 (Bach1) is a transcriptional repressor of the heme oxygenase (HO)-1 and β-globin genes (15,16). Heterodimers of the small Maf proteins and NF-E2-related factor 2 (Nrf2) activate HO-1 through binding to Maf-responsive elements. In contrast, heterodimers of the small Maf proteins and Bach1 repress Maf-responsive element-dependent transcription. The ability of Nrf2 to activate HO-1 expression is greatly reduced in the presence of Bach1 under normal conditions (16,17). However, under various stress conditions, such as inflammation, oxidative stress, or the presence of heme and cadmium, Bach1 detaches from the Maf-responsive elements , allowing Nrf2 and small Maf proteins to bind. This results in the transcriptional activation of target genes, such as HO-1 (18,19).

In Bach1-null (Bach1−/−) mice, the transcription of HO-1 is constitutively upregulated, thus leading to increased HO-1 protein levels and enzymatic activity under normal conditions in several organs, including the heart, lungs, and liver (16). Previous studies have shown that Bach1−/− mice are protected from conditions that are caused by oxidative stress, including atherosclerosis and myocardial ischemia/reperfusion injury (20,21). The underlining mechanism of the protection of Bach1−/− mice from oxidative stress can be explained, in part, by the upregulation of HO-1 activity (20); however, much remains to be elucidated. Bach1−/− adult mice have been shown to be well-protected against hyperoxic lung injury. This protection was demonstrated by studies showing that compared with WT mice, Bach1−/− mice survive significantly longer, have less tissue injury based on histological findings, less protein in their bronchoalveolar lavage fluid, and reduced levels of apoptosis (22). However, there are no previous reports investigating the effects of Bach1 deficiency on hyperoxia-induced lung injury in newborn mice.

Therefore, in this study, we tested the hypothesis that genetic ablation of the Bach1 gene ameliorates hyperoxia-induced lung injury in newborn mice via upregulation of its target genes.

Methods

Animals

All procedures and protocols were approved by the Animal Care and Use Committee of Saitama Medical University (Permit no. 1502). Bach1−/− mice, originally created by Sun et al. (16) and repeatedly backcrossed with C57BL/6 mice for at least 12 generations, were maintained in the animal facility at the Saitama Medical Center, Saitama Medical University. Heterozygous Bach1 knockout male and female mice were mated to produce Bach1−/− and WT littermates; genotyping was performed by PCR analysis of tail biopsies.

Neonatal Hyperoxic Exposure and Recovery

Neonatal pups were randomly assigned to normoxia (room air) or hyperoxia (95% O2). Exposure to hyperoxia was conducted for 96 h in a chamber (BioSpherix, Redfield, NY) that allowed for the continuous monitoring and regulation of oxygen and carbon dioxide. Dams were switched every 24 h between normoxia and hyperoxia. The inside of the chamber was kept at atmospheric pressure, and mice were exposed to a 12 h light–dark cycle.

Lung Tissue Collection

Mice were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). After the pulmonary artery was perfused with phosphate buffered saline, the right lung was excised and snap-frozen with liquid nitrogen for RNA and protein analysis. The left lung was inflated through the trachea with 10% neutral-buffered formalin (Sigma-Aldrich, St. Louis, MO) at 25 cm gravity pressure and allowed to fix for 1 min. The trachea was tied; the lung was then removed and fixed further overnight at 4°C. Lung tissue was paraffin-embedded and 5-mm thick sections were mounted onto glass slides.

Lung Histology and Morphometry

To assess distal airspace maturation, computer-aided morphometric analysis was performed using ImageJ software version 1.49 (NIH, Bethesda, MD) to measure the Lm and the number of secondary septa. Paraffin-embedded lung tissue sections were stained with hematoxylin and eosin. Lm, which is defined as the mean length of line segments on random test lines spanning the airspace between intersections of the line with the alveolar surface, was determined using light microscopy (23). Lm was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal, and five animals per condition at each time point were examined. Lm was obtained by dividing the total length of a line drawn across the lung section by the total number of intercepts, until the number of intercepts reached 50 per each field. Elastin staining was performed using an elastic stain kit (Abcam, Cambridge, MA) according to the manufacturer’s instructions. The number of secondary septa, where elastin was detected, was manually counted in six nonoverlapping fields of lung parenchyma in one tissue section per animal, and five animals per condition at each time point were examined. Lung tissue was also analyzed for terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) using the in situ Apoptosis Detection Kit (Takara Bio, Shiga Japan) according to the manufacturer’s protocol. The number of TUNEL-positive cells was manually counted in six nonoverlapping fields of lung parenchyma per animal, and five animals per condition at each time point were examined. Fields containing large vessels, conducting airways or sectioning artifacts were avoided for lung morphometry.

RNA Extraction and quantitative RT-PCR Analysis

Total RNA was extracted from five lung tissue samples per group as previously described (24). RNA (500 ng) was reverse-transcribed (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA), and cDNA was used in each PCR reaction with primers for HO-1, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 (Applied Biosystems) and TaqMan Universal PCR Master Mix (Applied Biosystems). Analyses were performed using an Applied Biosystems 7,500 Fast Real-Time PCR System. The relative mRNA expression levels were determined using the comparative critical threshold method, and each sample was normalized to beta-glucuronidase (Applied Biosystems).

Cytokine Measurements in Lung Tissue

Mouse IL-12, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, MCP-1, IL-10, and IL-6 levels were determined in lung tissue homogenates using the BD Cytometric Bead Array Mouse Inflammation Kit (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. The samples were analyzed on a BD FACSVerse flow cytometer (BD Biosciences).

Statistical Analysis

All values were expressed as the mean ± SEM or as box-and-whisker plots (the median, interquartile range, and range). Comparison between different groups was performed with SPSS software version 22.0 (SPSS, Chicago, IL) by one-way ANOVA followed by Tukey post-hoc test for analyzing parametric data or Kruskal–Wallis test followed by Mann–Whitney U-test for analyzing nonparametric data. P-values < 0.05 were considered to be significant.

Results

Alveolar Development is Impaired after Neonatal Hyperoxia in both WT and Bach1−/− Mice at 4 d of Age

In normal room air, mice develop well-organized terminal airways. In contrast, exposure of newborn mice to hyperoxia for 96 h impaired alveolar development, resulting in alveolar expansion and simplification ( Figure 1a ), and inhibited secondary septation ( Figure 1b ). The mean linear intercept (Lm) in both WT and Bach1−/− mouse lungs exposed to neonatal hyperoxia was significantly longer than that in normoxic control lungs at 4 d of age ( Figure 1c ). The number of secondary septa in the lungs was also significantly decreased after neonatal hyperoxia in both WT and Bach1−/− mice ( Figure 1d ). No significant differences in the Lm or the number of secondary septa were observed between 4-d-old WT and Bach1−/− mice exposed to either normoxia or hyperoxia.

Figure 1
figure 1

Alveolar development after neonatal hyperoxic exposure at 4 d of age. Exposure of newborn mice to hyperoxia for 96 h impaired alveolar development, resulting in alveolar expansion and simplification a) and inhibited secondary septation b. (a) hematoxylin and eosin (H&E) stained histological sections from mice on days 4, Scale bar = 100 µm (magnification: ×100). (b) Elastin stained histological sections from mice on days 4, Scale bar = 25 µm (magnification: ×400). Air: normoxia; O2: hyperoxia. (c) Lm on days 4 (n = 5 animals in each group). Lm was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O2 (filled bars). Lm in both WT and Bach1−/− mouse lungs exposed to neonatal hyperoxia was significantly longer than that in normoxic controls at 4 d of age. (d) Number of secondary septa on days 4 (n = 5 animals in each group). The number of secondary septa was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O2 (filled bars). The number of secondary septa in the lungs was significantly decreased after neonatal hyperoxia in both WT and Bach1−/− mice. Data are the mean ± SEM. Lm: mean linear intercept. Comparison between different groups was performed by one-way ANOVA followed by Tukey test. *P < 0.05 vs. Air.

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Impaired Alveolar Development in Bach1−/− Mice Recovers at 14 d of Age

When neonatal WT mice were exposed to hyperoxia for 4 d and then allowed to recover in normal room air for 10 d, they still had a significantly longer Lm and less secondary septa compared with their normoxic counterparts at 14 d of age. In contrast, despite impaired alveolarization after neonatal hyperoxia at 4 d of age, Bach1−/− mice had a significantly improved Lm and an increased number of secondary septa after room air recovery at 14 d of age compared with similarly exposed WT mice ( Figure 2 ).

Figure 2
figure 2

Alveolar development during recovery from neonatal hyperoxic exposure at 14 d of age. Impaired alveolar development in Bach1−/− mice recovers at 14 d of age. Bach1−/− mice had a significantly improved Lm and an increased number of secondary septa after recovery from hyperoxia compared with WT. (a) Hematoxylin and eosin (H&E) stained histological sections from mice on days 14, Scale bar = 100 µm (magnification: ×100). (b) Elastin stained histological sections from mice on days 14, Scale bar = 25 µm (magnification: ×400). Air: normoxia; O2: hyperoxia. (c) Lm on days 14 (n = 5 animals in each group). Lm was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O2 (filled bars). (d) Number of secondary septa on days 14 (n = 5 animals in each group). The number of secondary septa was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O2 (filled bars). Data are the mean ± SEM. Comparison between different groups was performed by one-way ANOVA followed by Tukey test. *P < 0.05 vs. Air, †P < 0.05 vs. WT O2.

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Hyperoxia-induced Apoptosis is Observed in the Lungs of both Bach1−/− and WT Mice at 4 d of Age, but not at 14 d of Age

To assess the antiapoptotic properties of the lungs of Bach1−/− mice, TUNEL assay was performed on lung sections of 4- and 14-d-old WT and Bach1−/− mice. The number of TUNEL-positive cells was significantly increased in the lungs of WT and Bach1−/− mice after hyperoxic exposure compared with their normoxic counterparts at 4 d of age ( Figure 3a , b ). However, no significant differences in the number of TUNEL-positive cells were observed between 4-d-old WT and Bach1−/− mice exposed to either normoxia or hyperoxia. At 14 d of age, there were no significant differences in the number of apoptotic cells in the lungs of WT and Bach1−/− mice exposed to neonatal hyperoxia compared with the normoxic control mice ( Figure 3a , c ).

Figure 3
figure 3

TUNEL staining of the lungs after neonatal hyperoxic exposure. Hyperoxia-induced apoptosis is observed in the lungs of both Bach1−/− and WT mice at 4 d of age, but not at 14 d of age. (a) TUNEL stained histological sections from animals, Scale bar = 50 µm (magnification: ×200). Air: normoxia; O2: hyperoxia. (b) Number of TUNEL positive cells on days 4 (n = 5 animals in each group). The number of TUNEL positive cells was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O2 (filled bars). (c) Number of TUNEL positive cells on days 14 (n = 5 animals in each group). The number of TUNEL positive cells was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O2 (filled bars). Data are the mean ± SEM. Comparison between different groups was performed by one-way ANOVA followed by Tukey test. *P < 0.05 vs. Air. TUNEL, terminal deoxynucleotidyl transferase nick-end labeling.

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Lung mRNA Expression Levels of HO-1 and IL-6 in Bach1−/− Mice Exposed to Neonatal Hyperoxia are Enhanced Compared with WT Mice

Because Bach1 is reported to be a transcriptional repressor of the HO-1 and IL-6 genes (22), lung mRNA expression levels of HO-1 and IL-6 were determined using q RT-PCR. Lung HO-1 and IL-6 mRNA expression levels were significantly increased in WT and Bach1−/− mice after a 4-d hyperoxic exposure, compared with normoxic controls. Bach1−/− mice exposed to neonatal hyperoxia also had significantly enhanced mRNA expression levels of HO-1 and IL-6 at 4 d of age compared with WT mice. After a 10-d recovery in room air (at 14 d of age), HO-1 and IL-6 mRNA expression levels were decreased to basal levels in WT and Bach1−/− mice ( Figure 4 ).

Figure 4
figure 4

Gene expression levels of HO-1 and IL-6 in the lungs after neonatal hyperoxic exposure. Lung mRNA expression levels of HO-1 (a, b) and IL-6 (c, d) in Bach1−/− mice exposed to neonatal hyperoxia are enhanced compared with WT mice. quantitative RT-PCR was performed on day 4 and on day 14 (n = 5 in each group). Animals were exposed to air (open bars) or O2 (filled bars). Data are the mean ± SEM. Comparison between different groups was performed by one-way ANOVA followed by Tukey test. *P < 0.05 vs. Air, **P < 0.01 vs. Air, †P < 0.05 vs. WT O2. HO-1, heme oxygenase; IL-6, interleukin-6.

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Effects of Bach1 Disruption on Inflammatory Cytokine Production in the Lungs after Exposure to Neonatal Hyperoxia

To evaluate the inflammatory response in the lungs during hyperoxic exposure, the inflammatory cytokine levels in 4-d-old WT and Bach1−/− mouse lungs exposed to either normoxia or hyperoxia for 4 d were measured using the Cytometric Bead Array Mouse Inflammation Kit. Furthermore, time course of changes in cytokine protein levels in the lungs after exposure to neonatal hyperoxia was examined. No significant differences were observed in the protein levels of TNF-α, IFN-γ, IL-10, or IL-12 in WT and Bach1−/− mouse lungs after either normoxic or hyperoxic exposure. However, both IL-6 and MCP-1 levels in the Bach1−/− mice lung were significantly higher than those of the WT mice lung following a 4-d hyperoxic exposure ( Figure 5 ). This increase in protein expression levels of IL-6 and MCP-1 was transient and these cytokine levels were decreased to the basal levels 24 h after exposure to hyperoxia (see Supplementary Figure S1 online).

Figure 5
figure 5

Protein levels of inflammatory cytokines in the lungs after neonatal hyperoxic exposure. Both IL-6 and MCP-1 levels in the Bach1−/− mice lung were significantly higher than those of the WT mice lung following a 4-d hyperoxic exposure. Cytometric bead array was performed on day 4 (n = 5 in each group). Animals were exposed to air (open bars) or O2 (filled bars). Median protein levels of IFN-ã (a), IL-12 (b), IL-6 (c), MCP-1 (d), IL-10 (e), and TNF-á (f) in the lung homogenates. Box = 25th and 75th percentiles; bars = min and max values. Comparison between different groups was performed by Kruskal–Wallis test followed by Mann–Whitney U-test.*P < 0.05 vs. Air. IFN, interferon; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; TNF, tumor necrosis factor.

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Lung mRNA Expression Levels of MCP-1 in Bach1−/− Mice Exposed to Neonatal Hyperoxia are Enhanced Compared with WT Mice

Because MCP-1 levels in the Bach1−/− mouse lungs were significantly higher than those in the lungs of WT mice, we also measured the mRNA expression levels of MCP-1 in the lungs using qRT-PCR. The MCP-1 mRNA expression level in the lungs of WT and Bach1−/− mice was significantly increased after 4 d of hyperoxic exposure compared with normoxic exposure. Furthermore, Bach1−/− mice exposed to neonatal hyperoxia had a significantly elevated mRNA expression level of MCP-1 at 4 d of age compared with WT mice. After 10 d of recovery in room air (at 14 d of age), the MCP-1 mRNA expression level was decreased to basal levels in WT and Bach1−/− mice ( Figure 6 ).

Figure 6
figure 6

Gene expression levels of MCP-1 in the lungs after neonatal hyperoxic exposure. Lung mRNA expression levels of MCP-1 in Bach1−/− mice exposed to neonatal hyperoxia are enhanced compared with WT mice. quantitative RT-PCR was performed on day4 (a) and on day 14 (b). Animals were exposed to air (open bars) or O2 (filled bars). Data are the mean ± SEM. Comparison between different groups was performed by one-way ANOVA followed by Tukey test. **P < 0.01 vs. Air, †P < 0.05 vs. WT O2. MCP-1, monocyte chemoattractant protein-1.

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Discussion

In this study, Bach1−/− mice were well-recovered from hyperoxic lung injury compared with WT mice. Bach1−/− mice showed an enhanced recovery from delayed alveolar development, caused by neonatal hyperoxia, back to normal developmental levels. Furthermore, mRNA and/or protein levels of HO-1, IL-6, and MCP-1 in lungs from Bach1−/− mice exposed to neonatal hyperoxia were significantly increased at 4 d of age compared with lungs from WT mice, suggesting that the recovery from hyperoxic lung injury observed in Bach1−/− mice may be due to the antioxidant/anti-inflammatory activity of HO-1 and/or transient proinflammatory cytokine overexpression that is strongly induced in Bach1−/− mice.

Mice with a targeted HO-1 mutation show partial penetrance of embryonic lethality, growth retardation and a deficiency in iron metabolism (25,26,27). HO-1 null mutant mice were found to have mild alveolar simplification, disorganization, and reduced secondary crest formation. Another study also demonstrated that HO-1-null mice showed exaggerated hyperoxia-induced hypoalveolarization (28). These experiments demonstrate that HO-1 is required for normal lung development. In contrast, HO-1 is well-known to be cytoprotective against redox stress and mitigate lung injury and alveolar simplification in hyperoxia-exposed neonatal mice. In a neonatal transgenic mouse model with constitutive lung-specific HO-1 overexpression, mice had attenuated pulmonary inflammation, arterial remodeling, right ventricular hypertrophy, pulmonary edema, hemosiderosis, and a decrease in the number of blood vessels (29). While some degree of HO activity is cytoprotective, HO-1 expression levels that are too high were associated with significant oxygen cytotoxicity (30). Another study demonstrated that mice expressing high levels of HO-1 had increased alveolar wall thickness with type II cell hyperproliferation and worsened pulmonary function after they recovered from hyperoxia (8). Overall, as regulators of cell proliferation and differentiation, moderate levels of HO-1 have a significant impact on both lung injury and lung repair processes in hyperoxia, suggesting that HO-1 expression levels in Bach1−/− mice, both during and after neonatal hyperoxia, might be optimal to recover from hyperoxia-induced lung injury.

In this study, higher levels of IL-6 were induced in the lungs of Bach1−/− mice during neonatal hyperoxic exposure compared with WT mice. Using transgenic adult mice that overexpress IL-6 in the lung, Ward et al. found that IL-6 markedly diminished hyperoxic lung injury and that this protection was associated with a marked decrease in hyperoxia-induced cell death and DNA fragmentation (31). Furthermore, Tanimoto et al. reported that the protective effects of IL-6 against hyperoxia in Bach1−/− mice may be mediated by the overexpression of IL-6 induced in the lungs after hyperoxic exposure (22). In contrast, in newborn mice, lung-targeted IL-6 overexpression was reported to cause significantly increased mortality, DNA injury, the induction of caspases, a cell death regulator, and the expression of angiogenic factors in hyperoxia (32).

In this study, despite IL-6 overexpression in the newborn lungs, Bach1−/− mice were well-recovered from lung injury induced by hyperoxic exposure. Two possibilities exist to explain this discrepancy. One is that HO-1 overexpression in the lungs of Bach1−/− mice is significantly more effective to recover from hyperoxic exposure compared with IL-6 overexpression. The other possibility is that transient overexpression of IL-6 is beneficial to recover from hyperoxia-induced lung injury, whereas permanent or prolonged overexpression is harmful.

MCP-1 has chemotactic, homing, and activating effects on leukocytes. It is typically used as a marker of leukocyte chemotaxis. Previous reports have identified the production of MCP-1, and subsequent leukocyte infiltration, as pivotal events in the course of newborn hyperoxic lung injury (33). In this study, compared with WT mice, Bach1−/− mice exposed to neonatal hyperoxia had significantly enhanced mRNA and protein levels of MCP-1 at 4 d of age and histologically better pulmonary outcomes during the recovery phase. Using C–C chemokine receptor 2 (CCR2) deficient mice, Okuma et al. found that MCP-1/CCR2 signaling is protective against hyperoxia-induced lung injury, likely by suppressing the induction of inducible nitric oxide synthase and the consequent production of reactive oxygen species by activated alveolar macrophages (34). In this study, we found that the overexpression of MCP-1 in addition to HO-1 and IL-6 overexpression in the lungs of Bach1−/− mice exposed to hyperoxia may play an important role in hyperoxic lung injury and the subsequent recovery process.

In conclusion, we found that Bach1−/− neonatal mice are well-recovered from hyperoxic lung injury during the recovery phase compared with WT mice. This recovery effect may be due to the antioxidant/anti-inflammatory activity of HO-1 and/or the transient overexpression of proinflammatory cytokines, such as IL-6 and MCP-1, which are strongly induced in Bach1−/− mice.

Statement of Financial Support

This work was supported by Saitama Medical Center Internal Research Grant for Young Physician Scientists (Grant no. 26F120) and Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (Grant no. 15K21334). The authors have no affiliations with or involvement in any organization or entity with any financial interest, or nonfinancial interest in the subject matter or materials discussed in this manuscript.