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Glutathione reductase deficiency alters lung development and hyperoxic responses in neonatal mice.

ABSTRACT: 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.

SUBMITTER: Robbins ME

PROVIDER: S-EPMC7708869 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Glutathione reductase deficiency alters lung development and hyperoxic responses in neonatal mice.

Robbins Mary E ME Cho Hye-Youn HY Hansen Jason M JM Luchsinger Joseph R JR Locy Morgan L ML Velten Markus M Kleeberger Steven R SR Rogers Lynette K LK Tipple Trent E TE

Redox biology 20201113

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, developm ...[more]

PMID: 33254076

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Project description:BACKGROUND:Exposure to high levels of oxygen (hyperoxia) after birth leads to lung injury. Our aims were to investigate the modulation of myeloid cell sub-populations and the reduction of fibrosis in the lungs following administration of human mesenchymal stem cells (hMSC) to neonatal mice exposed to hyperoxia. METHOD:Newborn mice were exposed to 90% O2 (hyperoxia) or 21% O2 (normoxia) from postnatal days 0-4. A sub-group of hyperoxia mice were injected intratracheally with 2.5X105 hMSCs. Using flow cytometry we assessed pulmonary immune cells at postnatal days 0, 4, 7 and 14. The following markers were chosen to identify these cells: CD45+ (leukocytes), Ly6C+Ly6G+ (granulocytes), CD11b+CD11c+ (macrophages); macrophage polarisation was assessed by F4/80 and CD206 expression. hMSCs expressing enhanced green fluorescent protein (eGFP) and firefly luciferase (fluc) were administered via the trachea at day 4. Lung macrophages in all groups were profiled using next generation sequencing (NGS) to assess alterations in macrophage phenotype. Pulmonary collagen deposition and morphometry were assessed at days 14 and 56 respectively. RESULTS:At day 4, hyperoxia increased the number of pulmonary Ly6C+Ly6G+ granulocytes and F4/80lowCD206low macrophages but decreased F4/80highCD206high macrophages. At days 7 and 14, hyperoxia increased numbers of CD45+ leukocytes, CD11b+CD11c+ alveolar macrophages and F4/80lowCD206low macrophages but decreased F4/80highCD206high macrophages. hMSCs administration ameliorated these effects of hyperoxia, notably reducing numbers of CD11b+CD11c+ and F4/80lowCD206low macrophages; in contrast, F4/80highCD206high macrophages were increased. Genes characteristic of anti-inflammatory 'M2' macrophages (Arg1, Stat6, Retnla, Mrc1, Il27ra, Chil3, and Il12b) were up-regulated, and pro-inflammatory 'M1' macrophages (Cd86, Stat1, Socs3, Slamf1, Tnf, Fcgr1, Il12b, Il6, Il1b, and Il27ra) were downregulated in isolated lung macrophages from hyperoxia-exposed mice administered hMSCs, compared to mice without hMSCs. Hydroxyproline assay at day 14 showed that the 2-fold increase in lung collagen following hyperoxia was reduced to control levels in mice administered hMSCs. By day 56 (early adulthood), hMSC administration had attenuated structural changes in hyperoxia-exposed lungs. CONCLUSIONS:Our findings suggest that hMSCs reduce neonatal lung injury caused by hyperoxia by modulation of macrophage phenotype. Not only did our cell-based therapy using hMSC induce structural repair, it limited the progression of pulmonary fibrosis.

Project description:Bronchopulmonary dysplasia (BPD) is characterized by a severe impairment in lung alveolarization and vascular development. We have previously shown that pulmonary angiogenesis is preserved in hyperoxia-exposed female mice accompanied by increased miR-30a expression, which is a proangiogenic miRNA. Also, miR-30a expression is decreased in human BPD. HIF-1α plays an essential role in postnatal lung development, especially in recovery from hyperoxic injury. Snai1 activation promotes pathological fibrosis through many mechanisms including Endo-MT, which may in turn adversely impact lung vascular development. Our objective was to test the hypothesis that higher miR-30a expression through HIF-1α decreases Snai1 expression in females and attenuates injury in the developing lung. Neonatal male and female mice (C57BL/6) were exposed to hyperoxia (P1-5, 0.95 FiO2) and euthanized on P21. Neonatal human pulmonary microvascular endothelial cells (HPMECs; 18-24-week gestation donors; 3/group either sex) were subjected to hyperoxia (95% O2 and 5% CO2) or normoxia (air and 5% CO2) up to 72 h. Snai1 expression was measured in HPMECs in vitro and in neonatal mouse lungs in vivo. Also, Snai1 expression was measured in HPMECs after miR-30a mimic and miR-30a inhibitor treatment. To further establish the potential regulation of miR-30a by Hif-1α, miR-30a expression after Hif-1α inhibition was measured in HPMECs. In vivo, Snai1 expression was decreased in neonatal female lungs compared to males at P7. Increased Snai1 expression was seen in male HPMECs upon exposure to hyperoxia in vitro. Treatment with the miR-30a mimic decreased Snai1 expression in HPMECs, while miR-30a inhibition significantly increased Snai1 expression in HPMECs. siRNA-mediated loss of Hif-1α expression in HPMECs decreased miR-30a expression. Hif-1α may lead to differential sex-specific miR-30a expression and may contribute to protection from hyperoxic lung injury in female neonatal mice through decreased Snai1 expression.

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Glutathione reductase deficiency alters lung development and hyperoxic responses in neonatal mice.

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Glutathione reductase deficiency alters lung development and hyperoxic responses in neonatal mice.

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