Neonatal hyperoxia increases sensitivity of adult mice to bleomycin-induced lung fibrosis

Abstract

Supplemental oxygen used to treat infants born prematurely constitutes a major risk factor for long-term deficits in lung function and host defense against respiratory infections. Likewise, neonatal oxygen exposure results in alveolar simplification in adult mice, and enhances leukocyte recruitment and fibrosis when adult mice are infected with a sublethal dose of influenza A virus. Because pulmonary fibrosis was not observed in infected adult mice exposed to room air as neonates, previous neonatal oxygen exposure may have reprogrammed how the adult lung responds to epithelial injury. By administering bleomycin to adult mice exposed to room air or hyperoxia as neonates, we tested the hypothesis that neonatal hyperoxia enhances fibrosis when the epithelium is injured by direct fibrotic stimulus. Increased sensitivity to bleomycin-induced lung fibrosis was observed in adult mice exposed to neonatal hyperoxia, and was associated with increased numbers of leukocytes and an accumulation of active transforming growth factor (TGF)-β1 in the lung. Fate mapping of the respiratory epithelium revealed that the epithelial-mesenchymal transition was not a significant source of fibroblasts in room air-exposed or oxygen-exposed mice treated with bleomycin. Instead, the treatment of mice with anti-Gr-1 antibody that depletes neutrophils and myeloid-derived suppressor cells reduced the early activation of TGF-β1 and attenuated hyperoxia-enhanced fibrosis. Because bleomycin and influenza A virus both cause epithelial injury, understanding how neonatal hyperoxia reprograms the epithelial response to these two different injurious agents could lead to new therapeutic opportunities for treating lung diseases attributed to prematurity.

Figure 1.

Neonatal hyperoxia increases bleomycin-induced morbidity and mortality. Bleomycin was administered to adult mice exposed to room air (RA) or hyperoxia (O₂) as neonates (n = 10 mice per group). (A) Mice were weighed before treatment with bleomycin (Day 0) and every other day after treatment for the next 28 days. (B) The mean percent body weight lost in mice exposed to hyperoxia was significantly greater on Days 26 and 28 (*P < 0.02), compared with siblings exposed to room air. (B) Survival of adult mice exposed to room air or hyperoxia as neonates, after treatment with bleomycin (P < 0.10).

Figure 2.

Neonatal hyperoxia increases bleomycin-induced pulmonary fibrosis. Bleomycin was administered to adult mice that had been exposed to room air (RA) or hyperoxia (O₂) as neonates (n = 3–5 mice per group per time point). (A) Soluble collagen in the left lung lobe was significantly increased in room air–exposed mice on Day 28 (^aP < 0.0002) and on Days 21 (^bP < 0.02) and 28 (^cP < 0.002) in hyperoxia-exposed mice, compared with Day 0 values. Less collagen was detected on Day 21 in room air–exposed mice compared with hyperoxia-exposed mice on Day 14 (^dP < 0.03). Columns with the same letter are not significantly different from each other; columns with different letters are significantly different from each other. (B) Representative images of lung tissue stained with trichrome. (C) Gross pathology of the lungs on Day 21 after treatment. Scale bar in B = 100 μm. d, day.

Figure 3.

Neonatal hyperoxia enhances leukocyte recruitment to lungs of adult mice treated with bleomycin. Bleomycin was administered intratracheally to adult mice that had been exposed to room air (RA) or hyperoxia (O₂) as neonates. Bronchoalveolar lavage (BAL) fluid was collected before treatment with bleomycin (Day 0) and on Days 3, 7, 14, 21, and 28 after treatment (n = 4–10 mice per group per time point). The total number of leukocytes (A), macrophages (B), lymphocytes (C), and neutrophils (D) in the BAL fluid of mice was significantly greater in mice exposed to neonatal hyperoxia, compared with mice exposed to room air. (*P < 0.02 for total cell counts, *P < 0.05 for macrophages, *P < 0.04 for lymphocytes, and *P < 0.03 for neutrophils). (E) Representative images of leukocytes collected on Day 7 after treatment.

Figure 4.

Anti–Gr-1 antibody depletes neutrophils and lymphocytes recruited to lungs of hyperoxia-exposed mice treated with bleomycin. Adult male mice exposed to room air (RA) or hyperoxia (O₂) as neonates received anti–Gr-1 antibody, and were then treated with bleomycin 2 hours later. Lungs (n = 5 mice per group) were lavaged on Day 3 after treatment. Columns with the same letter are not significantly different from each other; columns with different letters are significantly different from each other. (A) The total number of leukocytes in BAL fluid was significantly greater in mice exposed to hyperoxia, compared with mice exposed to room air (^bP < 0.003), but not when mice were treated with anti–Gr-1 antibody. (B) The total number of leukocytes in BAL fluid was not significantly greater in mice exposed to hyperoxia or treated with anti–Gr-1. (C) The total number of lymphocytes in BAL fluid was significantly greater in mice exposed to hyperoxia, compared with mice exposed to room air (^bP < 0.03), but not when mice were treated with anti–Gr-1 antibody. (D) The total number of neutrophils in BAL fluid was significantly greater in mice exposed to hyperoxia, compared with mice exposed to room air (^cP < 0.0001). The administration of anti–Gr-1 reduced the number of neutrophils recruited to the lungs in mice exposed to room air or hyperoxia (^bP < 0.009).

Figure 5.

Increased sensitivity to bleomycin-induced lung fibrosis is blocked by anti–Gr-1. Adult male mice exposed to room air (RA) or hyperoxia (O₂) as neonates received anti–Gr-1 antibody and were then treated with bleomycin 2 hours later. Lungs were harvested on Day 3 (A) or Day 21 (B and C) after treatment. Columns with the same letter are not significantly different from each other; columns with different letters are significantly different from each other. (A) Myeloperoxidase (MPO) activity was significantly greater in mice exposed to hyperoxia (^bP < 0.02), and was significantly decreased by anti–Gr-1 antibody (^cP < 0.02) compared with room air–exposed levels (n = 5 mice per group). (B) Soluble collagen was significantly increased on Day 21 after treatment in mice exposed to hyperoxia, compared with mice exposed to room air (^bP < 0.001), and was significantly suppressed by anti–Gr-1 antibody (^cP < 0.006) compared with room air–exposed mice (n = 5–9 mice per group). (C) Representative trichrome-stained images of lung tissue harvested on Day 21 from mice exposed to room air or hyperoxia, with or without previous treatment with anti–Gr-1 antibody. Scale bar = 100 μm.

Figure 6.

Pretreatment with anti–Gr-1 antibody inhibits the activation of transforming growth factor (TGF)–β1 in hyperoxia-exposed mice treated with bleomycin. Columns with the same letter are not significantly different from each other; columns with different letters are significantly different from each other. (A) Bleomycin was administered intratracheally to adult mice that had been exposed to room air (RA) or hyperoxia (O₂) as neonates (n = 3 mice per group). TGF-β1 mRNA (^bP < 0.01), active TGF-β1 in BAL fluid (^bP < 0.0001 on Day 3, and ^cP < 0.001 on Day 7), and total TGF-β1 (^cP < 0.01) significantly increased in mice treated with bleomycin. (B) Control IgG or anti–Gr-1antibody was injected into young adult mice exposed to room air or hyperoxia as neonates. Mice were then treated with bleomycin and killed 3 days later (n = 4–5 mice per group). TGF-β1 mRNA significantly increased (^bP < 0.02), but was significantly reduced by anti–Gr-1 antibody (^cP < 0.003). Active TGF-β1 significantly increased (^bP < 0.05), but was reduced by anti–Gr-1 antibody (^cP < 0.01). Total concentrations of TGF-β1 protein were not affected by neonatal hyperoxia or by anti–Gr-1 antibody on Day 3 after treatment (^aP = 0.2).

Figure 7.

Administration of anti–Gr-1 antibody after TGF-β1 is activated does not alleviate hyperoxia-induced lung fibrosis. Adult male mice exposed to room air (RA) or hyperoxia (O₂) as neonates received bleomycin intratracheally. Mice then received control IgG or anti–GR-1 antibody on Days 3 and 7 after treatment. Columns with the same letter are not significantly different from each other; columns with different letters are significantly different from each other. (A) Bleomycin significantly increased soluble collagen in oxygen-exposed mice treated with IgG or anti–GR-1 antibody (^bP < 0.05), compared with room air–exposed mice. (B) Representative images of trichrome-stained lung tissue from room air–exposed or oxygen-exposed mice, harvested 21 days after treatment with bleomycin. Scale bar = 100 μm.