Neonatal hyperoxia stimulates the expansion of alveolar epithelial type II cells

Abstract

Supplemental oxygen used to treat infants born prematurely disrupts angiogenesis and is a risk factor for persistent pulmonary disease later in life. Although it is unclear how neonatal oxygen affects development of the respiratory epithelium, alveolar simplification and depletion of type II cells has been observed in adult mice exposed to hyperoxia between postnatal Days 0 and 4. Because hyperoxia inhibits cell proliferation, we hypothesized that it depleted the adult lung of type II cells by inhibiting their proliferation at birth. Newborn mice were exposed to room air (RA) or hyperoxia, and the oxygen-exposed mice were recovered in RA. Hyperoxia stimulated mRNA expressed by type II (Sftpc, Abca3) and type I (T1α, Aquaporin 5) cells and inhibited Pecam expressed by endothelial cells. 5-Bromo-2'-deoxyuridine labeling and fate mapping with enhanced green fluorescence protein controlled statically by the Sftpc promoter or conditionally by the Scgb1a1 promoter revealed increased Sftpc and Abca3 mRNA seen on Day 4 reflected an increase in expansion of type II cells shortly after birth. When mice were returned to RA, this expanded population of type II cells was slowly depleted until few were detected by 8 weeks. These findings reveal that hyperoxia stimulates alveolar epithelial cell expansion when it disrupts angiogenesis. The loss of type II cells during recovery in RA may contribute to persistent pulmonary diseases such as those reported in children born preterm who were exposed to supplemental oxygen.

Figure 1.

Neonatal hyperoxia differentially affects alveolar epithelial protein expression. Newborn mice were exposed to room air (RA) or hyperoxia (100% oxygen [O₂]) through postnatal days (pnd)4, pnd7, and pnd10. (A) Lung homogenates were immunoblotted with antibodies against prosurfactant protein C (proSP-C), T1α, and platelet endothelial cell adhesion molecule (PECAM), with β-actin used as a loading control. Band intensities in mice exposed to RA (white bar) and hyperoxia (black bar) were graphed as mean ± SE fold over RA (n = 3–4 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001 when compared with RA. (B) Representative images of pnd4 and pnd10 lungs immunostained with antibody against proSP-C or ABCA3 (red), T1α (green), and 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar = 50 μm.

Figure 2.

Neonatal hyperoxia stimulates alveolar epithelial mRNA expression. Newborn mice were exposed to RA or O₂ through pnd4, pnd7, and pnd10. The mRNA expression of Sftpc, Abca3, T1α, Aqp5, Pecam, and 18S ribosomal RNA as a loading control were determined by quantitative RT-PCR. The mRNA expression in mice exposed to RA (white bar) and hyperoxia (black bar) were graphed as mean ± SE fold change over RA (n = 4 mice per group). *P < 0.05 and **P < 0.01 when compared with RA.

Figure 3.

Neonatal hyperoxia increases the number cells expressing thyroid transcription factor (TTF)-1. Newborn mice were exposed to RA or O₂ through pnd4 and pnd10. (A) Lungs were immunostained with antibody against TTF-1 (red) and Sftpc (green) and were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar = 50 μm. (B) The percentage of TTF-1 to DAPI–positive cells in mice exposed to RA (white bar) and hyperoxia (black bar) was graphed as mean ± SE fold change over RA (n = 5 mice per group). **P < 0.01 when compared with RA. (C) The mRNA expression of Ttf-1 and 18S ribosomal RNA as a loading control was determined by quantitative RT-PCR. The mRNA expression in mice exposed to RA (white bar) and hyperoxia (black bar) was graphed as mean ± SE fold change over RA (n = 5 mice per group). *P < 0.05 when compared with RA.

Figure 4.

Neonatal hyperoxia stimulates proliferation of type II cells. Newborn Sftpc-EGFP mice were exposed to RA or O₂. (A) Lungs were harvested daily through pnd4 and immunostained with antibody against BrdU (red) and enhanced green fluorescence protein (EGFP) (green) followed by counterstaining with DAPI. (B) The percentage of cells expressing BrdU and BrdU/EGFP on each day was determined and graphed as mean ± SE (n = 5 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001 when compared with RA on the same day. Scale bar, 30 μm.

Figure 5.

Neonatal hyperoxia stimulates lineage expansion of type II cells. (A) Cartoon model depicting administration of doxycycline to triple transgenic Scgb1a1-rtTA X (otet)₇CMV-Cre X mT/mG mice doxycycline between embryonic day (E)14.5 and E18.5 followed by exposure to RA or hyperoxia between birth and pnd4. (B) The percentage of EGFP to DAPI-positive cells in mice exposed to RA (white bar), 60% oxygen (gray bar), or 100% oxygen (black bar) was quantified and graphed as mean ± SE (n = 5 mice per group). **P < 0.01 when compared with RA. (C) Representative images of EGFP (green) and DAPI (blue) staining in lung tissues of mice exposed to RA, 60% oxygen, or 100% oxygen. Arrows point to airway. Scale bar, 200 μm. Dox, doxycycline.

Figure 6.

Expanded type II cells are depleted when mice are recovered in RA. (A) Lungs of Sftpc-EGFP mice exposed to RA or 100% O₂ between birth and pnd4 were stained with antibody against EGFP (green), Sftpc (red), and DAPI (blue). Note that virtually all EGFP cells express Sftpc. The percentage of EGFP to DAPI-positive cells was quantified in mice exposed to RA (white bar) or hyperoxia (closed bar) and graphed as mean ± SE fold change over RA (n = 4 mice per group). *P < 0.05 when compared with RA. (B) Lung homogenates were immunoblotted for EGFP and β-actin. Band intensities in mice exposed to RA and hyperoxia were quantified and graphed as mean ± SE fold (n = 4 mice per group). *P < 0.05 when compared with RA. (C) Lungs of Scgb1a1-rtTA X (otet)₇CMV-Cre X mT/mG mice exposed to RA or 60% O₂ between birth and pnd4 were stained with antibody against EGFP (green) and DAPI (blue). The percentage of EGFP to DAPI-positive cells was quantified in mice exposed to RA or hyperoxia and graphed as mean ± SE fold change over RA (n = 4 mice per group). **P < 0.01 when compared with RA. (D) Lung homogenates were immunoblotted with antibody against proSP-C, T1α, and β-actin. Band intensities in mice exposed to RA (white bar) and hyperoxia (black bar) were quantified and graphed as mean ± SE fold (n = 4 RA and n = 5 hyperoxia mice). *P < 0.compared with RA. Scale bar, 50 μm.

Figure 7.

Type II cells are depleted in adult mice exposed to neonatal hyperoxia. (A) Cartoon model showing how Scgb1a1-rtTA X (otet)₇CMV-Cre X mT/mG mice were exposed to RA or 100% O₂ between pnd0 and pnd4 and then administered doxycycline as adults. (B) Lungs of adult Scgb1a1-rtTA X (otet)₇CMV-Cre X mT/mG mice exposed to RA or O₂ as neonates and vehicle or doxycycline as adults were stained with antibody against proSP-C (red), EGFP (green), and DAPI (blue). The percentage of proSP-C to DAPI-positive cells and the percentage of EGFP to proSP-C–positive cells in mice exposed to RA or hyperoxia were graphed as mean ± SE fold (n = 5 mice per group). *P < 0.05 and **P < 0.01 when compared with RA. Scale bar, 50 μm. Dox, doxycycline.

Figure 8.

Cartoon model depicting the expansion of type II cells during exposure to hyperoxia followed by pruning during recovery in RA. Because type II cells are defenders of the alveolus, their loss in adult mice exposed to neonatal hyperoxia may impair host defense and ability to effectively repair after epithelial injury.