Preconditioning the immature lung with enhanced Nrf2 activity protects against oxidant-induced hypoalveolarization in mice

Abstract

Bronchopulmonary dysplasia (BPD) is a chronic disease of preterm babies with poor clinical outcomes. Nrf2 transcription factor is crucial for cytoprotective response, whereas Keap1-an endogenous inhibitor of Nrf2 signaling-dampens these protective responses. Nrf2-sufficient (wild type) newborn mice exposed to hyperoxia develop hypoalveolarization, which phenocopies human BPD, and Nrf2 deficiency worsens it. In this study, we used PND1 pups bearing bearing hypomorphic Keap1 floxed alleles (Keap1^f/f) with increased levels of Nrf2 to test the hypothesis that constitutive levels of Nrf2 in the premature lung are insufficient to mitigate hyperoxia-induced hypoalveolarization. Both wildtype and Keap1^f/f pups at PND1 were exposed to hyperoxia for 72 h and then allowed to recover at room air for two weeks (at PND18), sacrificed, and lung hypoalveolarization and inflammation assessed. Hyperoxia-induced lung hypoalveolarization was remarkably lower in Keap1^f/f pups than in wildtype counterparts (28.9% vs 2.4%, wildtype vs Keap1^f/f). Likewise, Keap1^f/f pups were protected against prolonged (96 h) hyperoxia-induced hypoalveolarization. However, there were no differences in hyperoxia-induced lung inflammatory response immediately after exposure or at PND18. Lack of hypoalveolarization in Keap1^f/f pups was accompanied by increased levels of expression of antioxidant genes and GSH as assessed immediately following hyperoxia. Keap1 knockdown resulted in upregulation of lung cell proliferation postnatally but had opposing effects following hyperoxia. Collectively, our study demonstrates that augmenting endogenous Nrf2 activation by targeting Keap1 may provide a physiological way to prevent hypoalveolarization associated with prematurity.

Figure 1

Increased expression of Nrf2 target genes in lungs of newborn Keap1 hypomorphs. (a) The schema represents the Keap1 mice bearing the heterozygous (f/wt) or homozygous (f/f) “lox P” sites (arrowheads) flanking the exons (gray boxes). The position of primers used for genotyping are indicated. (b) Total RNA was isolated from the lungs of 1-day-old (PND1) pups of wildtype (WT) and Keap1^f/f mice (n = 3), cDNA prepared, and qRT-PCR performed using gene-specific primers as indicated. (c) Immunoblot analyses of Nqo1 and Hmox1 in the lungs of WT and Keap1^f/f pups at PND1 (left). Uncropped immunoblots are presented in Supplementary Figure E1. Nqo1 and Hmox1 band intensities were quantified using β-actin band as a reference, and the values of the WT pups are represented as one unit (right).

Figure 2

Keap1 knockdown prevents hyperoxia-induced hypoalveolarization. Both wildtype (WT) and Keap1^f/f pups at PND1 were exposed to room air (RA) or hyperoxia (Hyp) for 72 h or 96 h, and pups were then allowed to recover at room air for 14 days or 13 days, respectively. Both 72 h and 96 h hyperoxia exposed mice were sacrificed at PND18, the left lung was fixed, sectioned, and stained with H&E. (a) Representative images of lung sections of WT and Keap1^f/f pups exposed to RA or Hyp at PND18 are shown (20×). Images captured with a Nikon Digital Camera are presented. Scale bar: 100 μm. (b) Serial H&E images of the lung Sects. (10x) were digitally captured and mean chord length (MCL) of alveolar region was analyzed by morphometry (n = 4–6).

Figure 3

Hyperoxia-induced lung inflammation is not different between wildtype and Keap1^f/f mice. Both wildtype (WT) and Keap1^f/f pups at PND1 were exposed to 72 h or 96 h hyperoxia and then allowed to recover at room air for 14 days or 13 days, respectively. Mice at PND18 were sacrificed, the right lung was lavaged, and total cells, macrophages, and neutrophils were enumerated as detailed in methods (n = 4–10).

Figure 4

Inflammatory cytokine gene expression in wildtype and Keap1^f/f pups exposed to 72 h hyperoxia. WT and Keap1^f/f pups at PND1 were exposed to room air (RA) or hyperoxia (Hyp) for 72 h and immediately sacrificed (i.e., at PND4), and lungs were harvested. Total RNA was isolated, cDNA prepared, and cytokine gene expression was determined by qRT-PCR using gene-specific primers as indicated (n = 6–8).

Figure 5

Hyperoxia-induced anti-oxidative gene expression is greater in Keap1^f/f than in WT pups. (a) Total RNA was isolated from the lungs of WT and Keap1^f/f neonatal pups (n = 6–8) exposed to room air (RA) or hyperoxia (Hyp) for 72 h as in Fig. 4 and subjected to qRT-PCR using gene-specific primers. (b) Immunoblot analyses of Nrf2, Nqo1, Hmox1 and Gclm in the lungs of WT and Keap1^f/f pups with and without hyperoxia exposure (n = 3–4). Uncropped immunoblots are presented in Figure E2. Protein band intensities quantified using β-actin band as a reference. The values of WT pups exposed to room air are represented as one unit.

Figure 6

Reduced levels of oxidative stress in the lungs of Keap1^f/f pups exposed to hyperoxia. Both wildtype (WT) and Keap1^f/f pups (n = 4) at PND1 were exposed to room air (RA) or hyperoxia (Hyp) for 72 h, and immediately sacrificed, the right lung was frozen and used to determine levels of reduced GSH (a), total GSH (reduced GSH plus oxidized GSH, GSSG) (b), and the ratio of GSH/GSSG (c).

Figure 7

Keap1 hypomorphism does not affect hyperoxia-induced DNA damage response pathway. As detailed above in Fig. 4, total RNA was isolated from the lungs of wildtype (WT) and Keap1^f/f pups (n = 3–4) exposed to room air (RA) or hyperoxia (Hyp) and subjected to qRT-PCR using gene specific primers as indicated.

Figure 8

Targeting Keap1 distinctly affects hyperoxia-induced lung cell damage and proliferation. WT and Keap1^f/f pups at PND1 (n = 4–5) were exposed to room air or hyperoxia for 72 h, then immediately sacrificed, the left lung was fixed, sectioned and immunostained with TUNEL or Ki-67 antibody. Images were counterstained with DAPI, captured and TUNEL-positive or Ki-67⁺ cells of the entire section were enumerated by Aperio scanning. (a) TUNEL-positive cells were enumerated by Aperio scanning. Left, total positive cells in the entire section; Right, % positively stained cells/total cells. (b) Quantification of Ki-67⁺ cells. Left, total positive cells in the entire section; Right, % positively stained cells/total cells.

Figure 9

Hyperoxia-induced alveolar simplification in Keap1^f/wt pups. Keap1^f/wt pups at PND1 were exposed to room air (RA) or hyperoxia (Hyp) for 72 h, allowed to recover at room air, and sacrificed at PND18. The left lung was fixed, sectioned, and stained with H&E. (a) A representative image of lung sections of the Keap1^f/wt neonatal mice exposed to RA or Hyp (20x). Images captured with Aperio Image Scope are presented. Scale bar: 100 μm. (b) The average MCL analyzed by morphometry (n = 3). Note that the MCL of the wildtype mice used for comparison are derived from Fig. 2B. (c) Nrf2 target gene expression in the lungs of room-air exposed 1-day-old (PND1) Keap1^f/wt pups (n = 4). The dotted line represent values of respective genes noted in WT pups as shown in Fig. 2. *, **, compared to WT mice.