Myeloid Nrf2 Protects against Neonatal Oxidant-Stress-Induced Lung Inflammation and Alveolar Simplification in Mice

Abstract

Bronchopulmonary dysplasia (BPD) is a chronic condition affecting preterm infants, characterized by lung alveolar simplification/hypoalveolarization and vascular remodeling. The nuclear factor erythroid 2 like 2 (Nfe2l2, or Nrf2) plays a critical role in the cytoprotective response to neonatal hyperoxia, and its global deficiency exacerbates hypoalveolarization in mice. The abnormal recruitment and activation of myeloid cells are associated with the pathogenesis of BPD. Therefore, we employed a genetic approach to investigate the role of myeloid Nrf2 in regulating hyperoxia-induced hypoalveolarization. Pups, both wild-type (Nrf2^f/f) and those with a myeloid Nrf2 deletion (abbreviated as Nrf2^∆/∆mye), were exposed to hyperoxia for 72 h at postnatal day 1 (Pnd1), and then sacrificed at either Pnd4 or Pnd18 following a two-week recovery period. We analyzed the hypoalveolarization, inflammation, and gene expression related to cytoprotective and inflammatory responses in the lungs of these pups. The hypoalveolarization induced by hyperoxia was significantly greater in Nrf2^∆/∆mye pups compared to their Nrf2^f/f counterparts (35.88% vs. 21.01%, respectively) and was accompanied by increased levels of inflammatory cells and IL-1β activation in the lungs. Antioxidant gene expression in response to neonatal hyperoxia was lower in Nrf2^∆/∆mye pups compared to their Nrf2^f/f counterparts. Furthermore, Nrf2-deficient macrophages exposed to hyperoxia exhibited markedly decreased cytoprotective gene expression and increased IL-1β levels compared to Nrf2-sufficient cells. Our findings demonstrate the crucial role of myeloid Nrf2 in mitigating hyperoxia-induced lung hypoalveolarization and inflammatory responses in neonatal mice.

Keywords: antioxidants; bronchopulmonary dysplasia; macrophages; neonatal lung injury.

Figure 1

(a) Wild-type (Nrf2^+/+) and Nrf2^–/– pups at Pnd1 were exposed to hyperoxia for 72 h and recovered at room air for 14 days as outlined. (b) Survival rates of neonatal hyperoxia-exposed Nrf2^+/+ and Nrf2^–/– pups in recovery. (c) Room air (RA) and 72 h hyperoxia-exposed and recovered Nrf2^+/+ and Nrf2^–/– pups at Pnd18 (2WKR) were sacrificed, and the left lung was fixed, sectioned and stained with H&E. A representative image of the lung sections is shown (20×). (d) Mean chord length (MCL) of the alveolar region of H&E images (10×) was analyzed by morphometry, as detailed in the methods. ***, ****, compared to respective RA controls; ^$$, compared to Nrf2^+/+ counterparts. Scale bars: 100 µm.

Figure 2

(a) Mice with Nrf2 deletion in myeloid cells (Nrf2^Δ/Δmye) were generated by crossing Nrf2-floxed mice with LysM2-Cre mice and genotyped. Nrf2 deletion in Nrf2^Δ/Δmye mice was characterized in tail DNA and BAL lung macrophages (MΦs) by PCR genotyping. (b) Newborn (Pnd1) pups from Nrf2^f/f (left panel) and Nrf2^Δ/Δmye mice (right panel) were exposed to 72 h hyperoxia and then recovered at room air for two weeks (2WKR), as in Figure 1a. Their survival rates were monitored for up to 14 days. (c) Pups exposed to room air or 72 h hyperoxia and sacrificed at Pnd18, and the left lung was fixed, sectioned, and stained with H&E. A representative image of the lung sections of both genotypes is shown (20×). (d) H&E images (10×) were analyzed to determine the mean chord length (MCL). ****, compared to respective genotype RA controls; ^$$$, compared to Nrf2^f/f counterparts. Scale bars: 100–101 µm.

Figure 3

(a) Room air or neonatal hyperoxia-exposed Nrf2^f/f and Nrf2^Δ/Δmye pups were sacrificed at Pnd18, the right lung was lavaged, and inflammatory cells were enumerated. (b) RNA was isolated from the lungs of Nrf2^f/f and Nrf2^Δ/Δmye pups as detailed in panel A, and cytokine gene expression was analyzed by qPCR. *, **, ***, ****, compared to respective RA controls; ^$, compared to Nrf2^f/f counterparts.

Figure 4

(a) Nrf2^Δ/Δmye and Nrf2^f/f pups were exposed to hyperoxia for 72 h and immediately sacrificed; the right lung was lavaged, and inflammatory cells were assessed. (b) RNA was isolated from the lungs of Nrf2^Δ/Δmye and Nrf2^f/f pups exposed to 72 h hyperoxia, and target gene expression was analyzed by qPCR. *, compared to respective RA controls, ^$, compared to Nrf2^f/f counterparts.

Figure 5

RNA was isolated from the lungs of Nrf2^Δ/Δmye and Nrf2^f/f pups exposed to neonatal hyperoxia and recovered at room air for 14 days (a) from the lungs of Nrf2^Δ/Δmye and Nrf2^f/f pups exposed to neonatal hyperoxia for 72 h and (b) Nrf2 putative target gene expression was analyzed by qPCR. **, compared to respective RA controls; ^$$, compared to Nrf2^f/f counterparts.

Figure 6

RNA isolated from the lungs of Nrf2^Δ/Δmye pups and Nrf2^f/f pups exposed to room air (RA) or 72h of hyperoxia at Pnd1 and sacrificed at Pnd4 was analyzed for Vegf, Fgf10, Notch1, and Notch2.

Figure 7

Freshly cultured BMDMs isolated from Nrf2^+/+ (WT) and Nrf2^–/– (NKO) mice were exposed to room air (RA) or hyperoxia for 14 h, and RNA was isolated. Both cytoprotective (a) and inflammatory cytokine (b) gene expression were determined by qPCR using gene-specific primers as indicated. *, ****, compared to respective genotype RA controls; ^$, ^$$, ^$$$, ^$$$$, compared to WT-BMDMs.