The NRF2 Activation and Antioxidative Response Are Not Impaired Overall during Hyperoxia-Induced Lung Epithelial Cell Death

Lung epithelial and endothelial cell death caused by pro-oxidant insults is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) patients. The NF-E2-related factor 2 (NRF2) activation in response to oxidant exposure is crucial to the induction of several antioxidative and cytoprotective enzymes that mitigate cellular stress. Since prolonged exposure to hyperoxia causes cell death, we hypothesized that chronic hyperoxia impairs NRF2 activation, resulting in cell death. To test this hypothesis, we exposed nonmalignant small airway epithelial cells (AECs) to acute (1–12 h) and chronic (36–48 h) hyperoxia and evaluated cell death, NRF2 nuclear accumulation and target gene expression, and NRF2 recruitment to the endogenous HMOX1 and NQO1 promoters. As expected, hyperoxia gradually induced death in AECs, noticeably and significantly by 36 h; ~60% of cells were dead by 48 h. However, we unexpectedly found increased expression levels of NRF2-regulated antioxidative genes and nuclear NRF2 in AECs exposed to chronic hyperoxia as compared to acute hyperoxia. Chromatin Immunoprecipitation (ChIP) assays revealed an increased recruitment of NRF2 to the endogenous HMOX1 and NQO1 promoters in AECs exposed to acute or chronic hyperoxia. Thus, our findings demonstrate that NRF2 activation and antioxidant gene expression are functional during hyperoxia-induced lung epithelial cell death and that chronic hyperoxia does not impair NRF2 signaling overall

Nrf2 Regulates Anti-Inflammatory A20 Deubiquitinase Induction by LPS in Macrophages in Contextual Manner

The aberrant regulation of inflammatory gene transcription following oxidant and inflammatory stimuli can culminate in unchecked systemic inflammation leading to organ dysfunction. The Nrf2 transcription factor dampens cellular stress and controls inflammation by upregulating antioxidant gene expression and TNFα-induced Protein 3 (TNFAIP3, aka A20) deubiquitinase by controlling NF-kB signaling dampens tissue inflammation. Here, we report that Nrf2 is required for A20 induction by inflammatory stimuli LPS in monocyte/bone marrow derived macrophages (MDMΦs) but not in lung-macrophages (LDMΦs). LPS-induced A20 expression was significantly lower in Nrf2−/− MDMΦs and was not restored by antioxidant supplementation. Nrf2 deficiency markedly impaired LPS-stimulated A20 mRNA expression Nrf2−/− MDMΦs and ChIP assays showed Nrf2 enrichment at the promoter Nrf2−/− MDMΦs upon LPS stimulation, demonstrating that Nrf2 directly regulates A20 expression. Contrary to MDMΦs, LPS-stimulated A20 expression was not largely impaired in Nrf2−/− LDMΦs ex vivo and in vivo and ChIP assays showed lack of increased Nrf2 binding at the A20 promoter in LDMΦ following LPS treatment. Collectively, these results demonstrate a crucial role for Nrf2 in optimal A20 transcriptional induction in macrophages by endotoxin, and this regulation occurs in a contextual manner

PI3K-AKT Signaling via Nrf2 Protects against Hyperoxia-Induced Acute Lung Injury, but Promotes Inflammation Post-Injury Independent of Nrf2 in Mice.

Lung epithelial and endothelial cell death accompanied by inflammation contributes to hyperoxia-induced acute lung injury (ALI). Impaired resolution of ALI can promote and/or perpetuate lung pathogenesis, including fibrosis. Previously, we have shown that the transcription factor Nrf2 induces cytoprotective gene expression and confers protection against hyperoxic lung injury, and that Nrf2-mediated signaling is also crucial for the restoration of lung homeostasis post-injury. Although we have reported that PI3K/AKT signaling is required for Nrf2 activation in lung epithelial cells, significance of the PI3K/AKT-Nrf2 crosstalk during hyperoxic lung injury and repair remains unclear. Thus, we evaluated this aspect using Nrf2 knockout (Nrf2(-/-)) and wild-type (Nrf2(+/+)) mouse models. Here, we show that pharmacologic inhibition of PI3K/AKT signaling increased lung inflammation and alveolar permeability in Nrf2(+/+) mice, accompanied by decreased expression of Nrf2-target genes such as Nqo1 and Hmox1. PI3K/AKT inhibition dampened hyperoxia-stimulated Nqo1 and Hmox1 expression in lung epithelial cells and alveolar macrophages. Contrasting with its protective effects, PI3K/AKT inhibition suppressed lung inflammation in Nrf2(+/+) mice during post-injury. In Nrf2(-/-) mice exposed to room-air, PI3K/AKT inhibition caused lung injury and inflammation, but it did not exaggerate hyperoxia-induced ALI. During post-injury, PI3K/AKT inhibition did not augment, but rather attenuated, lung inflammation in Nrf2(-/-) mice. These results suggest that PI3K/AKT-Nrf2 signaling is required to dampen hyperoxia-induced lung injury and inflammation. Paradoxically, the PI3K/AKT pathway promotes lung inflammation, independent of Nrf2, during post-injury

PI3K/AKT inhibition exacerbates hyperoxic lung injury in <i>Nrf2</i>^<i>+/+</i> mice.

<p>Mice were treated <i>i</i>.<i>p</i>. with LY294002 (LY) or DMSO (vehicle) 1 h prior to exposure and at 24 h intervals during hyperoxia exposure. Mice were exposed to 95% oxygen for 48 h at which time lungs were harvested. The BAL fluid was collected to measure both total cell counts and protein concentration, as markers of lung inflammation and lung injury (permeability), respectively. (A) Total protein in the BAL fluid of room air or hyperoxia-exposed mice treated with vehicle or LY. (B) Total cells in the BAL fluid of room air- or hyperoxia-exposed mice treated with vehicle or LY. (C) Total neutrophils and macrophages in the BAL fluid of vehicle- or LY-treated mice were exposed to room air or hyperoxia. BAL fluids (100 μl) were cytospun onto microslides and stained with Diff Quick stain kit. Differential cell counts were enumerated by counting a total number of 300 cells. The horizontal and vertical lines were plotted as median ± interquartile range for each group (n = 4–5). One-way ANOVA with Bonferroni corrections was performed for multiple group comparisons. *<i>P</i><u><</u> 0.05, hyperoxia vs room air; ^{<b><i>†</i></b>}<i>P</i><u><</u> 0.05, vehicle vs LY.</p

Hyperoxia-induced Nrf2-target gene expression in AKT1-depleted cells.

<p>(A) qRT-PCR analysis of hyperoxia-induced Nrf2-target gene expression in cells transfected with either scrambled siRNA (Scr-Si) or AKT1 siRNA. (A) Expression of AKT1 was calculated relative to Scr-Si-transfected room air-exposed samples. (B) Nrf2-target gene expression was calculated relative to Scr-Si-transfected room air-exposed samples. Values from the Scr-si transfected cells exposed to room air are considered as one unit. <i>p</i> ≤ 0.05, room air (RA) vs. hyperoxia (hyp); ^{<b><i>†</i></b>}<i>p</i> ≤ 0.05, Scr siRNA vs AKT1-siRNA. Data are expressed as mean ± SEM (n = 3–4).</p

PI3K/AKT inhibition attenuates lung inflammation in <i>Nrf2</i>^<i>+/+</i> and <i>Nrf2</i>^{<i>–/–</i>}mice post-injury.

<p>Cytospin slides of BAL cells were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129676#pone.0129676.g001" target="_blank">Fig 1</a>. Total cells, neutrophils and macrophages from room air or hyperoxia exposed <i>Nrf2</i>^{<b><i>+/+</i></b>} mice (A) and <i>Nrf2</i>^{<b><i>–/–</i></b>}mice (B) treated with vehicle or LY. *<i>P</i> = 0.05, hyperoxia vs room air; ^{<b><i>†</i></b>}<i>P</i> = 0.05, vehicle vs LY treated group.</p

The effects of PI3K/AKT inhibition on hyperoxic lung injury in <i>Nrf2</i>^<i>+/+</i> and <i>Nrf2</i>^{<i>–/–</i>}mice post-injury.

<p><i>Nrf2</i>^{<b><i>+/+</i></b>} and <i>Nrf2</i>^{<b><i>–/–</i></b>}mice were treated with vehicle or LY during hyperoxia as well as post-injury (recovery) from hyperoxia for 72 h at every 24 h intervals. Lung injury and inflammation was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129676#pone.0129676.g001" target="_blank">Fig 1</a>. Lung alveolar permeability of <i>Nrf2</i>^{<b><i>+/+</i></b>} (A) and <i>Nrf2</i>^{<b><i>–/–</i></b>}(B) mice post hyperoxic injury. The horizontal and vertical lines were plotted as median ± interquartile range for each group (n = 4–5). One-way ANOVA with Bonferroni corrections was performed for multiple group comparisons. *<i>P</i> = 0.05, hyperoxia vs room air; ^{<b><i>†</i></b>}<i>P</i> = 0.05, vehicle vs LY treated group.</p