Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

Recognition and clearance of apoptotic cells by phagocytes (also known as efferocytosis), primarily mediated by macrophages, are essential to terminate lung inflammatory responses and promote tissue repair after injury. The Nrf2 transcription factor is crucial for cytoprotection and host defense. Previously, we showed sustained neutrophilic lung inflammation in Nrf2-deficient (Nrf2−/−) mice after hyperoxia-induced lung injury in vivo, but the mechanisms underlying this abnormal phenotype remain unclear. To examine whether Nrf2 regulates apoptotic neutrophil clearance, we used the alveolar macrophages (AMФs) and bone-marrow-derived macrophages (BMDMФs) of wild-type (WT) and Nrf2−/− mice. We found that the efferocytic ability of AMФ was impaired in hyperoxia-exposed mice’s lungs, but the effect was more pronounced in Nrf2−/− mice. Importantly, AMФ-mediated efferocytosis remained impaired in Nrf2−/− mice recovering from injury but was restored to the basal state in the wild-type counterparts. Hyperoxia affected apoptotic neutrophil binding, not internalization, in both WT and Nrf2−/− BMDMФs, but the effect was more significant in the latter cells. Augmenting Nrf2 activity restored hyperoxia attenuated efferocytosis in WT, but not in Nrf2−/− macrophages. However, the loss of Nrf2 in neutrophils affected their uptake by WT macrophages. Collectively, these results demonstrate that Nrf2 is required for optimal macrophage-mediated efferocytosis and that activating Nrf2 may provide a physiological way to accelerate apoptotic cell clearance after oxidant injury

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

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

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 and inflammation in <i>Nrf2</i>^{<i>–/–</i>}mice.

<p><i>Nrf2</i>^{<b><i>–/–</i></b>}mice were treated with either vehicle or LY and subsequently exposed to hyperoxia or room air 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>. (A) Total protein in the BAL fluid of room air or hyperoxia exposed <i>Nrf2</i>^{<b><i>–/–</i></b>}mice treated with vehicle or LY. (B) Total cells in the BAL fluid of room air or hyperoxia exposed <i>Nrf2</i>^{<b><i>–/–</i></b>}mice treated with vehicle or LY. (C) Total neutrophils and macrophages in vehicle or LY-treated mice exposed to room air or hyperoxia. 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

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