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.