The Nrf2 Transcription Factor Promotes Efferocytosis by Activating Phosphatidyl Serine Receptor Tim‐4 Signaling during Oxidant‐Induced Lung Injury Repair
Authors: Reddy Narsa M , Tamatam Chandramohan R , Reddy Sekhar P 
Published: June 2021

Objective

Apoptotic cell clearance by macrophages (known as efferocytosis) is critical for the cessation of inflammatory responses and efficient tissue repair post injury. The Nrf2 transcription factor induces the expression of several cytoprotective genes in response to oxidant and toxicant stimuli. We have previously demonstrated that Nrf2‐deficiency impairs the resolution of hyperoxic lung injury, which was accompanied by epithelial cell death and persistent lung inflammation in mice. Here, we tested whether Nrf2 regulates efferocytosis mediated by alveolar macrophages (AMΦs) by upregulating the expression of phosphatidyl serine (PS) receptors that recognizes and internalizes the dead cells, and thereby resolves pro‐oxidant (hyperoxia)‐induced lung inflammation.

Methods

Both wild‐type (WT) and Nrf2‐null (Nrf2−/−) mice were exposed to room air or sub‐lethal (48 h) hyperoxia. Some mice with hyperoxic lung injury were allowed to recover for 72 h. We measured efferocytosis in vivo and ex vivo in AMΦs isolated from these mice. PS receptors expression was determined in AMΦs by qPCR. To determine whether Nrf2 deficiency impairs apoptotic cell binding and/or internalization, bone marrow derived macrophages (BMDMΦ) isolated from WT and Nrf2−/− mice were treated with or without actin destabilizer, cytochalasin D (15 μM for 20 min) prior to incubating them with apoptotic cells. The requirement of Tim‐4 for mediating efferocytosis is determined by knocking down Tim‐4 expression in BMDMΦ. To address whether Nrf2 activation will accelerates efferocytosis following oxidant stress, BMDMΦs isolated from WT mice exposed to hyperoxia were treated with Nrf2 activator, CDDO‐Im.

Results

We found impaired efferocytosis in AMΦs from room air or hyperoxia‐exposed Nrf2−/− mice compared to WT counterparts in vivo and ex vivo. Although the percent decrease (~40%) in efferocytosis is equal immediately after hyperoxia in both genotypes compared to room air controls, the percent efferocytosis reached to room air control levels in WT mice while it further declined in Nrf2−/− mice (~70%) after 72 h recovery. Similar differences in efferocytosis were found in ex vivo. Nrf2 deficiency impaired the binding as well as internalization of apoptotic cells in both room air and hyperoxia‐exposed BMDMΦs. Hyperoxia induced Tim‐4 expression in WT but not in Nrf2−/− AMΦs, whereas the expression of other PS receptors, brain angiogenesis inhibitor 1 (Bai‐1) and Stabilin 2 were not affected by hyperoxia. Si‐RNA mediated Tim‐4 knockdown in WT BMDMΦs resulted in impaired efferocytosis. Activation of Nrf2 enhanced Tim‐4 expression in WT BMDMΦs and reversed the suppressive effect of hyperoxia on efferocytosis.

Conclusions

Our findings suggest that Nrf2 is crucial for AMΦs mediated efferocytosis during the resolution of oxidant‐induced lung injury, and this transcription factor mediates this process through the activation of PS receptor, Tim‐4.

Support or Funding Information

Supported by RG‐272303‐N (to NMR) and RO1‐HL66109 (to SPR)