Nrf2 deficiency in myeloid cells accelerates atherosclerosis by promoting the inflammatory response and impairing efferocytosis

Introduction: Atherosclerotic lesions are the fundamental pathologies of cardiovascular diseases. The exact role of the nuclear factor erythroid 2-related factor 2 (NRF2) in macrophages in atherosclerosis remains uncertain.

Objectives: This study aimed to investigate the role of NRF2 in myeloid cells in the development of atherosclerosis.

Methods: Single-cell RNA Sequencing databases were used to explore the expression levels of NRF2 in human and murine atherosclerosis. Plaque areas, necrotic core size, instability index, and efferocytosis in aortic lesions were investigated in myeloid cell-specific Nrf2-knockout mice on an ApoE-deficient background (Nrf2(M)-KO; apoE^-/-). Transcriptomic, proteomic and chromatin immunoprecipitation (ChIP)-PCR analyses were used to unravel the underlying mechanism. Efferocytosis assays and mRNA levels were verified in primary macrophages and cell lines.

Results: NRF2 expression was upregulated in the macrophages of human and murine atherosclerotic arteries compared with their corresponding controls. Nrf2(M)-KO; apoE^-/- mice developed severe atherosclerosis lesions throughout the aorta and aortic sinus with concomitant increases in macrophage accumulation, inflammation, damage-associated molecular patterns release, necrotic core and apoptotic cell accumulation, suggesting that disrupted macrophage efferocytosis may be involved in the pathogenesis. In vitro and in vivo studies showed that Nrf2 deficiency inhibited macrophage efferocytosis. Transcriptomics, proteomics, and in vitro experiments were conducted in primary macrophages isolated from Nrf2(M)-KO mice to unravel the underlying mechanism. We demonstrated that Nrf2 binds to the Myh9 promoter, and that reduced Myh9 accumulation at the phagocytic cup underlies the defective efferocytosis seen in Nrf2-deficient macrophages. Pharmacological activation of NRF2 with 4-octyl itaconate alleviated inflammation and enhanced efferocytosis, but these restorative effects were abolished in Nrf2-KD cells, confirming the NRF2 dependence.

Conclusions: Myeloid-specific deletion of Nrf2 promotes inflammation and inhibits macrophage efferocytosis, thereby leading to the aggravation of atherosclerosis. NRF2 activation in macrophages could be a valuable strategy for preventing and treating atherosclerosis.

Atherosclerosis; Efferocytosis; Inflammation; Macrophage; NRF2.