Resolvin D1-mediated NOX2 inactivation rescues macrophages undertaking efferocytosis from oxidative stress-induced apoptosis

Abstract

Effective clearance of apoptotic cells by macrophages, termed efferocytosis, is pre-requisite for successful resolution of inflammation, and drives macrophage emigration to the draining lymph node, thereby promoting restoration of tissue homeostasis. During efferocytosis, engulfment of apopototic cells induces generation of reactive oxygen species in abundance. Macrophage apoptosis is an important feature of chronic inflammatory diseases including atherosclerosis. In the present study, we found that resolvin D1 (RvD1), one of endogenous pro-resolving lipid mediators derived from docosahexaenoic acid, prevented apoptosis of murine macrophage-like RAW264.7 cells engulfing apoptotic T cells. The inhibitory effect of RvD1 on efferocytosis-induced oxidative burst appears to be mediated by the inactivation of NADPH oxidase (NOX), a key enzyme involved in intracellular ROS production. In RvD1-treated macrophages, efferocytosis-induced phosphorylation of p47(phox) and association between p47(phox) and gp91(phox) were downregulated, resulting in abrogation of generation of superoxide anion and hydrogen peroxide. Furthermore, RvD1-mediated suppression of NOX activation was found to be dependent on cAMP-activated protein kinase (PKA) signaling. Besides inhibiting NOX activation, RvD1 rescued macrophages from oxidative stress-induced apoptosis by upregulating the expression of Bcl-xL and Bcl-2. However, knockdown of the RvD1 receptor, lipoxin A receptor/formyl-peptide receptor (ALX/FPR2), abolished the ability of RvD1 to activate cAMP-PKA signaling, to suppress NOX activation and to increase the expression of anti-apoptotic proteins, suggesting that ALX/FPR2 mediates the protective effect of RvD1 on effeocytosis-induced oxidative stress. Taken together, these findings indicate that RvD1 rescues macrophages from oxidative stress-induced apoptosis during efferocytosis through PKA-mediated repression of NOX activation and upregulation of anti-apoptotic protein expression.

Keywords: ALX/FPR2; Efferocytosis; NADPH oxidase; Oxidative stress-induced apoptosis; Resolvin D1.