PLAG enhances macrophage mobility for efferocytosis of apoptotic neutrophils via membrane redistribution of P2Y2

Neutrophil activity, including trapping of damage-associated molecular patterns by neutrophil extracellular traps (NETs), is an important response to microbial infection. Most activated neutrophils commit to apoptosis and are removed by activated macrophages in the process of efferocytosis. Improper clearance of apoptotic neutrophils often causes an unnecessary and exaggerated immune response and subsequent chronic inflammation. Effective macrophage mobility toward activated neutrophils, which is triggered by binding of 'find-me' signals to receptors such as P2Y2, is a crucial step for the timely clearance of apoptotic neutrophils. In this paper, we investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on efferocytosis and the underlying molecular mechanisms. In a coculture of apoptotic neutrophils with macrophages, PLAG treatment increased levels of efferocytosis of apoptotic neutrophils. PLAG induced faster translocation of P2Y2 from lipid rafts to nonlipid raft plasma membrane domains in macrophages. This repositioning of P2Y2 enables the polarization of the cytoskeleton by association of the receptor with cytoskeletal proteins such as alpha-tubulin and actin to improve the mobility of macrophages. The formation of vesicular, chylomicron-like structures by PLAG was a prerequisite for the induction of this macrophage activity, as none of these effects was seen when the vesicle receptor GPIHBP1 was absent. Taken together, these data showed that PLAG is a powerful immune resolvent that triggers the prompt clearance of apoptotic neutrophils by enhanced efferocytosis activity. PLAG could therefore be an effective lipid-based efferocytosis enhancer for use as a therapeutic drug to prevent inflammatory disease caused by uncontrolled immune responses.