Activation of PGE2 EP1 Receptor Promotes Microglial Phagocytosis Though CD36 Receptor Recycling.

Background and Purpose: We have shown that the deletion of prostaglandin E2 (PGE2) EP1 receptor undermined microglial phagocytosis in vivo. However, the related mechanism still remains unknown. CD36 is a type II scavenger receptor that mediates microglial phagocytosis. It was reported that microglial CD36 contributes to hematoma clearance after intracerebral hemorrhage (ICH). In this study, we sought to identify the effects of two EP1 agonists on microglial phagocytosis and CD36 receptor recycling in primary microglial cultures. Method: The EP1 agonists: 17-phenyl trinor-PGE2 (17-pt-PGE2) and ONO-DI-004 were used to treat primary microglial cultures, respectively. Fluorescent latex beads or CFSE-labeled red blood cells (CFSE-RBCs) were added to microglial cells at a concentration of ten beads/RBCs per cell for live-cell imaging. Immunocytochemistry was also performed with antibody against ionized calcium-binding adapter molecule 1 (Iba1, a marker to microglia). Furthermore, we analyzed CD36 receptor recycling by using an established receptor recycling assay. Result: The primary microglial cells were incubated with fluorescent latex beads and treated with the EP1 agonist 17-pt-PGE2 for 120 min. We found that 17-pt-PGE2 significantly increased the number of attached beads to microglial cells at the concentration of 10 μM from 40 min to 120 min of incubation. Furthermore, we analyzed CD36 receptor recycling using an established receptor recycling assay and the quantitative results showed that 10 μM of 17-pt-PGE2 treatment markedly increased the area of recycled CD36 receptor in microglial cells. We then treated primary microglial cells with another selective EP1 agonist ONO-DI-004 for 120 min at the concentration of 5 μM and 10 μM, respectively. The area of CFSE-RBCs after ice-cold PBS washing was markedly increased with 10 μM of ONO-DI-004 treatment. In the live-cell imaging experiment, 10 μM of ONO-DI-004 treatment also significantly increased the number of attached CFSE-RBCs to microglial cells from 25 min to 60 min of incubation. Conclusions: This study showed that the EP1 agonists: 17-pt-PGE2 and ONO-DI-004 can promote microglial phagocytosis in vitro and enhanced CD36 receptor recycling could mediate some of these effects.