Abstract TP317: Chemokine Production by Microglia Mediates Blood-Derived Monocytes Trafficking in Neuroinflammation

Abstract

Introduction: After ischemic stroke, microglia and recruited macrophages play major roles in neuroinflammation. We explored how these cells affect counterpart’s polarization and infiltration and revealed some chemokines and receptors can be important modulators of the interaction. Methods: BV2 (microglia cell line) were treated with lipopolysaccharides (LPS) or interleukin-4, and the supernatant was collected as M1 or M2 conditioned media of BV2. The supernatant of PMA differentiated THP-1 (monocyte cell line) followed by LPS treatment or interleukin-13 & IL-4co-treatment were collected as M1 or M2 conditioned media of THP-1. After BV2 or THP-1 cultured in conditioned media, the activation and polarization were assessed by ICC and confocal microscopy for CD11b, CD86 and CD206. Transwell inserts of 3 and 8 μm pore membrane were used for THP-1 infiltration and BV2 migration assay. M1/M2 conditioned media of BV2 and THP-1 and the brain tissue of ischemic mouse model were assessed by proteome profiler array (PPA) to find target cytokine and chemokine. THP-1 pretreated with antagonists of CCR2, CCR4, CXCR4 and CCR5 were used for infiltration assay, the expression of chemokine receptor in THP-1 were confirmed by western blotting. Results: Both THP-1 and BV2 expressed CD206 when cultured in M2 conditioned media of BV2 or THP-1. M2 conditioned media of BV2 increased the infiltratory ability of THP-1 while M1 conditioned media of THP-1 enhanced BV2 migration. After ischemic stroke, chemokines were significantly expressed at 3 days than at 7 days in the PPA and several chemokines such as CCL2, CCL3, CCL4 and CCL5 were match to the conditioned media of BV2 while their receptor CCR4, CCR5 shows high expression in THP-1. Antagonists of CCR2, CCR4 and CCR5 significantly reduced the infiltration ability of THP-1 to BV2 M2 conditioned media. Conclusion: CCL2, CCL3, CCL4, CCL5 and their receptors CCR4, CCR5 can be strong candidates of target proteins in new therapeutic strategies to acute brain inflammation by modulating the functions of microglia and monocytes. “This research was supported by the Brain Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(NRF-2016M3C7A1905098)“