Fig. 3.

Synaptamide suppresses LPS-induced inflammatory responses in neutrophils and macrophages in a GPR110/cAMP-dependent manner. Levels of GPR110 mRNA were determined by qPCR in primary microglia (Mi), peritoneal macrophage (Mc), neutrophil (Neu), peripheral blood mononuclear cells (PBMC), and platelets (Pla). Cell purity was determined by PCR using specific marker proteins (Iba-1 for microglia; Ly6G for neutrophil, F4/80 for macrophage) (a). WT- and GPR110 KO-derived neutrophils and macrophages were treated with 10 nM synaptamide for 15 min, and cAMP levels were measured (b). The cytokine/chemokine expression in the neutrophils and macrophages was determined by qPCR at 1 h after treatment of 100 ng/mL LPS followed by 10 nM synaptamide (c). Adenylyl cyclase inhibitor (SQ 22536, 10 μM) was added 30 min prior to LPS/synaptamide treatment. Levels of TNF and IL-1β mRNA were determined by qPCR after 1-h incubation in neutrophils and macrophages (d). Activity of transmigration was determined by Boyden chamber assay (at 30 min) after incubation. WT and GPR110 KO-derived neutrophils were incubated with LTB4 (30 nM)/CCL2 (10 ng/mL) or LPS (100 ng/mL) and/or 10 nM synaptamide (e). WT and GPR110 KO-derived peritoneal macrophages were incubated with LPS (100 ng/mL) and synaptamide (10 nM) for 2 h, and intracellular ROS level was determined by immunocytochemistry and ELISA using H2DCFDA (10 μM) (f). The data are expressed as the mean ± SEM (n = 3) representing at least three independent experiments. ns, the difference of means is not statistically significant