Regulation of Components of the Macrophage Inflammation Response by the G Protein-Coupled Estrogen Receptor

Abstract ID 130760 Poster Board 370 OBJECTIVES: Macrophages are critical for the inflammatory response of the innate immune system. The NLRP3 inflammasome is a multimeric protein complex consisting of NLRP3, apoptosis-associated speck-like protein, and pro-caspase-1 that functions to promote activation of caspase-1. Active caspase-1 cleaves pro-IL-1β and pro-IL-18 into their active secreted forms, which mediate the inflammatory response. The G protein-coupled estrogen receptor 1 (GPER, a.k.a. GPR30) is involved in the functions of many bodily systems. Genetic deletion of GPER is associated with cardiac dysfunctions that are reversed by inhibition of the NLRP3 inflammasome. Previous studies also suggest that GPER exerts anti-inflammatory effects that are protective in numerous animal disease models including stroke, atherosclerosis, cancer, and ulcerative colitis. However, whether and how GPER regulates the inflammatory response in macrophages is not well characterized. Here we report the effects of GPER expression and activation on several components of the inflammatory response in bone marrow-derived macrophages. METHODS: Progenitor cells were isolated from hind limb long bones of WT and GPER-/- adult male C57BL/6J mice and cultured in DMEM/F12 medium containing 10% fetal bovine serum and 20% vol/vol L929-cell conditioned medium for 7 days for differentiation into bone marrow-derived macrophages (BMDMs). Priming of the inflammasome was achieved by treatment with lipopolysaccharide for 4 hrs. Expression levels of inducible nitric oxide synthase, the NLRP3 inflammasome and interleukins were assessed using Western blotting and a pro-inflammatory array. Fura-2/AM and IPG-1/AM were used independently or simultaneously to measure intracellular Ca²⁺ and K⁺. RESULTS: Cell morphology and viability are similar between WT and GPER-/- BMDMs. Polarization towards proinflammatory subtype M1, as assessed by lipopolysaccharide-induced expression of inducible nitric oxide synthase, is also the same between WT and GPER-/- BMDMs. K⁺ efflux, an important component in the activation of the NLRP3 inflammasome, is increased in unprimed GPER-/- BMDMs. Intriguingly, acute treatment with 100 nM GPER agonist G-1 in unprimed conditions triggers robust Ca²⁺ signals in both WT and GPER-/- BMDMs, indicating a GPER-independent effect. Notably, the Ca²⁺ signal produced in GPER-/- BMDMs is substantially larger in GPER-/- than in WT BMDMs. Additionally, in the absence of extracellular Ca²⁺, G-1 does not trigger a Ca²⁺ response in either WT or GPER-/- BMDMs. However, 24-hr treatment with 100 nM G-1 significantly suppresses lipopolysaccharide-induced expression of the NLRP3 inflammasome, interleukin-1b, interleukin-6 and C-C motif chemokine ligand 5 (CCL5) in WT but not in GPER-/-BMDMs. CONCLUSIONS: In bone marrow-derived macrophages, GPER regulates multiple components of the inflammatory response. GPER expression and agonist G-1 exerts receptor-dependent effects that are predicted to suppress inflammation. However, in these cells, G-1 also produces GPER-independent action on intracellular Ca²⁺ signaling, which, intriguingly, is suppressed by GPER expression.