Laboratory of Molecular Signaling, National Institute of Alcohol Abuse and Alcoholism, 5625 Fishers Lane, Rm. 3N-07, Rockville, MD, 20852, USA.
J Neuroinflammation. 2019 Nov 15;16(1):225. doi: 10.1186/s12974-019-1621-2.
Neuroinflammation is a widely accepted underlying condition for various pathological processes in the brain. In a recent study, synaptamide, an endogenous metabolite derived from docosahexaenoic acid (DHA, 22:6n-3), was identified as a specific ligand to orphan adhesion G-protein-coupled receptor 110 (GPR110, ADGRF1). Synaptamide has been shown to suppress lipopolysaccharide (LPS)-induced neuroinflammation in mice, but involvement of GPR110 in this process has not been established. In this study, we investigated the possible immune regulatory role of GPR110 in mediating the anti-neuroinflammatory effects of synaptamide under a systemic inflammatory condition.
For in vitro studies, we assessed the role of GPR110 in synaptamide effects on LPS-induced inflammatory responses in adult primary mouse microglia, immortalized murine microglial cells (BV2), primary neutrophil, and peritoneal macrophage by using quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) as well as neutrophil migration and ROS production assays. To evaluate in vivo effects, wild-type (WT) and GPR110 knock-out (KO) mice were injected with LPS intraperitoneally (i.p.) or TNF intravenously (i.v.) followed by synaptamide (i.p.), and expression of proinflammatory mediators was measured by qPCR, ELISA, and western blot analysis. Activated microglia in the brain and NF-kB activation in cells were examined microscopically after immunostaining for Iba-1 and RelA, respectively.
Intraperitoneal (i.p.) administration of LPS increased TNF and IL-1β in the blood and induced pro-inflammatory cytokine expression in the brain. Subsequent i.p. injection of the GPR110 ligand synaptamide significantly reduced LPS-induced inflammatory responses in wild-type (WT) but not in GPR110 knock-out (KO) mice. In cultured microglia, synaptamide increased cAMP and inhibited LPS-induced proinflammatory cytokine expression by inhibiting the translocation of NF-κB subunit RelA into the nucleus. These effects were abolished by blocking synaptamide binding to GPR110 using an N-terminal targeting antibody. GPR110 expression was found to be high in neutrophils and macrophages where synaptamide also caused a GPR110-dependent increase in cAMP and inhibition of LPS-induced pro-inflammatory mediator expression. Intravenous injection of TNF, a pro-inflammatory cytokine that increases in the circulation after LPS treatment, elicited inflammatory responses in the brain which were dampened by the subsequent injection (i.p.) of synaptamide in a GPR110-dependent manner.
Our study demonstrates the immune-regulatory function of GPR110 in both brain and periphery, collectively contributing to the anti-neuroinflammatory effects of synaptamide under a systemic inflammatory condition. We suggest GPR110 activation as a novel therapeutic strategy to ameliorate inflammation in the brain as well as periphery.
神经炎症是大脑各种病理过程的一个被广泛认可的潜在条件。在最近的一项研究中，内源性代谢物突触酰胺，由二十二碳六烯酸（DHA，22:6n-3）衍生而来，被鉴定为孤儿粘附 G 蛋白偶联受体 110（GPR110，ADGRF1）的特定配体。突触酰胺已被证明可抑制脂多糖（LPS）诱导的小鼠神经炎症，但 GPR110 在这一过程中的参与尚未确定。在这项研究中，我们研究了 GPR110 在介导突触酰胺在全身炎症状态下的抗炎作用中的可能免疫调节作用。
对于体外研究，我们使用定量 PCR（qPCR）和酶联免疫吸附测定（ELISA）以及中性粒细胞迁移和 ROS 产生测定，评估了 GPR110 在突触酰胺对 LPS 诱导的成年原代小鼠小胶质细胞、永生化鼠小胶质细胞（BV2）、原代中性粒细胞和腹腔巨噬细胞炎症反应中的作用。为了评估体内效应，使用 LPS 腹腔内（i.p.）或 TNF 静脉内（i.v.）注射后，给予野生型（WT）和 GPR110 敲除（KO）小鼠突触酰胺（i.p.），并通过 qPCR、ELISA 和 Western blot 分析测量促炎介质的表达。通过免疫染色 Iba-1 和 RelA 分别检测大脑中活化的小胶质细胞和细胞中 NF-kB 的激活。
LPS 腹腔内（i.p.）给药增加了血液中的 TNF 和 IL-1β，并诱导了大脑中的促炎细胞因子表达。随后腹腔内注射 GPR110 配体突触酰胺可显著减轻 LPS 诱导的野生型（WT）而非 GPR110 敲除（KO）小鼠的炎症反应。在培养的小胶质细胞中，突触酰胺通过抑制 NF-κB 亚基 RelA 向核内易位，增加 cAMP 并抑制 LPS 诱导的促炎细胞因子表达。使用靶向 N 端的抗体阻断突触酰胺与 GPR110 的结合，可消除这些作用。在中性粒细胞和巨噬细胞中发现 GPR110 表达较高，突触酰胺也导致 GPR110 依赖性 cAMP 增加和 LPS 诱导的促炎介质表达抑制。促炎细胞因子 TNF 的静脉内（i.v.）注射，在 LPS 处理后增加了循环中的 TNF，随后在 GPR110 依赖性方式下，注射（i.p.）突触酰胺可减轻脑内的炎症反应。
我们的研究表明 GPR110 在大脑和外周组织中具有免疫调节功能，共同促成了在全身炎症状态下突触酰胺的抗炎作用。我们建议 GPR110 激活作为一种新的治疗策略，以改善大脑和外周组织的炎症。
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