BACKGROUND In the extracellular environment, lysophosphatidic acid (LPA) species are generated via autotaxin (ATX)-mediated hydrolysis of lysophospholipid precursors. Members of the LPA family are potent lipid mediators transmitting signals via six different G protein-coupled LPA receptors (LPAR1-6). The LPA signaling axis is indispensable for brain development and function of the nervous system; however, during damage of the central nervous system, LPA levels can increase and aberrant signaling events counteract brain function. Here, we investigated regulation of the ATX/LPA/LPAR axis in response to lipopolysaccharide-induced systemic inflammation in mice and potential neurotoxic polarization programs in LPA-activated primary murine microglia. METHODS In vivo, LPAR1-6 expression was established by qPCR in whole murine brain homogenates and in FACS-sorted microglia. ELISAs were used to quantitate LPA concentrations in the brain and cyto-/chemokine secretion from primary microglia in vitro. Transcription factor phosphorylation was analyzed by immunoblotting, and plasma membrane markers were analyzed by flow cytometry. We used MAPK inhibitors to study signal integration by the JNK, p38, and ERK1/2 branches in response to LPA-mediated activation of primary microglia. RESULTS Under acute and chronic inflammatory conditions, we observed a significant increase in LPA concentrations and differential regulation of LPAR, ATX (encoded by ENPP2), and cytosolic phospholipase A2 (encoded by PLA2G4A) gene expression in the brain and FACS-sorted microglia. During pathway analyses in vitro, the use of specific MAPK antagonists (SP600125, SB203580, and PD98059) revealed that JNK and p38 inhibition most efficiently attenuated LPA-induced phosphorylation of proinflammatory transcription factors (STAT1 and -3, p65, and c-Jun) and secretion of IL-6 and TNFα. All three inhibitors decreased LPA-mediated secretion of IL-1β, CXCL10, CXCL2, and CCL5. The plasma membrane marker CD40 was solely inhibited by SP600125 while all three inhibitors affected expression of CD86 and CD206. All MAPK antagonists reduced intracellular COX-2 and Arg1 as well as ROS and NO formation, and neurotoxicity of microglia-conditioned media. CONCLUSION In the present study, we show that systemic inflammation induces aberrant ATX/LPA/LPAR homeostasis in the murine brain. LPA-mediated polarization of primary microglia via MAPK-dependent pathways induces features reminiscent of a neurotoxic phenotype.

中文翻译：

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MAPK信号决定了溶血磷脂酸（LPA）引起的小胶质细胞炎症。

背景技术在细胞外环境中，溶血磷脂酸（LPA）物质是通过自分泌生物素（ATX）介导的溶血磷脂前体水解产生的。LPA家族的成员是有效的脂质介体，可通过六个不同的G蛋白偶联LPA受体（LPAR1-6）传递信号。LPA信号轴对于大脑发育和神经系统功能必不可少。但是，在中枢神经系统受损期间，LPA水平会升高，异常的信号事件会抵消大脑的功能。在这里，我们调查了ATX / LPA / LPAR轴的调节，以响应脂多糖诱导的小鼠全身炎症和LPA激活的原代小鼠小胶质细胞的潜在神经毒性极化程序。方法体内 通过qPCR在鼠全脑匀浆和FACS分选的小胶质细胞中建立了LPAR1-6表达。ELISA用于定量脑中LPA浓度和体外原发性小胶质细胞分泌的细胞/趋化因子。通过免疫印迹分析转录因子的磷酸化，并通过流式细胞术分析质膜标记。我们使用MAPK抑制剂来研究JLP，p38和ERK1 / 2分支对LPA介导的原发性小胶质细胞活化的信号整合。结果在急性和慢性炎症条件下，我们观察到大脑和FACS分类的小胶质细胞中LPA浓度显着增加，并且LPAR，ATX（由ENPP2编码）和胞质磷脂酶A2（由PLA2G4A编码）的基因表达明显不同。在体外途径分析中，使用特定的MAPK拮抗剂（SP600125，SB203580和PD98059）显示，JNK和p38抑制作用最有效地减弱了LPA诱导的促炎性转录因子（STAT1和-3，p65和c-Jun）的磷酸化以及IL-6的分泌和TNFα。所有这三种抑制剂均降低了LPA介导的IL-1β，CXCL10，CXCL2和CCL5的分泌。质膜标记CD40仅受SP600125抑制，而所有三种抑制剂均影响CD86和CD206的表达。所有MAPK拮抗剂均能减少细胞内COX-2和Arg1以及ROS和NO的形成以及小胶质细胞条件培养基的神经毒性。结论在本研究中，我们表明全身性炎症在鼠脑中诱导异常的ATX / LPA / LPAR稳态。