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首页> 外文期刊>Journal of neuroinflammation >Neuronal toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro
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Neuronal toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro

机译：神经元Toll样受体4信号传导诱导体外脑内皮细胞活化和中性粒细胞迁移

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Background The innate immune response in the brain is initiated by pathogen-associated molecular patterns (PAMPS) or danger-associated molecular patterns (DAMPS) produced in response to central nervous system (CNS) infection or injury. These molecules activate members of the Toll-like receptor (TLR) family, of which TLR4 is the receptor for bacterial lipopolysaccharide (LPS). Although neurons have been reported to express TLR4, the function of TLR4 activation in neurons remains unknown. Methods TLR4 mRNA expression in primary mouse glial and neuronal cultures was assessed by RT-PCR. Mouse mixed glial, neuronal or endothelial cell cultures were treated with LPS in the absence or the presence of a TLR4 specific antagonist (VIPER) or a specific JNK inhibitor (SP600125). Expression of inflammatory mediators was assayed by cytometric bead array (CBA) and ELISA. Activation of extracellular-signal regulated kinase 1/2 (ERK1/2), p38, c-Jun-N-terminal kinase (JNK) and c-Jun was assessed by Western blot. The effect of conditioned media of untreated- versus LPS-treated glial or neuronal cultures on endothelial activation was assessed by neutrophil transmigration assay, and immunocytochemistry and ELISA were used to measure expression of intercellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1). Results LPS induces strong release of the chemokines RANTES and CXCL1 (KC), tumor necrosis factor-α (TNFα) and IL-6 in primary mouse neuronal cultures. In contrast, LPS induced release of IL-1α, IL-1β and granulocyte-colony stimulating factor (G-CSF) in mixed glial, but not in neuronal cultures. LPS-induced neuronal KC expression and release were completely blocked by VIPER. In glial cultures, LPS induced activation of ERK1/2, p38 and JNK. In contrast, in neuronal cultures, LPS activated JNK but not ERK1/2 or p38, and the specific JNK inhibitor SP600125 significantly blocked LPS-induced KC expression and release. Finally, conditioned medium of LPS-treated neuronal cultures induced strong expression of ICAM-1 and VCAM-1 on endothelial cells, and induced infiltration of neutrophils across the endothelial monolayer, which was inhibited by VIPER. Conclusion These data demonstrate for the first time that neurons can play a role as key sensors of infection to initiate CNS inflammation.
机译：背景技术脑中的先天免疫应答是由病原体相关分子模式（PAMPS）或响应中枢神经系统（CNS）感染或损伤而产生的危险相关分子模式（DAMPS）引发的。这些分子激活Toll样受体（TLR）家族的成员，其中TLR4是细菌脂多糖（LPS）的受体。尽管已经报道了神经元表达TLR4，但是神经元中TLR4激活的功能仍然未知。方法采用RT-PCR技术检测小鼠胶质和神经元原代培养物中TLR4 mRNA的表达。在没有或没有TLR4特异性拮抗剂（VIPER）或特异性JNK抑制剂（SP600125）的情况下，用LPS处理小鼠混合的神经胶质，神经元或内皮细胞培养物。通过细胞计数珠阵列（CBA）和ELISA测定炎性介质的表达。通过蛋白质印迹评估细胞外信号调节激酶1/2（ERK1 / 2），p38，c-Jun-N-末端激酶（JNK）和c-Jun的激活。通过中性粒细胞迁移测定评估未处理和LPS处理的神经胶质或神经元培养条件培养基对内皮激活的影响，并使用免疫细胞化学和ELISA来测量细胞间粘附分子（ICAM-1）和血管粘附分子的表达（VCAM-1）。结果LPS诱导小鼠原代神经细胞培养物中的趋化因子RANTES和CXCL1（KC），肿瘤坏死因子-α（TNFα）和IL-6强烈释放。相反，LPS诱导混合胶质细胞释放IL-1α，IL-1β和粒细胞集落刺激因子（G-CSF），但在神经元培养物中则不释放。 LPS诱导的神经元KC表达和释放被VIPER完全阻断。在神经胶质细胞培养物中，LPS诱导ERK1 / 2，p38和JNK活化。相反，在神经元培养物中，LPS激活JNK，但不激活ERK1 / 2或p38，并且特定的JNK抑制剂SP600125显着阻断LPS诱导的KC表达和释放。最后，用LPS处理的神经元培养液的条件培养基诱导内皮细胞上ICAM-1和VCAM-1的强烈表达，并诱导嗜中性白细胞穿过内皮单层浸润，这被VIPER抑制。结论这些数据首次证明神经元可以作为感染的关键传感器，引发中枢神经系统炎症。

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