Exposure to Inflammatory Cytokines IL-1β and TNFα Induces Compromise and Death of Astrocytes; Implications for Chronic Neuroinflammation

BackgroundAstrocytes have critical roles in the human CNS in health and disease. They provide trophic support to neurons and are innate-immune cells with keys roles during states-of-inflammation. In addition, they have integral functions associated with maintaining the integrity of the blood-brain barrier. MethodsWe have used cytometric bead arrays and xCELLigence technology to monitor the to monitor the inflammatory response profiles and astrocyte compromise in real-time under various inflammatory conditions. Responses were compared to a variety of inflammatory cytokines known to be released in the CNS during neuroinflammation. Astrocyte compromise measured by xCELLigence was confirmed using ATP measurements, cleaved caspase 3 expression, assessment of nuclear morphology and cell death. ResultsInflammatory activation (IL-1β or TNFα) of astrocytes results in the transient production of key inflammatory mediators including IL-6, cell surface adhesion molecules, and various leukocyte chemoattractants. Following this phase, the NT2-astrocytes progressively become compromised, which is indicated by a loss of adhesion, appearance of apoptotic nuclei and reduction in ATP levels, followed by DEATH. The earliest signs of astrocyte compromise were observed between 24-48h post cytokine treatment. However, significant cell loss was not observed until at least 72h, where there was also an increase in the expression of cleaved-caspase 3. By 96 hours approximately 50% of the astrocytes were dead, with many of the remaining showing signs of compromise too. Numerous other inflammatory factors were tested, however these effects were only observed with IL-1β or TNFα treatment. ConclusionsHere we reveal direct sensitivity to mediators of the inflammatory milieu. We highlight the power of xCELLigence technology for revealing the early progressive compromise of the astrocytes, which occurs 24-48 hours prior to substantive cell loss. Death induced by IL-1β or TNFα is relevant clinically as these two cytokines are produced by various peripheral tissues and by resident brain cells.背景：星形胶质细胞（astrocytes）在健康与疾病状态下均对人类中枢神经系统（Central Nervous System, CNS）发挥关键调控作用。它们可为神经元提供营养支持，同时作为先天免疫细胞在炎症状态下扮演核心角色。此外，星形胶质细胞还在维持血脑屏障（blood-brain barrier）完整性方面具有不可或缺的功能。 方法：我们采用流式微球阵列（cytometric bead arrays）与xCELLigence技术，在多种炎症条件下实时监测星形胶质细胞的炎症应答谱与功能受损情况。将检测得到的应答情况与已知在神经炎症（neuroinflammation）期间于中枢神经系统中释放的多种炎症细胞因子（inflammatory cytokines）进行对比。通过三磷酸腺苷（ATP）水平检测、裂解型半胱氨酸天冬氨酸蛋白酶3（cleaved caspase 3）表达量分析、细胞核形态评估以及细胞死亡检测，对xCELLigence技术测得的星形胶质细胞受损情况进行验证。 结果：星形胶质细胞经炎症激活（白细胞介素1β（IL-1β）或肿瘤坏死因子α（TNFα））后，会短暂产生多种关键炎症介质，包括白细胞介素6（IL-6）、细胞表面黏附分子（cell surface adhesion molecules）以及各类白细胞趋化因子（leukocyte chemoattractants）。此阶段过后，NT2-星形胶质细胞（NT2-astrocytes）会逐渐出现功能受损，表现为黏附能力丧失、凋亡细胞核（apoptotic nuclei）出现以及ATP水平降低，最终发生细胞死亡。在细胞因子处理后的24-48小时即可观察到星形胶质细胞受损的最早迹象，但直至至少72小时才会出现显著的细胞丢失，此时裂解型半胱氨酸天冬氨酸蛋白酶3的表达量也会升高。至96小时时，约50%的星形胶质细胞已死亡，剩余细胞中也有多数表现出受损迹象。我们还检测了多种其他炎症因子，但仅在IL-1β或TNFα处理组中观察到了上述效应。 结论：本研究揭示了星形胶质细胞对炎症微环境（inflammatory milieu）介质的直接敏感性。我们证实了xCELLigence技术可有效揭示星形胶质细胞的早期进行性受损——该受损过程发生于实质性细胞丢失前的24-48小时。IL-1β与TNFα诱导的细胞死亡具有临床相关性，因为这两种细胞因子可由多种外周组织以及脑内常驻细胞产生。