Siddiqui Tamjeed A, Lively Starlee, Schlichter Lyanne C
Genes and Development Division, Krembil Research Institute, University Health Network, Toronto, Ontario, M5T 2S8, Canada.
Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
J Neuroinflammation. 2016 Mar 24;13(1):66. doi: 10.1186/s12974-016-0531-9.
Microglia are the "professional" phagocytes of the CNS. Phagocytosis is crucial for normal CNS development and maintenance, but it can be either beneficial or detrimental after injury or disease. For instance, white matter damage releases myelin debris that must be cleared by microglia in order for re-myelination to occur. However, phagocytosis can also produce damaging reactive oxygen species (ROS). Furthermore, microglia can acquire pro-inflammatory (M1) or anti-inflammatory (M2) activation states that affect cell functions. Although microglia are exposed to a changing cytokine environment after injury or disease, little is known about the molecular and functional consequences. Therefore, we applied several microglial activation paradigms, with or without myelin debris. We assessed (i) gene expression changes reflecting microglial activation and inflammatory states, and receptors and enzymes related to phagocytosis and ROS production, (ii) myelin phagocytosis and production of ROS, and (iii) expression and contributions of several ion channels that are considered potential targets for regulating microglial behavior.
Primary rat microglia were exposed to cytokines, individually or sequentially. First, responses to individual M1 or M2 stimuli were compared: IFN-γ plus TNF-α ("I + T"; M1 activation), interleukin-4 (M2a/alternative activation), and interleukin-10 (M2c/acquired deactivation). Second, sequential cytokine addition was used to assess microglia repolarization and cell functions. The paradigms were M2a→M1, M2c→M1, M1→M2a, and M1→M2c.
M1 stimulation increased pro-inflammatory genes, phagocytosis, and ROS, as well as expression of Kv1.3, KCa3.1, and Kir2.1 channels. M2a stimulation increased anti-inflammatory genes, ROS production, and Kv1.3 and KCa3.1 expression. Myelin phagocytosis enhanced the M1 profile and dampened the M2a profile, and both phagocytosis and ROS production were dependent on NOX enzymes and Kir2.1 and CRAC channels. Importantly, microglia showed some capacity for re-polarization between M1 and M2a states, based on gene expression changes, myelin phagocytosis, and ROS production.
In response to polarizing and re-polarizing cytokine treatments, microglia display complex changes in gene transcription profiles, phagocytic capacity, NOX-mediated ROS production, and in ion channels involved in microglial activation. Because these changes might affect microglia-mediated CNS inflammation, they should be considered in future experimental, pre-clinical studies.
小胶质细胞是中枢神经系统的“专职”吞噬细胞。吞噬作用对中枢神经系统的正常发育和维持至关重要，但在损伤或疾病后可能有益也可能有害。例如，白质损伤会释放髓磷脂碎片，小胶质细胞必须清除这些碎片才能发生再髓鞘化。然而，吞噬作用也会产生具有损害性的活性氧（ROS）。此外，小胶质细胞可获得影响细胞功能的促炎（M1）或抗炎（M2）激活状态。尽管小胶质细胞在损伤或疾病后会暴露于不断变化的细胞因子环境中，但对其分子和功能后果知之甚少。因此，我们应用了几种小胶质细胞激活模式，有无髓磷脂碎片的情况均有。我们评估了：（i）反映小胶质细胞激活和炎症状态以及与吞噬作用和ROS产生相关的受体和酶的基因表达变化；（ii）髓磷脂吞噬作用和ROS的产生；以及（iii）几种被认为是调节小胶质细胞行为潜在靶点的离子通道的表达和作用。
将原代大鼠小胶质细胞单独或依次暴露于细胞因子。首先，比较对单个M1或M2刺激的反应：干扰素-γ加肿瘤坏死因子-α（“I+T”；M1激活）、白细胞介素-4（M2a/替代性激活）和白细胞介素-10（M2c/获得性失活）。其次，采用依次添加细胞因子的方法评估小胶质细胞的复极化和细胞功能。模式为M2a→M1、M2c→M1、M1→M2a和M1→M2c。
M1刺激增加了促炎基因、吞噬作用和ROS，以及Kv1.3、KCa3.1和Kir2.1通道的表达。M2a刺激增加了抗炎基因、ROS产生以及Kv1.3和KCa3.1的表达。髓磷脂吞噬作用增强了M1特征并减弱了M2a特征，吞噬作用和ROS产生均依赖于NOX酶以及Kir2.1和CRAC通道。重要的是，基于基因表达变化、髓磷脂吞噬作用和ROS产生，小胶质细胞在M1和M2a状态之间显示出一定的复极化能力。
响应极化和再极化细胞因子处理，小胶质细胞在基因转录谱、吞噬能力、NOX介导的ROS产生以及参与小胶质细胞激活的离子通道方面表现出复杂变化。由于这些变化可能影响小胶质细胞介导的中枢神经系统炎症，因此在未来的实验性临床前研究中应予以考虑。
