Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics.
Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics.
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DOI：
10.1074/mcp.m115.053926
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发表时间：
2015-12
期刊：
影响因子：
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通讯作者：
Stevens SM Jr
中科院分区：
文献类型：
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作者：
Bell-Temin H;Culver-Cochran AE;Chaput D;Carlson CM;Kuehl M;Burkhardt BR;Bickford PC;Liu B;Stevens SM Jr
小胶质细胞是大脑中的常驻免疫细胞，已被证明根据其激活状态，表现出从神经营养到神经毒性的复杂作用。小胶质细胞可分为四个激活阶段，M1与经典(促炎)激活阶段最接近，以及交替激活阶段M2a、M2b和M2c。可供选择的激活阶段还没有通过无偏见的、全球范围的蛋白质表达谱进行全面分析。在这项研究中，BV2小鼠永生化的小胶质细胞被四个阶段的特异性激动剂刺激，并使用基于SILAC的蛋白质组学分析来定量4644个蛋白质组的总蛋白表达。在通过靶向细胞因子分析和激活状态的Western blotting验证了不同阶段的诱导后，数据揭示了对不同状态之间的相似和不同的新见解。这些数据识别了几个蛋白质组，它们在抗炎、促进愈合的激活状态下的表达可能是通过差异蛋白表达来抑制炎症激活，在M2a状态下包括CD74、Lyn、SQST1、TLR2和CD14。这些蛋白的差异表达促进愈合，限制吞噬作用，并通过Toll样受体级联限制活性氮物种的激活。M2c状态似乎集中在下调富含肌动蛋白的吞噬小体形成的关键成员SLP-76。此外，蛋白质组学数据发现了一个新的激活标记DAB2，它参与了网状蛋白介导的内吞作用，并且在M2a和M1或M2b状态之间存在显著差异。用不同激动剂刺激的小鼠原代小胶质细胞经经典和交替激活状态的Western印迹分析显示，DAB2的表达趋势与BV2细胞相似。
Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.