Abstract：
The major immune cells of the central nervous systems (CNS) are microglia and astrocytes, subsets of the glial cell population. The crosstalk between glia via soluble signaling molecules plays an indispensable role for neuropathologies, brain development as well as homeostasis. However, the investigation of the microglia-astrocyte crosstalk has been hampered due to the lack of suitable glial isolation methods. In this study, we investigated for the first time the crosstalk between highly purified Toll-like receptor (TLR)2-knock out (TLR2-KO) and wild-type (WT) microglia and astrocytes. We examined the crosstalk of TLR2-KO microglia and astrocytes in the presence of WT supernatants of the respective other glial cell type. Interestingly, we observed a significant TNF release by TLR2-KO astrocytes, which were activated with Pam3CSK4-stimulated WT microglial supernatants, strongly indicating a crosstalk between microglia and astrocytes after TLR2/1 activation. Furthermore, transcriptome analysis using RNA-seq revealed a wide range of significant up- and down-regulated genes such as Cd300, Tnfrsf9 or Lcn2, which might be involved in the molecular conversation between microglia and astrocytes. Finally, co-culturing microglia and astrocytes confirmed the prior results by demonstrating a significant TNF release by WT microglia co-cultured with TLR2-KO astrocytes. Our findings suggest a molecular TLR2/1-dependent conversation between highly pure activated microglia and astrocytes via signaling molecules. Furthermore, we demonstrate the first crosstalk experiments using ∼100% pure microglia and astrocyte mono-/co-cultures derived from mice with different genotypes highlighting the urgent need of efficient glial isolation protocols, which particularly holds true for astrocytes.
摘要：
中枢神经系统（CNS）的主要免疫细胞是小胶质细胞和星形胶质细胞，神经胶质细胞群的亚群。胶质细胞之间通过可溶性信号分子的串扰在神经病理学中起着不可或缺的作用，大脑发育和稳态。然而,由于缺乏合适的胶质细胞分离方法,小胶质细胞-星形胶质细胞串扰的研究受到阻碍.在这项研究中,我们首次研究了高度纯化的Toll样受体（TLR）2敲除（TLR2-KO）与野生型（WT）小胶质细胞和星形胶质细胞之间的串扰。我们检查了在其他神经胶质细胞类型的WT上清液存在下TLR2-KO小胶质细胞和星形胶质细胞的串扰。有趣的是,我们观察到TLR2-KO星形胶质细胞释放显著的TNF，用Pam3CSK4刺激的WT小胶质细胞上清液激活，强烈表明TLR2/1激活后小胶质细胞和星形胶质细胞之间的串扰。此外，使用RNA-seq的转录组分析揭示了广泛的显着上调和下调的基因，例如Cd300，Tnfrsf9或Lcn2，这些基因可能参与小胶质细胞和星形胶质细胞之间的分子对话。最后,共培养小胶质细胞和星形胶质细胞通过证明与TLR2-KO星形胶质细胞共培养的WT小胶质细胞的显著TNF释放证实了先前的结果。我们的发现表明，高纯度激活的小胶质细胞和星形胶质细胞之间通过信号分子进行分子TLR2/1依赖性对话。此外，我们展示了第一个串扰实验，使用100%纯的小胶质细胞和星形胶质细胞单/共培养物来自不同基因型的小鼠，突出了高效神经胶质分离方案的迫切需要，这对星形胶质细胞尤其适用。
