Abstract:
The ocular cytokine network plays pivotal roles in terms of the initiation and progression of retinal degeneration. Several types of immunocompetent cells such as microglia participate in inflammation, and a temporal transition in the molecular events of inflammation has been hypothesized. We previously found that the Csf2 gene was induced in the early phase of retinal degeneration. CSF2 participates in the transcriptional activation of several cytokines expressed by microglia; however, whether CSF2 is essential in this context is not known. In this work, we approach this question by using anti-CSF2 neutralizing bntibody and the protein synthesis inhibitor cycloheximide (CHX). We first revealed that CSF2 positively regulated the cytokine induction cascade using a CSF2-neutralizing antibody (anti-CSF2) to treat the microglial cell line that were activated by lipopolysaccharide (LPS). LPS or Lipid A stimulation in the presence of the protein synthesis inhibitor cycloheximide (CHX) led to cytokine superinduction, but suppression of the expression of a few cytokines was also noted in MG5 cells. To examine transitions of the molecular events within LPS-activated microglia, we next performed proteome analysis of MG5 cells stimulated with LPS for 0, 4, and 9 h. The Database for Annotation, Visualization, and Integrated Discovery analysis of differentially expressed proteins showed that various mRNA-modifying molecules were induced after LPS stimulation, in addition to molecules involved in inflammation. However, the numbers of common proteins founded in the comparison between the induced proteins of 4 and 9 h were only one-third and one-half of induced proteins at 4 and 9 h, respectively, suggesting dynamic transition of the induced proteins. LPS-induced mRNA-modifying proteins were almost completely suppressed by CHX, as expected, suggesting that transient induction of transcription-editing proteins plays an important role in terms of the phenotype of inflammation that develops in microglia after LPS stimulation.
摘要:
眼部细胞因子网络在视网膜变性的开始和进展方面起着关键作用。几种类型的免疫活性细胞,如小胶质细胞参与炎症,并且已经假设了炎症分子事件的时间转变。我们先前发现Csf2基因在视网膜变性的早期阶段被诱导。CSF2参与小胶质细胞表达的几种细胞因子的转录激活;然而,在这种情况下,CSF2是否必要尚不清楚。在这项工作中,我们通过使用抗CSF2中和抗体和蛋白质合成抑制剂环己酰亚胺(CHX)来解决这个问题。我们首先揭示了CSF2使用CSF2中和抗体(抗CSF2)正调节细胞因子诱导级联,以治疗由脂多糖(LPS)激活的小胶质细胞系。在蛋白质合成抑制剂环己酰亚胺(CHX)存在下的LPS或脂质A刺激导致细胞因子超诱导,但在MG5细胞中也注意到一些细胞因子的表达受到抑制。为了检查LPS激活的小胶质细胞内的分子事件的转变,我们接下来对用LPS刺激0、4和9小时的MG5细胞进行蛋白质组分析。可视化,和差异表达蛋白的整合发现分析表明,LPS刺激后诱导了各种mRNA修饰分子,除了参与炎症的分子。然而,在4和9h的诱导蛋白之间的比较中建立的常见蛋白的数量仅是4和9h的诱导蛋白的三分之一和二分之一,分别,提示诱导蛋白的动态转变。LPS诱导的mRNA修饰蛋白几乎完全被CHX抑制,正如预期的那样,提示转录编辑蛋白的瞬时诱导在LPS刺激后小胶质细胞中发展的炎症表型方面起着重要作用。