Glia maturation factor-gamma negatively modulates TLR4 signaling in macrophages

胶质细胞成熟因子-γ负调节巨噬细胞中的 TLR4 信号传导

• 批准号: 8149534
• 负责人: GRIFFIN RODGERS
• 金额: $ 69.25万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149534
• 关键词: Signal Transduction; TLR4 gene; glia maturation factor gamma; macrophage

Toll-like receptor 4 (TLR4) plays a critical role in innate immunity that recognize pathogenic molecules and trigger inflammatory response. However, excessive activation of TLR4 activation may contribute to pathogenesis of autoimmune and inflammatory diseases. Therefore, the negative regulation of TLR4-triggered inflammatory response has attracted much attention in recent years. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response. Glia maturation factor gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in macrophages immune response remains unclear. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction to LPS in macrophages by knockdown of GMFG using small-interfering RNA approach. We have demonstarted that knockdown of GMFG significantly enhanced LPS-induced production of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-8, and MCP-1 in human peripheral blood monocytes-derived macrophage as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay. Silencing of GMFG expression potentiates LPS-induced activation of p38, ERK1/2 and NF-kappaB signaling pathways by Western blot analysis. Moreover, luciferase assay revealed that gene silencing of GMFG promoted LPS-induced NF-kappaB activity for 2.5- to 4-fold. Furthermore, we found that TLR4 protein expression level were higher in GMFG-silenced macrophage compared with that of the control siRNA-transfected macrophages after stimulated with LPS for 1 hour. These results suggest that GMFG negatively regulation of TLR4 signaling-induced inflammatory cytokines by modulation of TLR4 expression levels and its down-stream NF-kappaB and p38 MAPK signaling pathway. In summary, we report that GMFG, in macrophage, function as a novel negative regulator that participates in the regulation of TLR4-signaling pathway, implicating that macrophage-specific modulation of GMFG may be beneficial in the treatment of inflammation as well as autoimmune disease.

Toll 样受体 4 (TLR4) 在识别致病分子并引发炎症反应的先天免疫中发挥着关键作用。然而，TLR4 的过度激活可能导致自身免疫性疾病和炎症性疾病的发病机制。因此，TLR4引发的炎症反应的负调控近年来备受关注。 脂多糖 (LPS) 激活 TLR4 信号通路会产生多种协调免疫反应的细胞因子和介质。 神经胶质成熟因子 γ (GMFG) 是调节肌动蛋白细胞骨架重组的 ADF/cofilin 蛋白家族的成员，优先在炎症细胞中表达，但其在巨噬细胞免疫反应中的功能仍不清楚。在这项研究中，我们通过使用小干扰RNA方法敲低GMFG，研究了GMFG是否参与了巨噬细胞中LPS炎症反应的分子事件。我们已经证明，通过实时定量测定，GMFG 的敲低显着增强了 LPS 诱导的促炎细胞因子和趋化因子的产生，包括人外周血单核细胞来源的巨噬细胞中的 TNF-α、IL-1β、IL-8 和 MCP-1 -PCR并通过酶联免疫吸附测定法证实。通过蛋白质印迹分析，GMFG 表达的沉默增强了 LPS 诱导的 p38、ERK1/2 和 NF-kappaB 信号通路的激活。此外，荧光素酶测定显示，GMFG 基因沉默可促进 LPS 诱导的 NF-κB 活性 2.5 至 4 倍。此外，我们发现用LPS刺激1小时后，与对照siRNA转染的巨噬细胞相比，GMFG沉默的巨噬细胞中的TLR4蛋白表达水平更高。这些结果表明，GMFG 通过调节 TLR4 表达水平及其下游 NF-κB 和 p38 MAPK 信号通路，对 TLR4 信号传导诱导的炎症细胞因子进行负调节。总之，我们报告巨噬细胞中的 GMFG 作为一种新型负调节因子参与 TLR4 信号通路的调节，这表明巨噬细胞特异性调节 GMFG 可能有益于炎症和自身免疫性疾病的治疗。