Glia maturation factor-gamma negatively modulates TLR4 signaling in macrophages

Toll样受体4(TLR4)在识别致病分子和触发炎症反应的先天免疫中起着关键作用。然而，TLR4的过度激活可能参与了自身免疫性疾病和炎症性疾病的发病。因此，近年来对TLR4引发的炎症反应的负性调控备受关注。 脂多糖(LPS)激活TLR4信号通路可产生多种细胞因子和调节免疫反应的介质。胶质成熟因子γ(GMFG)是ADF/cofilin家族中的一员，调节肌动蛋白的细胞骨架重组，主要表达于炎症细胞，但其在巨噬细胞免疫反应中的作用尚不清楚。在这项研究中，我们利用小干扰RNA的方法研究了GMFG是否参与了巨噬细胞对内毒素的炎症反应的分子事件。我们用实时定量聚合酶链式反应和酶联免疫吸附试验证实，GMFG基因敲除可显著增强内毒素诱导的人外周血单核细胞来源的巨噬细胞产生促炎细胞因子和趋化因子，包括肿瘤坏死因子-α、白介素1-β、白介素8和单核细胞趋化蛋白-1。Western印迹分析显示，沉默GMFG表达可增强内毒素诱导的p38、ERK1/2和NF-kappaB信号通路的激活。此外，荧光素酶分析显示，GMFG的基因沉默使内毒素诱导的NF-kappaB活性增加了2.5-4倍。此外，我们还发现GMFG沉默的巨噬细胞在脂多糖刺激1h后，TLR4蛋白的表达水平明显高于对照组。提示GMFG通过调节TLR4的表达水平及其下游的NF-kappaB和p38MAPK信号通路，对TLR4信号诱导的炎性细胞因子产生负性调节。综上所述，我们报道了GMFG在巨噬细胞中作为一种新的负性调节因子参与TLR4信号通路的调节，暗示巨噬细胞特异性调节GMFG在治疗炎症和自身免疫性疾病方面可能是有益的。

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.