A Liquid Culture System Model For Adult Hematopoiesis At The Molecular Level

中性粒细胞是循环中最丰富的白细胞，在特异性免疫应答发生之前提供针对细菌病原体的主要先天免疫防御功能。 脂多糖（LPS）激活TLR 4信号通路导致产生一系列广泛的细胞因子和介质，协调免疫反应。TLR 4介导的炎症反应受到严格的调控，由于TLR 4的过度激活可导致多种炎症性疾病，因此对TLR 4引发的炎症反应的负调控备受关注。胶质细胞成熟因子γ（GMFG）是一种新的肌动蛋白细胞骨架重组因子，优先在炎症细胞中表达，但其在白细胞中的功能尚不清楚。在这项研究中，我们研究了GMFG是否参与炎症反应的分子事件，以及GMFG如何介导中性粒细胞对LPS的炎症反应。我们在这里显示，小干扰RNA介导的敲低GMFG表达的白细胞表现出大于80%的抑制内源性表达水平的促炎细胞因子和趋化因子，包括TNF-α，IL-1 β，IL-6和IL-8使用定量RT-PCR。这些减少的细胞因子产生可以通过用TLR 4激动剂LPS刺激而部分恢复，但与非靶向沉默对照细胞相比，GMFG细胞的沉默水平显著较低（TNF-α的抑制率为71%+/-3.9%，IL-1 β的抑制率为68%+/-6.9%，IL-6的抑制率为50%+/-4.3%，IL-8的抑制率为40%+/-3.1%）。通过Western印迹分析，GMFG表达的敲低导致磷酸化p38 MAPK水平降低以及NF-κ B抑制。这些数据表明GMFG的内源性表达是基础细胞因子和趋化因子应答及其对LPS刺激的诱导应答所必需的。此外，免疫荧光分析显示，GMFG基因沉默抑制LPS诱导的髓系HL-60和THP-1细胞中NF-κ B的核转位。与此相反，瞬时过表达GMFG显着增加内源性促炎细胞因子的水平，1.82.2倍，磷酸化p38 MAPK和核转位的NF-κ B在LPS刺激的情况下。这些结果表明，GMFG介导的炎症细胞因子的调节通过激活p38 MAPK和NF-κ B信号通路发生。总之，我们的研究结果确定GMFG作为一种新的因素，参与调节TLR 4介导的控制细胞因子和趋化因子的表达，通过p38 MAPK和NF-κ B途径。这些数据，如果在体内实验中证实，暗示GMFG作为一个潜在的目标，在嗜中性粒细胞介导的自身免疫性疾病的治疗干预。

Neutrophils are the most abundant leukocytes in the circulation and provide a primary innate immune defense function against bacterial pathogens before the development of a specific immune response. 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. The TLR4-mediated inflammatory response is tightly regulated, since excessive activation of TLR4 may induce a variety of inflammatory diseases, so the negative regulation of TLR-triggered inflammatory response attracts much attention. Glia maturation factor gamma (GMFG), a novel factor in actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in leucocytes is undefined. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction and how GMFG mediated neutrophils inflammatory response to LPS. We show here that small interfering RNA-mediated knockdown of GMFG expression in leukocytes exhibited greater than 80% inhibition of the endogenous expression levels of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-6, and IL-8 using quantitative RT-PCR. These decreased cytokine productions could be partially restored by stimulation with TLR4 agonist LPS, but exhibit significantly lower level in silencing of GMFG cells compared to non-targeting silencing control cells (inhibition by 71%+/-3.9% of TNF-alpha, 68%+/-6.9% of IL-1beta, 50%+/-4.3% of IL-6 and 40%+/-3.1% of IL-8). Knockdown of GMFG expression results in decreased phospho-p38 MAPK levels as well as suppressed NF-kappaB by Western blot analysis. These data suggest that endogenous expression of GMFG is required for basal cytokine and chemokine response and their induced response to LPS stimulation. Moreover, immunofluorescence analysis revealed that gene silencing of GMFG inhibited LPS-induced nuclear translocation of NF-kappaB in myeloid HL-60 and THP-1 cells. In contrast, transient overexpression of GMFG significantly enhanced endogenous level of proinflammatory cytokines by 1.82.2 fold, phosphor-p38 MAPK and nuclear translocation of NF-kappaB in the absence of LPS stimulation. These results suggest that GMFG-mediated regulation of inflammatory cytokines occurs through activation of p38 MAPK and NF-kappaB signaling pathway. Taken together, our findings identify GMFG as a novel factor that participates in the regulation of TLR4-mediated control of cytokine and chemokine expression through the p38 MAPK and NF-kappaB pathway. These data, if confirmed in in vivo experiments, implicates GMFG as a potential target for therapeutic intervention in neutrophil-mediated autoimmune disease.