Glia maturation factor-gamma modulation of signaling pathways in macrophages

沙门氏菌是一种细胞内细菌病原体，在膜结合腔内复制并改变宿主铁代谢以维持自身生存。吞噬细胞内的持续生存和复制是沙门氏菌感染发病机制的核心。巨噬细胞在调节铁代谢以确保体内铁的充足性和控制病原体细胞内增殖的铁的可用性方面发挥关键作用。然而，沙门氏菌诱导的巨噬细胞铁代谢变化与该病原体在巨噬细胞内存活和复制机制的关系尚不清楚。因此，确定参与沙门氏菌细胞内存活和复制的宿主因素对于设计更有效的抗菌药物至关重要。神经胶质成熟因子γ (GMFG)是一种新的肌动蛋白相关蛋白-2/3 （Arp2/3）复合物的调节因子，主要在炎症细胞中表达。我们之前已经证明GMFG负向调节tlr4诱导的促炎信号，但其在巨噬细胞对细胞内细菌感染的反应中的功能尚不清楚。在这项研究中，我们通过小干扰RNA （RNAi）技术来研究GMFG在沙门氏菌感染的小鼠巨噬细胞中的作用。我们发现，与对照siRNA转染的Raw264.7巨噬细胞相比，敲低GMFG显著提高了感染后24小时细胞内沙门氏菌的生长数量（>3倍，p< 0.008）。然而，感染后4小时观察到的细菌生长数量没有显著差异，表明GMFG不影响细菌的吞噬作用。感染后24小时，免疫荧光显微镜也显示在GMFG敲除的巨噬细胞中有沙门氏菌的积累。敲低GMFG可显著降低Raw264.7巨噬细胞中铁输出铁转运蛋白水平，提高铁储存铁蛋白- l水平。敲除gmfg的巨噬细胞细胞内铁含量比正常巨噬细胞高1.9倍，p< 0.05)。这些观察结果表明，GMFG对沙门氏菌胞内生长的调节作用可能是通过调节巨噬细胞铁代谢来实现的。此外，与之前的研究一致，我们发现感染沙门氏菌的Raw264.7巨噬细胞增加了铁转运蛋白铁转运蛋白和铁蛋白的表达，这表明这是宿主通过限制细胞内微生物对铁的获取来防御细胞内微生物感染的防御策略。虽然沙门氏菌感染24小时后，GMFG蛋白水平没有明显改变，但感染沙门氏菌的GMFG敲除巨噬细胞与对照组相比，细胞内铁含量和铁储存蛋白铁蛋白均有所增加。进一步分析沙门氏菌感染的gmfg敲除巨噬细胞中细胞因子的表达，发现在感染后24小时，促炎tnf - α mRNA（1.86倍，p<0.05）和抗炎IL-10 mRNA（2.2倍，p<0.0）的表达增强，这与细胞内沙门氏菌复制的增加是平行的。我们的研究结果表明，GMFG调节了感染沙门氏菌的小鼠巨噬细胞铁代谢和免疫功能的关键途径。

Salmonella is an intracellular bacterial pathogen that replicates within membrane-bound compartment and alters host iron metabolism for its own survival. Persistent survival and replication within phagocytes is central to the pathogenesis of Salmonella infections. Macrophages play a critical role in regulating iron metabolism for securing body iron sufficiency and controlling the availability of iron for intracellular proliferation of pathogens. However, the relationship of Salmonella- induced changes of macrophage iron metabolism to the survival and replication mechanism of this pathogen within macrophages remains poorly understood. Thus, it is critical to identify the host factors involved in the intracellular survival and replication of Salmonella in order to design more-efficient antimicrobial therapeutics. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously demonstrated that GMFG negatively regulate TLR4-induced proinflammatory signaling, but its function in macrophage response to intracellular bacteria infection remains unclear. In this study, we investigated the role of GMFG in Salmonella-infected murine macrophages by using small-interfering RNA (RNAi) techniques to knockdown GMFG. We found that knockdown of GMFG significantly enhanced the numbers of intracellular Salmonella growth (>3-fold, p< 0.008) at 24 hr postinfection compared with control siRNA transfected Raw264.7 macrophages. However, there was no significant difference in growth numbers of bacteria observed at 4 hr postinfection, indicating that GMFG does not influence bacterial phagocytosis. Immunofluorescence microscopy also revealed an accumulation of Salmonella, in GMFG knockdown macrophages at 24 hr postinfection. Knockdown of GMFG results in marked decreased the iron exporter ferroportin protein levels and increased iron storage ferritin-L protein levels in Raw264.7 macrophages. Further, the intracellular iron content was elevated in GMFG-knockdown macrophages compared with control macrophages (1.9-fold, p< 0.05). These observations indicate that regulatory impact of GMFG in Salmonella intracellular growth may be through modulation of macrophage iron metabolism. Moreover, consistent with previous studies, we found that Raw264.7 macrophage infections with Salmonella increase the expression of the iron transporter ferroportin and ferritin, indicating this is the host defense strategy against infection with intracellular microbes by limiting their access to iron. Although there was no marked altered in GMFG protein level after 24hr infection with Salmonella, GMFG knockdown macrophage infected with Salmonella displayed increased the intracellular iron content and iron storage protein ferritin compared with control macrophages. Further analysis of cytokines expression in Salmonella-infected GMFG-knockdown macrophage revealed enhanced the proinflammatory TNF-alpha mRNA (1.86-fold, p<0.05) and anti-inflammatory IL-10 mRNA (2.2-fold, p< 0.0) at 24 hr postinfection, which is paralleled with increased intracellular Salmonella replication. Our results suggest that GMFG modulation of crucial pathways of macrophage iron metabolism and immune function in murine macrophages infected with Salmonella.