Glia maturation factor-gamma modulation of signaling pathways in macrophages

巨噬细胞信号通路的神经胶质成熟因子-γ调节

• 批准号: 9157363
• 负责人: GRIFFIN RODGERS
• 金额: $ 56.41万
• 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/9157363
• 关键词: Actins; Address; Anti-Inflammatory Agents; Anti-inflammatory; Bacteria; Binding; Biological Process; Blood; Categories; Cell Lineage; Cells; Clinic; Complex; Data; Development; Disease; Dysmyelopoietic Syndromes; Erythroid; Exposure to; Ferritin; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic Transcription; Global Change; Granulopoiesis; Growth; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Host Defense; Immunofluorescence Microscopy; Infection; Inflammatory; Integration Host Factors; Interleukin-10; Iron; L-ferritin; Leukemic Cell; Liquid substance; Maps; Membrane; Messenger RNA; Methods; Microbe; Molecular Profiling; Mus; Mutation; Myelogenous; Myeloproliferation; Oncogenes; Ontology; Pathogenesis; Pathway interactions; Phagocytes; Phagocytosis; Play; Population; Proteins; RNA Interference; Role; Salmonella; Salmonella infections; Secure; Signal Pathway; Signal Transduction; Small Interfering RNA; Stem cells; Structure; Suggestion; System; TLR4 gene; Techniques; Therapeutic; Time; Trees; Tumor Necrosis Factor-alpha; Two-Dimensional Gel Electrophoresis; antimicrobial; cell type; cytokine; design; glia maturation factor gamma; human TNF protein; immune function; iron metabolism; leukemia; macrophage; metal transporting protein 1; novel; pathogen; progenitor; response; stem; tool; two-dimensional

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.

沙门氏菌是一种细胞内细菌病原体，其在膜结合区室内复制并改变宿主的铁代谢以使其自身存活。在吞噬细胞内的持续存活和复制是沙门氏菌感染的发病机制的核心。巨噬细胞在调节铁代谢以确保机体铁充足和控制病原体细胞内增殖所需的铁的可用性方面起着关键作用。然而，沙门氏菌诱导的巨噬细胞铁代谢的变化与该病原体在巨噬细胞内的存活和复制机制的关系仍然知之甚少。因此，确定沙门氏菌细胞内存活和复制所涉及的宿主因子以设计更有效的抗微生物治疗剂是至关重要的。胶质细胞成熟因子γ（GMFG）是肌动蛋白相关蛋白2/3（Arp 2/3）复合物的一种新的调节因子，主要在炎性细胞中表达。我们先前已经证明GMFG负性调节TLR 4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，我们通过使用小干扰RNA（RNAi）技术敲除GMFG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Raw 264.7巨噬细胞相比，GMFG的敲低在感染后24小时显著增加了细胞内沙门氏菌生长的数量（>3倍，p< 0.008）。然而，在感染后4小时观察到的细菌生长数没有显著差异，表明GMFG不影响细菌的吞噬作用。免疫荧光显微镜也显示沙门氏菌的积累，在GMFG敲除巨噬细胞在感染后24小时。GMFG的敲低导致Raw 264.7巨噬细胞中铁输出转运蛋白蛋白水平显著降低和铁储存铁蛋白-L蛋白水平增加。此外，与对照巨噬细胞相比，GMFG敲减的巨噬细胞中的细胞内铁含量升高（1.9倍，p< 0.05）。这些观察结果表明，GMFG在沙门氏菌细胞内生长的调节作用可能是通过调节巨噬细胞铁代谢。此外，与以前的研究一致，我们发现沙门氏菌感染Raw 264.7巨噬细胞增加了铁转运蛋白和铁蛋白的表达，表明这是宿主通过限制其获得铁来对抗细胞内微生物感染的防御策略。尽管沙门氏菌感染24小时后GMFG蛋白水平没有明显变化，但与对照巨噬细胞相比，沙门氏菌感染GMFG敲除的巨噬细胞显示细胞内铁含量和铁储存蛋白铁蛋白增加。对沙门氏菌感染的GMFG敲除巨噬细胞中细胞因子表达的进一步分析显示，感染后24小时，促炎性TNF-alpha mRNA（1.86倍，p<0.05）和抗炎性IL-10 mRNA（2.2倍，p< 0.0）增强，这与细胞内沙门氏菌复制的增加平行。我们的研究结果表明，GMFG调节巨噬细胞铁代谢和免疫功能的关键途径在感染沙门氏菌的小鼠巨噬细胞。