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研究生:徐湘庭
研究生(外文):Hsiang-Ting Hsu
論文名稱:第三號誘餌受體所調控之第二型巨噬細胞於流行性感冒病毒感染中所扮演的角色
論文名稱(外文):The Role of DcR3-induced M2 Macrophage in Influenza Virus Infection
指導教授:謝世良
指導教授(外文):Shie-Liang Hsieh
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:76
中文關鍵詞:第三號誘餌受體流行性感冒病毒第二型巨噬細胞
外文關鍵詞:Decoy Receptor 3Influenza VirusM2 macrophage
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中文摘要
第三號誘餌受體(Decoy receptor 3, DcR3)是腫瘤壞死因子超家族(tumor necrosis factor receptor superfamily)的成員,高量表現於許多癌細胞與發炎組織。它會透過與其配體的結合來中和它們原有的多樣性生物功能,因此被認為具有強大的生物活性。其配體包含FasL, LIGHT及TL1A,第三號誘餌受體能透過結合FasL來抑制由Fas-FasL所引起的細胞凋亡,透過中和LIGHT來抑制T細胞活化,並透過與TL1A結合來促進血管新生。除了與上述三種專一性配體的結合之外,第三號誘餌受體亦可藉由與單核球(monocyte)細胞膜表面上的硫酸乙醯肝素蛋白多糖(Heparin sulfate proteoglycans, HSPG)進行交互作用後,調控巨噬細胞(macrophage)及樹突細胞(dendritic cell, DC)的分化、促進蝕骨細胞(osteoclast)的形成,以及刺激樹突細胞的凋亡。除卻上述功能,最近我們利用微陣列技術(microarray)針對第三號誘餌受體處理後的巨噬細胞上的基因表現進行分析,發現它可以促進M2巨噬細胞的形成。為了進一步瞭解M2巨噬細胞在病毒感染的致病機轉中所扮演的角色,我們分別建立了活體外及活體內的實驗模型。在活體外的實驗結果中,我們發現M2巨噬細胞在A型流行性感冒病毒的感染後所產生的細胞激素(cytokine)總量較低,且M2巨噬細胞上負責辨識外來病毒的受體(receptor)以及感應受器(sensor) 包含TLR7、TLR9、MDA5,以及RIG-I的表現量皆受到抑制。我們觀察到的第三號誘餌受體所進行的調控,可能是透過與細胞膜表面上的硫酸乙醯肝素蛋白多糖結合,並引起p38和ERK的活化所造成。為了進一步觀察第三號誘餌受體在活體內所造成的影響,我們利用小鼠適應型A型流感病毒PR/8進行小鼠的感染。實驗結果顯示,在小鼠受感染的肺組織中,浸入肺部的嗜中性球(neutrophils)與巨噬細胞(macrophage)的細胞群(cell population)分佈模式,在感染後不同時間點上有明顯的差異。此外,兩組小鼠受到感染後之死亡率雖沒有顯著差異,但基因轉殖鼠開始死亡的時間點卻有延遲的現象。有趣的是,雖然野生型和基因轉殖鼠肺部沖洗液中包含TNF��, IL-6和IL-1�珛市P炎性細胞激素(proinflammatory cytokine)的表現量並沒有顯著差異,然而基因轉殖鼠的IFN�蛌穛{量卻在感染後第七天明顯高於野生型小鼠。儘管目前並不瞭解IFN�蚺W升所代表的意義,但綜合實驗結果的觀察,我們認為在流感病毒感染後,第三號誘餌受體能透過誘導M2巨噬細胞的產生來調控浸入肺部組織的免疫細胞,並有效地降低急性的免疫反應,使得受感染的老鼠免除高度的發炎反應所引起的死亡,但由於第三號誘餌受體無法抑制受感染的小鼠肺部中病毒複製,因此僅具有延遲老鼠病程發展的效果。
ABSTRACT
Decoy receptor 3 (DcR3), a soluble TNFR, is over-expressed in various tumors and inflammatory tissues. By neutralizing the biological functions of its ligands, including FasL, LIGHT and TL1A, DcR3 is now considered as a potent biological modulator. DcR3 can block Fas-FasL-mediated cell apoptosis by neutralizing FasL, and it can also neutralize LIGHT to reduce T cell activation, as well as to induce angiogenesis via neutralization of TL1A. In addition, DcR3 interacts with heparin sulfate proteoglycans (HSPGs) on the cell surface of monocyte to modulate activation and differentiation of dendritic cell (DC) and macrophage, as well as to induce the formation of osteoclast and DC apoptosis. More recently, by analyzing the gene expression profile of DcR3.Fc-treated human monocyte-derived macrophage (hMDM) with microarray technology, we found that DcR3 induces the differentiation of monocytes into M2 macrophage (alternatively-activated macrophage). To understand the role of M2 macrophage in the pathogenesis of virus infection, we set up both an in vitro and an in vivo model system to address this question. In our in vitro system, we found that DcR3-treated M2-like macrophage had a weaker cytokine response towards type A influenza virus (H1N1) infection. Furthermore, the expression of several viral receptors, such as TLR7 and TLR9, as well as the viral sensors, including MDA5 and RIG-I was down-regulated. The modulatory effects we observed here may be via DcR3-HSPG interaction to activate p38 and ERK. To further analyze the effect of DcR3 on virus infection in vivo, we inoculated DcR3-transgenic (DcR3-Tg) mice and wild type non-transgenic littermate (WT) with a murine-adapted PR/8 strain of type A influenza virus (H1N1). We found that the pattern of infiltrated neutrophils and macrophages in the infected-lungs was different between WT and DcR3-Tg mice at several time points post infection. Moreover, compared to the WT mice, the lethality of DcR3-Tg mice was delayed even though the final survival rate was approximately the same, . Interestingly, while the level of proinflammatory cytokines in the bronchial alveolar lavage (BAL) fluid including TNF��, IL-6 and IL-1���nis similar, the level of IFN�� in DcR3-Tg mice was significantly higher than that of the WT mice at 7 days post infection. Even though the meaning of upregulating IFN�� secretion is still unknown, this observation suggests that DcR3 may attenuate host response to influenza virus infection via inducing M2 macrophages to delay immune cell infiltration to pulmonary tissues. However, DcR3 was unable to reduce mortality because of its inability to suppress viral replication in the infected lungs.
TABLE OF CONTENTS

中文摘要 1

ABSTRACT 3

INTRODUCTION
1. Tumor Necrosis Factor (TNF) and TNF Receptor (TNFR) Superfamilies 5
1.1 TNF Superfamilies
1.2 TNF Receptor Superfamily
2. Decoy Receptor 3 (DcR3) 7
2.1 Expression of DcR3
2.2 Ligands of DcR3
2.3 Immuno-modulatory Effects of DcR3
3. Macrophage 10
3.1 M1 and M2 Macrophage
3.2 DcR3-induced M2 Macrophage and Viral Infection
4. Influenza Virus 12
4.1 Pathogenesis of Influenza Virus Infection
4.2 The Role of Macrophage in Influenza Virus Infection

OBJECTIVES 15

MATERIALS 16
1. Reagents and Solutions
2. Culture Medium
3. Cell Lines
4. Virus Strains

METHODS 23
1. Preparation of Plasmid DNA
2. Reverse-transcriptase Polymerase Chain Reaction (RT-PCR)
3. Electrophoresis
4. Western Blotting
5. Purification of Recombinant Proteins
6. Cell Isolation and In Vitro Culture Condition
7. Flow Cytometry
8. ELISA
9. Preparation and Quantification of Influenza Virus
10. Macrophages Infected by Influenza Virus PR/8 Strain In Vitro
11. Infection of PR/8 Influenza Virus
12. Bronchial Alveolar Lavage
13. Histochemical Staining
14. Statistical Evaluation

RESULTS
1. Modulatory Effects of DcR3.Fc on Influenza Virus-Infected hMDMs 36
1.1 DcR3.Fc-mediated effects on cell apoptosis of influenza virus-infected hMDMs
1.2 DcR3.Fc-mediated effects on cytokine secretion of influenza-infected hMDMs
2. Mechanism of DcR3.Fc-mediated Effects on Influenza Virus-Infected hMDMs 38
2.1 DcR3.Fc-mediated suppressive effects were via interaction with HSPG
2.2 DcR3.Fc suppresses expressions of viral sensors and receptors
2.3 DcR3.Fc suppresses cytokine secretion via activation of MAP kinases
3. Investigating DcR3-mediated Effect on Influenza Virus Infection In Vivo 41
3.1 In vivo model system for influenza virus infection
3.2 Differential response of wild type and DcR3 transgenic mice to influenza virus infection

DISCUSSION 46

REFERENCES 51

FIGURES 55

APPENDIX 75
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