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研究生：	王立群 Wang, Li-Chun
論文名稱：	擬交感神經藥物經由調節細胞產生氧活性分子影響巨噬細胞之極化 Sympathomimetic drugs affect macrophage polarization through regulating cellular reactive oxygen species production
指導教授：	謝奇璋 Shieh, Chi-Chang
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	37
中文關鍵詞：	氧活性分子、NADPH氧化酶 2 (NOX2) 、擬交感神經藥物、可樂定、特布他林、巨噬細胞極化
外文關鍵詞：	ROS, NADPH oxidase 2 (NOX2), sympathomimetic drugs, clonidine, terbutaline, macrophage polarization
相關次數：	點閱：191 下載：0
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由白血球的NADPH氧化酶2 ( NOX2) 所產生的氧活性分子 (reactive oxygen species, ROS) 在非特異性宿主防禦中扮演重要的角色。先前的研究表示缺乏 NOX2所介導的ROS會抑制巨噬細胞 (macrophage) 分化成M2表型。腎上腺素能受體促效劑 (adrenergic receptor agonists, AR agonists) 是擬交感神經藥物 (sympathomimetic drugs)。根據報導，AR agonists 可以調節巨噬細胞極化。α-AR agonists 促使巨噬細胞分化成M1表型，而 β-AR agonists 促使巨噬細胞分化成M2表型。我們之前的研究顯示了相似的結果，也就是β-AR agonists 特布他林 (terbutaline, Ter) 在HL-60細胞中促使ROS產生，而且這種現象會被NOX抑制劑阻斷。然而，對於AR agonists誘導的ROS產生和巨噬細胞極化之間的關係仍然未知。在這項研究中，我們假設AR agonists會促使NOX2所介導的ROS產生以驅動野生型的骨髓來源的巨噬細胞 (bone marrow-derived macrophage, BMDM) 往M2表型極化。我們發現可樂定 (clonidine, Clo) 和Ter可以在BMDM顯著增加NOX2所介導的ROS。另外，我們的實驗結果顯示，Ter可能經由NOX2依賴性途徑促進BMDM極化成M2表型，而Clo刺激經由NOX2非依賴性途徑抑制M2巨噬細胞極化。我們研究可能有望揭示AR agonists通過氧化還原調節巨噬細胞分化的關鍵機制。這些發現可能為Ter在過敏性氣喘中的免疫調節活性提供新的見解。

Reactive oxygen species (ROS) produced by leukocyte NADPH oxidase 2 (NOX2) plays an essential role in nonspecific host defense. Previous studies indicated that the lack of NOX2-mediated ROS production inhibits macrophage differentiation into M2 phenotype. Adrenergic receptor (AR) agonists are sympathomimetic drugs. These agonists have been reported to modulate the immune system. In previous studies, AR agonists were found to regulate macrophage polarization. α-AR agonists promote macrophage differentiation toward M1 phenotype while β-AR agonists promote macrophage differentiation toward M2 phenotype. Our previous data showed a similar result that β2-AR agonist terbutaline (Ter) promoted ROS production in HL-60 cells, and the phenomena was blocked by NOX inhibitor. However, the relationship between AR-agonists-induced ROS production and macrophage polarization is still unknown. In this study, we hypothesized that AR agonists promote NOX2-mediated ROS production to drive the polarization of bone marrow-derived macrophages (BMDM) toward M2 phenotype in wild-type mice. We found that clonidine (Clo) and Ter significantly increase NOX2-mediated ROS production in BMDM. Our data showed that Ter may promote BMDM polarization toward M2 phenotype though the NOX2-dependent pathway, whereas Clo stimulation inhibited M2 macrophage polarization through NOX2-independent pathway. Our research may promise to reveal a key mechanism that AR agonists modulate macrophage differentiation through redox regulation. These findings may provide new insights into the immunomodulation activity of Ter in allergic asthma.

Abstract I
中文摘要 II
誌 謝 III
Abbreviations	IV
Chapter 1 Introduction 1
1.1 The role of NADPH oxidases in phagocytes in innate immunity 1
1.2 The regulation of NADPH oxidase 2-mediated ROS in macrophage polarization 2
1.3 The signaling and function of sympathomimetic drugs through adrenergic receptors 3
1.4 The role of sympathomimetic drugs in macrophage polarization 4
1.5 The effect of sympathomimetic drugs to induce NOX-mediated ROS 5
1.6 The aim of this study	5
Chapter 2 Materials and methods	6
2.1 Reagents 7
2.2 Cell culture 7
2.3 ROS measurement 8
2.4 Flow cytometry analysis 8
2.5 ELISA 9
2.6 Statistical analysis 9
Chapter 3 Results 10
3.1 Clo and Ter induced NOX2-mediated ROS production 11
3.2 Clo did not modulate M2 macrophage polarization through NOX2-dependent pathway 12
3.3 Ter treatment may promote M2 macrophage polarization through NOX2-produced ROS 13
Chapter 4 Discussion	15
4.1 The efficient concentrations are different between Clo and Ter to generate ROS 16
4.2 The effect of Clo in macrophage polarization 16
4.3 Ter promotes M2 macrophage polarization	17
4.4 Clo and Ter may induce NOX2-mediated ROS production through non-canonical signaling pathways 18
4.5 Ter-induced M2 macrophage polarization may play a role in allergic asthma 18
4.6 The limitations of this study 19
Chapter 5 Figures and tables 20
Figure 1 21
Figure 1. ROS production in response to several sympathomimetic drugs. 22
Figure 2 23
Figure 2. The effects of Clo- and Ter-induced ROS production at different time points and dose response in WT BMDM. 24
Figure 3 25
Figure 3. Clo alone treatment did not significantly affect M1/M2 surface markers CD86 and CD206 expression respectively in WT and Ncf1-/- BMDM.	26
Figure 4 27
Figure 4. Clo co-stimulated with LPS/IFN-γ modulated cytokine responses in BMDM. 28
Figure 5 29
Figure 5. Ter alone treatment triggered WT BMDM to upregulate CD206 expression via NOX2-mediated ROS. 30
Figure 6 31
Figure 6. Ter co-stimulated with LPS/IFN-γ modulated cytokine responses in BMDM. 32
Chapter 6 References 33
                                    

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