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研究生：	凌倫翎 Ling, Lun-Ling
論文名稱：	菸鹼醯胺腺嘌呤二核甘酸磷酸氧化酶缺陷之肺部嗜中性粒細胞和巨噬細胞上程序性死亡配體1之低表現影響自然淋巴球細胞群並加重過敏性肺部炎症 NOX2-deficient Pulmonary Neutrophils and Macrophages Have Lower PD-L1 Expression and May Aggravate Allergic Lung Inflammation through Affecting ILC Populations
指導教授：	謝奇璋 Shieh, Chi-Chang
學位類別：	碩士 Master
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2023
畢業學年度：	111
語文別：	英文
論文頁數：	55
中文關鍵詞：	嗜中性白血球、自然免疫類淋巴球細胞、巨噬細胞、巨噬細胞極化、程序性死亡蛋白1 、程序性細胞死亡蛋白配體 1 、過敏性氣喘
外文關鍵詞：	Neutrophils, Innate lymphoid cells, Macrophages, M1/M2 polarization, PD-1, PD-L1, Allergic asthma
相關次數：	點閱：146 下載：0
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Abstract I
中文摘要 III
致謝 V
Abbreviations VI
Chapter 1. Introduction 1
1.1 The exacerbation of allergic asthma 2
1.1.1 Dermatophagoides pteronyssinus (Der p) and LPS initiates allergic inflammation in the lung 2
1.2 Reactive oxygen species (ROS) and NADPH OXIDASE 2(NOX2) 3
1.2.1 NADPH oxidase 2 is an essential regulator in allergen-induced airway inflammation 3
1.3 The NOX2-derived ROS may regulate PD-L1 expression 4
1.4 The role of PD-1 in innate lymphoid cells (ILCs) 4
1.5 The role of PD-L1 in macrophage polarization	5
1.6 Research goals 6
Chapter 2. Materials and Methods	7
2.1 Experimental animal 8
2.2 Allergen exposure experiment	8
2.2.1 Preparation of Der p extract 8
2.2.2 Preparation of Der p extract along with LPS	9
2.2.3 Administration of Der p extract along with LPS exposure experiment	9
2.3 Measurement of airway responsiveness 9
2.4 Lung tissue staining, and histology examination 9
2.4.1 Hematoxylin and eosin (H&E) staining	10
2.5 Enzyme-linked immunosorbent assay (ELISA) 10
2.5.1 Measurement of cytokines 10
2.6 Isolation of pulmonary cell from lung tissue by flow cytometric analysis 10
2.6.1 Detection of macrophage phenotypes and cytokines by flow cytometric analysis 11
2.6.2 Detection of pulmonary ILC subsets by flow cytometric analysis 11
2.6.3 Detection of PD-1 and PD-L1 expressions by flow cytometric analysis 12
2.7 Statistical analysis 13
Chapter 3. Results 14
3.1 Der p extract-LPS stimulations induced more weight loss and more severe AHR in Cybb-/- mice when compared with WT mice 15
3.2 Der p extract-LPS stimulations promoted more cell infiltration in the inflammatory lung of Cybb-/- mice when compared with WT mice 15
3.3 Elevated levels of IL-17A and IL-1β were in asthmatic lung of Cybb-/- mice 16
3.4 Cybb-/- mice exhibited increased numbers of pulmonary neutrophils and AMs in response to Der p extract-LPS stimulations 16
3.5 Cybb-/- mice showed higher induction of pulmonary ILC2s and ILC3s upon exposure to Der p extract-LPS stimulations 17
3.6 The pulmonary neutrophils in the asthmatic lung of Cybb-/- mice exhibited lower PD-L1 expression when compared with WT mice	18
3.7 Cybb-/- AMs showed higher level of TNF-α in comparison with WT mice  19
3.8 The Cybb-/- AMs primarily comprised of M1 macrophages while WT AMs mainly comprised of M2 macrophages 19
3.9 Cybb-/- M1 AMs expressed lower level of PD-L1 when compared with WT M1 AMs 20
Chapter 4. Discussion 22
4.1 Redox regulated PD-L1 expressions on neutrophil and AM to modulate downstream cell responses 23
4.2 Oxidative stress elicits macrophage polarization through regulating PD-L1 expression 24
4.3 The cell interaction between neutrophils/macrophages and ILCs in exacerbated lung inflammation 25
Chapter 5. Figure and Legends 27
Figure 1 28
Cybb-/- mice showed enhanced AHR while lowed body weight after challenged with Der p extract-LPS stimulations when compared with WT mice on day 12 29
Figure 2 30
Cybb-/- mice expressed enhanced inflammatory immune cell infiltration and type 3 lung inflammation after Der p-LPS stimulations 31
Figure 3 32
The gating strategies of pulmonary neutrophil, alveolar macrophage, interstitial macrophage and eosinophil by flow cytometry in lung tissue of WT and Cybb-/- mice	33
Figure 4 34
Increased levels neutrophil, eosinophil and macrophage were detected in inflammatory lung of WT and Cybb-/- mice 35
Figure 5 36
Pulmonary ILC1, ILC2, and ILC3 by flow cytometry in lung tissue of WT and Cybb-/- mice 37
Figure 6 38
The levels of ILC2 and ILC3 increased in allergic lung inflammation of WT and Cybb-/- mice 39
Figure 7 40
The geometric means of PD-L1 expressions on neutrophil in lung inflammation from WT and Cybb-/- mice 41
Figure 8 42
The percentages of TNF-α and Arg-1 in total AM in lung inflammation from WT and Cybb-/- mice 43
Figure 9 44
The percentages of M0, M1, and M2 AMs and their PD-L1 expressions in Der p-LPS induced asthmatic lung from WT and Cybb-/- mice 45
Figure 10	46
Cybb-/- mice result in decreased PD-L1 expression on pulmonary neutrophils and AM polarization towards the M1 phenotype with reduced PD-L1 expression, leading to the exacerbation of lung inflammation and increased ILC3s	46
Table 1 47
Representative gating strategy for the identification of ILCs in the lung tissue from WT and Cybb-/- inflammatory lungs	 47
Table 2 48
Antibodies for detecting cell populations, flow cytometry analysis 48
Chapter 6. References 50
                                    

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