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系統識別號 U0026-0708201923324600
論文名稱(中文) 過敏原所引起的肺部發炎中白血球菸醯胺腺嘌呤二核苷酸磷酸氧化酶於調節自然免疫類淋巴球細胞所扮演的角色
論文名稱(英文) The role of phagocytic NADPH oxidase in the regulation of innate lymphoid cells in Dermatophagoides pteronyssinus-induced lung inflammation
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 107
學期 2
出版年 108
研究生(中文) 李瑞珊
研究生(英文) Ruei-Shan Li
學號 S96061066
學位類別 碩士
語文別 英文
論文頁數 62頁
口試委員 指導教授-謝奇璋
口試委員-林秋烽
口試委員-凌斌
口試委員-許育祥
中文關鍵字 過敏性氣喘  氧自由基  菸醯胺腺嘌呤二核苷酸磷酸氧化脢  自然免疫類淋巴球細胞  塵蟎 
英文關鍵字 Allergic asthma  Reactive oxygen species (ROS)  NADPH oxidase  Innate lymphoid cell (ILCs)  Dermatophagoides pteronyssinus (Der p)  Ovalbumin (OVA) 
學科別分類
中文摘要 塵蟎為導致過敏性慢性發炎氣喘的主要原因之一,過敏性氣喘的病理特徵為呼吸急促、胸悶、支氣管腫大且造成免疫系統失調導致肺部發炎反應。我們先前的研究發現,菸醯胺腺嘌呤二核苷酸(NADPH氧化酶)是肺部組織發炎反應的重要調節者,其免疫反應特徵在於其調節Th2和Th17免疫反應而造成嚴重的肺部發炎。自然免疫類淋巴球細胞(ILC),是2010年被發現的新型淋巴球,具有T淋巴球或B淋巴球等後天免疫細胞不同的特性。目前已有研究發現,自然免疫類淋巴球細胞對於發炎反應的引發和調控扮演重要角色。在其失衡的形況下,會導致慢性發炎和自體免疫疾病。我們的研究認為NADPH缺陷的吞噬細胞氧化酶的氧化還原失衡的變化可能會影響早期自然免疫細胞聚集和其後伴隨的特異性免疫反應所造成的肺部發炎。在本研究中我們假設白血球NADPH氧化酶會藉由影響自然免疫類性淋巴球細胞活化進而引發下游一連串免疫反應機制,最終導致過敏原誘發肺部發炎的病徵。在實驗設計上, 我們利用過敏原Der p或OVA 刺激8-9週齡的野生型和白血球NADPH氧化酶2缺陷型小鼠引起致敏以建立過敏原誘發的氣喘。首先,我們測試Der p或OVA刺激是否引起野生型和白血球NADPH氧化酶2缺陷型小鼠過敏性氣喘病徵。結果發現Der p刺激會引起野生型和白血球NADPH氧化酶2缺陷型小鼠高反應性的氣喘病徵、過敏原IgE增加且產生大量免疫細胞及黏膜浸潤在支氣管間。而在OVA刺激下會引起野生型小鼠高反應性的氣喘病徵、過敏原IgE增加且產生大量免疫細胞及黏膜浸潤在支氣管間,但白血球NADPH氧化酶2缺陷型小鼠則無此症狀。進一步檢測野生型和白血球NADPH氧化酶2缺陷型小鼠在Der p或OVA刺激後的肺部組織中之第2型細胞因子(包括IL-4, 5, 13)或免疫細胞(eosinophils)及第17型炎症細胞因子(包括IL-17) 或免疫細胞(eosinophils及neutrophils)。我們發現IL-4, 5, 13及eosinophils在Der p刺激的組別中,野生型和白血球NADPH氧化酶2缺陷型小鼠均會增加。此外,促發炎因子IL-17和eosinophils及neutrophils在Der p刺激的白血球NADPH氧化酶2缺陷型小鼠更有顯著增加。而在OVA刺激下會誘發野生型小鼠的IL-4, -5, -13及eosinophils產生,但白血球NADPH氧化酶2缺陷型小鼠則無此現象。最後我們利用細胞表面標記表達和細胞內細胞因子評估在Der p-或OVA誘導肺發炎的野生型和白血球NADPH氧化酶2缺陷型小鼠之肺部的ILC亞群。結果顯示ILC2s在Der p刺激的組別中,野生型和白血球NADPH氧化酶2缺陷型小鼠均會增加。而在OVA刺激下會誘發野生型小鼠的ILC2s增加,但白血球NADPH氧化酶2缺陷型小鼠則無此現象。
因此,在我們的研究結果顯示,在Der p和OVA誘導的肺部發炎反應中,吞噬性白血球NADPH氧化酶可能會調控自然免疫類淋巴球細胞的活化且影響下游免疫機制(包括Th2和Th17免疫反應)。
英文摘要 Asthma is a chronic inflammatory disease of the airway induced by exogenous allergens. The most important allergen in Taiwan is house dust mites (HDMs) which affects people in all ages including children. The characteristics of asthma are airway inflammation and airway hyperresponsiveness (AHR). In asthma, immune cells, particular eosinophils and T cells, infiltrate into airways and increase the expression of type 2 inflammatory cytokines. Previous studies indicated that phagocytic NADPH oxidase (NOX2) is an essential regulator in allergen-induced airway inflammation characterized by a severe lung inflammation with Th17 immune response, and decreased lung inflammation in the absence of NOX2. Changes in the redox balance by defective NOX2 may affect lung inflammation in term of early innate immune cell activation/recruitment and the subsequent specific immune responses. Innate lymphoid cell (ILCs) have been known to play an important role in the development of lymphoid tissues and in the initiation of inflammation in response to infection or tissue damage. Therefore, we used allergens including Dermatophagoides pteronyssinus (Der p) and ovalbumin (OVA) to stimulate NOX2-deficient mice to address the hypothesis that Der p or OVA-induced allergic lung inflammation are affected in NOX2 deficient mice due to different ILC-mediated immune responses. In this study, allergen-induced asthma models had been established by intranasal administration of Der p or OVA in WT and Ncf1-/- mice at 8-9 weeks of age. First, we investigated the role of NOX2 in airway inflammation by using WT and Ncf1-/- mice with Der p or OVA exposure. We found that Der p induced AHR and increased IgE production, inflammatory cells and mucus infiltration around the bronchiole in WT and Ncf1-/- mice. OVA exposure induced AHR and increased IgE production, inflammatory cells and mucosa infiltration around the bronchiole in WT mice but not in Ncf1-/- mice. Second, we determined the effects of Der p or OVA on induction of Th2 cytokines (including IL‐4, 5, 13) and Th17 cytokines (IL-17) production in the lung of WT and Ncf1-/- mice. Moreover, we analyzed the immune cell of the lung tissue by flow cytometry to identify lung inflammation of WT and Ncf1-/- mice. We found that after Der p exposure, eosinophils and type 2 cytokines are induced in WT mice, and then neutrophils and IL-17 are increased in Ncf1-/- mice with Der p exposure. After OVA exposure, eosinophils were induced in WT mice but not in Ncf1-/- mice. Finally, we studied the cell surface markers expression to characterize the ILC subsets in the lung of Der p or OVA- induced mice in both WT and Ncf1-/- mice. We found that ILC2s number increased in lung tissue after Der p exposure in WT and Ncf1-/- mice. ILC2s number also increased in lung tissue after OVA exposure in WT but not in Ncf1-/- mice. Our data indicate that changes in ILCs activity may have a role in phagocytic NADPH oxidase-mediated immune regulation in Der p and OVA-induced airway inflammation.
論文目次 Contents
Abstract.......IV
中文摘要.......VI
Abbreviations.......VIII
Chapter 1 Introduction.......1
1.1 Allergic asthma.......2
1.2 The immunity system of allergic asthma.......2
1.2.1 Ovalbumin (OVA) induced allergic asthma.......2
1.2.2 Toluene diisocyanate (TDI) induced allergic asthma.......3
1.2.3 Dermatophagoides pteronyssinus (Der p) induced allergic asthma.......3
1.3 Phagocytic NADPH oxidase system.......5
1.4 Phagocytic NADPH is an essential regulator in allergen-induced airway inflammation.......7
1.5 Innate lymphoid cell (ILCs).......8
1.6 The role of ILCs in asthma disease.......9
1.7 Research Goals.......10
Chapter 2 Materials and Methods.......11
2.1 Experimental animals.......12
2.2 Allergen exposure experiment.......12
2.2.1 Preparation of Der p.......12
2.2.2 Der p exposure experiment.......13
2.2.3 OVA exposure experiment.......13
2.3 Measurement of airway responsiveness.......13
2.4 Lung tissue collection, staining and histology examination.......14
2.4.1 Hematoxylin and eosin stain.......14
2.4.2 Periodic Acid-Schiff stain.......14
2.4.3 Immunofluorescence stain.......15
2.4.4 Immunohistochemistry stain.......16
2.5 Enzyme-linked immunosorbent assay (ELISA).......17
2.5.1 IgE quantification.......17
2.5.2 Measurement of cytokines.......17
2.6 Luminex suspension bead array.......18
2.7 Leukocyte isolation by flow cytometric analysis.......19
2.8 ILCs detection by flow cytometric analysis.......19
2.8.1 Isolation of ILC2 by flow cytometric analysis.......20
2.9 Statistical analysis.......20
Chapter 3 Results.......21
3.1 Der p induced more airway hyperreactivity and IgE production in lung tissue Ncf1-/- mice than WT mice, whereas OVA induced hyperreactivity and IgE production in WT mice but not Ncf1-/- mice.......22
3.2 Der p induced leukocyte infiltration and mucus formation in lung tissues of Ncf1-/- mice but not OVA exposure.......24
3.3 Der p- induced IL-4, IL-5, IL-13, and IL-1β level in lung tissue of WT and Ncf1- / - mice, more IL-33 and IL-17A production in Ncf1- / - mice when compared with WT mice.......25
3.4 Der p and OVA exposure tended to induce eosinophils in lung tissues of WT mice, whereas Der p exposure induced eosinophils and neutrophils in lung tissue of Ncf1-/- mice but not OVA exposure.......26
3.5 Increased eosinophils in WT and Ncf1-/- mice with Der p or OVA stimulation, whereas more neutrophils induced in Ncf1-/- mice with Der p exposure than WT mice.......28
3.6 Ncf1-/- mice increased more ILC2 production after Der p stimulation when compared with WT mice, OVA induced ILC2 in WT mice but not in Ncf1-/- mice.......29
Chapter 4 Discussion.......30
4.1 Respiratory allergy as a chronic inflammation in the lung.......31
4.2 NOX2 is a crucial regulator in allergen-induced lung inflammation.......32
4.3 Oxidant stress modulates different immune responses in allergen-induced lung inflammation.......33
4.4 Oxidant stress regulated ILCs activation in allergen-induced asthma.......35
4.5 Implication and potential application of the results in this study.......36
Chapter 5 Figure and Legends.......38
Figure 2. 39
Der p, but not OVA, induced airway hyperreactivity and IgE production in lung tissue of Ncf1-/- mice........40
Figure 3........41
Derp, but not OVA, induced leukocytes infiltration and mucus formation in lung tissues of Ncf1-/- mice........43
Figure 4........44
Der p-induced lung inflammation in Ncf1- / - mice is through Th2 cytokines and Th17 cytokines........45
Figure 5........46
Der p and OVA exposure tended to induce eosinophils in lung tissues of wild type mice, whereas Der p exposure induced eosinophils and neutrophils in lung tissue of Ncf1-/- mice but not OVA exposure........47
Figure 6........48
Der p and OVA exposure tended to induce eosinophils in lung tissues of wild type mice, whereas Der p induced neutrophils infiltration in lung tissue of Ncf1-/- mice........49
Figure 7........50
Leukocyte gating strategy by flow cytometry in lung cell of WT mice and Ncf1 - / - mice........51
Figure 8........52
Der p and OVA induced more eosinophil infiltration in lung tissues of Ncf1-/- mice, and Der p induced neutrophils in lung tissue of Ncf1-/- mice........53
Figure 9........54
ILC2 are increased in the lung tissue of Ncf1-/- mice with Der p stimulation but not OVA stimulation........55
Table 2........56
Antibodies for depletion of lineage-positive cells, flow cytometry analysis.......56
Chapter 6 References.......58
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