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研究生：	詹子頤 Chan, Tzu-Yi
論文名稱：	探討嗜中性白血球活化於菸醯胺腺嘌呤二磷酸氧化酶缺陷小鼠中調控介白素-1和自然免疫類淋巴球細胞在血清誘發小鼠關節炎中的角色 The role of leukocyte NADPH oxidase in the regulation of IL-1 family cytokines and innate lymphoid cells in serum-induced arthritis
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：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	64
中文關鍵詞：	活性氧化物、介白素-1 、自然免疫類淋巴球細胞、氧化壓力、關節炎
外文關鍵詞：	IL-1 family cytokines, reactive oxygen species, innate lymphoid cell, NADPH oxidase, serum-induced arthritis
相關次數：	點閱：136 下載：5
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類風濕性關節炎(RA)是一種慢性炎症，為可造成人類關節傷害的主要疾病之一。這種炎症是由自身免疫系統失調引致免疫系統攻擊關節。在我們實驗室先前的研究中發現，當老鼠體內缺乏了白血球菸醯胺腺嘌呤二磷酸氧化酶的情況下利用血清誘發關節炎後，除了有大量白血球驅化激素外也有大量的介白素-1β產生，造成發炎反應明顯比起野生型老鼠來的嚴重。這樣的實驗結果告訴我們氧化壓力的調控對於發炎反應的重要性。
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。依所分泌介白素種類，可分為三大類。此三類細胞，雖由相同的前驅細胞(precursor cell)分化而來，但如何產生這些自然免疫類淋巴球細胞、或發生階段的分化機制，仍須進一步的探討。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。
在本實驗中，我們主要探討氧化壓力(ROS)在小鼠關節炎中調節自然免疫類淋巴球細胞與介白素-1的角色與其參與發炎反應的相關機轉。在實驗設計上，我們利用血清誘發關節炎於野生型老鼠與白血球菸醯胺腺嘌呤二磷酸氧化酶缺失鼠中，結果發現於血清誘發關節炎中白血球菸醯胺腺嘌呤二磷酸氧化酶缺失鼠有較嚴重的發炎反應，在組織學上的表現也發現關節腔內有較高的嗜中性白血球浸潤。比較兩組關節炎小鼠中細胞介素的表現，發現介白素-1β、介白素-18和介白素-23的表現量均有上升，但介白素-33和介白素-25的表現量則下降。進一步將三群自然免疫類淋巴球細胞從關節腔中分離出來，則發現當老鼠體內缺乏了白血球菸醯胺腺嘌呤二磷酸氧化酶的情況下，會使得第三型自然免疫類淋巴球細胞的表現量上升。由此結果推斷在血清誘發關節炎中，氧化分子透過調控細胞介素參與自然免疫類淋巴球細胞的分化，進一步造成不同嚴重程度的關節發炎。未來仍需進一步分析其中機制，此研究結果能更清楚了解白血球菸醯胺腺嘌呤二磷酸氧化酶在關節炎中對於先天性免疫調控的重要性。

The chronic autoimmune disease rheumatoid arthritis (RA) is characterized by infiltration of leukocytes into synovial tissue, proliferation of synoviocytes, and degra-dation of bone and cartilage. The role of ROS in the pathogenic process of autoimmune arthritis has been debated. Some studies supported a proinflammatory effect of oxidant stress in arthritis while others found evidences for a role in immunoregulation. Our previous studies showed that ROS act as negative feedback to regulate IL-1β-mediated inﬂammation, accounting for the more severe arthritis in the absence of leukocyte NADPH oxidase (NOX2), which is responsible for producing most of the ROS in inflammatory tissues.
Innate lymphoid cells (ILCs) have been postulated to be important in the regulation of barrier homeostasis. It has been reported that ILC-derived cytokines are important for the induction and regulation of inflammation. Emerging data indicated that ILCs not only have protective functions but can have detrimental effects when dysregulated, leading to chronic inflammation and autoimmune diseases.
In this study, we aimed to investigate the role of redox regulation in immune-mediated joint inflammation and explored the potential role of innate lymphoid cells and IL-1 family cytokines-related mechanisms in this process. We performed experiments to characterize innate lymphoid cells in the WT and Ncf1-/- arthritic joints and detected the IL-1 family cytokines along with other cytokines relevant to innate lymphoid cell function and development.
We found that serum-induced joint inﬂammation was more severe in Ncf1-/- mice. Injection of arthritogenic serum induced higher levels of IL-1β, IL-18 and IL-23 but lower level of IL-25 and IL-33 expression. Furthermore, populations of lineage-negative CD45 positive cells were identified in the synovial fluid and tissue of diseased joints. Comparison of ILCs populations between WT and Ncf1-/- arthritic mice showed that ILC3 were more abundant in the joint of NOX2 deficient mice and ILC1 were more abundant in WT mice. The phagocytic NADPH oxidase hence appears to be an esential regulator of ILC3 populations switching in the inflammatory tissues of arthritis.
Given the facts that the ILCs are capable of rapid cytokine production and leukocyte-produced ROS appear to affect these ILC populations in the inflammatory joints, the differences in ILCs may be a critical factor in the more severe arthritis in NOX2 deficient animals. We will continue our ongoing investigations to reveal novel redox-sensitive immunoregulatory mechanisms mediated by ILCs and unravel the role IL-1 family cytokines in this important process.

Contents
Abstract.....7
中文摘要.....9
Abbreviations.....11
Chapter 1  Introduction.....13
1.1 Immunological basis of rheumatoid arthritis.....14
1.2 Oxidative stress in RA.....14
1.2 Leukocyte NADPH oxidase system......15
1.3 Interleukin-1 family cytokines (IL 1β, IL-18, and IL-33) in the regulation of RA.....16
1.4 Innate lymphoid cells (ILCs).....18
1.5 The role of ILCs in rheumatic diseases.....20
1.6 Research Goals.....20
Chapter 2  Materials and Methods.....21
2.1 Experimental animals.....22
2.2 Serum-induced arthritis.....23
2.3 Preparation of protein extracts from wrists and ankles.....24
2.4 Histology and immunohistochemistry.....24
2.4.1 Hematoxylin and eosin stain.....24
2.4.2 Immunohistochemical.....25
2.5 Measurement of cytokines by Enzyme-linked immunosorbent assay (ELISA).....25
2.6 Luminexsuspension bead array......26
2.7 ILCs detection by flow cytometric analysis.....27
2.7.1 Isolation of ILCs by flow cytometric analysis and cell sorter.....28
2.7.2 Cultures ILCs.....28
2.8 Intracellular Cytokine Staining.....28
2.9 Statistical analysis.....29
Chapter 3  Results......30
3.1 Serum-induced joint inflammation was more severe in NOX2-deficient mice.....31
3.1.1 Serum-induced arthritis was a neutrophil-dominant tissue inflammation in both Ncf1- / - and WT mice......31
3.1.2 Injection of arthritogenic serum induced higher levels of IL-1β and IL-18 in NOX2 deficient mice.....32
3.2 Characterization of distinct subsets of ILCs in serum-induced arthritis mice.....33
3.2.1 NOX2 deficient mice increased the ILC3 production and ILC1 were more abundant in WT mice.....34
3.2.2 Increased levels of intracellular interleukin-17A-producing ILC3 in Ncf1-/- disease joints and IFN-γ-producing ILC1 in WT disease joints.....35
3.3 IL-23 and IL-1β treatment is sufficient to drive differentiation of ILC1 toward ILC3 in leukocytes from mice with serum-induced arthritis.....35
Chapter 4  Discussion.....37
4.1 Oxidant stress regulated ILCs activation in serum-induced arthritis mice.....39
4.2 Redox modulate ILCs transdifferentiation through IL-1 family cytokines.....39
Chapter 5  Figure and Legends.....41
Figure 1......42
Increased severity of serum-induced arthritis in Ncf1-/- mice.....43
Figure 2......44
Serum-induced arthritis is a neutrophil-dominant tissue inflammation.....45
Figure 3........46
Different cytokines expression profiles in serum-induced arthritis mouse model.......47
Figure 4.........48
Representative gating strategy for the identification of ILCs in the synovial fluid and tissue of WT and Ncf1 - / - arthritic joints.......50
Figure 5.........51
The level of ILCs (ILC1, ILC2, ILC3) in the joints leukocytes from WT and Ncf1 - / - serum-induced arthritis mice.........52
Figure 6.........53
Expression of intracellular interleukin-17A-producing ILC3 and IFN-γ-producing ILC1 in the joints leukocytes from WT and Ncf1 - / - serum-induced arthritis mice.......54
Figure 7........55
Exogenous IL-1β and IL-23 stimulation abolished the difference between WT and Ncf1-/- ILC3 populations in joints leukocytes........57
Figure 8........58
Ncf1-/- arthritic mice lead to increased ILC3 expression and transdifferentiation of ILC1s into IL17A-producing ILC3-like cells generates a highly inflammatory immune cell.....58
Chapter 6  References.......59
                                    

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校內：2019-08-01公開
校外：2019-08-01公開

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