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系統識別號 U0026-1907201711380500
論文名稱(中文) 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
論文名稱(英文) The role of leukocyte NADPH oxidase in the regulation of IL-1 family cytokines and innate lymphoid cells in serum-induced arthritis
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 105
學期 2
出版年 106
研究生(中文) 詹子頤
研究生(英文) Tzu-Yi Chan
學號 s96044030
學位類別 碩士
語文別 英文
論文頁數 64頁
口試委員 指導教授-謝奇璋
口試委員-許育祥
口試委員-洪志興
中文關鍵字 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 
英文關鍵字 IL-1 family cytokines  reactive oxygen species  innate lymphoid cell  NADPH oxidase  serum-induced arthritis 
學科別分類
中文摘要 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。
英文摘要 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.
論文目次 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
參考文獻 1.McInnes IB, Schett G: The pathogenesis of rheumatoid arthritis. The New England journal of medicine 2011, 365(23):2205-2219.
2.Gizinski AM, Fox DA: T cell subsets and their role in the pathogenesis of rheumatic disease. Curr Opin Rheumatol 2014, 26(2):204-210.
3.Klareskog L, Amara K, Malmstrom V: Adaptive immunity in rheumatoid arthritis: anticitrulline and other antibodies in the pathogenesis of rheumatoid arthritis. Curr Opin Rheumatol 2014, 26(1):72-79.
4.McInnes IB, Schett G: Cytokines in the pathogenesis of rheumatoid arthritis. Nature reviews Immunology 2007, 7(6):429-442.
5.Bekiaris V, Sedy JR, Rossetti M, Spreafico R, Sharma S, Rhode-Kurnow A, Ware BC, Huang N, Macauley MG, Norris PS et al: Human CD4+CD3- innate-like T cells provide a source of TNF and lymphotoxin-alphabeta and are elevated in rheumatoid arthritis. Journal of immunology 2013, 191(9):4611-4618.
6.Cascao R, Rosario HS, Souto-Carneiro MM, Fonseca JE: Neutrophils in rheumatoid arthritis: More than simple final effectors. Autoimmunity reviews 2010, 9(8):531-535.
7.Wink DA, Hines HB, Cheng RY, Switzer CH, Flores-Santana W, Vitek MP, Ridnour LA, Colton CA: Nitric oxide and redox mechanisms in the immune response. Journal of leukocyte biology 2011, 89(6):873-891.
8.Hitchon CA, El-Gabalawy HS: Oxidation in rheumatoid arthritis. Arthritis research & therapy 2004, 6(6):265-278.
9.Biemond P, Swaak AJ, Koster JF: Protective factors against oxygen free radicals and hydrogen peroxide in rheumatoid arthritis synovial fluid. Arthritis and rheumatism 1984, 27(7):760-765.
10.Holmdahl R, Sareila O, Olsson LM, Backdahl L, Wing K: Ncf1 polymorphism reveals oxidative regulation of autoimmune chronic inflammation. Immunological reviews 2016, 269(1):228-247.
11.Ozturk HS, Cimen MY, Cimen OB, Kacmaz M, Durak I: Oxidant/antioxidant status of plasma samples from patients with rheumatoid arthritis. Rheumatology international 1999, 19(1-2):35-37.
12.Jaswal S, Mehta HC, Sood AK, Kaur J: Antioxidant status in rheumatoid arthritis and role of antioxidant therapy. Clinica chimica acta; international journal of clinical chemistry 2003, 338(1-2):123-129.
13.Liljander M, Andersson A, Holmdahl R, Mattsson R: Increased susceptibility to collagen-induced arthritis in female mice carrying congenic Cia40/Pregq2 fragments. Arthritis research & therapy 2008, 10(4):R88.
14.Nauseef WM: Biological roles for the NOX family NADPH oxidases. The Journal of biological chemistry 2008, 283(25):16961-16965.
15.Babior BM: NADPH oxidase. Current opinion in immunology 2004, 16(1):42-47.
16.Cachat J, Deffert C, Hugues S, Krause KH: Phagocyte NADPH oxidase and specific immunity. Clinical science 2015, 128(10):635-648.
17.Gardiner GJ, Deffit SN, McLetchie S, Perez L, Walline CC, Blum JS: A role for NADPH oxidase in antigen presentation. Frontiers in immunology 2013, 4:295.
18.Bernink JH, Peters CP, Munneke M, te Velde AA, Meijer SL, Weijer K, Hreggvidsdottir HS, Heinsbroek SE, Legrand N, Buskens CJ et al: Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nature immunology 2013, 14(3):221-229.
19.Huang YF, Lo PC, Yen CL, Nigrovic PA, Chao WC, Wang WZ, Hsu GC, Tsai YS, Shieh CC: Redox Regulation of Pro-IL-1beta Processing May Contribute to the Increased Severity of Serum-Induced Arthritis in NOX2-Deficient Mice. Antioxidants & redox signaling 2015, 23(12):973-984.
20.Sims JE, Smith DE: The IL-1 family: regulators of immunity. Nature reviews Immunology 2010, 10(2):89-102.
21.Spits H, Artis D, Colonna M, Diefenbach A, Di Santo JP, Eberl G, Koyasu S, Locksley RM, McKenzie AN, Mebius RE et al: Innate lymphoid cells--a proposal for uniform nomenclature. Nature reviews Immunology 2013, 13(2):145-149.
22.Kim HY, Lee HJ, Chang YJ, Pichavant M, Shore SA, Fitzgerald KA, Iwakura Y, Israel E, Bolger K, Faul J et al: Interleukin-17-producing innate lymphoid cells and the NLRP3 inflammasome facilitate obesity-associated airway hyperreactivity. Nature medicine 2014, 20(1):54-61.
23.Schett G, Dayer JM, Manger B: Interleukin-1 function and role in rheumatic disease. Nature reviews Rheumatology 2016, 12(1):14-24.
24.Sedimbi SK, Hagglof T, Karlsson MC: IL-18 in inflammatory and autoimmune disease. Cellular and molecular life sciences : CMLS 2013, 70(24):4795-4808.
25.Amin MA, Mansfield PJ, Pakozdi A, Campbell PL, Ahmed S, Martinez RJ, Koch AE: Interleukin-18 induces angiogenic factors in rheumatoid arthritis synovial tissue fibroblasts via distinct signaling pathways. Arthritis and rheumatism 2007, 56(6):1787-1797.
26.Morel JC, Park CC, Kumar P, Koch AE: Interleukin-18 induces rheumatoid arthritis synovial fibroblast CXC chemokine production through NFkappaB activation. Laboratory investigation; a journal of technical methods and pathology 2001, 81(10):1371-1383.
27.Miller AM: Role of IL-33 in inflammation and disease. Journal of inflammation 2011, 8(1):22.
28.Xu D, Jiang HR, Kewin P, Li Y, Mu R, Fraser AR, Pitman N, Kurowska-Stolarska M, McKenzie AN, McInnes IB et al: IL-33 exacerbates antigen-induced arthritis by activating mast cells. Proceedings of the National Academy of Sciences of the United States of America 2008, 105(31):10913-10918.
29.Leung BP, Xu D, Culshaw S, McInnes IB, Liew FY: A novel therapy of murine collagen-induced arthritis with soluble T1/ST2. Journal of immunology 2004, 173(1):145-150.
30.Palmer G, Talabot-Ayer D, Lamacchia C, Toy D, Seemayer CA, Viatte S, Finckh A, Smith DE, Gabay C: Inhibition of interleukin-33 signaling attenuates the severity of experimental arthritis. Arthritis and rheumatism 2009, 60(3):738-749.
31.Ali S, Mohs A, Thomas M, Klare J, Ross R, Schmitz ML, Martin MU: The dual function cytokine IL-33 interacts with the transcription factor NF-kappaB to dampen NF-kappaB-stimulated gene transcription. Journal of immunology 2011, 187(4):1609-1616.
32.Morita H, Moro K, Koyasu S: Innate lymphoid cells in allergic and nonallergic inflammation. The Journal of allergy and clinical immunology 2016, 138(5):1253-1264.
33.Shikhagaie MM, Germar K, Bal SM, Ros XR, Spits H: Innate lymphoid cells in autoimmunity: emerging regulators in rheumatic diseases. Nature reviews Rheumatology 2017, 13(3):164-173.
34.Spits H, Di Santo JP: The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling. Nature immunology 2011, 12(1):21-27.
35.Klose CS, Flach M, Mohle L, Rogell L, Hoyler T, Ebert K, Fabiunke C, Pfeifer D, Sexl V, Fonseca-Pereira D et al: Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages. Cell 2014, 157(2):340-356.
36.Hazenberg MD, Spits H: Human innate lymphoid cells. Blood 2014, 124(5):700-709.
37.Artis D, Spits H: The biology of innate lymphoid cells. Nature 2015, 517(7534):293-301.
38.Sonnenberg GF, Monticelli LA, Elloso MM, Fouser LA, Artis D: CD4(+) lymphoid tissue-inducer cells promote innate immunity in the gut. Immunity 2011, 34(1):122-134.
39.Vonarbourg C, Mortha A, Bui VL, Hernandez PP, Kiss EA, Hoyler T, Flach M, Bengsch B, Thimme R, Holscher C et al: Regulated expression of nuclear receptor RORgammat confers distinct functional fates to NK cell receptor-expressing RORgammat(+) innate lymphocytes. Immunity 2010, 33(5):736-751.
40.Bernink JH, Krabbendam L, Germar K, de Jong E, Gronke K, Kofoed-Nielsen M, Munneke JM, Hazenberg MD, Villaudy J, Buskens CJ et al: Interleukin-12 and -23 Control Plasticity of CD127(+) Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria. Immunity 2015, 43(1):146-160.
41.Mizuno S, Mikami Y, Kamada N, Handa T, Hayashi A, Sato T, Matsuoka K, Matano M, Ohta Y, Sugita A et al: Cross-talk between RORgammat+ innate lymphoid cells and intestinal macrophages induces mucosal IL-22 production in Crohn's disease. Inflammatory bowel diseases 2014, 20(8):1426-1434.
42.Wenink MH, Leijten EFA, Cupedo T, Radstake T: Review: Innate Lymphoid Cells: Sparking Inflammatory Rheumatic Disease? Arthritis Rheumatol 2017, 69(5):885-897.
43.Ren J, Feng Z, Lv Z, Chen X, Li J: Natural killer-22 cells in the synovial fluid of patients with rheumatoid arthritis are an innate source of interleukin 22 and tumor necrosis factor-alpha. The Journal of rheumatology 2011, 38(10):2112-2118.
44.Dalbeth N, Callan MF: A subset of natural killer cells is greatly expanded within inflamed joints. Arthritis and rheumatism 2002, 46(7):1763-1772.
45.Mangialaio S, Ji H, Korganow AS, Kouskoff V, Benoist C, Mathis D: The arthritogenic T cell receptor and its ligand in a model of spontaneous arthritis. Arthritis and rheumatism 1999, 42(12):2517-2523.
46.Kelkka T, Hultqvist M, Nandakumar KS, Holmdahl R: Enhancement of antibody-induced arthritis via Toll-like receptor 2 stimulation is regulated by granulocyte reactive oxygen species. The American journal of pathology 2012, 181(1):141-150.
47.Wright HL, Moots RJ, Bucknall RC, Edwards SW: Neutrophil function in inflammation and inflammatory diseases. Rheumatology 2010, 49(9):1618-1631.
48.Neill DR, Wong SH, Bellosi A, Flynn RJ, Daly M, Langford TK, Bucks C, Kane CM, Fallon PG, Pannell R et al: Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 2010, 464(7293):1367-1370.
49.Price AE, Liang HE, Sullivan BM, Reinhardt RL, Eisley CJ, Erle DJ, Locksley RM: Systemically dispersed innate IL-13-expressing cells in type 2 immunity. Proceedings of the National Academy of Sciences of the United States of America 2010, 107(25):11489-11494.
50.Bal SM, Bernink JH, Nagasawa M, Groot J, Shikhagaie MM, Golebski K, van Drunen CM, Lutter R, Jonkers RE, Hombrink P et al: IL-1beta, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs. Nature immunology 2016, 17(6):636-645.
51.Leijten EFA, van Kempen TS, Boes M, Michels-van Amelsfort JMR, Hijnen D, Hartgring SAY, van Roon JAG, Wenink MH, Radstake TRDJ: Enrichment of Activated Group 3 Innate Lymphoid Cells in Psoriatic Arthritis Synovial Fluid. Arthritis Rheumatol 2015, 67(10):2673-2678.
52.Ivanov, II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, Cua DJ, Littman DR: The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 2006, 126(6):1121-1133.
53.Lazarevic V, Chen X, Shim JH, Hwang ES, Jang E, Bolm AN, Oukka M, Kuchroo VK, Glimcher LH: T-bet represses T(H)17 differentiation by preventing Runx1-mediated activation of the gene encoding RORgammat. Nature immunology 2011, 12(1):96-104.
54.Bjorklund AK, Forkel M, Picelli S, Konya V, Theorell J, Friberg D, Sandberg R, Mjosberg J: The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing. Nature immunology 2016, 17(4):451-460.
55.Ohne Y, Silver JS, Thompson-Snipes L, Collet MA, Blanck JP, Cantarel BL, Copenhaver AM, Humbles AA, Liu YJ: IL-1 is a critical regulator of group 2 innate lymphoid cell function and plasticity. Nature immunology 2016, 17(6):646-655.
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