臺灣博碩士論文加值系統

指導教授推薦書
口試委員會審定書
致謝 iii
摘要 iv
Abstract v
目錄 vi
圖目錄 ix
附錄目錄 xi
第一章 背景 1
1.1 葡萄膜炎 1
1.2 實驗性自體免疫葡萄膜炎 3
1.3 第一型與第二型輔助型T細胞 (Th1/Th2) 5
1.4 輔助型T細胞-17 (Th17)與介白素-17 (IL-17)及IL-17受器 (IL-17 receptor) 7
1.5 IL-17扮演抗發炎角色 11
1.6 Adoptive transfer EAU (tEAU) 12
1.7 研究動機與目的 13
第二章 研究方法 15
2.1 實驗設計 15
2.2 材料方法 18
2.2.1 免疫小鼠 18
2.2.2 IRBP備製 18
2.2.3 CFA佐劑 18
2.2.4 Pertussis toxin (PTX ) 18
2.2.5 IRBP-CFA emulsification 19
2.2.6 Antigen injection 19
2.2.7 小鼠脾臟細胞採集與前處理 19
2.2.8 小鼠週邊血採集與前處理 20
2.2.9 細胞外染色 20
2.2.10 細胞內染色 20
2.2.11 Treg染色 21
2.2.12 酵素連結免疫吸附法 (Enzyme-linked immunosorbent assay) 22
2.2.13 tEAU 23
第三章 實驗結果 24
3.1 比較WT、IL-17KO小鼠的疾病誘發率、發炎趨勢及疾病嚴重度 24
3.2 比較IL-17KO小鼠在EAU初期與後期時免疫反應的差異 25
3.3 Adoptive transfer (tEAU)發炎趨勢 31
3.4 a7tEAU和a28tEAU的疾病發展及免疫反應 33
第四章 討論 40
第五章 參考文獻 46
附圖 57
附錄 81


圖目錄
圖一、 Wild-type (WT)和IL-17KO之EAU小鼠疾病誘發率與發炎曲線 57
圖二、 Wild-type和IL-17KO之EAU小鼠疾病嚴重程度在各天數所佔百分比 59
圖三、 分析EAU小鼠脾臟細胞中細胞激素IFN-γ和IL-17的分泌量 61
圖四、 分析EAU小鼠脾臟細胞以Neutralization antibody於 in vitro環境中其細胞激素IFN-γ的分泌量 63
圖五、 分析EAU小鼠脾臟細胞以Neutralization antibody於 in vitro環境中其細胞激素IL-17的分泌量 65
圖六、 分析EAU小鼠脾臟細胞IFN-γ所佔比例 66
圖七、 分析EAU小鼠周邊血白血球IFN-γ所佔比例 67
圖八、 分析EAU小鼠脾臟細胞和周邊血白血球中IL-17所佔比例 69
圖九、 tEAU發炎曲線 70
圖十、 a7tEAU和a28tEAU發炎趨勢 71
圖十一、 a7tEAU及a28tEAU小鼠初期與後期時具抗原專一性T細胞所產生之細胞激素IFN-γ分泌量 73
圖十二、 a7tEAU及a28tEAU小鼠初期與後期時具抗原專一性T 細胞所產生之細胞激素IL-17分泌量 75
圖十三、 a7tEAU和a28tEAU小鼠脾臟細胞輔助型和毒殺型T細 胞中表現IFN-γ+和IL-17+的比例分析 77
圖十四、 a7tEAU和a28tEAU小鼠周邊血白血球細胞輔助型和毒 殺型T細胞中表現IFN-γ+和IL-17+的比例分析 79



附錄目錄

附錄一、 小鼠眼底評分參考 81
附錄二、 WT及IL-17KO之EAU小鼠於7天眼底攝影 82
附錄三、 WT及IL-17KO之EAU眼底發炎評估標準 83
附錄四、 分析EAU小鼠脾臟細胞中Treg的比例 85
附錄五、 分析a7tEAU和a28tEAU小鼠脾臟細胞中Treg比例 86
附錄六、 tEAU小鼠實驗誘發流程 87

1. Nussenblatt, R.B., et al., Intraocular inflammatory disease (uveitis) and the use of oral tolerance: a status report. Ann N Y Acad Sci, 1996. 778: p. 325-37.
2. Ke, Y., et al., Retinal Astrocytes respond to IL-17 differently than Retinal Pigment Epithelial cells. J Leukoc Biol, 2009. 86(6): p. 1377-84.
3. Amadi-Obi, A., et al., TH17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1. Nat Med, 2007. 13(6): p. 711-8.
4. Nussenblatt, R.B., Basic and clinical immunology in uveitis. Jpn J Ophthalmol, 1987. 31(3): p. 368-74.
5. Silver, P.B., et al., The requirement for pertussis to induce EAU is strain-dependent: B10.RIII, but not B10.A mice, develop EAU and Th1 responses to IRBP without pertussis treatment. Invest Ophthalmol Vis Sci, 1999. 40(12): p. 2898-905.
6. Sudweeks, J.D., et al., Locus controlling Bordetella pertussis-induced histamine sensitization (Bphs), an autoimmune disease-susceptibility gene, maps distal to T-cell receptor beta-chain gene on mouse chromosome 6. Proc Natl Acad Sci U S A, 1993. 90(8): p. 3700-4.
7. Luger, D. and R.R. Caspi, New perspectives on effector mechanisms in uveitis. Semin Immunopathol, 2008. 30(2): p. 135-43.
8. Caspi, R.R., et al., T cell lines mediating experimental autoimmune uveoretinitis (EAU) in the rat. J Immunol, 1986. 136(3): p. 928-33.
9. Weaver, C.T., et al., Th17: an effector CD4 T cell lineage with regulatory T cell ties. Immunity, 2006. 24(6): p. 677-88.
10. Damsker, J.M., A.M. Hansen, and R.R. Caspi, Th1 and Th17 cells: adversaries and collaborators. Ann N Y Acad Sci, 2010. 1183: p. 211-21.
11. Harrington, L.E., et al., Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol, 2005. 6(11): p. 1123-32.
12. Veldhoen, M., et al., TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity, 2006. 24(2): p. 179-89.
13. Park, H., et al., A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol, 2005. 6(11): p. 1133-41.
14. Mosmann, T.R. and R.L. Coffman, TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol, 1989. 7: p. 145-73.
15. Alvaro-Gracia, J.M., N.J. Zvaifler, and G.S. Firestein, Cytokines in chronic inflammatory arthritis. V. Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on HLA-DR expression, proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor production by rheumatoid arthritis synoviocytes. J Clin Invest, 1990. 86(6): p. 1790-8.
16. Ferber, I.A., et al., Mice with a disrupted IFN-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J Immunol, 1996. 156(1): p. 5-7.
17. Jones, L.S., et al., IFN-gamma-deficient mice develop experimental autoimmune uveitis in the context of a deviant effector response. J Immunol, 1997. 158(12): p. 5997-6005.
18. Dalton, D.K., et al., Multiple defects of immune cell function in mice with disrupted interferon-gamma genes. Science, 1993. 259(5102): p. 1739-42.
19. Cua, D.J., et al., Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature, 2003. 421(6924): p. 744-8.
20. Zhang, G.X., et al., Induction of experimental autoimmune encephalomyelitis in IL-12 receptor-beta 2-deficient mice: IL-12 responsiveness is not required in the pathogenesis of inflammatory demyelination in the central nervous system. J Immunol, 2003. 170(4): p. 2153-60.
21. Korn, T., et al., IL-17 and Th17 Cells. Annu Rev Immunol, 2009. 27: p. 485-517.
22. Aggarwal, S., et al., Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J Biol Chem, 2003. 278(3): p. 1910-4.
23. Langrish, C.L., et al., IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med, 2005. 201(2): p. 233-40.
24. Hirano, T., K. Ishihara, and M. Hibi, Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene, 2000. 19(21): p. 2548-56.
25. Akira, S., et al., Biology of multifunctional cytokines: IL 6 and related molecules (IL 1 and TNF). FASEB J, 1990. 4(11): p. 2860-7.
26. Korn, T., et al., IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells. Nature, 2007. 448(7152): p. 484-7.
27. Wei, L., et al., IL-21 is produced by Th17 cells and drives IL-17 production in a STAT3-dependent manner. J Biol Chem, 2007. 282(48): p. 34605-10.
28. Ouyang, W., J.K. Kolls, and Y. Zheng, The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity, 2008. 28(4): p. 454-67.
29. Mangan, P.R., et al., Transforming growth factor-beta induces development of the T(H)17 lineage. Nature, 2006. 441(7090): p. 231-4.
30. Nurieva, R., et al., Essential autocrine regulation by IL-21 in the generation of inflammatory T cells. Nature, 2007. 448(7152): p. 480-3.
31. Zhou, L., et al., TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature, 2008. 453(7192): p. 236-40.
32. Manel, N., D. Unutmaz, and D.R. Littman, The differentiation of human T(H)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor RORgammat. Nat Immunol, 2008. 9(6): p. 641-9.
33. Yang, L., et al., IL-21 and TGF-beta are required for differentiation of human T(H)17 cells. Nature, 2008. 454(7202): p. 350-2.
34. Luger, D., et al., Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J Exp Med, 2008. 205(4): p. 799-810.
35. Koenders, M.I., et al., Induction of cartilage damage by overexpression of T cell interleukin-17A in experimental arthritis in mice deficient in interleukin-1. Arthritis Rheum, 2005. 52(3): p. 975-83.
36. Elson, C.O., et al., Monoclonal anti-interleukin 23 reverses active colitis in a T cell-mediated model in mice. Gastroenterology, 2007. 132(7): p. 2359-70.
37. Hue, S., et al., Interleukin-23 drives innate and T cell-mediated intestinal inflammation. J Exp Med, 2006. 203(11): p. 2473-83.
38. Ye, P., et al., Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. J Exp Med, 2001. 194(4): p. 519-27.
39. Higgins, S.C., et al., TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells. J Immunol, 2006. 177(11): p. 7980-9.
40. Cua, D.J. and C.M. Tato, Innate IL-17-producing cells: the sentinels of the immune system. Nat Rev Immunol, 2010. 10(7): p. 479-89.
41. Huber, M., et al., IL-17A secretion by CD8+ T cells supports Th17-mediated autoimmune encephalomyelitis. J Clin Invest, 2013. 123(1): p. 247-60.
42. Nigam, P., et al., Loss of IL-17-producing CD8 T cells during late chronic stage of pathogenic simian immunodeficiency virus infection. J Immunol, 2011. 186(2): p. 745-53.
43. Rachitskaya, A.V., et al., Cutting edge: NKT cells constitutively express IL-23 receptor and RORgammat and rapidly produce IL-17 upon receptor ligation in an IL-6-independent fashion. J Immunol, 2008. 180(8): p. 5167-71.
44. Lockhart, E., A.M. Green, and J.L. Flynn, IL-17 production is dominated by gammadelta T cells rather than CD4 T cells during Mycobacterium tuberculosis infection. J Immunol, 2006. 177(7): p. 4662-9.
45. Yao, Z., et al., Herpesvirus saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor. J Immunol, 2011. 187(9): p. 4392-402.
46. Toy, D., et al., Cutting edge: interleukin 17 signals through a heteromeric receptor complex. J Immunol, 2006. 177(1): p. 36-9.
47. Yu, J.J. and S.L. Gaffen, Interleukin-17: a novel inflammatory cytokine that bridges innate and adaptive immunity. Front Biosci, 2008. 13: p. 170-7.
48. Mellett, M., et al., Orphan receptor IL-17RD tunes IL-17A signalling and is required for neutrophilia. Nat Commun, 2012. 3: p. 1119.
49. Kagami, S., et al., Circulating Th17, Th22, and Th1 cells are increased in psoriasis. J Invest Dermatol, 2010. 130(5): p. 1373-83.
50. Kobayashi, T., et al., IL23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn's disease. Gut, 2008. 57(12): p. 1682-9.
51. McAllister, F., et al., Role of IL-17A, IL-17F, and the IL-17 receptor in regulating growth-related oncogene-alpha and granulocyte colony-stimulating factor in bronchial epithelium: implications for airway inflammation in cystic fibrosis. J Immunol, 2005. 175(1): p. 404-12.
52. Komiyama, Y., et al., IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis. J Immunol, 2006. 177(1): p. 566-73.
53. Rizzo, H.L., et al., IL-23-mediated psoriasis-like epidermal hyperplasia is dependent on IL-17A. J Immunol, 2011. 186(3): p. 1495-502.
54. Smith, E., et al., IL-17A inhibits the expansion of IL-17A-producing T cells in mice through "short-loop" inhibition via IL-17 receptor. J Immunol, 2008. 181(2): p. 1357-64.
55. Lin, P., E.B. Suhler, and J.T. Rosenbaum, The future of uveitis treatment. Ophthalmology, 2014. 121(1): p. 365-76.
56. O'Connor, W., Jr., et al., A protective function for interleukin 17A in T cell-mediated intestinal inflammation. Nat Immunol, 2009. 10(6): p. 603-9.
57. Ke, Y., et al., Anti-inflammatory role of IL-17 in experimental autoimmune uveitis. J Immunol, 2009. 182(5): p. 3183-90.
58. Dick, A.D., et al., Secukinumab in the treatment of noninfectious uveitis: results of three randomized, controlled clinical trials. Ophthalmology, 2013. 120(4): p. 777-87.
59. Namba, K., et al., Identification of a peptide inducing experimental autoimmune uveoretinitis (EAU) in H-2Ak-carrying mice. Clin Exp Immunol, 1998. 111(2): p. 442-9.
60. Rizzo, L.V., et al., Establishment and characterization of a murine CD4+ T cell line and clone that induce experimental autoimmune uveoretinitis in B10.A mice. J Immunol, 1996. 156(4): p. 1654-60.
61. Shao, H., et al., Severe chronic experimental autoimmune uveitis (EAU) of the C57BL/6 mouse induced by adoptive transfer of IRBP1-20-specific T cells. Exp Eye Res, 2006. 82(2): p. 323-31.
62. Yoshimura, T., et al., Differential roles for IFN-gamma and IL-17 in experimental autoimmune uveoretinitis. Int Immunol, 2008. 20(2): p. 209-14.
63. Sonoda, K.H., et al., WSX-1 plays a significant role for the initiation of experimental autoimmune uveitis. Int Immunol, 2007. 19(1): p. 93-8.
64. Caspi, R.R., T. Kuwabara, and R.B. Nussenblatt, Characterization of a suppressor cell line which downgrades experimental autoimmune uveoretinitis in the rat. J Immunol, 1988. 140(8): p. 2579-84.
65. Shevach, E.M., Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity, 2009. 30(5): p. 636-45.
66. Josefowicz, S.Z. and A. Rudensky, Control of regulatory T cell lineage commitment and maintenance. Immunity, 2009. 30(5): p. 616-25.
67. Chen, L., et al., Diminished frequency and function of CD4+CD25high regulatory T cells associated with active uveitis in Vogt-Koyanagi-Harada syndrome. Invest Ophthalmol Vis Sci, 2008. 49(8): p. 3475-82.
68. Jiang, H.R., L. Lumsden, and J.V. Forrester, Macrophages and dendritic cells in IRBP-induced experimental autoimmune uveoretinitis in B10RIII mice. Invest Ophthalmol Vis Sci, 1999. 40(13): p. 3177-85.
69. Avichezer, D., et al., Residues 1-20 of IRBP and whole IRBP elicit different uveitogenic and immunological responses in interferon gamma deficient mice. Exp Eye Res, 2000. 71(2): p. 111-8.
70. Avichezer, D., et al., Interphotoreceptor retinoid-binding protein (IRBP)-deficient C57BL/6 mice have enhanced immunological and immunopathogenic responses to IRBP and an altered recognition of IRBP epitopes. J Autoimmun, 2003. 21(3): p. 185-94.