臺灣博碩士論文加值系統

多發性硬化症(MS)是由第一型輔助型T細胞(Th1)與分泌介白素-17(IL-17)之輔助型T細胞(Th17)調控慢性自體免疫疾病，其致病原因尙不明瞭且非僅由單一基因或是環境因子造成。實驗性自體免疫腦脊髓炎小鼠(EAE)是常用於研究多發性硬化症的動物模式，由於抗原呈現細胞(APC)呈現髓鞘抗原刺激T細胞，促使T細胞釋放發炎介質與細胞激素，如:干擾素-γ(IFN-γ)與IL-17，而導致髓鞘與中樞神經系統被破壞。近來研究發現表基因體調控參與不同T細胞族群之分化，實驗室先前研究顯示，EAE小鼠前處理甲基化轉移酶抑制劑5-aza-2′-deoxycytidine (5-Aza)後，可藉由提升CD4+Foxp3+表達量抑制病症產生。除調節性輔助型T細胞(Treg)以外，其他T細胞表基因體模式尚未被詳細探討﹔因此，我們針對IFN-γ與IL-17A這兩種細胞激素，探討其DNA甲基化程度是否伴隨EAE疾病而改變。本研究第一部份我們將Th1與Th17細胞分化並處理5-Aza，接著利用亞硫酸鹽焦磷酸定序偵測IFN-γ DNA不同區域甲基化程度，包含intron1、 promoter -53CG、 CNS-6、CNS-22、CNS-54 與IL-17A 的CNS2。結果發現處理5-Aza後，Th1/Th17極化中分別於CNS-6與IL-17A之CNS2第六個與第八個CpG位置皆有顯著性差異。在蛋白質表現上，Th1極化過程處理5-Aza後發現CD4+ T細胞的IFN-γ 表現量增加﹔然而Th17極化過程中處理5-Aza後IL-17A表達量呈現抑制作用。此現象是由於5-Aza處理後，Th17有較高表現7-AAD與annexin-V，這些細胞走向細胞凋亡而導致。第二部分，我們誘導EAE老鼠並於不同疾病時期蒐集周邊血單核細胞，結果於DNA甲基化程度上發現IFN-γ intron1 於EAE疾病高峰時呈現去甲基化，年齡大(10周)的小鼠去甲基化現象較年齡小(6-8周)明顯且疾病較為嚴重。IFN-γ CNS-6 於EAE臨床疾病分數為1時呈現去甲基化﹔IL-17A CNS2第8與第10個位置於EAE小鼠發病初期與疾病到達高峰時皆呈現去甲基化現象。總結以上實驗結果顯示IFN-γ的CNS-6與IL-17A的CNS2 這些cis elements可用於預測EAE小鼠發病早期以及給予適當治療時機。

Multiple sclerosis (MS) is a Th1/Th17 CD4+ T cell-mediated chronic autoimmune disease caused by genetic and/or environmental factors. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model to study the MS. Because of myelin antigens presented by antigen presenting cells (APCs) and stimulated T cells that released inflammatory mediators and cytokines such as IFN-γ and IL-17 will destroy the myelin and central nerve system (CNS). In recent study, epigenetic regulation has been shown to involve in distinct T helper lineages differentiation and polarization. In our previous study, EAE mice pre treated with 5-aza-2′-deoxycytidine(5-Aza), an inhibitor of DNA nucleotide methylation transferase (DNMT), inhibited the disease through upregulated the expression of CD4+Foxp3+.In addition to the Treg cell, other T cells epigenetic pattern has not been detailed study in EAE model and MS. Therefore, we aimed to identify the cytokines of IFN-γ and IL-17A in DNA methylation pattern in vitro and in vivo EAE model. First part in this study, we carried out in vitro Th1 and Th17 polarization under 5-Aza treatment. We used bisulfite pyrosequencing to detect the regions of IFN-γ methylation pattern including intron1, promoter-53CG, CNS-6, CNS-22, CNS-54 and CNS2 of IL-17A. The CNS-6 in Th1 polarization and CNS2 site6 and site8 of IL-17A in Th17 polarization had significant difference after treated with 5-Aza. In Th1 polarization, protein level of IFN-γ was increased in CD4+ after treated with 5-Aza, however IL-17A was downregulated in Th17 polarization. The downregulation of Th17 cells were through cell apoptosis because more 7-AAD and annexin-V double positive cells was observed after treated with 5-Aza. Second part, we evaluated the methylation pattern of cytokine in different disease status in peripheral blood mononuclear cells (PBMC). In DNA level, we found the intron1 of IFN-γ was demethylated when EAE disease at peak. Older mice (more than 10 weeks) showed more demethylation and severe disease score than younger mice ( 6-8 weeks). CNS-6 of IFN-γ was demethylated when EAE score=1. Site8 and site10 in CNS2 of IL-17A was demethylated when EAE score at onset and peak. Summary, the above data show 5-Aza treatment will increase IFN-γ expression while inhibit IL-17A expression through Th17 apoptosis, and show these cis elements including CNS-6 of IFN-γ and CNS2 of IL-17A can be a tool to predict the time for EAE mice therapeutic treatment at early disease stage.

目錄
中文摘要(第ⅱ頁)
英文摘要(第ⅲ頁)
縮寫對照表(第Ⅷ頁)
一、緒論(第9頁)
1.1多發性硬化症(Mutiple Sclerosis)(第9頁)
1.2實驗性自體免疫腦脊髓炎小鼠模式(Experimental autoimmune encephalomyelitis)(第10頁)
1.3輔助型T細胞(T helper cells)(第10頁)
1.4第一型輔助型T細胞(Type1 T helper cells)(第10頁)
1.5第二型輔助型T細胞(Type2 T helper cells)(第11頁)
1.6第十七型輔助型T細胞(IL-17-secreting T helper cells)(第11頁)
1.7調節性輔助型T細胞(Regulatory T helper cells)(第12頁)
1.8對位基因體學(Epigenetics)與DNA甲基化(DNA methylation)(第12頁)
1.9 DNA甲基化與免疫(第13頁)
1.10 DNA甲基化與調節性輔助型T細胞(第14頁)
1.11去甲基化試劑5-Aza-2′-deoxycytidine(5-Aza)(第15頁)
二、研究目的(第16頁)
三、材料與方法(第17頁)
3.1.小鼠(第17頁)
3.2.實驗性自體免疫腦脊髓炎小鼠模式誘導(第17頁)
3.3.培養基、溶液與試劑(第18頁)
3.4.純化與分離小鼠T 細胞(第19頁)
3.5.細胞內細胞激素染色法(Intra-cellular cytokine staining)(第21頁)
3.6.細胞凋亡染色(第23頁)
3.7. DNA甲基化分析(第23頁)
3.8.小鼠脊椎染色(第28頁)
3.9.染色分析方法(第31頁)
3.10.數據分析(第31頁)
四、實驗結果(第32頁)
4.1.第一型輔助型T細胞極化過程中處理5-Aza之IFN-γ DNA甲基化表現(第32頁)
4.2.第十七型輔助型T細胞極化過程中處理5-Aza 後IL-17A DNA甲基化表現(第37頁)
4.3.第一型輔助型T細胞極化過程中處理5-Aza 後IFN-γ蛋白質表現(第38頁)
4.4.第十七型輔助型T細胞極化過程中處理5-Aza後IL-17A與IFN-γ蛋白質表現(第38頁)
4.5.第十七型輔助型T細胞極化過程處理5-Aza後進行細胞凋亡標記之染色(第38頁)
4.6.實驗性自體免疫腦脊髓炎小鼠模式中偵測IFN-γ不同cis element DNA甲基化狀態(第39頁)
4.7.實驗性自體免疫腦脊髓炎小鼠模式中偵測IL-17A不同cis element DNA甲基化狀態(第42頁)
4.8.利用蘇木素(Hematoxylin)-伊紅(Eosin)與Luxol Fast blue於實驗性自體免疫腦脊髓炎小鼠脊椎進行組織染色(第42頁)
五、實驗討論(第43頁)
六、圖表(第47頁)
七、文獻(第86頁)

1.A. Alonso, M. A. Hernan, Temporal trends in the incidence of multiple sclerosis: a systematic review. Neurology 71, 129-135 (2008).
2.D. H. Mahad, B. D. Trapp, H. Lassmann, Pathological mechanisms in progressive multiple sclerosis. The Lancet Neurology 14, 183-193 (2015).
3.B. Weinstock-Guttman, M. Ramanathan, R. Zivadinov, Interferon-beta treatment for relapsing multiple sclerosis. Expert Opin Biol Ther 8, 1435-1447 (2008).
4.L. S. H. W. A. SHEREMATA, Magnetic resonance imaging of spinal cord lesions in multiple sclerosis. (1989).
5.R. A. O'Connor et al., Cutting Edge: Th1 Cells Facilitate the Entry of Th17 Cells to the Central Nervous System during Experimental Autoimmune Encephalomyelitis. The Journal of Immunology 181, 3750-3754 (2008).
6.C. S. Constantinescu, N. Farooqi, K. O'Brien, B. Gran, Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol 164, 1079-1106 (2011).
7.J. M. Fletcher, S. J. Lalor, C. M. Sweeney, N. Tubridy, K. H. Mills, T cells in multiple sclerosis and experimental autoimmune encephalomyelitis. Clin Exp Immunol 162, 1-11 (2010).
8.R. V. Luckheeram, R. Zhou, A. D. Verma, B. Xia, CD4(+)T cells: differentiation and functions. Clin Dev Immunol 2012, 925135 (2012).
9.S. Romagnani, Th1/Th2 cells. Inflamm Bowel Dis 5, 285-294 (1999).
10.G. H. Stummvoll et al., Th1, Th2, and Th17 Effector T Cell-Induced Autoimmune Gastritis Differs in Pathological Pattern and in Susceptibility to Suppression by Regulatory T Cells. The Journal of Immunology 181, 1908-1916 (2008).
11.S. S. Deo, K. J. Mistry, A. M. Kakade, P. V. Niphadkar, Role played by Th2 type cytokines in IgE mediated allergy and asthma. Lung India 27, 66-71 (2010).
12.T. Korn, E. Bettelli, M. Oukka, V. K. Kuchroo, IL-17 and Th17 Cells. Annu Rev Immunol 27, 485-517 (2009).
13.S. Sakaguchi, K. Wing, M. Miyara, Regulatory T cells - a brief history and perspective. Eur J Immunol 37 Suppl 1, S116-123 (2007).
14.J. D. Fontenot, M. A. Gavin, A. Y. Rudensky, Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4, 330-336 (2003).
15.S. Hori, T. Nomura, S. Sakaguchi, Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057-1061 (2003).
16.C. R. Grant, R. Liberal, G. Mieli-Vergani, D. Vergani, M. S. Longhi, Regulatory T-cells in autoimmune diseases: challenges, controversies and--yet--unanswered questions. Autoimmun Rev 14, 105-116 (2015).
17.P. A. Jones, S. B. Baylin, The epigenomics of cancer. Cell 128, 683-692 (2007).
18.M. Okano, D. W. Bell, D. A. Haber, E. Li, DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247-257 (1999).
19.H. Leonhardt, A. W. Page, H. U. Weier, T. H. Bestor, A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71, 865-873 (1992).
20.N. Rivlin, R. Brosh, M. Oren, V. Rotter, Mutations in the p53 Tumor Suppressor Gene: Important Milestones at the Various Steps of Tumorigenesis. Genes Cancer 2, 466-474 (2011).
21.B. Suarez-Alvarez, R. M. Rodriguez, M. F. Fraga, C. Lopez-Larrea, DNA methylation: a promising landscape for immune system-related diseases. Trends Genet 28, 506-514 (2012).
22.S. D. Bos et al., Genome-wide DNA methylation profiles indicate CD8+ T cell hypermethylation in multiple sclerosis. PLoS One 10, e0117403 (2015).
23.P. C. Janson et al., Profiling of CD4+ T cells with epigenetic immune lineage analysis. J Immunol 186, 92-102 (2011).
24.Q. Lu et al., Demethylation of ITGAL (CD11a) regulatory sequences in systemic lupus erythematosus. Arthritis Rheum 46, 1282-1291 (2002).
25.P. Matatiele, M. Tikly, G. Tarr, M. Gulumian, DNA methylation similarities in genes of black South Africans with systemic lupus erythematosus and systemic sclerosis. J Biomed Sci 22, 34 (2015).
26.N. J. Bernard, Rheumatoid arthritis: Who knows why regulatory T cells are defective in RA ... IDO. Nat Rev Rheumatol 10, 381 (2014).
27.J. K. Wiencke et al., The DNA methylation profile of activated human natural killer cells. Epigenetics 11, 363-380 (2016).
28.R. C. Hardison, Conserved noncoding sequences are reliable guides to regulatory elements. Trends Genet 16, 369-372 (2000).
29.J. R. Schoenborn et al., Comprehensive epigenetic profiling identifies multiple distal regulatory elements directing transcription of the gene encoding interferon-gamma. Nat Immunol 8, 732-742 (2007).
30.G. R. Lee, S. T. Kim, C. G. Spilianakis, P. E. Fields, R. A. Flavell, T helper cell differentiation: regulation by cis elements and epigenetics. Immunity 24, 369-379 (2006).
31.R. D. Hatton et al., A distal conserved sequence element controls Ifng gene expression by T cells and NK cells. Immunity 25, 717-729 (2006).
32.A. Balasubramani et al., Deletion of a conserved cis-element in the Ifng locus highlights the role of acute histone acetylation in modulating inducible gene transcription. PLoS Genet 10, e1003969 (2014).
33.J. K. Polansky et al., DNA methylation controls Foxp3 gene expression. Eur J Immunol 38, 1654-1663 (2008).
34.R. M. Thomas, H. Sai, A. D. Wells, Conserved intergenic elements and DNA methylation cooperate to regulate transcription at the il17 locus. J Biol Chem 287, 25049-25059 (2012).
35.K. Raj, G. J. Mufti, Azacytidine (Vidaza(R)) in the treatment of myelodysplastic syndromes. Ther Clin Risk Manag 2, 377-388 (2006).
36.S. F. Ziegler, J. H. Buckner, FOXP3 and the regulation of Treg/Th17 differentiation. Microbes Infect 11, 594-598 (2009).
37.M. Kleinewietfeld, D. A. Hafler, The plasticity of human Treg and Th17 cells and its role in autoimmunity. Semin Immunol 25, 305-312 (2013).
38.M. W. Chan et al., Low-dose 5-aza-2'-deoxycytidine pretreatment inhibits experimental autoimmune encephalomyelitis by induction of regulatory T cells. Mol Med 20, 248-256 (2014).
39.B. Jones, J. Chen, Inhibition of IFN-gamma transcription by site-specific methylation during T helper cell development. EMBO J 25, 2443-2452 (2006).
40.C. M. Tato et al., Cutting Edge: Innate production of IFN-gamma by NK cells is independent of epigenetic modification of the IFN-gamma promoter. J Immunol 173, 1514-1517 (2004).
41.M. Inoue et al., Interferon-beta therapy against EAE is effective only when development of the disease depends on the NLRP3 inflammasome. Sci Signal 5, ra38 (2012).
42.G. Arellano, P. A. Ottum, L. I. Reyes, P. I. Burgos, R. Naves, Stage-Specific Role of Interferon-Gamma in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis. Front Immunol 6, 492 (2015).
43.S. S. Yoshinobu Okuda , Takehiko Yanagihara, The pattern of cytokine gene expression in lymphoid organs and peripheral blood mononuclear cells of mice with experimental allergic encephalomyelitis. Journal of Neuroimmunology, (1998).