臺灣博碩士論文加值系統

研究背景：根據過去的研究已知不論是在人類或是老鼠動物實驗均顯示第一型干擾素與全身性紅斑狼瘡關係密切，尤其是干擾素�恁C接受干擾素治療的病人有一部份會產生自體免疫抗體，少數病人甚至發生全身性紅斑狼瘡的臨床症狀。病情活躍的全身性紅斑狼瘡病人血清中第一型干擾素濃度是升高的，且其濃度與狼瘡的疾病活動度成正相關。另外在全身性紅斑狼瘡病人週邊血液單核白血球中第一型干擾素誘導基因的表現也證實是提高的，且其表現也同樣地與狼瘡的疾病活動度成正相關。干擾素調控因子家族是一種轉錄因子，其主要作用是影響第一型干擾素的誘發與功能表現，尤其是干擾素調控因子3、5、7在第一型干擾素誘發的調控上佔最重要的角色，已有研究指出干擾素調控因子5的基因多型性與全身性紅斑狼瘡的發病有密切關係。干擾素調控因子的基因表現量與全身性紅斑狼瘡的關係迄今未有相關的研究發表，因此本研究主要就是要來探討兩者之間可能的關係。
研究方法：51位成年全身性紅斑狼瘡患者，其診斷都有符合美國風濕病醫學會全身性紅斑狼瘡分類標準中四項要件以上。病人不得罹患近期感染性疾病、癌症及病毒性肝炎。另外以65位年齡性別相當的成年健康者作為實驗控制組。抽取全身性紅斑狼瘡患者和實驗控制組健康成人的血液先分離出週邊血液單核白血球，以定量聚合酶連鎖反應方法測其週邊血液單核白血球干擾素調控因子3、5、7的基因表現；同時以酶免疫吸附法測量所有志願者血清中干擾素�悛瑪@度，並依全身性紅斑狼瘡疾病活動度量表計算當時患者狼瘡的疾病活動度。此外，92位成年全身性紅斑狼瘡患者，病人不得罹患病毒性肝炎及癌症；另92位年齡性別相當的成年健康者作為實驗控制組，以ABI公司的TaqMan基因型鑑定則用來進行干擾素調控因子7的基因多型性研究。
研究結果：全身性紅斑狼瘡患者血清中干擾素�悛瑪@度明顯高於實驗控制組健康成人。其干擾素調控因子5及干擾素調控因子7在全身性紅斑狼瘡病人週邊血液單核白血球的基因表現增加，在狼瘡腎炎患者特別高，尤其是干擾素調控因子7的基因表現特別高，且和患者血清中干擾素�悛瑪@度及其狼瘡的疾病活動度成正相關。但是全身性紅斑狼瘡患者週邊血液單核白血球的干擾素調控因子3基因表現並未有意義的增加。干擾素調控因子7的基因多型性研究發現rs1061501單一核苷酸基因多型性TT基因型與T對偶基因在全身性紅斑狼瘡患者明顯較健康成人多。
結論：雖然研究樣本數不多且實驗結果可能受到患者使用藥物〈如類固醇與免疫抑制劑〉的影響，本研究首先發現干擾素調控因子5及干擾素調控因子7在全身性紅斑狼瘡病人週邊血液單核白血球的基因表現增加且和患者當時的狼瘡疾病活動度成正相關。干擾素調控因子5及干擾素調控因子7可能因過度表現促使干擾素�悀尷c增加而影響紅斑狼瘡病的發病與疾病活性，此研究提供了全身性紅斑狼瘡致病機轉及治療上一個新的思考方向。另外，本研究亦發現干擾素調控因子7的基因多型性與全身性紅斑狼瘡的相關性，可見干擾素調控因子7在全身性紅斑狼瘡的致病機轉及疾病活性均佔有重要的角色。

Background: Recent studies in humans and murine models suggest that type-I interferons (IFNs), especially interferon-alpha (IFN��), are important for the initiation and potentiation of systemic lupus erythematosus (SLE) activity. Some patients received IFN therapy developed autoantibodies and some of them even evolved into clinical SLE. Serum levels of IFN�� are elevated in patients with active SLE, and positively correlated with SLE disease activity. In addition, the expression of type-I IFN-inducible genes has been demonstrated to be elevated in peripheral blood mononuclear cells (PBMCs) in patients with SLE, and also positively correlated with SLE disease activity. Interferon regulatory factors (IRFs) are a family of transcriptional factors mediating induction of IFNs and diverse IFN responses. Especially IRF3, 5, and 7 play key roles in regulation of the type-I IFN induction. To our knowledge, the investigation regarding the association between IRFs and SLE is limited. Therefore, the proposed research is aimed to examine the association between IRFs and SLE.
Methods: 51 adult SLE patients fulfilled at least four criteria of the American College of Rheumatology for SLE classifications were enrolled in this study. Subjects with a current or recent infection including viral hepatitis and malignancy were excluded. Another 65 age- and gender-matched healthy hospital employees were used as the control group. We check the levels of IRF3, 5, and 7 gene expressions of peripheral blood mononuclear cells (PBMCs) by quantitative real time polymerase chain reaction (PCR) methods. Simultaneously, their serum IFN�� levels were measured by enzyme linked immunosorbent assay (ELISA), and SLE disease activity index (SLEDAI) score were assessed and recorded. In addition, 92 SLE patients (viral hepatitis and malignancy excluded) and 92 healthy controls were enrolled for IRF7 polymorphisms study. IRF7 single-nucleotide polymorphism (SNP) research was manipulated according to ABI (Applied biosystems incorporated) TaqMan genotyping assays.
Results: In concordance with previous reports, our study again showed that patients with SLE had significantly higher IFN�� levels in serum than healthy controls (1.84�b1.12 vs 0.39�b0.26 pg/ml, p<0.001). Among the 3 IRF genes studied, we found that the expression of IRF5 and IRF7 gene w significantly increased in patients with SLE compared with those of healthy controls, especially IRF7. However, there was no significant difference regarding the expression of IRF3 between SLE patients and normal controls. In addition, the expression of IRF5 and IRF7 gene was particularly higher in patients with lupus nephritis, and positively correlated with serum IFN�� levels and SLEDAI score in patients with SLE. IRF7 rs1061501 and IRF7 rs1061502 SNPs were examined. The results showed that IRF7 rs1061501 SNP TT genotype and T allele were significantly raised in patients with SLE compared with the control group.
Conclusions: Although the power of the results might be limited due to the small sample size and medications effect such as glucocorticoids and immunosuppressants. However, our study is the first to demonstrate the increased expression of IRF5 and IRF7 gene in patients with SLE, especially in patients with lupus nephritis. Meanwhile, expression of IRF5 and IRF7 gene was positively correlated with serum IFN�� levels and lupus disease activity. The study suggests that the dysregulation of IRF5 and IRF7 might mediate the excessive production of IFN�� cytokine which then exerts a crucial effect in the pathogenesis and disease activity of human SLE. Besides, The IRF7 rs1061501 SNP study revealed that TT genotype and T allele are risk genetic marker for SLE was also a novel finding. The result may offer new etiological and therapeutic insights into SLE.

Abstract I
中文摘要 III
誌謝 V
Table of contents VI
List of tables VIII
List of figures IX
Abbreviations XI
Introduction
1. SLE 1
2. Type-I IFN and SLE 1
3. Type-I IFN-inducible genes and SLE 3
4. IRFs 3
5. IRF3, IRF5, IRF7, and Type-I IFN 4
6. IRF5 polymorphisms and SLE 7
Aims and significance of the study 8
Research design and methods
Patients and samples 9
Separation PBMC and serum 9
Blood RNA stabilization and purification 9
Reverse transcription 10
PCR primers 10
IRF3, 5, and 7 real-time PCR 10
Serum INF�� level 11
SLE disease activity and flare up 11
Lupus nephritis 12
Subjects for IRF7 SNPs study 12
Genomic DNA extraction 12
IRF7 Polymorphisms genotyping 13
ABI TaqMan SNP genotyping assay 13
Statistical analysis 15
Results
1. Baseline characteristics between SLE group and control group 17
2. Patients with SLE had significantly increased IFN�� serum levels 17
3. The expression of IRF5 and IRF7 genes were significantly raised in patients with SLE, especially IRF7 17
4. The expression of IRF5 and IRF7 genes were particularly elevated in SLE patients with lupus nephritis 18
5. The correlations between expression level of IRF5 in PBMC and lupus disease activity 18
6. The correlations between expression level of IRF7 in PBMC and lupus disease activity 19
7. IRF7 polymorphisms and SLE 19
Discussions 21
Conclusions 26
References 27
Tables 33
Figures 43
Curriculum vitae 54

1. Fessel WJ. Epidemiology of systemic lupus erythematosus. Rheumatic Disease Clinics of North America 14:15-23, 1998.
2. Huang MX, Zhang L, Shi KX. The epidemiology of systemic lupus erythematosus in Shanghai. Chinese Journal of Internal Medicine 24:451-3, 1985.
3. Ronnblom L. Eloranta ML. Alm GV. The type I interferon system in systemic lupus erythematosus. Arthritis & Rheumatism 54(2):408-20, 2006.
4. Koutouzov S, Mathian A, Dalloul A. Type-I interferons and systemic lupus erythematosus. Autoimmun Review 5:554-62, 2006.
5. Pascual V. Farkas L. Banchereau J. Systemic lupus erythematosus: all roads lead to type I interferons. Current Opinion in Immunology 18(6):676-82, 2006.
6. Niewold TB. Swedler WI. Systemic lupus erythematosus arising during interferon-alpha therapy for cryoglobulinemic vasculitis associated with hepatitis C. Clinical Rheumatology 24(2):178-81, 2005.
7. Hooks JH, Moutsopoulos HM, Geis SA, Stahl NI, Decker JL, Notkins AL. Immune interferon in the circulation of patients with autoimmune disease. New England Journal of Medicine 301:5-8, 1979.
8. Ytterberg SR, Schnitzer TJ. Serum interferon levels in patients with systemic lupus erythematosus. Arthritis & Rheumatism 25:401-6, 1982.
9. Kim T, Kanayama Y, Negoro N, Okamura M, Takeda T, Inoue T. Serum levels of interferons in patients with systemic lupus erythematosus. Clinical and Experimental Immunology 70:562-9, 1987.
10. Banchereau J. Pascual V. Type I interferon in systemic lupus erythematosus and other autoimmune diseases. Immunity 25(3):383-92, 2006.
11. Niewold TB, Hua J, Lehman TJ, Harley JB, Crow MK. High serum IFN-alpha activity is a heritable risk factor for systemic lupus erythematosus. Genes Immun 8:492-502, 2007.
12. Lorant Farkas, Klaus Beiske, Fridtjof Lund-Johansen, Per Brandtzaeg and Frode L. Jahnsen. Plasmacytoid Dendritic Cells (Natural Interferon-��/��-Producing Cells) Accumulate in Cutaneous Lupus Erythematosus Lesions. American Journal of Pathology 159:237-43, 2001.
13. Ronnbblom L, Alm GV. The natural interferon-alpha producing cells in systemic lupus erythematosus. Human Immunology 63:1181-93, 2002.
14. Selmi C. Lleo A. Zuin M. Podda M. Rossaro L. Gershwin ME. Interferon alpha and its contribution to autoimmunity. Current Opinion in Investigational Drugs 7(5):451-6, 2006.
15. Kyriakos A. Kirou, Christina Lee, Sandhya George, Kyriakos Louca, Margeret G.E. Peterson, Mary K. Crow. Activation of interferon-�� pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis & Rheumatism 52(5):1491-503, 2005.
16. MC Dall’Era, PM Cardarelli, BT Preston, A Witte, JC Davis Jr. Type I interferon correlates with serological and clinical manifestations of SLE. Annals of Rheumatic Disease 64:1692-7, 2005.
17. Xeubing Feng et al. Association of increased interferon-inducible gene expression with disease activity and lupus nephritis in patients with systemic lupus erythematosus. Arthritis & Rheumatism 54(9):2591-62, 2006.
18. Honda K, Yanai H, Takaoka A, Taniguchi T. Regulation of the type I IFN induction: a current view. Int Immunol 17:1367-78, 2005.
19. Lars Rönnblom, Gunnar V Alm. Systemic lupus erythematosus and the type I interferon system. Arthritis Research & Therapy 5(2):68-75, 2003.
20. Sasai M. Matsumoto M. Seya T. The kinase complex responsible for IRF-3-mediated IFN-beta production in myeloid dendritic cells (mDC). Journal of Biochemistry 139(2):171-5, 2006.
21. Solis M. Goubau D. Romieu-Mourez R. Genin P. Civas A. Hiscott J. Distinct functions of IRF-3 and IRF-7 in IFN-alpha gene regulation and control of anti-tumor activity in primary macrophages. Biochemical Pharmacology 72(11):1469-76, 2006.
22. Hiscott J. Nguyen TL. Arguello M. Nakhaei P. Paz S. Manipulation of the nuclear factor-kappaB pathway and the innate immune response by viruses. Oncogene 25(51):6844-67, 2006.
23. Honda K. Takaoka A. Taniguchi T. Type I interferon gene induction by the interferon regulatory factor family of transcription factors. Immunity 25(3):349-60, 2006.
24. Honda K. Taniguchi T. IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nature Reviews. Immunology 6(9):644-58, 2006.
25. Barnes B, Lubyova B, Pitha PM. On the role of IRF in host defense. J Interferon Cytokine Res 22:59-71, 2002.
26. Suhara, W. et al. Analyses of virus-induced homomeric and heteromeric protein associations between IRF-3 and coactivator CBP/p300. Journal of Biochemistry (Tokyo) 128:301-7, 2000.
27. Lin, R., Heylbroeck, C., Pitha, P. M. & Hiscott, J. Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. Molecular and Cellular Biology 18:2986-96, 1998.
28. Yoneyama, M. et al. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300. The EMBO Journal 17:1087-95, 1998.
29. Betsy Barnes, Barbora Lubyova, Paula M. Pitha. Review: On the Role of IRF in Host Defense. Journal of Interferon & Cytokine Research 22(1): 59-71, 2002.
30. Tailor P. Tamura T. Ozato K. IRF family proteins and type I interferon induction in dendritic cells. Cell Research 16(2):134-40, 2006.
31. Graham RR, Kozyrev SV, Baechler EC, Reddy MV, Plenge RM, Bauer JW, et al. A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus. Nat Genet 38:550-5, 2006.
32. Graham RR, Kyogoku C, Sigurdsson S, Vlasova IA, Davies LR, Baechler EC, et al. Three functional variants of IFN regulatory factor 5 (IRF5) define risk and protective haplotypes for human lupus. Proc Natl Acad Sci USA 104:6758-63, 2007
33. Kozyrev SV, Lewen S, Reddy PM, Pons-Estel B, Witte T, Junker P, et al. Structural insertion/deletion variation in IRF5 is associated with a risk haplotype and defines the precise IRF5 isoforms expressed in systemic lupus erythematosus. Arthritis Rheumatism 56:1234-41, 2007.
34. Niewold TB, Kelly JA, Flesch MH, Espinoza LR, Harley JB, Crow MK. Association of the IRF5 risk haplotype with high serum interferon-alpha activity in systemic lupus erythematosus patients. Arthritis Rheumatism 58:2481-7, 2008.
35. Sigurdsson S, Goring HH, Kristjansdottir G, Milani L, Nordmark G, Sandling JK, et al. Comprehensive evaluation of the genetic variants of interferon regulatory factor 5 (IRF5) reveals a novel 5 bp length polymorphism as strong risk factor for systemic lupus erythematosus. Hum Mol Genet17:872-81, 2008.
36. Shin HD, Sung YK, Choi CB, Lee SO, Lee HW, Bae SC. Replication of the genetic effects of IFN regulatory factor 5 (IRF5) on systemic lupus erythematosus in a Korean population. Arthritis Res Ther 9:R32, 2007.
37. Ferreiro-Neira I, Calaza M, Alonso-Perez E, Marchini M, Scorza R, Sebastiani GD, et al. Opposed independent effects and epistasis in the complex association of IRF5 to SLE. Genes Immun 8:429-38, 2007.
38. Okabe Y, Kawane K, Nagata S. IFN regulatory factor (IRF) 3/7-dependent and -independent gene induction by mammalian DNA that escapes degradation. Eur J Immunol 38:3150-8, 2008.
39. Yasuda K, Richez C, Maciaszek JW, Agrawal N, Akira S, Marshak-Rothstein A, et al. Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production. J Immunol 178:6876-85, 2007.
40. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus [letter]. Arthritis & Rheumatism 40:1725, 1997.
41. Michelle Petri et al. Combined Oral Contraceptives in Women with Systemic Lupus Erythematosus. New England Journal of Medicine 353:2550-8, 2005.
42. Wan-Uk Kim, Antoine Sreih, Richard Bucala. Toll-like receptors in systemic lupus erythematosus prospects fortherapeutic intervention. Autoimmunity review 8:204-8, 2009.
43. Hiscott J, Nguyen TL, Arguello M, Nakhaei P, Paz S. Manipulation of the nuclear factor-kappaB pathway and the innate immune response by viruses. Oncogene 25:6844-67, 2006.
44. Moynagh PN. TLR signalling and activation of IRFs: revisiting old friends from the NF-kappaB pathway. Trends Immunol 26:469-76, 2005.
45. Rahman AH, Eisenberg RA. The role of toll-like receptors in systemic lupus erythematosus. Springer Semin Immunopathol 28:131-43, 2006.
46. Romieu-Mourez R, Solis M, Nardin A, et al. Distinct roles for IFN regulatory factor (IRF)-3 and IRF-7 in the activation of antitumor properties of human macrophages. Cancer Res 66:10576-85, 2006.
47. Barnes BJ, Richards J, Mancl M, Hanash S, Beretta L, Pitha PM. Global and distinct targets of IRF-5 and IRF-7 during innate response to viral infection. J Biol Chem 279:45194-207, 2004.
48. Hijikata M, Mishiro S, Miyamoto C, Furuichi Y, Hashimoto M, Ohta Y. Genetic polymorphism of the MxA gene promoter and interferon responsiveness of hepatitis C patients: revisited by analyzing two SNP sites (_123 and _88) in vivo and in vitro. Intervirology 44:379-82, 2001.