臺灣博碩士論文加值系統

原發性膽汁性膽管炎 (Primary biliary cholangitis, PBC)是一種肝臟自體免疫疾病。先前研究指出在早期診斷的PBC病患血清中發現interleukin (IL)-21的高量表現。IL-21隸屬於IL-2 family，此家族是由一群多效性cytokine組成，可以調控許多不同細胞的功能。然而IL-21如何影響PBC疾病病程尚未被研究清楚，因此我們使用PBC小鼠動物模式以探討IL-21在自體肝臟發炎的情況下之角色。我們每兩週以腹腔注射的方式給予小鼠2-octynoic acid-OVA與complete/incomplete Freund’s adjuvant的混和物使其誘發PBC，在第一週時注射Adeno-associated virus (AAV)-mIL-21使小鼠肝臟表現高量mIL-21，而五週及十一週時犧牲小鼠並進一步分析各組之間的差異。PBC的標誌是血清中存在專一性辨識pyruvate dehydrogenase E2 complex (PDC-E2)的antimitochondrial antibody (AMA)。本實驗中，每個組別均測得AMA表現，因此表示小鼠成功誘發PBC。我們發現給予AAV-mIL-21的組別有較高的liver mononuclear cells (LMNCs) 浸潤，肝臟中促進發炎的IFN-γ亦顯著升高。此外，T、CD8+ T與follicular helper T (Tfh) cells的百分比在mIL-21存在的情況下顯著增加。我們更進一步發現CD8+ T cells高度活化並且高度表現granzyme B。另一方面，我們從Masson’s trichrome stain染色的病理切片觀察到AAV-mIL-21組呈現較嚴重的肝臟纖維化，而collagen III的qPCR結果亦證實了此現象。因此，我們認為IL-21藉由活化CD8+ T cells與促進肝臟纖維化而加劇PBC病程。

Primary biliary cholangitis (PBC) is an autoimmune liver disease. Previous study showed that serum interleukin (IL)-21 is highly expressed in early diagnosed PBC patients. IL-21 is a member of IL-2 family which has been reported to be a pleiotropic cytokine that mediates numerous cell types. However, the mechanism of IL-21 in PBC remains unclear. In this study, we used a mouse model of PBC to evaluate the roles of IL-21 in autoimmune liver inflammation. Mice were immunized with 2-octynoic acid-OVA mixed with complete/incomplete Freund’s adjuvant biweekly through i.p. to form PBC. Adeno-associated virus (AAV)-mIL-21 was given at week 1 by i.v. to express mIL-21 in liver. Mice were euthanized to examine the differences among groups. Antimitochondrial antibody (AMA) against pyruvate dehydrogenase E2 complex (PDC-E2), a specific hallmark of PBC, was detected in all groups. The numbers of liver mononuclear cells (LMNCs), mRNA expression level of proinflammatory cytokine IFN-γ increased in AAV-mIL-21 injected group. In addition, the percentage of T, CD8+ T, follicular helper T (Tfh) cells increased with the existence of mIL-21. Furthermore, CD8+ T cells were highly activated and granzyme B were further upregulated in early-stage PBC. Moreover, the histopathology sections stained by Masson’s trichrome stain showed exacerbated liver fibrosis in AAV-mIL-21 injected group and mRNA expression levels of collagen III revealed the data. The results indicate that IL-21 may exacerbate PBC by upregulating CD8+ T cells and further enhance liver fibrosis.

誌謝..........i
摘要..........ii
Abstract..........iii
Abbreviation..........iv
Content of Figures..........vii
Content of Tables..........viii
Chapter 1. Introduction..........1
1.1 Primary biliary cholangitis (PBC) ..........2
1.2 Cell dysregulation in PBC..........3
1.3 Xenobiotic-induced PBC mouse model..........5
1.4 Adeno-associated virus..........5
1.5 IL-2 family cytokines..........6
1.6 Biological functions of IL-21..........6
1.7 IL-21 and NK cells..........7
1.8 IL-21 and NKT cell ..........7
1.9 IL-21 and CD4+ T cells..........8
1.10 IL-21 and CD8+ T cells..........8
1.11 IL-21 and follicular helper T cells..........9
1.12 IL-21 and B cells..........9
1.13 IL-21 in autoimmune diseases..........10
1.14 Therapeutic potential of IL-21..........11
1.15 IL-21 and fibrosis..........11
1.16 Specific aims..........12
Chapter 2. Materials and Methods..........13
2.1 Mice..........14
2.2 Experiment protocol of the effect of mIL-21 to PBC..........14
2.3 RNA extraction..........14
2.4 cDNA synthesis..........15
2.5 Quantitative PCR (qPCR) ..........16
2.6 AAV-mIL-21 packaging ..........16
2.7 AAV purification..........17
2.8 AAV concentration and titration..........17
2.9 2-OA-OVA production..........18
2.10 Xenobiotics (2-OA-OVA)-induced mouse model of PBC..........18
2.11 AAV injection..........19
2.12 Serum sampling..........19
2.13 Liver perfusion and pathological section..........19
2.14 LMNCs purification..........19
2.15 Cell surface marker analysis..........20
2.16 Intracellular cytokine production activation..........21
2.17 Intracellular staining..........21
2.18 Serum cytokine analysis by ELISA..........22
2.19 Serum anti-mPDC-E2 IgM and IgG level..........22
2.20 Statistical analysis..........23
Chapter 3. Results..........24
3.1 mIL-21 and mIL-21R expression levels in liver under PBC mouse model..........25
3.2 Serum AMA production level..........25
3.3 Cell subsets analysis..........25
3.4 Analysis of T cell subsets..........26
3.5 Cell activation marker analysis..........26
3.6 CD8+ T cells cytotoxic activities analysis..........26
3.7 Analysis of Tfh and PD-1+ T cells..........27
3.8 Analysis of the correlation between Tfh cells and AMA levels..........27
3.9 Analysis of B cell subsets..........27
3.10 mRNA expression levels in liver..........28
3.11 Experiment of long term effect of mIL-21 in PBC pathogenesis..........28
3.12 mRNA expression levels in liver..........29
Chapter 4. Discussion..........30
Figures and Tables..........36
References..........53
Appendix ..........58

1.Cheung, A.C., et al., Time to make the change from ‘primary biliary cirrhosis’ to ‘primary biliary cholangitis’. Can J Gastroenterol Hepatol, 2015. 29.
2.Kaplan, M.M. and M.E. Gershwin, Primary biliary cirrhosis. N Engl J Med, 2005. 353(12): p. 1261-73.
3.Bergasa, N.V., J.K. Mehlman, and E.A. Jones, Pruritus and fatigue in primary biliary cirrhosis. Baillieres Best Pract Res Clin Gastroenterol, 2000. 14(4): p. 643-55.
4.Gershwin, M.E., et al., Molecular considerations of primary biliary cirrhosis. 1998: p. p. 40–52.
5.Bittencourt, P.L., et al., Prevalence of immune disturbances and chronic liver disease in family members of patients with primary biliary cirrhosis. J Gastroenterol Hepatol, 2004. 19(8): p. 873-8.
6.Mason, A.L. and G. Zhang, Linking human beta retrovirus infection with primary biliary cirrhosis. Gastroenterol Clin Biol, 2010. 34(6-7): p. 359-66.
7.Selmi, C. and M.E. Gershwin, Bacteria and human autoimmunity: the case of primary biliary cirrhosis. Curr Opin Rheumatol, 2004. 16(4): p. 406-10.
8.Leung, P.S., et al., Immunization with a xenobiotic 6-bromohexanoate bovine serum albumin conjugate induces antimitochondrial antibodies. J Immunol, 2003. 170(10): p. 5326-32.
9.Poupon, R.E., R. Poupon, and B. Balkau, Ursodiol for the long-term treatment of primary biliary cirrhosis. The UDCA-PBC Study Group. N Engl J Med, 1994. 330(19): p. 1342-7.
10.Nevens, F., et al., A Placebo-Controlled Trial of Obeticholic Acid in Primary Biliary Cholangitis. N Engl J Med, 2016. 375(7): p. 631-43.
11.Shimoda, S., et al., Natural killer cells regulate T cell immune responses in primary biliary cirrhosis. Hepatology, 2015. 62(6): p. 1817-27.
12.Wu, S.J., et al., Innate immunity and primary biliary cirrhosis: activated invariant natural killer T cells exacerbate murine autoimmune cholangitis and fibrosis. Hepatology, 2011. 53(3): p. 915-25.
13.Wang, L., et al., CXCR5+ CD4+ T follicular helper cells participate in the pathogenesis of primary biliary cirrhosis. Hepatology, 2015. 61(2): p. 627-38.
14.Wang, L., et al., Increased numbers of circulating ICOS(+) follicular helper T and CD38(+) plasma cells in patients with newly diagnosed primary biliary cirrhosis. Dig Dis Sci, 2015. 60(2): p. 405-13.
15.Li, Y., et al., Chemokine (C-X-C motif) ligand 13 promotes intrahepatic chemokine (C-X-C motif) receptor 5+ lymphocyte homing and aberrant B-cell immune responses in primary biliary cirrhosis. Hepatology, 2015. 61(6): p. 1998-2007.
16.Kita, H., et al., Quantitative and functional analysis of PDC-E2-specific autoreactive cytotoxic T lymphocytes in primary biliary cirrhosis. J Clin Invest, 2002. 109(9): p. 1231-40.
17.Ma, H.D., et al., Chemokine receptor CXCR3 deficiency exacerbates murine autoimmune cholangitis by promoting pathogenic CD8+ T cell activation. J Autoimmun, 2017. 78: p. 19-28.
18.Wakabayashi, K., et al., Loss of tolerance in C57BL/6 mice to the autoantigen E2 subunit of pyruvate dehydrogenase by a xenobiotic with ensuing biliary ductular disease. Hepatology, 2008. 48(2): p. 531-40.
19.Amano, K., et al., Chemical xenobiotics and mitochondrial autoantigens in primary biliary cirrhosis: identification of antibodies against a common environmental, cosmetic, and food additive, 2-octynoic acid. J Immunol, 2005. 174(9): p. 5874-83.
20.Syu, B.J., et al., Dual Roles of IFN-gamma and IL-4 in the Natural History of Murine Autoimmune Cholangitis: IL-30 and Implications for Precision Medicine. Sci Rep, 2016. 6: p. 34884.
21.Daya, S. and K.I. Berns, Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev, 2008. 21(4): p. 583-93.
22.Deyle, D.R. and D.W. Russell, Adeno-associated virus vector integration. Curr Opin Mol Ther, 2009. 11(4): p. 442-7.
23.Grimm, D., et al., In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol, 2008. 82(12): p. 5887-911.
24.Rochman, Y., R. Spolski, and W.J. Leonard, New insights into the regulation of T cells by gamma(c) family cytokines. Nat Rev Immunol, 2009. 9(7): p. 480-90.
25.Gharibi, T., et al., Biological effects of IL-21 on different immune cells and its role in autoimmune diseases. Immunobiology, 2016. 221(2): p. 357-67.
26.Leonard, W.J. and R. Spolski, Interleukin-21: a modulator of lymphoid proliferation, apoptosis and differentiation. Nat Rev Immunol, 2005. 5(9): p. 688-98.
27.Ettinger, R., et al., IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells. J Immunol, 2005. 175(12): p. 7867-79.
28.Spolski, R. and W.J. Leonard, The Yin and Yang of interleukin-21 in allergy, autoimmunity and cancer. Curr Opin Immunol, 2008. 20(3): p. 295-301.
29.Mehta, D.S., et al., IL-21 induces the apoptosis of resting and activated primary B cells. J Immunol, 2003. 170(8): p. 4111-8.
30.Ozaki, K., et al., Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6. J Immunol, 2004. 173(9): p. 5361-71.
31.Caruso, R., et al., A functional role for interleukin-21 in promoting the synthesis of the T-cell chemoattractant, MIP-3alpha, by gut epithelial cells. Gastroenterology, 2007. 132(1): p. 166-75.
32.Kinter, A.L., et al., The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands. J Immunol, 2008. 181(10): p. 6738-46.
33.Parrish-Novak, J., et al., Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature, 2000. 408(6808): p. 57-63.
34.Spolski, R. and W.J. Leonard, Interleukin-21: a double-edged sword with therapeutic potential. Nat Rev Drug Discov, 2014. 13(5): p. 379-95.
35.Caprioli, F., et al., Autocrine regulation of IL-21 production in human T lymphocytes. J Immunol, 2008. 180(3): p. 1800-7.
36.Kwon, H., et al., Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors. Immunity, 2009. 31(6): p. 941-52.
37.Zeng, R., et al., Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function. J Exp Med, 2005. 201(1): p. 139-48.
38.Kasaian, M.T., et al., IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity. Immunity, 2002. 16(4): p. 559-69.
39.Frederiksen, K.S., et al., IL-21 induces in vivo immune activation of NK cells and CD8(+) T cells in patients with metastatic melanoma and renal cell carcinoma. Cancer Immunol Immunother, 2008. 57(10): p. 1439-49.
40.Godfrey, D.I. and M. Kronenberg, Going both ways: immune regulation via CD1d-dependent NKT cells. J Clin Invest, 2004. 114(10): p. 1379-88.
41.Smyth, M.J., et al., Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer. J Exp Med, 2005. 201(12): p. 1973-85.
42.Coquet, J.M., et al., IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production. J Immunol, 2007. 178(5): p. 2827-34.
43.Mukherjee, S., P.K. Maiti, and D. Nandi, Role of CD80, CD86, and CTLA4 on mouse CD4(+) T lymphocytes in enhancing cell-cycle progression and survival after activation with PMA and ionomycin. J Leukoc Biol, 2002. 72(5): p. 921-31.
44.Attridge, K., et al., IL-21 promotes CD4 T cell responses by phosphatidylinositol 3-kinase-dependent upregulation of CD86 on B cells. J Immunol, 2014. 192(5): p. 2195-201.
45.Spolski, R. and W.J. Leonard, Interleukin-21: basic biology and implications for cancer and autoimmunity. Annu Rev Immunol, 2008. 26: p. 57-79.
46.Mittal, A., et al., IL-27 induction of IL-21 from human CD8+ T cells induces granzyme B in an autocrine manner. Immunol Cell Biol, 2012. 90(8): p. 831-5.
47.Brodeur, T.Y., et al., IL-21 Promotes Pulmonary Fibrosis through the Induction of Profibrotic CD8+ T Cells. J Immunol, 2015. 195(11): p. 5251-60.
48.Qi, H., T follicular helper cells in space-time. Nat Rev Immunol, 2016. 16(10): p. 612-25.
49.Forster, R., et al., A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen. Cell, 1996. 87(6): p. 1037-47.
50.Nutt, S.L. and D.M. Tarlinton, Germinal center B and follicular helper T cells: siblings, cousins or just good friends? Nat Immunol, 2011. 12(6): p. 472-7.
51.Ozaki, K., et al., A critical role for IL-21 in regulating immunoglobulin production. Science, 2002. 298(5598): p. 1630-4.
52.Feng, P.H., Systemic lupus erythematosus: the face of Asia. Ann N Y Acad Sci, 2007. 1108: p. 114-20.
53.Sawalha, A.H., et al., Genetic association of interleukin-21 polymorphisms with systemic lupus erythematosus. Ann Rheum Dis, 2008. 67(4): p. 458-61.
54.Herber, D., et al., IL-21 has a pathogenic role in a lupus-prone mouse model and its blockade with IL-21R.Fc reduces disease progression. J Immunol, 2007. 178(6): p. 3822-30.
55.Arnett, F.C., et al., The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum, 1988. 31(3): p. 315-24.
56.McInnes, I.B. and G. Schett, The pathogenesis of rheumatoid arthritis. N Engl J Med, 2011. 365(23): p. 2205-19.
57.Gharibi, T., et al., Investigation of IL-21 gene polymorphisms (rs2221903, rs2055979) in cases with multiple sclerosis of Azerbaijan, Northwest Iran. Am J Clin Exp Immunol, 2015. 4(1): p. 7-14.
58.Tedder, T.F. and W.J. Leonard, Autoimmunity: regulatory B cells--IL-35 and IL-21 regulate the regulators. Nat Rev Rheumatol, 2014. 10(8): p. 452-3.
59.Yoshizaki, A., et al., Regulatory B cells control T-cell autoimmunity through IL-21-dependent cognate interactions. Nature, 2012. 491(7423): p. 264-8.
60.Lei, L., et al., Elevated frequencies of CD4(+) IL-21(+) T, CD4(+) IL-21R(+) T and IL-21(+) Th17 cells, and increased levels of IL-21 in bleomycin-induced mice may be associated with dermal and pulmonary inflammation and fibrosis. Int J Rheum Dis, 2016. 19(4): p. 392-404.
61.Pan, Q., et al., Increased levels of IL-21 responses are associated with the severity of liver injury in patients with chronic active hepatitis B. J Viral Hepat, 2014. 21(9): p. e78-88.
62.McClure, C., et al., Production and titering of recombinant adeno-associated viral vectors. J Vis Exp, 2011(57): p. e3348.
63.Shipkova, M. and E. Wieland, Surface markers of lymphocyte activation and markers of cell proliferation. Clin Chim Acta, 2012. 413(17-18): p. 1338-49.
64.Fogel, L.A., et al., Markers of nonselective and specific NK cell activation. J Immunol, 2013. 190(12): p. 6269-76.
65.Pesce, J., et al., The IL-21 receptor augments Th2 effector function and alternative macrophage activation. J Clin Invest, 2006. 116(7): p. 2044-55.
66.Wynn, T.A., Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat Rev Immunol, 2004. 4(8): p. 583-94.
67.Wynn, T.A., Integrating mechanisms of pulmonary fibrosis. J Exp Med, 2011. 208(7): p. 1339-50.
68.Nagano, T., et al., Cytokine profile in the liver of primary biliary cirrhosis. J Clin Immunol, 1999. 19(6): p. 422-7.
69.Kaech, S.M. and W. Cui, Transcriptional control of effector and memory CD8+ T cell differentiation. Nat Rev Immunol, 2012. 12(11): p. 749-61.
70.Lee, J., et al., Interferon gamma suppresses collagen-induced arthritis by regulation of Th17 through the induction of indoleamine-2,3-deoxygenase. PLoS One, 2013. 8(4): p. e60900.
71.Crotty, S., Follicular helper CD4 T cells (TFH). Annu Rev Immunol, 2011. 29: p. 621-63.
72.Chen, L. and X. Han, Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future. J Clin Invest, 2015. 125(9): p. 3384-91.
73.Parry, R.V., et al., CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Mol Cell Biol, 2005. 25(21): p. 9543-53.
74.Dong, H., et al., B7-H1 determines accumulation and deletion of intrahepatic CD8(+) T lymphocytes. Immunity, 2004. 20(3): p. 327-36.
75.Chang, C.H., et al., Innate immunity drives xenobiotic-induced murine autoimmune cholangitis. Clin Exp Immunol, 2014. 177(2): p. 373-80.