探討invariant NKT細胞的活化對於原發性膽道硬化症之影響

The Effects of Activation of Invariant NKT Cells on the Pathogenesis of Primary Biliary Cirrhosis

吳希傑(Si-Jie Wu)

指導教授 ： 莊雅惠

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02551

摘要

PBC為慢性膽汁鬱積肝臟的自體免疫疾病，患者肝臟門脈三角出現自體反應淋巴球浸潤以及血清中可測得抗粒線體自體抗體，嚴重者導致肝臟衰竭。iNKT細胞在經由抗原呈獻細胞表面分子CD1d呈獻抗原 (glycolipid) 活化後，會在短時間內分泌大量細胞激素，如IL-4及IFN-g等，調控其他免疫細胞。在PBC患者肝臟內有較多iNKT細胞，且其膽道上皮細胞亦表現較多CD1d分子，推測iNKT細胞可能會影響疾病的發生。我們先前發現dnTGFbRII PBC小鼠肝臟含有高量過度活化且分泌高量IFN-g的iNKT細胞。再者，iNKT細胞缺乏之dnTGFbRII小鼠的肝臟淋巴球浸潤減少且膽道發炎減輕，推測iNKT細胞可能在早期藉由IFN-g的分泌而促使疾病加重。在此，我們進一步探討iNKT cells活化對此疾病的影響。我們以靜脈注射a-GalCer活化iNKT cells，並觀察其對於xenobiotics (2-OA BSA) 致敏之PBC小鼠疾病進程變化。
　　我們在初期以靜脈注射給予2-OA BSA致敏小鼠a-GalCer，活化小鼠體內iNKT細胞，12週後觀察晚期PBC小鼠肝臟病理變化以及血清中AMAs效價高低。結果顯示給予a-GalCer之PBC小鼠，血清anti-PDC-E2 IgG以及IgM效價增高、肝臟門脈區發炎和膽道破壞嚴重，甚至出現肝臟肉芽腫及纖維化。分析其肝臟單核細胞組成，發現執行毒殺功能之CD8+ T cells顯著增加，且肝臟出現Th1與Th17細胞激素微環境。進一步分析致敏4週後之結果，給予a-GalCer的2-OA BSA致敏小鼠已加快PBC疾病進程，包括血清AMAs效價顯著升高及肝臟門脈三角更多淋巴球浸潤及膽道破壞，同時肝臟中CD8+ T cells亦已顯著增加。深入探討其機制，我們發現給予a-GalCer至2-OA BSA引起之PBC小鼠可促使肝臟DCs表面MHC class I分子表現量增加，可能因此導致更多CD8+ T cells遷移至肝臟，造成肝臟膽道破壞加重。此外，我們利用pichia pastoris酵母菌表現mPDC-E2蛋白片段，證實在2-OA BSA致敏之PBC小鼠體內有mPDC-E2-specific auto-reactive T cells。
　　由此研究結果，我們在xenobiotic (2-OA BSA) 引起之PBC小鼠模式中，再以a-GalCer活化iNKT cells可加快及加重PBC疾病進程，且產生更為相似PBC病人的肝臟肉芽腫及纖維化的病徵。因此，未來我們可在此一小鼠模式下， 配合a-GalCer的給予，使成為更好的PBC小鼠模式。加上mPDC-E2之製備及證明小鼠體內有auto-reactive T cells之存在，且此auto-reactive T cells亦是對抗與人類相同的自體抗原 (PDC-E2) ，給予a-GalCer 之2-OA BSA致敏小鼠將可廣泛用於研究PBC疾病之自體免疫機轉及治療的應用。

關鍵字

原發性膽道硬化症 ； iNKT cells ； a-GalCer ； 2-OA BSA致敏小鼠 ； mPDC-E2 ； mPDC-E2-specific auto-reactive T cells

並列摘要

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver autoimmune disease. The characteristics of PBC are the presence of anti-mitochondrial auto-Abs, the presence of auto-reactive lymphocyte infiltration in the portal triad of liver, and the destruction of biliary epithelial cells. The destroyed liver would become fibrosis and then failure at the end stage of disease. Invariant NKT (iNKT) cells, an unconventional T cells, are activated by glycolipids, such as a-galactosylceramide (a-GalCer), presented by CD1d molecule on antigen presenting cells. After activation, iNKT cells immediately secrete a variety of cytokines, such as IL-4 and IFN-g, to regulate downstream immune cells. In PBC patients, both the frequency of iNKT cells and CD1d expression on bile duct epithelium were increased, suggesting that iNKT cells are correlated with the onset of PBC. Our previous study demonstrated that the increased number of hyper-reactive liver iNKT cells which secreted more IFN-g than IL-4 was noted in dnTGFbRII PBC mice. However, in NKT cell deficient dnTGFbRII mice, lymphocyte infiltration and bile duct damage in liver were alleviated. These results suggested that iNKT cells exacerbated liver bile duct injury by IFN-g production in early stage. In this study, we investigated the effects of activating iNKT cells in PBC. We intravenously injected a-GalCer to activate iNKT cells and evaluated the PBC progress in xenobiotic (2-OA BSA) -induced PBC mouse model.
We report herein a-GalCer injection exacerbated autoimmune cholangitis in 2-OA BSA immunized mice, including increased AMA production, portal inflammation, bile duct damage, granuloma formation as well as fibrosis, and Th1 and Th17 prone immune responses. Liver total mononuclear cells were significantly increased in a-GalCer-injected 2-OA BSA immunized mice. Importantly, significantly increased CD8+ T cells in the liver were noted in a-GalCer-injected 2-OA BSA immunized mice. Moreover, a-GalCer intravenous injection up-regulated the antigen presenting capacity of dendritic cells (DCs) in 2-OA BSA immunized mice. These results suggest that iNKT cell activation by a-GalCer administration exacerbate PBC by either secreting IFN-g or stimulating the maturation of DCs and subsequently recruiting CD8+ T cells to destroy bile ducts. In addition, we also identified mPDC-E2 specific auto-reactive T cells in 2-OA BSA-immunized mice.
In conclusion, our results demonstrated that administrat ion with a-GalCer to activate iNKT cells in 2-OA BSA immunized mice exacerbate profound liver injury. Moreover, the features of a-GalCer injected 2-OA BSA immunized mice were more similar to that of patients of PBC, such as the presence of granuloma and fibrosis. Meanwhile, we also expressed mPDC-E2 and defined the presence of auto-reactive T cells directed against PDC-E2, the immune dominant autoantigen of human PBC. Therefore, a-GalCer administration in 2-OA BSA immunized mice could be a better mouse model for studying the mechanism of autoimmune pathogenesis and therapeutic strategies of PBC.

並列關鍵字

primary biliary cirrhosis ； iNKT cells ； a-GalCer ； 2-OA BSA-induced PBC mouse model ； mPDC-E2 specific autoreactive T cells

參考文獻

1. Kaplan MM. 1996. Primary biliary cirrhosis. N Engl J Med 335: 1570-80

2. Kaplan MM, Gershwin ME. 2005. Primary biliary cirrhosis. N Engl J Med

3. Wong RK, Lim SG, Wee A, Chan YH, Aung MO, Wai CT. 2008. Primary biliary cirrhosis in Singapore: evaluation of demography, prognostic factors and natural course in a multi-ethnic population. J Gastroenterol Hepatol 23: 599-605

4. Su CW, Hung HH, Huo TI, Huang YH, Li CP, Lin HC, Lee PC, Lee SD, Wu JC. 2008. Natural history and prognostic factors of primary biliary cirrhosis in Taiwan: a follow-up study up to 18 years. Liver Int 28: 1305-13

5. Jones DE. 2008. Pathogenesis of primary biliary cirrhosis. Postgrad Med J 84: 23-33