2011-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647439摘要：肝臟是一個最有趣的免疫器官因為它可以選擇性的對抗原引起免疫性或耐受性。原發性膽道硬化症(PBC)是一種進行性自體免疫性肝臟疾病，其特徵為出現抗粒線體抗體和肝門脈區內小膽管被免疫性的破壞，主要是CD8 T細胞的浸潤及破壞膽管。然而引起自體反應性T細胞聚集至門脈區的先期因素則還不清楚。我們實驗室已發展出一種注射2-octynoic acid (2-OA) 結合BSA使小鼠產生類似PBC病徵的高量抗粒線體抗體及自體免疫性膽管炎的小鼠模式。初步實驗我們以a-galactosylceramide(a-GalCer) 活化iNKT 細胞在2-OA BSA致敏的小鼠上，發現致敏2-OA BSA小鼠原有的自體免疫性膽管炎更嚴重，包括抗粒線體抗體的量更高，更顯著的CD8 T細胞浸潤，門脈區發炎，肉芽腫產生及膽管的破壞。最重要的是產生肝纖維化，這是在其他PBC的小鼠模式上不曾發現的，這結果強調先天性免疫對於PBC發生的重要性。此研究計劃的主要目的是探討先天性免疫，特別是iNKT 細胞，在自體免疫性膽管炎及肝纖維化的致病機轉。四個子目標如下:子目標一：確定iNKT細胞在自體免疫性膽管炎及肝纖維化的角色。1.分析CD1d缺陷小鼠給予2-OA BSA致敏後的自體免疫性膽管炎及肝纖維化之影響。2.分析利用其他醣蛋白給予2-OA BSA致敏小鼠後的自體免疫性膽管炎及肝纖維化之影響。子目標二：探討a-GalCer注射導致的肝纖維化與2-OA BSA致敏的免疫反應有無關係。1.分析只給予a-GalCer或a-GalCer加上CFA之小鼠是否產生肝纖維化。子目標三：探討給予a-GalCer小鼠的自體反應性T細胞的百分比和功能以及樹突細胞之角色。1.建構及表現小鼠PDC-E2以確認自體抗原特異性T細胞。2.確認CD4細胞、CD8細胞及B細胞抗原決定位。3.決定小鼠肝臟內自體抗原特異性T細胞之百分比。4.評估自體抗原特異性T細胞的功能。5.研究DC的成熟及呈獻抗原的能力。子目標四：抗發炎及抗纖維化的IL-10對於抑制自體免疫性膽管炎及肝纖維化的研究。1.分析給予IL-10對於 2-OA BSA致敏小鼠的自體免疫性膽管炎及肝纖維化之影響。2.分析給予IL-10 siRNA對於 2-OA BSA致敏小鼠的自體免疫性膽管炎及肝纖維化之影響。3.分析IL-10缺陷小鼠給予2-OA BSA致敏後的自體免疫性膽管炎及肝纖維化之影響。4.分析給予IL-10的小鼠是否產生Foxp3+ 調控型T細胞或 分泌IL-10的細胞。藉由此研究，我們將可了解iNKT 細胞在免疫引起的肝臟損傷的角色及先天性免疫如何影響後天性免疫而導致肝損傷。另外，我們也可了解抗發炎及抗纖維化的IL-10在抑制自體免疫性膽管炎及肝纖維化的角色。<br> Abstract: Livers are most interesting immunologic organs because they are able to selectively induce immunity or tolerance to antigens. Primary biliary cirrhosis (PBC) is an organ-specific autoimmune liver disease characterized by the presence of anti-mitochondrial antibodies (AMAs) and the destruction of small intrahepatic bile ducts with portal inflammation. CD8+ T cells have been shown the predominant cellular infiltrates in the damage of bile ducts. However, the early events in the induction of autoreactive T cell recruitment to portal areas remain unknown. Our laboratory has developed a murine model of PBC based upon immunization with 2-octynoic acid (2-OA) coupled to BSA. Such mice develop high titer anti-mitochondrial antibodies (AMAs) and autoimmune cholangitis. We have recently demonstrated that invariant natural killer T (iNKT) cell activation by a-galactosylceramide (a-GalCer) leads to a profound exacerbation of portal disease in 2-OA BSA immunized mice, including increased production of AMAs, a significant increase in CD8+ T cell infiltrates, portal inflammation, granuloma formation and bile duct damage. Furthermore, a-GalCer treated mice have evidence of fibrosis, a feature not previously reported in the murine models. These results are critical and emphasize the role of innate immunity in the natural history of PBC. The overall goal of the current application is to further delineate the underlying mechanism of innate immunity, especially iNKT cells, in autoimmune cholangitis and liver fibrosis by 2-OA BSA immunized mice. The specific aims are as follows:Specific Aim 1. Determine the role of iNKT cell in autoimmune cholangitis and liver fibrosis. a.Analyze autoimmune cholangitis and liver fibrosis in CD1d-/- mice immunized with 2-OA BSA.b.Analyze autoimmune cholangitis and liver fibrosis in other glycolipid compound injected 2-OA BSA immunized B6 mice.Specific Aim 2. Determine the direct or indirect effects of a-CalCer injection in liver fibrosis.a.Analyze autoimmune cholangitis and liver fibrosis in a-CalCer injected mice or a-CalCer and adjuvant CFA injected mice.Specific Aim 3. Determine the frequency and function of autoreactive T cells and the role of dendritic cells in a-GalCer treated mice. a.Construct and express mouse PDC-E2 as an autoantigen to identify the autoreactive T cells.b.Epitopes mapping of CD4 T, CD8 T and B cells in mPDC-E2.c.Determine the frequency of antigen-specific CD4+ and CD8+ T cells in the liver. d.Assay the function of autoreactive T cells. e.Determine the maturation of dendritic cells.Specific Aim 4. Determine if IL-10, an anti-inflammatory and anti-fibrotic cytokine, inhibits autoimmune cholangitis and liver fibrosis in 2-OA BSA immunized mice.a.Analyze autoimmune cholangitis and liver fibrosis in 2-OA BSA immunized mice administered with IL-10.b.Analyze autoimmune cholangitis and liver fibrosis in 2-OA BSA immunized mice administered with IL-10 siRNA.c.Analyze autoimmune cholangitis and liver fibrosis in IL-10-/- mice immunized with 2-OA BSA.d.Analyze Foxp3+ Tregs and IL-10 secreting cells in mice treated with IL-10.By this study, we will clarify the role of iNKT cells in the pathogenesis of immune-mediated liver injury and fibrosis and how innate immunity affects acquired immunity and then leads to liver injury. In addition, we will clarify the role of anti-inflammatory and anti-fibrotic IL-10 in autoimmune cholangitis and/or liver fibrosis.原發性膽道硬化症肝臟自體免疫疾病iNKT細胞xenobiotics介白質10Primary biliary cirrhosisliver autoimmune diseaseinvariant NKT cellsxenobioticsIL-10