2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643174摘要：目的此計畫中，我們將集中探討在慢性B型肝炎病毒感染時先天性免疫力的誘發及病毒核心蛋白在調節適應性免疫之T細胞反應的角色，我們將探索由B型肝炎病毒誘發之可打破免疫耐性的先天性免疫與重要因子，同時我們亦將探討由病毒核心蛋白所誘發之先天性免疫的分子機制，最後我們亦將在小鼠模式中研究由先天性免疫反應到適應性T細胞反應與分化及肝臟浸潤淋巴球細胞素產生之間的連結。背景B型肝炎病毒的持續性感染會導致慢性肝炎，肝硬化及肝癌，但對於病毒的清除或無法清除而造成持續性感染的免疫機制則相當不清楚。藉由高壓水柱注射具複製力之HBV DNA於免疫健全小鼠體內，我們已經建立B型肝炎病毒持續性感染的小鼠模式。初步的結果顯示在B型肝炎病毒帶原小鼠的肝臟浸潤淋巴球表現較高的PD-1+/CD8+及CD4+FoxP3+調節性T細胞；並且缺乏病毒核心蛋白則使小鼠無法清除病毒並伴隨表現較高PD-1之浸潤性T淋巴球而導致小鼠持續性B型肝炎病毒感染。這些結果顯示B型肝炎病毒核心蛋白可能在誘發宿主對抗病毒的免疫反應上扮演重要角色。B型肝炎病毒誘發的免疫反應被認為是與肝臟內先天性免疫系統作用所啟動，由此觀點，我們假設較弱的先天性免疫反應與較強的調節性T細胞活性導致B型肝炎病毒感染時產生無效的免疫反應，為了回答此問題，我們準備利用B型肝炎病毒持續性感染的小鼠模式研究在病毒感染時核心蛋白在先天性免疫力的誘發上所扮演的角色。研究目標1. 在小鼠模式中定義出先天性免疫系統(細胞內辨認受體)在B型肝炎病毒誘發之免疫反應的角色2. 研究肝臟內先天性免疫細胞反應與CD4 T細胞分化，並探討何種分子連結病毒核心蛋白誘發之先天性免疫與適應性免疫3. 於小鼠模式中發展分子標的誘發對抗B型肝炎病毒之免疫反應與破壞病毒持續性之免疫療法4. 小鼠模式的發現將於土撥鼠模式及B型肝炎帶原者中應證。研究方法1. 在小鼠模式中定義出先天性免疫系統在B型肝炎病毒誘發之免疫反應的角色將藉由高壓水柱注射TLRs, MYD88, TRIF, RIG-I/MDA5, NLRs 及 ASC的shRNA以及基因剔除小鼠和阻斷性抗體例如anti-PD-1，以釐清先天性免疫細胞在B型肝炎病毒慢性持續性感染的小鼠模式誘發免疫抑制的角色。2. 研究不同病毒核心蛋白突變株誘發之CD4 T細胞分化與肝臟內先天性免疫細胞反應。於此小鼠模式中將探討不同病毒核心蛋白突變株誘發之Th1,Th2, Th17, Treg的分化及T細胞分化所需專一性之轉錄因子T-bet, GATA-3, RORγT, FoxP3。3. 欲了解關鍵因子於病毒核心蛋白所誘發之先天性免疫及適應性免疫反應Foxp3/Treg and RORγT/Th17產生，微陣列及蛋白質體方法將用來尋找核心蛋白所誘發之關鍵因子。4. 為了將B型肝炎病毒於小鼠中誘發之免疫機轉轉譯於日後能破壞人類帶原者之病毒持續性，我們將利用已尋獲之分子標的在小鼠模式中研究可誘發先天性免疫或打破T細胞對病毒不反應性之免疫療法，以便於日後能於慢性B型肝炎感染者中破壞病毒持續性感染。<br> Abstract: Objectives: In this project, we will focus in induction of innate immunity, and the role of HBV core protein in modulating adaptive immunity in regulation of T cell response in chronic HBV infection. We will explore the innate immunity and critical factors induced by HBV in breaking immune tolerance in chronic HBV infection. Meanwhile, we will investigate the molecular mechanism of induction of innate immunity by HBV core protein. Finally, the linking from innate immunity to adaptive T cell response and T cell subset differentiation and cytokine production in the liver infiltrating lymphocytes in mice animal model will be investigated.Background: Persistent HBV infection results in chronic hepatitis, liver cirrhosis, and heptocellular carcinoma (HCC). However, the immune mechanism leads to tolerance or clearance of HBV is still not clear. We have established the mouse model for HBV persistence in immunocompetent mice by hydrodynamic injection of replication-competent HBV DNA. In the murine model, our preliminary data showed that there were increased PD1-expressing CD8+ T cells in livers and increased CD4+ FoxP3+ regulatory T (Treg) cells in the carrier mice. Furthermore, the core-null HBV viral construct resulted in loss of ability to clear virus, with increased PD-1 expressing T cells infiltration, leading to persistent HBV infection in mice, indicating HBV core protein may play an important role in induction of host immune response to HBV. The induction of immune response by HBV is thought to initiate via interaction with innate immunity by HBV in the liver. In this regard, we hypothesize that the poor innate immune response and the strong induction of Treg cells to HBV leads to ineffective immune response towards HBV infection. To address this, we intend to study role of HBV core protein in induction of innate immunity to HBV infection in the chronic HBV mice animal model.Specific Aims:1. Define the role of the innate immune system (intracellular PRR) in induction immune response against HBV in the mice animal model.2. To investigate the responding innate immune cells within liver, and differentiation of the CD4 T cells subsets, and to investigate the molecules in linking of innate immunity to adaptive immune response to HBV core.3. To study the immune therapy in mice animal model by using molecular targets to induce immune response to HBV and to abolish the viral persistence.4: To validate the discoveries from mouse animals in the chronic hepatitis B patients and in woodchuck HBV infection animal model.Approaches:1. Define the role of the innate immune system in induction immune response against HBV infection via using hydrodynamic injection of shRNAs against TLRs, MYD88, TRIF, RIG-I/MD5, NLRs and ASC as well as the gene knock out mice and blocking antibodies, such as anti-PD-1 mAbs to verify the role of the innate immune cells in inducing immune suppression in chronic persistent HBV infection mice animal model.2. For study the induction of CD4 T cells subsets in response to different HBV core mutant constructs, the responding innate immune cells within liver will defined. The differentiation of Th1,Th2, Th17, Treg and expression of T cell specific differentiation transcription factors, Tbet, GATA-3, RORγT, FoxP3 induced by HBV core mutant constructs will be analyzed in the mice animal model.3. To investigate the key molecules in induction of innate immunity by HBV core as well as the induction of Foxp3/Treg and RORγT/Th17 in adaptive immunity. The micrarray and proteomics approach will be used to identify key candidate activators of induction of innate immunity by HBV core and inducing differentiation of T cell subsets.4. In order to translate the immune mechanism in induction of immune response to HBV in mice to further abolish viral persistence in human, we will study the immune therapy in mice animal model by using molecular targets identified to either induce innate immunity, or to break the T cell tolerance to HBV, and to further abolish the viral persistence in chronic HBV infection.B型肝炎病毒先天性免疫力小鼠動物模式T細胞反應Hepatitis B virusinnate immunitymice animal modelT cell response