摘要：腎移植之後發生的急性排斥，有一半是抗體性排斥，目前的免疫抑制藥物減少了抗體性排斥，相對地卻發現抗體性排斥增加了。發炎反應是抗體產生過程中很重要的步驟，從National Center for Biotechnology Information(NCBI)的網站檢索發現，以LPS 刺激Raw264.7 細胞時Znrf1 的表現增加，ZNRF1 是E3 泛素接合酶(ubiquitin ligase)，泛素化(Ubiquitination)控制NF-"B 的活化，而NF-"B 是發炎和免疫反應最重要的控制分子。我們先前的研究發現ZNRF1 控制巨噬細胞的發炎反應，ZNRF1 可以與E2-ubiquitin 的硫酯中間物(thioester intermediate)結合，催化泛素從E2 轉化到基質蛋白上的離氨基酸(lysine)。在RAW264.7細胞中ZNRF1 與lysosomal-associated membrane protein 1 (LAMP1)共同定位，而且剔除巨噬細胞的ZNRF1 導致一個核內體標記early endosome antigen 1 (EEA1)的累積。巨噬細胞在呈現抗原給T 細胞時，需要MHC II 與抗原結合，並在多泡體multivesicular bodies (MVBs)內運送，問題是我們並不瞭解MHC II 與抗原結合後是如何被運送的。所以本計畫目的在於研究ZNRF1 在MHC II 與抗原運送的角色，並進一步探討抗體性移植排斥的分子機制。我們計畫運用Znrf1 基因剔除的老鼠經輸血產生排斥抗體的動物模型，初步我們發現Znrf1 基因剔除的老鼠經輸血後產生較強的抗體與細胞性反應。進一步探討ZNRF1 相關的分子機制將有助於了解移植排斥的免疫反應，期望能找到有效治療或預防抗體性排斥的新方法。本計畫有三項工作目標:一: 確認 ZNRF1控制MHC II運送的分子機制二: 探討ZNRF1對淋巴細胞反應的影響三: 以小鼠心移植模型探討ZNRF1在移植排斥的角色
Abstract: Antibody-mediated rejection is responsible for up to half of acute rejection episodes in patients whoreceived renal transplantation1, 2. The use of calcineurin inhibitor-based immunosuppression has decreasedthe occurrence of T cell mediated graft rejection; however, the observation of antibody-mediated rejectionepisodes is increased.Inflammation is an important component of innate immunity to initiate adaptive immune response andproduce antigen-specific antibodies. We explored the microarray datasets from Gene Expression Omnibus onthe web site of National Center for Biotechnology Information (NCBI) and discovered that the expression ofan E3 ubiquitin ligase, Znrf1, was up-regulated in LPS treated Bone marrow derived macrophages andRaw264.7 cells. Ubiquitination regulates the activation of nuclear factor (NF)-"B, which is considered to bethe master regulator of inflammation and immune responses. In our previous studies, we found that an E3ubiquitin ligase, ZNRF1, would regulate innate inflammatory response in macrophages (Fig .1;submitted). ZNRF1 functions as an ubiquitin E3 ligase. E3 ubiquitin ligases bind an E2-ubiquitin thioesterintermediate and catalyze transfer of ubiquitin from the E2 to a lysine on a substrate protein. In ourpreliminary results, we found that ZNRF1 would be co-localized with a lysosome marker,lysosomal-associated membrane protein 1 (LAMP1), in RAW264.7 cells and deletion of ZNRF1 inmacrophages would lead to accumulation of an endosomal marker, early endosome antigen 1 (EEA1). Inresponse to microbial and inflammatory stimuli, macrophages initiate primary immune responses bypresenting pathogen-derived antigens in association with MHC II to T lymphocytes which become activatedand elicit downstream effects. Once peptide loaded to MHC II, MHC II with peptide complex is transportedto intraluminal vesicles of multivesicular bodies (MVBs) in the endocytic pathway for further antigenpresentation and activation of lymphocytes. Our understanding to MHC II is always the missing piece of thepuzzle by lacking of definition of the MHC II trafficking pattern in intracellular compartments, and theircommunication with vesicles. In this proposal, we aim to determine the mechanism of ZNRF1 on theadjusting of MHC II and to investigate transplant immunity in depth.In order to study the influence of ZNRF1 on the response of lymphocytes and the production ofantibodies upon challenged with antigens in ZNRF1 knock-out mice, we used the model of transfusion withblood from MHC mismatched donor and detected donor-specific antibodies and the proliferation oflymphocytes in mix-lymphocyte culture. In our preliminary results, we found that the production ofdonor-specific antibodies and the proliferation of lymphocytes were higher in ZNRF1 knock-out mice aftertransfusion with blood from MHC mismatched donor. The finding in our studies may provide us newknowledge to understand the molecular mechanism of rejection and lead us a new way to treat rejectionepisodes in patients received organ transplantation.There are three specific aims in this proposal:Aim 1: Identify the mechanism of ZNRF1 in the regulation of class II major histocompatibilitycomplex (MHC) moleculesAim 2: Investigate the influence of ZNRF1 on the responses of lymphocytesAim 3: Study the role of ZNRF1 in allograft rejection using the model of heterotropic hearttransplantation in mice.