分子伴侣介导的自噬在PBC胆管特异性损伤中的作用和机制研究

Primary biliary cholangitis (PBC) is a typical organ-specific autoimmune disease of unknown etiology. A major unanswered question in the pathogenesis of PBC is why small bile ducts are selectively damaged when mitochondria are found in nearly all nucleated cell types. Chaperone-mediated autophagy (CMA) is process where cytosolic substrates that possess a KFERQ-like motif are recognized by hsp70 and directly delivered to lysosomal receptor LAMP-2A together with other co-chaperones followed by degradation into lysosomes. Given the important roles of lysosomal compartments in antigen presentation and immunity, any dysfunction of CMA may be implicated in disordered intracellular antigen processing. We have previously reported that increased LAMP-2 was evident in PBC patients and LAMP-2 deficiency causes a phenotype of early intrahepatic cholestasis. Further data demonstrated that the accumulation of autophagic vacuoles was observed in RBE cells treated with sodium glycochenodeoxycholate. In addition, the abnormal expression of PDC-E2, a mitochondrial autoantigen, was associated with increased expression of LAMP-2A and hsp70 in the treated RBE cells. In this study, we hypothesized that CMA dysfunction may be implicated in disordered mitochondrial autoantigen production and further involved in bile duct injury in PBC patients. Therefore, the present study aims to further clarify the potential roles and mechanisms of CMA in bile duct injury in PBC, which may help illuminate the pathogenesis of PBC and find the novel therapeutic targets in PBC patients.

PBC是一种典型的器官特异性自身免疫性疾病，发病机制至今不清。为什么免疫系统特异性攻击肝内胆管上皮细胞，是PBC研究领域的重要问题。CMA是自噬类型之一，其发生以溶酶体为中心，目前研究认为CMA异常可能与抗原异常产生相关。然而CMA在PBC中的作用未见报道。在国科金资助下，本课题组率先发现CMA关键分子LAMP-2在PBC患者中高表达，且LAMP-2异常与PBC胆汁淤积现象密切相关。进一步预实验结果发现，胆管癌细胞系RBE接受胆汁酸盐刺激后，自噬体形成增加，且CMA相关元件LAMP-2A和hsp70表达增加同时伴有线粒体自身抗原PDC-E2表达异常。据此我们的科学假设是：PBC患者胆管上皮CMA异常参与线粒体自身抗原异常表达，进而诱发机体免疫失衡，引起胆管特异性损伤。本课题拟进一步探讨胆管上皮细胞CMA异常与线粒体抗原表达的相关性及CMA影响线粒体抗原产生在PBC胆管损伤中的作用和机制。

原发性胆子性胆管炎（Primary biliary cholangitis，PBC）是一种由自身免疫介导的慢性胆汁淤积性肝病，其发病机制尚未完全阐明，免疫细胞的过度活化损伤胆管上皮是PBC发生发展的关键。近年来研究显示自噬在维持机体正常的免疫功能中发挥重要作用，溶酶体功能异常可导致免疫功能异常，尤其是自身免疫的发生。课题组前期通过肝脏膜小体分离技术及抗原鉴定，发现相关酶蛋白2(Lamp-2 )的肝脏表达和血清水平与PBC的发生发展密切相关，其作为分子伴侣介导的自噬（CMA）主要效应分子，可能通过自噬参与PBC的自身免疫失衡，从而促进胆管特异性损伤。本项目基于PBC患者的临床样本，通过体内外实验，围绕CMA/LAMP-2A在PBC淋巴细胞活化中的作用及与PBC胆管损伤的关系进行了研究，发现：1.PBC中存在CD4+T细胞的异常活化：与健康对照相比，PBC中ICOS+/HLA-DR+亚群比例升高，CD4+T细胞分泌的IFN-γ、IL-4和IL-17增多；2.CD4+T细胞中LAMP-2A水平表达及在活化中的作用：无论是正常还是何种疾病状态，CD4+T细胞活化后，无论是ICOS+CD4+T细胞和HLA-DR+CD4+T细胞中LAMP-2A表达水平与未活化细胞相比均具有显著差异；3. LAMP-2A通过CMA途径影响PBC患者CD4+T的活化：PBC外周血naïve T细胞较健康对照者的LAMP-2A表达差异显著，且具有更强的增殖能力，而在敲减LAMP-2A后，细胞的增殖能力和分泌IFN-γ、IL-2等细胞因子的能力均受到显著影响；4.PBC中CD4+T细胞CMA异常与疾病的胆管损伤等联系紧密：胆管损伤严重的患者中naïveCD4+T细胞LAMP-2A表达水平与胆管损伤较轻的患者存在显著差异，淋巴细胞亚群和细胞因子水平具有同样的相关性，同时UDCA的应答与治疗前患者T细胞的CMA水平也存在显著相关性。这些结果提示PBC患者中的CMA水平异常可能通过影响淋巴细胞的活化，参与疾病的发生发展，围绕CMA和免疫失衡进行深入研究可能为PBC的机制阐明和靶点开发提供新的思路。

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