JMJD3通过表观遗传调控诱导Kupffer细胞表型重塑在大鼠肝移植后免疫耐受形成中的作用

The phenotype and function of activated Kupffer cells (KCs) are the key factors to induce immune tolerance after liver transplantation. Recently we found that up-regulation of JAK-STAT6 signal pathway in KCs can induce M2 polarization and immune tolerance, but the mechanism needs to be further studied. Recently it is reported that histone demethylase JMJD3 can remove the inhibition of H3K27Me3 on the transcription of M2 type genes and induce polarization of macrophage to M2 type. The process was regulated by STAT6. However, the molecular mechanism is not clear. So far, studies on JMJD3 have not been involved in the field of organ transplantation. Therefore we assume that JMJD3 may be the intranuclear target of JAK-STAT6 downstream signaling, enhanced expression of JMJD3 may induce M2 phenotype remodeling and maintainance through the JAK-STAT6 signaling, thereby facilitating immune tolerance after liver transplantation. In this study, we will separate KCs and establish liver transplantation model in rats. JMJD3 was set as the breakthrough point, and gene regulation, chromatin immunoprecipitation sequencing technology, thin layer chromatography analysis were used to explore JAK-STAT6-JMJD3 pathway’s mechanism in the phenotype remodeling of KCs. Through the study, we look forward to elucidate the mechanism of immune tolerance in liver transplantation.

Kupffer 细胞（KCs）活化后表型及功能状态与肝移植术后免疫耐受微环境形成密切相关。课题组前期发现：上调KCs中JAK-STAT6信号通路活性，可促进 KCs 向M2型极化并诱导免疫耐受形成，但其核内信号机制尚不清楚。新近发现：核内组蛋白去甲基化酶-JMJD3具有特异性解除H3K27Me3对巨噬细胞M2型基因转录的抑制作用，且该过程受STAT6调节。但相关研究尚未涉及移植领域，更未涉及KCs。我们推测：JMJD3可能是JAK-STAT6信号通路下游的核内直接调控靶点，增强JMJD3表达可诱导KCs 向M2型极化并维持其功能，有利于免疫耐受微环境形成。本项目选择肝内KCs为靶细胞，以JMJD3为切入点，分离KCs并建立肝移植模型，采用基因调控、染色质免疫共沉淀测序、薄层色谱分析等方法，初步探讨JMJD3对活化KCs表型重塑的影响，为阐明移植肝脏免疫耐受的形成机制做一些探索性工作。

Kupffer 细胞（KCs）活化后表型及功能状态与肝移植术后免疫耐受微环境形成密切相关。本研究主要探讨了主要探讨了JMJD3沉默或过表达对KCs表型重塑和免疫功能的影响；以及JMJD3介导KCs表型重塑在大鼠肝移植免疫耐受诱导及维持中的作用。过表达KCs中的JMJD3能促进KCs分化为M2型，增加抗炎因子IL-10的表达，降低促炎因子IL-1β，TNF－α，IFN-γ的水平，同时抑制淋巴细胞的增殖，促进淋巴细胞的凋亡，敲低KCs中的JMJD3得到相反的结果；CHIP实验检测发现，在诱导M2型KCs极化的过程中，磷酸化STAT6的表达增加，且直接结合到JMJD3的启动子区，同时M2标记基因的H3K27me3的富集水平下降，敲除JMJD3后得到相反的结论，以上表明M2型KCs激活时依赖STAT6-STAT3通路，且依赖于JMJD3去甲基作用。复合材料包裹AdV-JMJD3，成功过表达大鼠肝脏KCs的JMJD3，在建立大鼠肝移植急性排斥反应模型，通过检测血清中ALT、AST水平，HE染色检测坏死，炎症细胞浸润，空泡化情况，证实增加JMJD3能减轻了大鼠肝移植排斥反应。观察术后生存率也发现KCs中的JMJD3能提高大鼠肝移植的生成率。我们的研究表明JMJD3在KCs的激活过程中发挥作用，激活JMJD3能减少肝移植后的肝脏损伤提高生存率。STAT6-JMJD3可能是治疗肝移植后急性排斥反应，增加免疫耐受的治疗靶点。