IL-12/IL-23p40表观遗传修饰调控T细胞分化在克罗恩病发病机制和治疗策略中的作用研究

Crohn's disease (CD) is an immune disorder of intestine that is dominated by over-reactive Th1 and Th17 signaling pathways. IL-12 and IL-23, which share a common subunit called p40 (expressed by IL12B gene), are the major drivers for the differentiation Th1 and Th17, respectively, and play a major role in the pathogenesis of CD. Therefore, blocking p40 subunit can simultaneously prevent over-activation of both IL-12/Th1 and TL-23/Th17 pathways. The highly successful therapeutic outcomes of CD in recent clinical trials on some fully humanized monoclonal antibodies targeting the IL-12/23-p40 subunit, further reinforced this concept. IL-12 and IL-23 are predominantly derived from macrophages, which are major antigen-present cells of innate immunity involved in the pathogenesis of Crohn's. In our preliminary experiments, we demonstrated that epigenetic modifications were involved in the pathogenesis of CD, and that methylation of IL12B promoter controlled the p40 expression. Our data would strongly suggest a feasibility of developing a novel therapeutic strategy for CD by controlling p40 expression through epigenetic manipulation. In this study, we will comprehensively analyze and determine the epigenetic status of IL12B gene, including DNA methylation and histone acetylation in colonic macrophages of CD patients and chronic TNBS-induced mouse model of CD by methylation-specific PCR and ChIP. To manipulate the epigenetic alterations, we propose two independent approaches, one is conventional drug-mediated and the other is a novel gene- and cell-specific targeting, to evaluate their influences on the IL-12B methylation and p40 expression and the down-stream regulation on Th1 and Th17 differentiation of T cells (both in vitro and in vivo). TAL, transcription activator-like (TAL) effector and construct, is the most recently developed novel gene-target technology to mediate efficiently modification of specific genes of interest. The specifically TAL-targeted gene in our study is IL-12B. The TAL-methyltransferase will be constructed and delivered into macrophages in both cell culture model (in vitro) and mouse model of CD (in vivo), using a highly efficient and stable lentivirus expression system. The effects of the conventional drugs and TAL-methyltransferase on the methylation of IL-12B promoter, p40 expression, Th1/Th17 differentiation of T cells, as well as therapeutic efficacy on CD mouse model will be systemically evaluated. Successful completion of our proposed study will not only lead to the elucidation of a novel molecular mechanism of pathogenesis of CD, but also provide a new therapeutic target for developing novel strategies of managing CD through epigenetic-based gene-targeted technology.

克罗恩病（CD）发病机制与Th1和Th17细胞介导的免疫反应密切相关。IL-12和IL-23是调节Th1和Th17分化的关键因子，主要由巨噬细胞分泌，两者共享亚单位p40 (由IL12B基因表达)。前期研究显示，表观遗传机制参与了CD的发生发展，IL12B基因启动子甲基化是影响其表达的主要机制，但该基因表观遗传修饰与CD发病及病程进展的关系尚不明确，改变其表观遗传状态能否发挥治疗效应也有待确认。本项目拟深刻解析CD患者和小鼠模型中肠黏膜巨噬细胞IL12B基因启动子DNA甲基化和组蛋白乙酰化状态，运用转录激活子样（TAL）效应因子这种全新的基因靶向技术，建立靶向巨噬细胞的特异性转导体系（TAL甲基化酶），在细胞和整体水平研究干预IL12B启动子甲基化对T细胞分化的影响和对CD小鼠的疗效，为深入揭示CD的发病机制及运用表观遗传学理论和基因靶向技术建立新的治疗策略提供实验依据和全新的应用前景。

克罗恩病（CD）发病机制与Th1和Th17细胞介导的免疫反应密切相关。IL-12和IL-23是调节Th1和Th17分化的关键因子，主要由巨噬细胞分泌，两者共享亚单位p40 (由IL12B基因表达)。前期研究显示，表观遗传机制参与了CD的发生发展，IL12B基因启动子甲基化是影响其表达的主要机制，但该基因表观遗传修饰与CD发病及病程进展的关系尚不明确，改变其表观遗传状态能否发挥治疗效应也有待确认。本课题重点研究人类巨噬细胞株和小鼠肠炎模型中IL12B基因启动子DNA甲基化和组蛋白乙酰化状态，并运用表观遗传学理论和一种全新的基因靶向技术探讨新的治疗策略。我们研究发现，IL-12B基因在小鼠和人类巨噬细胞株以及小鼠肠炎模型中表达均增高；MSP分析显示IL12B基因启动子DNA甲基化状态无明显改变，提示IL-12B基因启动子DNA甲基化可能不是其基因表达改变的主要机制之一；与对照组相比，IL-12B基因H3K9Ac, H3K27me3 表达异常，提示组蛋白修饰可能是IL-12B基因表达改变的主要机制，为进一步丰富表观遗传理论、并明确肠道炎症中免疫紊乱的发病机制提供了理论依据。进一步的，我们运用转录激活子样（TAL）效应因子，成功构建靶向人类巨噬细胞IL12B基因的IL12B-TAL-核酸修饰酶质粒，可成功调节IL12B基因转录，并进一步筛选出IL12B-TAL-3Ac1质粒，通过诱导IL12B基因启动子甲基化而抑制其转录，基因表达降低。我们的研究明确了炎症状态下IL12B基因表达异常的机制，并探讨了一种新的治疗策略，即通过TAL这种基因靶向技术，可特异性诱导IL12B基因表观遗传改变，从而调控基因表达。本课题为深入揭示CD的发病机制及运用表观遗传学理论和基因靶向技术建立新的治疗策略提供实验依据和全新的应用前景。