乙酰化调控TLR2基因表达在结核病发病中的作用及机制研究

Mycobacterium tuberculosis(Mtb) is the causative agent of tuberculosis, responsible for approximately 3 million deaths a year across the world.Monocyte/macrophage system contributes to the early identification of Mycobacterium tuberculosis and the incidence of tuberculosis. Therefore, we studied.the monocyte/macrophage cell surface molecules-Toll-like receptors(TLRs) and their signaling pathway proteins in the extracellular which directly contact with the protein antigen of Mycobacterium tuberculosis in the first stage.Of the whole family of TLRs,TLR2 exerts an important role in the anti-tuberculosis immune response..Recent years,the link between bacteria and host chromatin remodeling is an emerging topic.Bacteria provoke histone modifications and chromatin remodeling in infected cells,thereby altering the host's transcriptional program.Histone acetyltransferase (HAT) and histone deacetylase(HDAC), which modulate the acetylation status of proteins, act not only on histones, but also on transcription factors in regulating gene transcription. p300 is a kind of HAT,which can coactivator for several transcriptional factors.One group published that p300 is so important for CCL5 production by epithelial cells infected with M. bovis BCG that limit mycobacterial infection..In our previous work,we found that BCG infection can improve the acetylated level of total proteins and expression of p300 protein, at the same time upregulate TLR2 mRNA expression in human monocytic cells U937.Later,we used delphinidin(DP),a specific inhibitor of p300,to downregulate the acetylated level of U937 cells,finding that the expression of TLR2 also downregulated in parallel.Analysis of TLR2 gene promoter using TRANSFAC-TESS program indicated a response element-C/EBPα.Moreover,p300 is reporterd to regulate gene expression through C/EBPα and histone acetylation pathways. Therefore,we infer that BCG infection may lead to the change of protein acetylated level, through p300-C/EBPα/histone pathways regulating TLR2 gene expression..In this study,we propose to 1)detect acetylation regulating the expression of TLR2 gene and downstream proinflammatory cytokines during BCG infection.2)clarify the mechanism of P300-C/EBPα/histone acetylation regulating the expression of TLR2.3)dectect the expression of TLR2 gene and downstream proinflammatory cytokines,expression and activity of p300,and the acetylated level of histone and C/EBPα proteins in BCG infected mouse.Our results may represent a new insights into the of pathogenesis of tuberculosis.

TLR2在抗结核免疫中发挥重要作用，但其表达调控机制尚未见报道。课题组前期研究发现用BCG感染人单核细胞后，TLR2表达水平与细胞乙酰化水平呈正相关；理论预测TLR2基因启动子区参与乙酰化调控的C/EBPa反应元件。本项目拟深入探索BCG感染通过影响乙酰化而参与调节TLR2基因表达的机制。首先检测BCG感染通过增强乙酰化影响TLR2及下游炎性因子表达；然后鉴定BCG感染对p300表达和活性的影响以及p300对C/EBPα和组蛋白乙酰化的调节，从而证明p300-C/EBPα/组蛋白途径是BCG感染上调TLR2 基因表达的一种作用机制；最后通过检测BCG感染小鼠体内TLR2基因及其下游炎性因子表达水平，p300表达和活性、组蛋白和c-Myc乙酰化水平,体内鉴定上述机制。本课题从表观遗传学的角度探讨TLR2基因在结核病发病中的调控机制，为结核病的防治提供新思路。

TLR2在抗结核免疫中发挥重要作用，但具体调控机制尚不明确。本研究鉴定了在BCG感染过程中，通过改变细胞内乙酰化水平, 从而参与调节TLR2基因表达的机制。一方面，我们证明了在分化成熟的THP-1细胞系中, BCG感染能够上调p300蛋白表达水平和组蛋白H3乙酰化水平，加入p300特异性抑制剂delphinidin后，组蛋白H3乙酰化水平降低，提示BCG感染通过p300途径导致蛋白质乙酰化水平发生改变。随后，我们应用组蛋白去乙酰化酶抑制剂( Trichostatin A，TSA)处理人THP-1细胞，发现TSA以浓度依赖方式上调TLR2表达水平。同时我们发现TSA可以上调组蛋白H3乙酰化水平，使TLR2启动子区H3乙酰化水平明显上升，提示TSA通过组蛋白H3乙酰化影响TLR2基因表达，这是乙酰化调控TLR2基因表达的一种可能机制。另一方面，我们鉴定了TLR2启动子区的的一个AP-2α反应元件，并证明AP-2α乙酰化可通过增强其与TLR2启动子的结合，增加转录活性而上调TLR2基因表达。我们的研究结果证明了在BCG感染过程中，通过p300表达水平和H3组蛋白及AP-2α乙酰化水平的改变对TLR2基因表达水平及启动子活性的影响，从而阐明一种BCG感染通过乙酰化影响TLR2基因表达调控的机制。本课题从表观遗传学的角度探讨TLR2基因在结核病发病中的调控机制，为结核病的防治提供新思路。