模式识别受体中最为重要的Toll样受体，能够通过识别病原体，传递感染信号入核，不但能够诱导抗炎细胞因子表达，还能够诱导树突状细胞（DC）成熟，启动天然免疫和适应性免疫应答。感染信号的活化和转录因子的诱导是正调控炎症反应的主要方式，目前研究得较为深入，而TLR信号抑制的转录因子，特别是通过表观遗传调控来抑制转录因子方面的研究，尚待开展。本课题组前期通过建立人DC细胞成熟的表观组学图谱和转录组，发现在DC成熟过程中，LPS信号诱导的HSF4启动子区DNA甲基化水平增加，由此抑制了HSF4的表达。本课题拟研究TLR信号如何特异性介导DNA甲基化抑制HSF4的表达，HSF4又如何参与调控天然免疫，包括炎症因子的分泌、DC的成熟及T细胞活化，从而揭示感染信号由外而内，通过表观遗传因子特异性地调控关键性转录因子，建立天然免疫细胞活化和成熟的特异性功能状态的分子生物学机制。

Toll-like receptor is the most important pattern recognition receptors (PRR). Through identifying pathogens and passing the infection signals into nuclear, Toll-like receptor is not only able to induce the expression of anti-inflammatory cytokines, but also able to induce mature dendritic cells (DC), and activate the innate immune and adapt immune responses. The activation of infection signals and the induction of transcription factors are the important way to positively regulate the inflammatory response, which is extensively studied. However, the study of transcription factors inhibited by TLR signaling, especially through epigenetic regulation to inhibit the transcription factor, remains unknown. In our study, through establishing dynamic epigenomes and transcriptome for the process of human DC maturation, we found that the DNA methylation level was increased in the promoter region of HSF4 during the induction of LPS, thus inhibiting the HSF4 expression. This study intends to investigate how TLR signaling specificity mediated DNA methylation to inhibit the expression of HSF4, how HSF4 involved in the regulation of innate immunity, including the secretion of inflammatory cytokines, the maturation of DC and T-cell activation. Through such research, we may reveal the regulation mechanisms of the infection signals transducting from outside to inside, and the molecular biological mechanisms of epigenetic regulating the key transcription factors in innate immune cell activation and maturation.

本项目综合应用细胞生物学、免疫学、分子生物学和化学生物学领域中的通用方法作为研究手段，包括稳定诱导体外DC细胞分化，siRNA干扰，实时定量PCR，ELISA，Western Blot，免疫共沉淀，慢病毒表达体系和人/鼠原代细胞的提取和培养等。同时利用成熟的DNA甲基化检测、ChIP-seq等表观遗传研究体系和定量蛋白质组学平台开展课题研究。研究结果发现，在人源和鼠源DC成熟及巨噬细胞活化的过程中，转录因子HSF4启动子区域呈现高甲基化，伴有HSF4蛋白表达水平下降。进一步研究发现，甲基转移酶DNMT3a负责调控该区域DNA甲基化，转录因子AP-1与DNMT3a形成复合物，共同调节HSF4启动子区的甲基化程度。通过分析HSF4在免疫功能调节中的作用，我们发现HSF4能够调节DC成熟进程，影响巨噬细胞吞噬能力、细胞活化和抗原呈递作用，并改变细胞因子IL-18，IL-6，IL-10和TNF-α的分泌和表达水平，这些因子与溶酶体的活性密切相关。后期我们利用蛋白质组学研究巨噬细胞活化中溶酶体蛋白含量的变化及蛋白网络调控机制，发现SRC蛋白在溶酶体应答巨噬细胞激活中发挥重要作用。此外，我们还在溶酶体中鉴定出300个潜在的糖基化位点，其中CD16和ITGAM在巨噬细胞激活过程中糖基化水平发生改变，提示糖基化在调节免疫应答方面扮演着重要作用。