T-bet-microRNAs相互作用在Tim-3抑制慢性HCV感染者单核巨噬细胞功能中的作用机制

Persistent viral infection has been a serious problem in the prevention and treatment of infectious diseases. Negative immunomodulator Tim-3 plays a pivotal role in negative regulation of Toll-like receptor (TLR)-mediated innate immune responses. While it is clear that Tim-3 is up-regulated on monocyte/macrophages(M/MФ) during HCV infection, little is known about the transcription factors that control its expression. Recent studies revealed that negative immunomodulators' expression can be controlled by the transcription factor T-bet. And some microRNAs affect T-bet and negative immunomodulators' expression by regulating their mRNA translation. Based on our innovative preliminary studies on Tim-3 expression and function on HCV infected M/MФ, in this project, by employing a newly established in vitro HCV culture system, we will further explore the regulatory effects of T-bet and miRNAs on Tim-3 transcription/translation in M/MФ during HCV infection. To this end, purified M/MФ from HCV-infected and control subjects, M/MФ treated with HCV core protein, and M/MФ incubated with HCV+/- Huh-7.5 hepatocytes, will be subjected to measure T-bet and microRNAs expression levels. The interrelationship between T-bet and microRNAs will be evaluated. The signaling transduction pathway will be analyzed and M/MФ functions will be detected. These experiments will provide new information regarding Tim-3 transcriptional/translational regulation during HCV infection by T-bet and miRNAs and might develop novel immunotherapy targets to combat this global epidemic viral infection.

持续病毒感染是传染病防治面临的重大问题，Tim-3等负性调节因子网络的抑制作用是慢性HCV持续感染的分子基础。然而，在慢性HCV感染者单核巨噬细胞（M/MФ）调控Tim-3表达的转录机制还不清楚。近期在小鼠的研究揭示了T-bet和microRNAs参与了Tim-3的负性调节。基于我们前期对慢性HCV感染者M/MФ中Tim-3表达和功能的研究，依赖已建立的HCV体外细胞培养体系，提出本课题研究思路：选择慢性HCV感染者和健康者M/MФ、HCV core抗原刺激的M/MФ，及与HCV+ Huh-7.5肝细胞共培养的M/MФ，检测T-bet和miRNAs在M/MФ的表达水平及相互作用关系；探索T-bet和miRNAs对Tim-3表达和慢性HCV感染者M/MФ功能的影响，分析信号转导途径分子。以揭示T-bet-miRNAs相互作用在Tim-3抑制慢性HCV感染者M/MФ功能中的作用机制。

丙型肝炎病毒感染可以使宿主免疫失调，进而导致感染慢性化。我们前期研究发现负性调节因子Tim-3在慢性HCV感染者表达上调，影响固有免疫细胞功能，然而，在慢性 HCV 感染者单核巨噬细胞（M/Mϕ）调控 Tim-3 表达和功能的转录调控机制还不清楚。本研究明确了在HCV患者M/Mϕ中转录因子T-bet、Tim-3与miR-155之间的关系以及对M/Mϕ功能的影响。研究发现，慢性丙型肝炎患者CD14+ M/Mϕ中，T-bet表达显著上调，并且与Tim-3表达水平呈正相关。单核细胞系THP-1及健康志愿者（HS）CD14+ M/Mϕ分别与HCV+ Huh7.5细胞共培养或者HCV core蛋白刺激后，T-bet表达水平上调。在CD14+ M/Mϕ将JNK信号通路抑制后，T-bet和Tim-3表达水平显著下调。THP-1细胞表达T-bet被抑制后，Tim-3表达水平降低，IL-12 分泌增加，STAT-1磷酸化水平恢复。以上结果表明，HCV感染后，T-bet通过JNK通路，与HCV core/ gC1qR相互作用，上调Tim-3表达，使M/Mϕ功能被抑制。项目还研究了miRNAs对T-bet和Tim-3的影响，结果发现，与HS相比，慢性丙型肝炎患者CD14+ M/Mϕ中miR-155表达上调。分别用LPS、HCV core蛋白、NS3/4刺激THP-1，miR-155表达同样上调。进一步用miR-155 mimics和miR-155 inhibitor分别转染健康志愿者和HCV患者PBMC，发现经过上调或者抑制miR-155表达后，T-bet和Tim-3的表达水平同样被上调或者抑制。在THP-1细胞中上调miR-155的表达水平，可引起JNK水平上调，说明HCV感染后，miR-155可能通过激活JNK通路来影响T-bet和Tim-3的表达。本研究还分析了慢性HCV感染者外周血CD14+ M/Mϕ亚群分布，发现慢性HCV感染者单核细胞比例及其表面负性调节因子Tim-3和PD-1以及胞内抑制因子IL-10表达发生改变。综上述，本研究阐明了 T-bet、Tim-3、miRNAs 相互作用在抑制慢性 HCV 感染者 M/Mϕ功能的作用机制，为将 T-bet、miRNAs 应用于靶向干预或治疗HCV及其它慢性肝炎病毒感染奠定了基础。