DNA病毒感染引发天然免疫应答的分子机制研究

Innate immunity is the first line of defense against microbial invasions via a set of pattern-recognition molecules. Activation of the innate immune system will lead to the induction of inflammatory cytokines and many other effective factors against different pathogens. Malfunction of the innate immunity is involved in a variety of diseases, such as endotoxic shock, IBD and some cancers due to the hypersensitivity of innate immunity, and many immune-deficiency-related diseases due to hyposensitivity of the innate immunity...We have established a powerful cDNA library screening system to search systematically for new genes that play essential roles in the innate immunity. We identified ERIS/ STING/MITA as an essential player in anti-DNA viral innate immunity, and more recently, ERIS-STAT6-TBK1 was also found to play a critical role in controling the expression a set of chemokines for immune cell homing. Using Yeast two hybrid screening, immuno-purification and Mass-spec-based protein identification techniques, we are also interested in identifying new genes participating in the formation of the signal complexes, such as RIG-I/Mda5/MAVS complex, and STING/TBK1 complex, during the activation of the innate immune system...Based on our previous work, we will work on the following aspects of innate immunity:..I. Identification and characterization of a Golgi protein Z1 that regulates ERIS-mediated innate immunity against DNA viral infection...II. Identification and characterization of an E3 ubiquitin ligase required for IRF3 K63-ubiquitination and activation...III. characterization of the physiololigical function of the new defined signaling cascade leading to STAT6 activation and the existance of other ERIS-STATx cascade, as well as the possible function in anti-viral innate immunity.

天然免疫与人类的健康、疾病密切相关。天然免疫的不足或过度皆可引起严重的疾病。十几年来人们对天然免疫的研究取得了许多重大突破，可是对来自于病原体基因组DNA（如病毒DNA）所激活的天然免疫应答的机制还很不清楚。我们的前期工作发现了ERIS及ERIS-TBK1-STAT6对于DNA病毒激活的天然免疫反应至关重要，以此为基础，近期又发现多个可能在此通路中起重要作用的新基因。本项目拟围绕这些分子，研究它们在细胞受到DNA病毒感染后引发天然免疫应答中的作用。重点研究1）高尔基体蛋白Z1及其相关蛋白帮助ERIS进行天然免疫应答的机制；2）IRF3特异性K63位泛素化E3连接酶IUK63活化IRF3的分子机制；3）STAT6及其它STATx在天然免疫中的生理作用。本项目不仅能丰富我们对免疫应答及调控的细胞与分子机制的认识，而且可以对设计研究下一代新型抗感染药物提供可能的靶标。

近期在细胞中发现了能够激活STING的环化双核苷酸c-di-GMP、c-di-AMP及cGAMP，它们构成了一类全新的第二信使，在细胞的代谢、生长、趋药性及抵抗感染等很多方面具有非常重要的作用。利用真核筛选体系，我们首次发现了三个特异性降解3’3’-cGAMP的磷酸二酯酶，填补了该第二信使被降解的空白，为临床药物的研发提供了新的作用靶点。我们发现炎性小体能负调控I型干扰素通路，炎性小体激活后活化的Caspase-1能够招募并切割cGAS蛋白，使cGAS功能受到极大的丧失，导致cGAMP合成减少，从而抑制DNA病毒感染诱导的cGAS-STING信号通路的活化。首次揭示cGAS蛋白N端序列在识别细胞质DNA并活化天然免疫中发挥重要的生理功能，改变了人们对cGAS功能机制的理解。我们还研究了抗病毒天然免疫信号通路中重要激酶TBK1及IKKα/β/IKKε的活化机制。发现E3泛素连接酶TRAF蛋白在此通路中至关重要，并表明不同TRAF蛋白在机体应对RNA病毒的免疫反应中起不同作用。TRAF蛋白与TBK1/IKKε形成复合体，通过与MAVS蛋白中的TRAF结合序列招募TBK1/IKKε到MAVS，进而促进激酶的自磷酸化及激活。我们还发现没有NFκB/NEMO活化的情况下，细胞仍产生少量的I-型干扰素。这种IKKα/β和TBK1/IKKε相互活化的机制同样存在于由DNA病毒感染而激活的cGAS–STING通路中。我们首次发现低浓度的血清培养可以强烈促进细胞对DNA病毒的反应，血清饥饿使以往对细胞质DNA没有反应的细胞株对DNA病毒产生很强的反应。以此为基础，我们从遗传学角度发现并证明NEMO及NFκB对cGAS- STING激活至关重要，NEMO-IKKα/β复合体通过结合E3泛素化连接酶TRIM32和TRIM56合成的泛素链而被活化。研究表明，RLR-MAVS和cGAS–STING通路不但在抗病毒天然免疫反应中发挥重要作用，在肿瘤的发生，迁移及免疫治疗方面也非常重要。传统的研究认为IKKα/β介导的NFκB通路对肿瘤的迁移很重要，而TBK1/IKKε介导的I型干扰素通路在肿瘤的免疫治疗中发挥重要作用。我们发现的IKKα/β和TBK1/IKKε相互活化的机制可能会为肿瘤的免疫治疗及抗肿瘤药物的研发提供新的切入点和方向。本项目研究取得多项重大的成果，在项目资助下共发表论文10篇。