【作者】 张鹏飞；

【导师】 吴缅；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2019， 博士

【摘要】 免疫系统是用来保护机体免受内外源感染和损伤的一系列蛋白、细胞、组织和器官的集合,可以简单地分为固有免疫和适应性免疫。进化上保守的固有免疫系统是机体抵御病原体入侵和感染的第一道防线,主要通过免疫细胞上表达的模式识别受体来感应内外源危险信号,产生介导细胞间通讯的细胞因子并招募大量免疫细胞到感染或者炎症部位发挥抵抗外源感染或者内源损伤的功能。炎症小体是机体固有免疫的重要一环,主要在巨噬细胞和树突细胞等免疫细胞的细胞质中组装和激活,可以感知内源危险相关分子模式和外源病原相关分子模式,从而控制炎症反应来抵御病原体感染。炎症小体是由PRRs、ASC和pro-caspase-1构成的多聚蛋白复合物,激活后可以活化caspase-1并促进IL-1β和IL-18的成熟和分泌,还伴随有细胞焦亡,进一步通过级联效应促进机体炎症反应。巨噬细胞中炎症小体激活一般需要两个信号步骤:启动信号和激活信号。虽然炎症小体充分激活可以消除病原体和损伤细胞,但是炎症小体失调会导致自身免疫性疾病、癌症、神经退行性疾病和其他一些相关疾病。因此,研究并阐明各种各样炎症小体结构、组装、激活以及相关分子机制是很重要的,既具有理论价值又具有应用前景。长非编码RNA是一类长度大于200个核苷酸而且不具有编码蛋白质能力的RNA,其在各种疾病发生发展和生物发育过程中都具有多种生物学功能。长非编码RNA也具有调控炎症小体激活的功能,已知影响炎症小体激活的lncRNA主要是Gm4419、lincRNA-Cox2和lincRNA-EPS。它们都作用于炎症小体激活的第一信号启动阶段,影响炎症小体相关蛋白NLRP3、ASC和IL-1β等的表达,从而促进NLRP3炎症小体激活。目前还没有发现直接参与并调控炎症小体组装的lncRNA,所以深入研究和阐明lncRNA在各种炎症小体组装过程中的作用有助于深入理解炎症小体的激活机制。鉴于目前还没发现直接参与炎症小体组装的lncRNA,本文将鉴定直接参与炎症小体组装的lncRNA并深入研究其调控炎症小体激活的分子机制。我们通过NLRP3紫外交联免疫沉淀结合高通量测序发现,长非编码RNA Neatl可能结合在NLRP3炎症小体上。进一步研究发现,在小鼠巨噬细胞中Neatl可以结合在NLRP3、NLRC4和AIM2等经典炎症小体上,并促进这些炎症小体的组装和激活,从而促进了其下游的caspase-1活化、细胞因子成熟和细胞焦亡。随后在分子机制方面的研究发现,Neatl可以通过其5’端区域结合在pro-caspase-1上的p20结构域,并促进相关炎症小体的组装。此外,Neatl也结合在caspase-1加工生成的p20亚基上,并随之结合在成熟的caspase-1异源四聚体上,增加caspase-1的稳定性和酶活性。在各种炎症小体激活信号的作用下,Neatl从旁斑中释放出来,并转移到细胞质中参与炎症小体组装激活。本研究还发现了低氧通过HIF-2α激活的Neatl调控炎症小体激活的新通路。此外,基于Neatl基因敲除小鼠体内炎症模型的研究也证明了,Neatl具有促进体内炎症小体激活并增强相关炎症反应的作用。综上所述,本研究发现并证实了 lncRNA在炎症小体组装和激活中的关键作用,揭示了 Neatl是多种炎症小体激活信号下游的共同介导分子,它在固有免疫反应中发挥着重要作用。更多还原

【Abstract】 The immune system,that could protect the body against invaders,is made up of virous types of proteins,cells,tissues,and organs.The immune system is composed of two major subdivisions:innate immunity and adaptive immunity.The evolutionarily conserved innate immune system is the host’s first line of defense against invasion or infection by pathogens.The innate immune system relies on the pattern recognition receptors expressed by innate immune cells to rapidly recognize and respond to signals derived from the invading pathogens or injured self-cells.In order to defend against the infection and damage,the innate immunity mainly recruits immune cells to sites of infection and inflammation through the production of cytokines,that are small proteins involved in cell-to-cell communication.Inflammasomes are a group of multicomponent signaling platforms composed of PRRs,ASC,and pro-caspase-1.They are assembled and located in the cytoplasm of immune cells,including macrophages and dendritic cells.Inflammasomes play an important role in innate immune,because they control inflammatory response and anti-pathogen defense against a wide range of infection and damage signals by responding to a wide variety of stimuli,such as pathogen-associated molecular patterns and damage-associated molecular patterns.Upon activation,the sensor proteins bind to and induce the oligomerization of a common adaptor protein ASC,.Oligomerized ASC recruits pro-caspase-1,facilitating its auto-processing into the mature subunits.Active caspase-1,composed of mature subunits,mediates proteolytic maturation of pro-inflammatory cytokines IL-1β and IL-18 and elicits pyroptosis.In macrophages,inflammasome activation generally requires two signals:priming and activating.While adequate inflammasome activation is crucial for the elimination of pathogens and damaged cells,dysregulation of inflammasome contributes to autoimmune,cancer,neurodegenerative disorders,and other diseases.Therefore,it is very important to study and clarify the structure,assembly,activation,and associated molecular mechanisms of various inflammasomes,which has both theoretical value and application prospect.Long non-coding RNAs are defined as transcripts longer than 200 nucleotides but lacking significant protein coding capacity,and have a variety of biological functions in some diseases and biological development.In the context of innate immunity,while a few long non-coding RNAs have been implicated in regulation of inflammasome,including Gm4419,lincRNA-Cox2,and lincRNA-EPS3.All above long non-coding RNAs mainly function on the first step of inflammasome activation,and affect the expression of NLRP3,ASC,and IL-1β to promote the activation of NLRP3 inflammasome.However,no long non-coding RNA has been reported to directly involve in inflammasome assembly.Therefore,further study and clarification of the role of long non-coding RNAs in the assembly process of various inflammasomes will help us greatly to furtherly understand the mechanism of inflammasome activation.In order to investigate long non-coding RNAs associated with inflammasomes,we set out to identify long non-coding RNAs that are associated with the NLRP3 inflammasome in murine immortalized bone marrow-derived macrophages.We performed the NLRP3 CLIP sequencing and analysis,and found that Neatl might bind to the NLRP3 inflammasome.Furtherly,we found that Neatl promoted the activation of NLRP3,NLRC4,and AIM2 inflammasomes and enhances caspase-1 activation,cytokine production,and pyroptotic cell death.Mechanistically,Neatl binds to the p20 domain of pro-caspase-1 via its 5’ region,and facilitates the assembly of inflammasomes.Furthermore,Neatl also stabilizes the mature caspase-1 through p20 subunit and increases caspase-1 protease activity.In response to various inflammasome-activating signals,Neatl is released from paraspeckles and translocated to the cytoplasm to participate in inflammasome assembly and activation.In this study,we found that hypoxia-induced activation of the NLRP3,NLRC4,and AIM2 inflammasomes and associated inflammatory responses were in part due to HIF-2a-mediated upregulation of Neatl.This is a novel pathway.We also observed the overt defects of Neat1-/-mice in alum-induced peritonitis and flagellin-induced pneumonia,which emphasizes an important function for Neatl in the activations of the canonical inflammasomes,the subsequent accumulation of immune cells and the associated symptoms in vivo.Our findings establish a direct role for long non-coding RNAs in regulating inflammasomes and suggest that Neatl may represent a downstream convergence point for inflammasome stimuli.Neatl plays an important role in innate immune response.更多还原