炎性小体研究进展

炎性小体是由胞浆内模式识别受体组装的多蛋白复合体,是宿主先天免疫系统的重要组成部分。在细胞应对外界危险信号时,炎性小体能激活半胱天冬酶-1,通过调节白介素-1β和白介素-18等促炎性细胞因子的成熟与释放,参与先天性免疫防御。炎性小体活性异常与人类先天性和获得性炎症性疾病密切相关,炎性小体在免疫应答中具有重要作用。综述了炎性小体的组成、功能、激活方式及其与疾病的相关性。     

炎性体与器官移植

炎性体是指存在于细胞质内能够激活半胱天冬酶-1（caspase-1）的大分子复合物,活化的caspase-1通过酶切白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）前体分子而生成具有生物学活性的IL-1β和IL-18.近来研究发现,炎性体分子NLRP1、NLRP2、NLRP5、CARD8、CASP5的基因型与移植的临床结果相关,心脏移植排斥反应时ASC和IL-1β的表达升高,缺血再灌注损伤中NLRP3炎性体激活增加IL-1β分泌,表明炎性体的激活与移植排斥反应和缺血再灌注损伤密切相关,但诱导炎性体活化的配体和参与的炎性体分子有待进一步研究.     

细胞调亡是半胱天冬酶参与的复杂过程

自从发现线虫(Caenorhabditis elegan)的CED蛋白与哺乳动物的白介素-1β转换酶(ICE)有相似作用以来[1],已有13种半胱天冬酶家族成员先后被发现或克隆[2](表1).半胱天冬酶(caspase)分为启动型和执行型,前者接受死亡信号、启动凋亡或激活下游的分子,后者则降解细胞骨架、蛋白质、核酸等.     

细胞凋亡是半胱天冬酶参与的复杂过程

自从发现线虫 (Ｃａｅｎｏｒｈａｂｄｉｔｉｓｅｌｅｇａｎ)的ＣＥＤ蛋白与哺乳动物的白介素 1 β转换酶(ＩＣＥ)有相似作用以来[1] ,已有 1 3种半胱天冬酶家族成员先后被发现或克隆[2 ] (表 1 )。半胱天冬酶 (ｃａｓｐａｓｅ)分为启动型和执行型 ,前者接受死亡信号、启动凋亡或激活下游的分子 ,后者则降解细胞骨架、蛋白质、核酸等。半胱天冬酶如何启动和实施凋亡、受哪些因素调节等问题是近年来研究的热点。凋亡是由多种因素引起、多分子参与的复杂过程 ,本文就其目前的研究进展作一简要报道。表 1　现已发现十三种半胱天冬酶 ( 1998)1 .…     

NLRP3 炎性小体研究新进展

NLRP3 炎性小体是一种分子量约为700Kda 的大分子多蛋白复合体,能被多种病原相关的分子模式或损伤相关的分子模式 活化,对固有免疫系统免疫功能的发挥具有极其重要的作用。但如果其被过度激活则可通过活化的半胱天冬酶-1 持续地将 pro-IL-1茁和pro-IL-18 剪切为成熟的IL-1茁和IL-18,进而激活下游信号转导通路,产生大量的炎性介质,引起机体发生严重的炎 症反应,最终促进多种炎症性疾病的发生与发展,如Muckle-Wells综合征、2 型糖尿病、非酒精性脂肪肝、动脉粥样硬化、炎症性肠 病和阿尔兹海默病等。因此,对NLRP3 炎性小体进行深入的研究不仅有助于阐释固有免疫系统如何有效地发挥其免疫功能,而 且作为系列炎症反应的核心,NLRP3 炎性小体还可能成为多种炎症性疾病防治的新靶点。我们就NLRP3 炎性小体的结构与功 能,激活与调控,分布与疾病的近期研究作一综述。     

NLRP3炎性小体研究新进展

NLRP3炎性小体是一种分子量约为700Kda的大分子多蛋白复合体,能被多种病原相关的分子模式或损伤相关的分子模式活化,对固有免疫系统免疫功能的发挥具有极其重要的作用。但如果其被过度激活则可通过活化的半胱天冬酶-1持续地将pro-IL-1β和pro-IL-18剪切为成熟的IL-1β和IL-18,进而激活下游信号转导通路,产生大量的炎性介质,引起机体发生严重的炎症反应,最终促进多种炎症性疾病的发生与发展,如Muckle—wells综合征、2型糖尿病、非酒精性脂肪肝、动脉粥样硬化、炎症性肠病和阿尔兹海默病等。因此,对NLRP3炎性小体进行深入的研究不仅有助于阐释固有免疫系统如何有效地发挥其免疫功能,而且作为系列炎症反应的核心,NLRP3炎性小体：还可能成为多种炎症性疾病防治的新靶点。我们就NLRP3炎性小体的结构与功能,激活与调控,分布与疾病的近期研究作一综：违。     

半胱天冬酶（caspase）

半胱天冬酶是ＩＣＥ／ＣＥＤ－３蛋白酶家族的总称,在细胞凋亡中起着关键的作用,迄今已鉴定的人类半胱天冬梅共有１１种,其底物可分为４类,半胱天冬酶的激活机制已初见端倪,有非自然抑制剂和自然抑制剂,抑制半胱天冬酶活性作为神经变性疾病的治疗途径已开始探索。     

半胱天冬酶在艾滋病发病机制中的作用

艾滋病的基本病理机制是CD4 淋巴细胞的减少和多种免疫功能的紊乱。在感染过程中,无论CD4 、CD8 等免疫细胞的凋亡还是病毒蛋白的加工,天冬氨酸酶都有重要的作用。主要对半胱天冬酶在艾滋病发病机制的作用作了一些探讨。     

细胞焦亡机制及与疾病的关系

细胞焦亡(pyroptosis)是近年来发现的一种区别于细胞凋亡的促炎程序性死亡方式。焦亡途径包括半胱天冬酶(caspase)-1介导的经典焦亡途径和Caspase-4/5/11介导的非经典焦亡途径。细胞焦亡涉及多种炎性小体的激活,如核苷酸结合寡聚化结构域样受体蛋白3(NLR pyrin domain containing 3,NLRP3)、核苷酸结合寡聚化结构域样受体蛋白C4 (NLR containing a caspase recruitment domain 4,NLRC4)以及黑色素瘤缺乏因子2 (absent in melanoma 2,AIM2)等。Gasdermin-D(GSDMD)是参与细胞焦亡的关键切割蛋白,最终导致膜蛋白通道开放、膜孔形成、白细胞介素(interleukins,ILs)释放,从而扩大炎症反应。细胞焦亡介导许多疾病如感染性疾病、神经系统疾病、心血管疾病、代谢性疾病以及炎症免疫性疾病等。本文综述了细胞焦亡机制及与疾病关系的研究进展。     

NLRP1炎性体

核苷酸结合寡聚化结构域样受体蛋白1(NLRP1)炎性体是NLRP1在识别胞内病原相关分子模式(PAMP)后与凋亡相关斑点样蛋白(ASC)以及半胱天冬氨酸酶(Caspase-1、Caspase-5)前体等分子结合形成的蛋白复合物,活化后促进IL-1β、IL-18、IL-33等炎症因子的成熟和释放,在先天性免疫中发挥重要作用。本文主要介绍了NLRP1炎性体的组成、激活机制、信号通路、负向调控及生物学功能,综述了NLRP1炎性体在炭疽、弓形虫病、泛发型白癜风、肠炎、牛皮癣等疾病中作用的研究进展。     

Critical roles of ASC inflammasomes in caspase-1 activation and host innate resistance to Streptococcus pneumoniae infection

Streptococcus pneumoniae is a Gram-positive, extracellular bacterium that is responsible for significant mortality and morbidity worldwide. Pneumolysin (PLY), a cytolysin produced by all clinical isolates of the pneumococcus, is one of the most important virulence factors of this pathogen. We have previously reported that PLY is an essential factor for activation of caspase-1 and consequent secretion of IL-1β and IL-18 in macrophages infected with S. pneumoniae. However, the host molecular factors involved in caspase-1 activation are still unclear. To further elucidate the mechanism of caspase-1 activation in macrophages infected with S. pneumoniae, we examined the involvement of inflammasomes in inducing this cellular response. Our study revealed that apoptosis-associated specklike protein containing a caspase recruitment domain (ASC), an adaptor protein for inflammasome receptors such as nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2), is essentially required for the induction of caspase-1 activation by S. pneumoniae. Caspase-1 activation was partially impaired in NLRP3(-/-) macrophages, whereas knockdown and knockout of AIM2 resulted in a clear decrease in caspase-1 activation in response to S. pneumoniae. These results suggest that ASC inflammasomes, including AIM2 and NLRP3, are critical for caspase-1 activation induced by S. pneumoniae. Furthermore, ASC(-/-) mice were more susceptible than wild-type mice to S. pneumoniae, with impaired secretion of IL-1β and IL-18 into the bronchoalveolar lavage after intranasal infection, suggesting that ASC inflammasomes contribute to the protection of host from infection with PLY-producing S. pneumoniae.     

炎症小体在对抗微生物感染中的作用

炎症小体(inflammasomes)是由胞浆内模式识别受体(PRRs)参与组装的多蛋白复合物,是天然免疫系统的重要组成部分。炎症小体能够识别病原相关分子模式(PAMPs)或者宿主来源的危险信号分子(DAMPs),招募和激活促炎症蛋白酶Caspase-1。活化的Caspase-1切割IL-1β和IL-18的前体,产生相应的成熟细胞因子。炎症小体的活化还能够诱导细胞的炎症坏死(pyroptosis)。目前已经确定多种炎症小体参与了针对多种病原体的宿主防御反应,病原体也已经进化出多种相应的机制来抑制炎症小体的活化。该文总结了炎症小体在抗感染免疫研究领域的最新进展,重点讨论了炎症小体对细菌、病毒、真菌和寄生虫的识别,以及炎症小体的活化在宿主抗感染过程中所发挥的作用。     

Functional screening of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-kappaB and caspase-1

PYRIN-containing Apaf-1-like proteins (PYPAFs) are a recently identified family of proteins thought to function in apoptotic and inflammatory signaling pathways. PYPAF1 and PYPAF7 proteins have been found to assemble with the PYRIN–CARD protein ASC and coordinate the activation of NF-κB and pro-caspase-1. To determine if other PYPAF family members function in pro-inflammatory signaling pathways, we screened five other PYPAF proteins (PYPAF2, PYPAF3, PYPAF4, PYPAF5 and PYPAF6) for their ability to activate NF-κB and pro-caspase-1. Co-expression of PYPAF5 with ASC results in a synergistic activation of NF-κB and the recruitment of PYPAF5 to punctate structures in the cytoplasm. The expression of PYPAF5 is highly restricted to granulocytes and T-cells, indicating a role for this protein in inflammatory signaling. In contrast, PYPAF2, PYPAF3, PYPAF4 and PYPAF6 failed to colocalize with ASC and activate NF-κB. PYPAF5 also synergistically activated caspase-1-dependent cytokine processing when co-expressed with ASC. These findings suggest that PYPAF5 functions in immune cells to coordinate the transduction of pro-inflammatory signals to the activation of NF-κB and pro-caspase-1.     

The central inflammasome adaptor protein ASC activates the inflammasome after transition from a soluble to an insoluble state

Apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) (ASC) is a 22 kDa protein that functions as the central adaptor for inflammasome assembly. ASC forms insoluble specks in monocytes undergoing pyroptosis, and the polymerization of ASC provides a template of CARDs that leads to proximity-mediated autoactivation of caspase-1 in canonical inflammasomes. However, specks are insoluble protein complexes, and solubility is typically important for protein function. Therefore, we sought to define whether ASC specks comprise active inflammasome complexes or are simply the end stage of exhausted ASC polymers. Using a THP-1 cell–lysing model of caspase-1 activation that is ASC dependent, we compared caspase-1 activation induced by preassembled insoluble ASC specks and soluble monomeric forms of ASC. Unexpectedly, after controlling for the concentration dependence of ASC oligomerization, we found that only insoluble forms of ASC promoted caspase-1 autocatalysis. This link to insolubility was recapitulated with recombinant ASC. We show that purified recombinant ASC spontaneously precipitated and was functional, whereas the maltose-binding protein–ASC fusion to ASC (promoting enhanced solubility) was inactive until induced to insolubility by binding to amylose beads. This functional link to insolubility also held true for the Y146A mutation of the CARD of ASC, which avoids insolubility and caspase-1 activation. Thus, we conclude that the role of ASC insolubility in inflammasome function is inextricably linked to its pyrin domain–mediated and CARD-mediated polymerizations. These findings will support future studies into the molecular mechanisms controlling ASC solubility.     

Angiostrongylus cantonensis activates inflammasomes in meningoencephalitic BALB/c mice

Angiostrongylus cantonensis is a metastrongyloid nematode that causes eosinophilic meningoencephalitis in humans. A high infestation of A. cantonensis can cause permanent brain damage or even death. The inflammasome is an oligomeric molecular platform that can detect microbial pathogens and activate inflammatory cytokines. The recognition of larval surface antigens by pattern recognition receptors (PRRs) can cause oligomerization of the NOD-like receptor (NLR) or absent in melanoma 2 (AIM2) with the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) to form a caspase-1-activating scaffold. Activated caspase-1 converts pro-inflammatory cytokines into their mature, active forms. Helminths infection has been shown to activate NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasomes. In this study, we aimed to investigate the mechanism of inflammasome activation upon A. cantonensis infection in a mouse model. This study provides evidence that A. cantonensis infection can activate NLRP1B and NLRC4 inflammasomes and promote pyroptosis to cause meningoencephalitis.     

Promyelocytic Leukemia Protein Interacts with the Apoptosis-associated Speck-like Protein to Limit Inflammasome Activation

The apoptosis-associated speck-like protein containing a caspase-activating recruitment domain (ASC) is an essential component of several inflammasomes, multiprotein complexes that regulate caspase-1 activation and inflammation. We report here an interaction between promyelocytic leukemia protein (PML) and ASC. We observed enhanced formation of ASC dimers in PML-deficient macrophages. These macrophages also display enhanced levels of ASC in the cytosol. Furthermore, IL-1β production was markedly enhanced in these macrophages in response to both NLRP3 and AIM2 inflammasome activation and following bone marrow-derived macrophage infection with herpes simplex virus-1 (HSV-1) and Salmonella typhimurium. Collectively, our data indicate that PML limits ASC function, retaining ASC in the nucleus.     

The NLR adaptor ASC/PYCARD regulates DUSP10, mitogen-activated protein kinase (MAPK), and chemokine induction independent of the inflammasome

ASC/PYCARD is a common adaptor for a diverse set of inflammasomes that activate caspase-1, most prominently the NLR-based inflammasome. Mounting evidence indicates that ASC and these NLRs also elicit non-overlapping functions, but the molecular basis for this difference is unclear. To address this, we performed microarray and network analysis of ASC shRNA knockdown cells. In pathogen-infected cells, an ASC-dependent interactome is centered on the mitogen-activated protein kinase (MAPK) ERK and on multiple chemokines. ASC did not affect the expression of MAPK but affected its phosphorylation by pathogens and Toll-like receptor agonists via suppression of the dual-specificity phosphatase, DUSP10/MKP5. Chemokine induction, DUSP function, and MAPK phosphorylation were independent of caspase-1 and IL-1β. MAPK activation by pathogen was abrogated in Asc(-/-) but not Nlrp3(-/-), Nlrc4(-/-), or Casp1(-/-) macrophages. These results demonstrate a function for ASC that is distinct from the inflammasome in modulating MAPK activity and chemokine expression and further identify DUSP10 as a novel ASC target.     

Identification of an ASC oligomerization inhibitor for the treatment of inflammatory diseases

The ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) protein is an scaffold component of different inflammasomes, intracellular multiprotein platforms of the innate immune system that are activated in response to pathogens or intracellular damage. The formation of ASC specks, initiated by different inflammasome receptors, promotes the recruitment and activation of procaspase-1, thereby triggering pyroptotic inflammatory cell death and pro-inflammatory cytokine release. Here we describe MM01 as the first-in-class small-molecule inhibitor of ASC that interferes with ASC speck formation. MM01 inhibition of ASC oligomerization prevents activation of procaspase-1 in vitro and inhibits the activation of different ASC-dependent inflammasomes in cell lines and primary cultures. Furthermore, MM01 inhibits inflammation in vivo in a mouse model of inflammasome-induced peritonitis. Overall, we highlight MM01 as a novel broad-spectrum inflammasome inhibitor for the potential treatment of multifactorial diseases involving the dysregulation of multiple inflammasomes.Subject terms: Small molecules, Target validation     

AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC

Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2- and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome ‘specks'' in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.     

Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion

Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1β (IL-1β) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1β in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1β secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd^−/− cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd^−/− cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.