NLRP3炎性小体激活调控机制研究新进展

NLRP3炎性小体是一种胞内多蛋白复合体,主要由NOD样受体家族成员NLRP3、接头蛋白ASC以及前体半胱天冬酶1(pro-caspase-1)组成,该炎性小体可以通过激活caspase-1促进促炎因子白细胞介素-1 beta(IL-1β)和IL-18的分泌以及细胞焦亡(pyroptosis)的形成.NLRP3炎性小体的激活在很多自身免疫性及自身炎症性疾病中扮演着重要的角色,因此,深入探究NLRP3炎性小体激活的调控机制可为NLRP3炎性小体相关疾病的治疗提供更多新的思路.     

NOD样受体蛋白3炎症小体在急性肺损伤/急性呼吸窘迫综合征中的作用机制研究进展

急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)是由多种非心源性肺内外因素引起的急性进行性呼吸衰竭,发病核心为过度放大或失控的炎症反应,目前没有特效的治疗药物.NOD样受体蛋白3(nucleotide-binding domain (NOD)-like receptor protein 3,NLRP3)炎症小体是细胞受到刺激时形成的多蛋白复合体,活化后导致细胞焦亡及IL-1β、IL-18等产生,在多种感染性、炎症性疾病中起重要作用.引起肺损伤的多种因素均可导致NLRP3炎症小体形成、活化,有研究提示,与ALI/ARDS中的过度炎症反应有关.因而深入研究NLRP3炎症小体在ALI/ARDS中的作用,对于进一步阐明ALI/ARDS的发病机制有重要意义,甚至有望成为治疗ALI/ARDS的新靶点.     

柯萨奇病毒B3(CVB3)通过激活NADK2促进小鼠巨噬细胞NLRP3的活化

目的 研究在柯萨奇病毒B3(CVB3)刺激下，小鼠巨噬细胞中含pyrin结构域NOD样受体家族蛋白3(NLRP3)表达的变化及机制。方法 CVB3刺激RAW264.7细胞、小鼠原代(骨髓来源或腹腔)巨噬细胞以及感染NLRP3短发夹RNA(shRNA-NLRP3)慢病毒的RAW264.7细胞，实时荧光定量PCR检测NLRP3和白细胞介素1β(IL-1β)表达水平；ELISA检测细胞上清中IL-1β的含量；Western blot法检测NLRP3蛋白的变化，免疫共沉淀(Co-IP)法检测NLRP3相互作用蛋白的表达。结果CVB3刺激RAW264.7细胞、骨髓来源或腹腔巨噬细胞后，NLRP3和IL-1β表达明显升高；下调NLRP3后，IL-1β分泌明显减少；NLRP3抗体Co-IP所得复合物银染，组间差异蛋白经质谱分析鉴定为烟酰胺腺嘌呤二核苷酸激酶2(NADK2),证明CVB3诱导NADK2与NLRP3相互作用。结论 CVB3通过激活NADK2促进巨噬细胞NLRP3活化，增加炎症因子IL-1β表达和释放。     

NLRP3炎性小体在动脉粥样硬化发生发展中的作用

NLRP3炎性小体是一种包含胞内受体(主要是NOD样受体)、半胱天冬氨酸前体和凋亡相关斑点样蛋白的蛋白质复合体.该复合体最初只是被描述为一种影响感染和炎症过程的复合体,它的活化引起半胱天冬氨酶-1的激活并剪切加工底物白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-18(interleukin-18,IL-18),从而引起炎症反应;此外,炎性小体的激活过程对有氧糖酵解(瓦伯格效应,Warburg effect)有着重要的影响,这同样可以促进炎症的发生.随后的证据表明炎性小体的活化还影响很多代谢紊乱包括动脉粥样硬化(atherosclerosis, AS)、2型糖尿病、痛风和肥胖等.本综述将探讨AS与炎症、NLRP3炎性小体活化的关联性,以及瓦伯格效应如何关联炎症反应及炎性小体的激活.     

炎症环境下牙髓成纤维细胞中NOD样受体蛋白3炎症体相关分子表达的调节

背景:NOD样受体蛋白3炎症体是细胞抵御外来病原入侵的重要炎性因子,前期研究已证实其在牙髓成纤维细胞中有表达,然而炎症环境下牙髓成纤维细胞NOD样受体蛋白3炎症体相关分子的转录调节机制尚不清楚。目的:阐明炎症环境下牙髓成纤维细胞中NOD样受体蛋白3炎症体相关分子的转录调节机制。方法:选择第4代牙髓成纤维细胞,分6组:A组正常培养,不进行任何处理;B组加入脂多糖刺激6 h;C组加入Toll样受体4特异性抑制剂处理1 h,再加入Toll样受体4特异性抑制剂与脂多糖混合溶液处理6 h;D组加入髓样分化因子88特异性抑制剂处理1 h,再加入髓样分化因子88特异性抑制剂与脂多糖混合溶液处理6 h;E组加入核因子κB特异性抑制剂处理1 h,再加入核因子κB特异性抑制剂与脂多糖混合溶液处理6 h;F组加入髓样分化因子88特异性抑制剂阴性对照处理1 h,再加入髓样分化因子88特异性抑制剂阴性对照与脂多糖混合溶液处理6 h。采用RT-PCR检测NOD样受体蛋白3、Caspase-1与白细胞介素1βmRNA的表达,Western blot检测NOD样受体蛋白3与Caspase-1蛋白表达,ELISA法检测...     

NOD1和NOD2介导的信号通路及其抗病毒免疫应答研究进展

NOD1和NOD 2蛋白为胞浆内模式识别受体,其识别进入胞内的细菌胞壁及其降解产物,介导NF-κB和MAPKs信号途径,产生相关效应分子,介导了抗病原微生物免疫应答.近年来的最新研究发现,NOD1受体还通过ISGF3信号途径诱导产生1型干扰素,NOD2受体能识别ssRNA和病毒基因组ssRNA,通过MAVs信号途径激活IRF3,诱导产生1型干扰素,1型干扰素又可正向调控NOD1和NOD2功能性表达.NOD1和NOD2通过介导新的信号途径诱导产生大量的1型干扰素,并参与抗病毒固有免疫应答.因而,对NOD1和NOD2介导的抗病毒免疫应答新认识,将为防治病毒感染性疾病的研究提供新策略.     

α-微管蛋白乙酰化调控NLRP3炎症小体活化的研究进展

NLRP3炎症小体在固有免疫系统中发挥重要作用,介导caspase-1激活并促进炎症因子IL-1β分泌应答机体的病原微生物感染和细胞损伤,但异常活化的NLRP3炎症小体也会导致多种炎症性疾病的发生。NLRP3炎症小体的激活涉及多种细胞内分子和信号通路,导致细胞内离子流动变化、线粒体功能紊乱和溶酶体损伤等一系列事件发生,然而NLRP3炎症小体组装的详细调控机制尚不清楚。本综述着重介绍α-微管蛋白的乙酰化介导线粒体通过动力蛋白转运到内质网附近,从而促进线粒体上的ASC与内质网上的NLRP3接触,诱导NLRP3炎症小体组装。     

NOD1/2介导的信号通路及其抗病原微生物免疫应答的研究进展

NOD1/2蛋白为胞质内的模式识别受体,识别进入胞内的细菌胞壁及其降解产物,介导NF-κB和丝裂原活化蛋白激酶(MAPK)信号途径。NOD1受体还通过干扰素刺激基因因子3(ISGF3)信号途径诱导产生1型干扰素,NOD2受体能识别ssRNA和病毒基因组ssRNA,通过线粒体抗病毒信号蛋白(MAV)信号途径而激活干扰素调节因子3(IRF3)。它们分别参与了抗细菌、抗病毒、抗寄生虫等病原微生物免疫应答。对NOD1和NOD2受体的进一步认识,为研究相关病原感染和慢性炎症性疾病的防治措施提供了新的思路。     

NOD1、NOD2:两个重要的天然免疫胞内识别受体

机体对微生物入侵的免疫炎症应答过程中模式识别受体(patternrecognitionreceptors,PRRs)是启动机体天然免疫应答机制的关键,主要在获得性免疫系统被活化之前发挥抗感染作用。NOD1和NOD2这两个蛋白分子作为一种新的胞内识别受体参与了细胞凋亡和核因子NF-κB的活化,并与一些炎症性疾病密切相关,在天然免疫中发挥了重要的作用。     

含pyrin结构域NOD样受体家族3(NLRP3)炎性体抑制剂及其作用机制研究进展

含pyrin结构域NOD样受体家族3(NLRP3)炎性体是由NLRP3、凋亡相关斑点样蛋白(ASC)及胱天蛋白酶1(caspase-1)组成的蛋白复合体,主要介导白细胞介素1β(IL-1β)和IL-18的前体成熟参与炎症反应。NLRP3炎性体的异常活化与代谢紊乱、神经退行性疾病、自身免疫性疾病等炎症性疾病的发生发展关系密切。寻找并开发NLRP3炎性体的有效抑制剂成为治疗炎症性疾病的新策略。我们总结了已报道的在NLRP3炎性体活化的起始阶段和激活阶段发挥作用的抑制剂及其作用机制,期望为开发以NLRP3炎性体为靶点的抗炎药物提供新思路。     

The crosstalk between TLR2 and NOD2 in Aspergillus fumigatus keratitis

Innate immunity is considered to be critical in the pathogenesis of fungal keratitis. Pattern recognition receptors (PRRs) recognize conserved microbial structures called pathogen-associated molecular patterns (PAMPS), thereby initiating the innate immunity. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (leucine-rich repeat-containing receptors, NLRs) are two major PRR families. The crosstalk between TLR2 and NOD2 is not completely understood, and their interrelationship in Aspergillus fumigates keratitis is still unclear. To our surprise, we found herein that NOD2 and TLR2 were increased by A. fumigatus conidia in immortalized human corneal epithelial cells (HCECs). In addition, NOD2 expression was up-regulated by its agonist muramyl dipeptide (MDP), along with receptor interacting protein 2 (RIP2), nuclear factor κB (NFκB)-p65, inhibitor of NFκB (IκB)-α, and multiple inflammatory cytokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor α (TNF-α). Interestingly, zymosan, a TLR2 agonist, promoted the expression of NOD2 and RIP2 in a TLR2-dependent manner. Furthermore, we demonstrated that the increased expression of NOD2 and RIP2 caused by A. fumigatus conidia occurred in part through a TLR2-dependent pathway. However, zymosan pretreatment decreased NOD2 and RIP2 expression along with the MDP induced secretion of inflammatory cytokines in HCECs. In agreement, NOD2 knockdown by small interfering RNA (siRNA) reduced the release of IL-6, IL-8 and TNF-α induced by A. fumigatus conidia. These findings suggest the existence of complex interactions between TLR2 and NOD2 in HCECs inflammatory response against A. fumigatus infection.     

Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1)

Nucleotide-binding oligomerization domain 1 (NOD1) is an imperative cytoplasmic pattern recognition receptor (PRR) and considered as a key member of the NOD-like receptor (NLR) family which plays a critical role in innate immunity through sensing microbial components derived from bacterial peptidoglycan. In the current study, the full-length of duck NOD1 (duNOD1) cDNA from duck embryo fibroblasts (DEFs) was cloned. Multiple sequence alignment and phylogenetic analysis demonstrated that duNOD1 exhibited a strong evolutionary relationship with chicken and rock pigeon NOD1. Tissue-specific expression analysis showed that duNOD1 was widely distributed in various organs, with the highest expression observed in the liver. Furthermore, duNOD1 overexpression induced NF-κB activation in DEFs and the CARD domain is crucial for duNOD1-mediated NF-κB activation. In addition, silencing the duNOD1 decreased the activity of NF-κB in DEFs stimulated by iE-DAP. Overexpression of duNOD1 significantly increased the expression of TNF-α, IL-6, and RANTES in DEFs. These findings highlight the crucial role of duNOD1 as an intracellular sensor in duck innate immune system.     

Increased inflammatory responsiveness of peripheral blood mononuclear cells (PBMCs) to in vitro NOD2 ligand stimulation in patients with ankylosing spondylitis

Abstract

Background: Ankylosing spondylitis (AS) is a common debilitating rheumatic disease in which the innate immune components especially the Interleukin (IL)-23/IL-17 axis related genes play important role in its pathogenesis. Nucleotide binding oligomerization domain-containing protein (NOD)2, as an innate receptor, is critical for IL-23 production in cells. Therefore, we aimed to stimulate NOD2 signaling and study its effects on cytokine production in peripheral blood mononuclear cells (PBMC) of these patients.

Methods: PBMCs from 18 patients with active AS and 18 healthy individuals were separated by Ficoll-Hypaque density gradient centrifugation and cultured in the presence of muramyl dipeptide (MDP), as NOD2 ligand. Quantitative expression analysis of NOD1, NOD2, RIPK2, SLC15A4, NLRP1, NLRP3, IL23A, IL17A, IL1B, and TNFA genes was performed using Real-time polymerase chain reaction (PCR). Finally, protein changes of IL23A and IL17A expression were validated using enzyme linked immunosorbent assay (ELISA).

Results: Apart from NOD1 that tend to be downregulated in the controls, all the selected genes showed overexpression in response to MDP in cells from the studied groups. Except RIPK2, all the genes had higher expression changes upon MDP stimulation in the AS population. Overexpression of IL23A and IL17A were confirmed at protein levels using ELISA. The strong positive correlation between NLRP3 and NOD2 was decreased after stimulation but new correlations between NLRP3 and IL1B, RIPK2 and SLC15A4 were observed after treatment.

Conclusions: This study indicated that AS PBMCs were hyper-responsive to MDP stimulation. This observation implies an important role of NOD2 in the pathogenesis of inflammatory diseases including AS.     

NOD蛋白与肺部疾病

天然免疫系统通过识别模式识别受体(patternrecognition receptors,PRR)介导的病原相关分子模式(pathogen-associated molecular patterns,PAMP)是抗微生物宿主防御的第1道防线,在机体感染早期发挥重要的保护性作用。在近来的研究中证实除Toll样受体(toll-like receptors,TLRs)     

固有免疫中TLR和NOD的研究现状

固有免疫是机体防御感染性疾病的第一道防线.随着模式识别理论的提出和Toll样受体(TLR),细胞质内的核苷酸结合寡聚化结构城(NOD)的发现,为了解机体识别细菌和炎症防御机制方面开阔了视野.了解TLR和NOD的结构、表达、分布和信号途径,相互影响和与疾病的关系以及研究TLR和NOD的意义很重要.     

Normal responses to specific NOD1-activating peptidoglycan agonists in the presence of the NOD2 frameshift and other mutations in Crohn's disease

Both NOD2/CARD15 alleles are mutated in approximately 10% of Crohn's disease patients, causing loss of functional responses to low-dose muropeptide agonists. We hypothesized that NOD2 mutations may also impair NOD1/CARD4 responses, supported by data suggesting NOD2 1007fs/1007fs patients had reduced responses to a putative NOD1 agonist, diaminopimelic acid-containing muramyl tripeptide (M-TriDAP). We measured peripheral blood mononuclear cell (n = 8 NOD2 wild type, n = 4 1007fs/1007fs, n = 6 702Trp/1007fs, n = 5 702Trp/702Trp, n = 3 908Arg/1007fs) responses to NOD1 agonists alone (IL-8/TNF-alpha), and agonist enhancement of lipopolysaccharide (LPS) responses (IL-1beta). Significant responses were seen with M-TriDAP at 10 nM (as with NOD2 agonists), but only at > or =100 nM with FK565/TriDAP. M-TriDAP induced IL-8/TNF-alpha secretion, and enhancement of LPS IL-1beta responses was significantly reduced between NOD2 double mutation carriers versus healthy controls, whereas there was no difference with FK565 or TriDAP stimulation, or between 1007fs/1007fs cells and other genotypes. M-TriDAP contains both NOD1 (gamma-D-Glu-mesoDAP) and NOD2 (MurNAc-L-Ala-D-Glu) minimal structures whereas FK565/TriDAP contain only NOD1 activating structures. M-TriDAP has dual NOD1/NOD2 agonist activity in primary cells, possibly due to different intracellular peptidoglycan processing compared to the HEK293 cell system typically used for agonist specificity studies. Responses to specific NOD1 agonists are unaffected by NOD2 genotype, suggesting independent action of the NOD1 and NOD2 pathways.     

Absence of NOD2 receptor predisposes to intestinal inflammation by a deregulation in the immune response in hosts that are unable to control gut dysbiosis

Mutations in NOD2 predisposes to Inflammatory Bowel Diseases. Therefore, we evaluated the role of this innate receptor in the modulation of immunity in face of host microbiota changes. NOD2^?/? mice presented higher susceptibility to experimental colitis than WT, with increased CD4 and CD8 T lymphocytes in the spleen. NOD2 deficiency also led to reduced Th17-related cytokines in the colon, with overall augmented IFN-γ in the gut and spleen. Nonetheless, there was increased frequency of CD4⁺IL-4⁺ cells in the mesenteric lymph nodes besides elevated CTLA-4 and FoxP3 regulatory markers in the spleen of NOD2^-/- mice, although it did not result in more efficient control of gut inflammation. Indeed, these animals also had augmented IL-1β and IL-5 in the peritoneum, indicating that this receptor may be important to control bacteria translocation too. Microbiota exchanging between cohoused WT and NOD2^?/? mice led to colitis worsening in the absence of the receptor, while antibiotic therapy in WT mice abrogated this effect. Then, not only the genetic mutation confers increased susceptibility to inflammation, but it is also influenced by the microbiota harbored by the host. Finally, NOD2^-/- mice are more prone to intestinal inflammation due to deregulated immune response and increased susceptibility to colitogenic bacteria.     

NOD1 and NOD2 regulation of pulmonary innate immunity to Legionella pneumophila

The role of nucleotide‐binding oligomerization domain‐1 (NOD1) and nucleotide‐binding oligomerization domain‐2 (NOD2), cytoplasmic receptors which detect bacterial cell wall molecules, in pulmonary innate immune responses is poorly understood. We determined that both NOD1 and NOD2 detect heat‐killed Legionella and stimulate NF‐κb and IFN‐β promoter activity using an in vitro luciferase reporter system. We next infected NOD1‐ and NOD2‐deficient animals with aerosolized Legionella pneumophila. At 3 days post infection, Nod1^?/? mice had impaired bacterial clearance compared to WT controls. In addition, at 4 h and 24 h, Nod1^?/? mice had impaired neutrophil recruitment to the alveolar space. In contrast, increased lung neutrophils were seen in the Nod2^?/? animals at 24 h. Analysis of cytokine production at 4 h post infection revealed a significant decrease in proinflammatory cytokines in the Nod1^?/? animals when compared to WT animals. In contrast, increased 4‐h proinflammatory cytokines were seen in the Nod2^?/? animals. Furthermore, the lungs of both Nod1^?/? and Nod2^?/? mice had significantly increased pro‐inflammatory cytokine levels at 24 h, suggesting possible suppressive roles for later stages of infection. Together, our data suggest that although both NOD1 and NOD2 can detect Legionella, these receptors modulate the in vivo pulmonary immune response differently.