UCP2-induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis

Cellular lipid metabolism has been linked to immune responses; however, the precise mechanisms by which de novo fatty acid synthesis can regulate inflammatory responses remain unclear. The NLRP3 inflammasome serves as a platform for caspase-1–dependent maturation and secretion of proinflammatory cytokines. Here, we demonstrated that the mitochondrial uncoupling protein-2 (UCP2) regulates NLRP3-mediated caspase-1 activation through the stimulation of lipid synthesis in macrophages. UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis. Moreover, UCP2 expression was increased in human sepsis. Consistently, UCP2-deficient mice displayed impaired lipid synthesis and decreased production of IL-1β and IL-18 in response to LPS challenge. In macrophages, UCP2 deficiency suppressed NLRP3-mediated caspase-1 activation and NLRP3 expression associated with inhibition of lipid synthesis. In UCP2-deficient macrophages, inhibition of lipid synthesis resulted from the downregulation of fatty acid synthase (FASN), a key regulator of fatty acid synthesis. FASN inhibition by shRNA and treatment with the chemical inhibitors C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation and inhibited NLRP3 and pro–IL-1β gene expression in macrophages. In conclusion, our results suggest that UCP2 regulates the NLRP3 inflammasome by inducing the lipid synthesis pathway in macrophages. These results identify UCP2 as a potential therapeutic target in inflammatory diseases such as sepsis.     

NLRP3 inflammasome induces CD4+ T cell loss in chronically HIV-1–infected patients

Chronic HIV-1 infection is generally characterized by progressive CD4⁺ T cell depletion due to direct and bystander death that is closely associated with persistent HIV-1 replication and an inflammatory environment in vivo. The mechanisms underlying the loss of CD4⁺ T cells in patients with chronic HIV-1 infection are incompletely understood. In this study, we simultaneously monitored caspase-1 and caspase-3 activation in circulating CD4⁺ T cells, which revealed that pyroptotic and apoptotic CD4⁺ T cells are distinct cell populations with different phenotypic characteristics. Levels of pyroptosis and apoptosis in CD4⁺ T cells were significantly elevated during chronic HIV-1 infection, and decreased following effective antiretroviral therapy. Notably, the occurrence of pyroptosis was further confirmed by elevated gasdermin D activation in lymph nodes of HIV-1–infected individuals. Mechanistically, caspase-1 activation closely correlated with the inflammatory marker expression and was shown to occur through NLRP3 inflammasome activation driven by virus-dependent and/or -independent ROS production, while caspase-3 activation in CD4⁺ T cells was more closely related to T cell activation status. Hence, our findings show that NLRP3-dependent pyroptosis plays an essential role in CD4⁺ T cell loss in HIV-1–infected patients and implicate pyroptosis signaling as a target for anti–HIV-1 treatment.     

ASC deglutathionylation is a checkpoint for NLRP3 inflammasome activation

Activation of NLRP3 inflammasome is precisely controlled to avoid excessive activation. Although multiple molecules regulating NLRP3 inflammasome activation have been revealed, the checkpoints governing NLRP3 inflammasome activation remain elusive. Here, we show that activation of NLRP3 inflammasome is governed by GSTO1-promoted ASC deglutathionylation in macrophages. Glutathionylation of ASC inhibits ASC oligomerization and thus represses activation of NLRP3 inflammasome in macrophages, unless GSTO1 binds ASC and deglutathionylates ASC at ER, under control of mitochondrial ROS and triacylglyceride synthesis. In macrophages expressing ASC^C171A, a mutant ASC without glutathionylation site, activation of NLRP3 inflammasome is GSTO1 independent, ROS independent, and signal 2 less dependent. Moreover, Asc^C171A mice exhibit NLRP3-dependent hyperinflammation in vivo. Our results demonstrate that glutathionylation of ASC represses NLRP3 inflammasome activation, and GSTO1-promoted ASC deglutathionylation at ER, under metabolic control, is a checkpoint for activating NLRP3 inflammasome.     

NLRP3炎症小体与心肌缺血再灌注损伤的研究进展

及时再通阻塞的冠状动脉是降低急性心肌梗死患者死亡率的关键,但实现再灌注的同时可能对缺血的心肌造成二次损伤,即心肌缺血再灌注损伤(myocardial ischemia reperfusion injury, MIRI)。核苷酸结合寡聚化结构域样受体蛋白3(nucleotide binding oligomerization domain-like receptor protein 3,NLRP3)炎症小体通过促进心肌细胞焦亡、级联放大炎症反应和破坏心肌血管内皮细胞,参与了MIRI的整个过程,引起了临床广泛关注。目前,以NLRP3炎症小体及其调节因子为药物靶点的相关研究正在如火如荼地开展,有望为MIRI的预防和治疗提供新的思路。     

Divergence of IL-1, IL-18, and cell death in NLRP3 inflammasomopathies

The inflammasome is a cytoplasmic multiprotein complex that promotes proinflammatory cytokine maturation in response to host- and pathogen-derived signals. Missense mutations in cryopyrin (NLRP3) result in a hyperactive inflammasome that drives overproduction of the proinflammatory cytokines IL-1β and IL-18, leading to the cryopyrin-associated periodic syndromes (CAPS) disease spectrum. Mouse lines harboring CAPS-associated mutations in Nlrp3 have elevated levels of IL-1β and IL-18 and closely mimic human disease. To examine the role of inflammasome-driven IL-18 in murine CAPS, we bred Nlrp3 mutations onto an Il18r-null background. Deletion of Il18r resulted in partial phenotypic rescue that abolished skin and visceral disease in young mice and normalized serum cytokines to a greater extent than breeding to Il1r-null mice. Significant systemic inflammation developed in aging Nlrp3 mutant Il18r-null mice, indicating that IL-1 and IL-18 drive pathology at different stages of the disease process. Ongoing inflammation in double-cytokine knockout CAPS mice implicated a role for caspase-1–mediated pyroptosis and confirmed that CAPS is inflammasome dependent. Our results have important implications for patients with CAPS and residual disease, emphasizing the need to explore other NLRP3-mediated pathways and the potential for inflammasome-targeted therapy.     

TRPC6靶向miR-214负调控Caspase-1表达以改善肾缺血再灌注损伤的机制研究

目的探讨TRPC6对肾缺血再灌注损伤（RIRI）的缓解作用及潜在作用机制。 方法建立氧糖剥离再灌注（OGD/R HK-2）模型细胞和缺血再灌注（I/R）模型小鼠后,采用ELISA方法检测白介素（IL）-1β和IL-18水平,通过RT-qPCR、Western blot和免疫组化检测caspase-1表达,并利用CCK-8和流式细胞仪检测细胞活力和焦亡情况。此外,通过双荧光素酶报告基因检测TRPC6与miR-214之间的相互作用。 结果TRPC6在构建的OGD/R HK-2细胞和I/R模型小鼠中表达明显升高。同时,下调caspase-1可增强OGD/R HK-2细胞的活力,抑制炎症水平和细胞焦亡。TRPC6高表达能减轻I/R模型小鼠肾损伤并且下调caspase-1水平。从机制探究,我们发现TRPC6可通过调控miR-214下调caspase-1,并改善RIRI。 结论TRPC6通过靶向调控miR-214下调caspase-1缓解RIRI,TRPC6/miR-214/caspase-1的作用通路可能为RIRI的治疗提供关键线索。     

Atorvastatin suppresses NLRP3 inflammasome activation via TLR4/MyD88/NF-κB signaling in PMA-stimulated THP-1 monocytes

AimsIncreasing evidence shows that NLRP3 inflammasome is closely associated with the progression of atherosclerosis. The purpose of the present study was to evaluate the effects of atorvastatin on NLRP3 inflammasome in PMA-stimulated THP-1 cells and explore its underlying mechanism.MethodsHuman monocytic THP-1 cells were pretreated with atorvastatin for 1 h and then induced by PMA for 48 h. Total protein was collected for real-time PCR and Western blot analysis. Cytokine IL-1β release was detected by ELISA assay. And the NF-κB p65 translocation was detected by cellular NF-κB translocation kit.ResultsIt was shown that atorvastatin significantly reduced the expression of NLRP3, the cleavage of caspase-1 and IL-1β in PMA-induced THP-1 cells. Moreover, Bay (a NF-κB inhibitor) treatment greatly suppressed the expression of NLRP3, the cleavage of caspase-1 and IL-1β in PMA-induced THP-1 cells, suggesting that the activation of NF-κB pathway takes part in regulating the expression of NLRP3 inflammasome. In addition, atorvastatin markedly inhibited the up-regulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and the activation of nuclear factor kappa b (NF-κB) in PMA-stimulated THP-1 cells.ConclusionsAtorvastatin exerts an anti-inflammatory effect by inhibiting NLRP3 inflammasome through suppressing TLR4/MyD88/NF-κB pathway in PMA-induced THP-1 monocytes.     

Uropathogenic Escherichia coli strain CFT073 disrupts NLRP3 inflammasome activation

Successful bacterial pathogens produce an array of virulence factors that allow subversion of the immune system and persistence within the host. For example, uropathogenic Escherichia coli strains, such as CFT073, express Toll/IL-1 receptor–containing (TIR-containing) protein C (TcpC), which impairs TLR signaling, thereby suppressing innate immunity in the urinary tract and enhancing persistence in the kidneys. Here, we have reported that TcpC also reduces secretion of IL-1β by directly interacting with the NACHT leucin-rich repeat PYD protein 3 (NLRP3) inflammasome, which is crucial for recognition of pathogens within the cytosol. At a low MOI, IL-1β secretion was minimal in CFT073-infected macrophages; however, IL-1β release was markedly increased in macrophages infected with CFT073 lacking tcpC. Induction of IL-1β secretion by CFT073 and tcpC–deficient CFT073 required the NLRP3 inflammasome. TcpC attenuated activation of the NLRP3 inflammasome by binding both NLRP3 and caspase-1 and thereby preventing processing and activation of caspase-1. Moreover, in a murine urinary tract infection model, CFT073 infection rapidly induced expression of the NLRP3 inflammasome in the bladder mucosa; however, the presence of TcpC in WT CFT073 reduced IL-1β levels in the urine of infected mice. Together, these findings illustrate how uropathogenic E. coli use the multifunctional virulence factor TcpC to attenuate innate immune responses in the urinary tract.     

NLRP3炎症小体作为肾脏疾病潜在治疗靶标的研究进展

急性肾损伤(acute kidney injury,AKI)和慢性肾脏病(chronic kidney disease, CKD)是肾脏常见病理状态。最近,大量证据表明肾脏疾病与炎症小体之间存在关联,尤其是核苷酸结合寡聚化结构域(nucleotide-binding oligomerization domain, NOD)样受体家族含pyrin结构域蛋白3(NOD-like receptor family pyrin domain-containing protein 3,NLRP3)炎症小体。 经典的NLRP3炎症小体是由NOD样受体(NOD-like receptors, NLRs)、凋亡相关的斑点样蛋白(apoptosis-associated speck-like protein containing a CARD, ASC)和半胱氨酸天冬氨酸蛋白酶1 (Caspase-1)组成的多蛋白复合物。大量的研究探索了NLRP3炎症小体在肾脏疾病发生发展中的机制以及在肾脏疾病中针对NLRP3炎症小体的治疗。本文综述了近几年NLRP3炎症小体在肾脏疾病中的作用的最新发现以及以NLRP3炎症小体为靶标的最新治疗策略。     

Helicobacter urease–induced activation of the TLR2/NLRP3/IL-18 axis protects against asthma

Inflammasome activation and caspase-1–dependent (CASP1-dependent) processing and secretion of IL-1β and IL-18 are critical events at the interface of the bacterial pathogen Helicobacter pylori with its host. Whereas IL-1β promotes Th1 and Th17 responses and gastric immunopathology, IL-18 is required for Treg differentiation, H. pylori persistence, and protection against allergic asthma, which is a hallmark of H. pylori–infected mice and humans. Here, we show that inflammasome activation in DCs requires the cytoplasmic sensor NLRP3 as well as induction of TLR2 signaling by H. pylori. Screening of an H. pylori transposon mutant library revealed that pro–IL-1β expression is induced by LPS from H. pylori, while the urease B subunit (UreB) is required for NLRP3 inflammasome licensing. UreB activates the TLR2-dependent expression of NLRP3, which represents a rate-limiting step in NLRP3 inflammasome assembly. ureB-deficient H. pylori mutants were defective for CASP1 activation in murine bone marrow–derived DCs, splenic DCs, and human blood-derived DCs. Despite colonizing the murine stomach, ureB mutants failed to induce IL-1β and IL-18 secretion and to promote Treg responses. Unlike WT H. pylori, ureB mutants were incapable of conferring protection against allergen-induced asthma in murine models. Together, these results indicate that the TLR2/NLRP3/CASP1/IL-18 axis is critical to H. pylori–specific immune regulation.     

Inflammasome recognition of influenza virus is essential for adaptive immune responses

Influenza virus infection is recognized by the innate immune system through Toll like receptor (TLR) 7 and retinoic acid inducible gene I. These two recognition pathways lead to the activation of type I interferons and resistance to infection. In addition, TLR signals are required for the CD4 T cell and IgG2a, but not cytotoxic T lymphocyte, responses to influenza virus infection. In contrast, the role of NOD-like receptors (NLRs) in viral recognition and induction of adaptive immunity to influenza virus is unknown. We demonstrate that respiratory infection with influenza virus results in the activation of NLR inflammasomes in the lung. Although NLRP3 was required for inflammasome activation in certain cell types, CD4 and CD8 T cell responses, as well as mucosal IgA secretion and systemic IgG responses, required ASC and caspase-1 but not NLRP3. Consequently, ASC, caspase-1, and IL-1R, but not NLRP3, were required for protective immunity against flu challenge. Furthermore, we show that caspase-1 inflammasome activation in the hematopoietic, but not stromal, compartment was required to induce protective antiviral immunity. These results demonstrate that in addition to the TLR pathways, ASC inflammasomes play a central role in adaptive immunity to influenza virus.     

肌萎缩侧索硬化蛋白激活小胶质细胞NLRP3炎性体

小胶质细胞NLRP3炎症小体激活正在成为神经退行性变期间神经炎症的关键因素。β-淀粉样蛋白和α-突触核蛋白等致病蛋白的聚集体可触发小胶质细胞NLRP3激活,并导致半胱氨酸天冬氨酸酶激活和白介素-1β分泌。半胱氨酸天冬氨酸酶和白介素-1β均促进肌萎缩侧索硬化(ALS)小鼠SOD1G93A模型的疾病进展,这提示小胶质细胞NLRP3在该进程中起作用。然而,先前的研究表明SOD1G93A模型小鼠的小胶质细胞并不表达NLRP3,并且SOD1G93A蛋白在小胶质细胞中产生白介素-1β不依赖于NLRP3。本研究展示了使用Nlrp3-GFP基因敲入小鼠,在SOD1G93A小鼠中小胶质细胞表达NLRP3。结果表明,聚集和可溶性SOD1G93A均以剂量和时间依赖性方式激活小鼠原代小胶质细胞中的炎性体,导致半胱氨酸天冬氨酸酶和白介素-1β裂解、ASC斑点形成和白介素-1β分泌。重要的是,SOD1G93A不能诱导缺乏Nlrp3的小胶质细胞或用特异性NLRP3抑制剂MCC950预处理的小胶质细胞分泌白介素-1β,这证实NLRP3是介导SOD1诱导的小胶质细胞白介素-1β分泌的关键炎性体复合物。在TDP-43Q331K ALS小鼠模型中也观察到小胶质细胞NLRP3上调,并且TDP-43野生型和突变蛋白也能以NLRP3依赖性方式激活小胶质细胞炎症小体。从机制上,本研究确定活性氧簇和ATP的产生是SOD1G93A介导的NLRP3激活所需的关键事件。总之,本研究的数据表明ALS小胶质细胞表达NLRP3,病理性ALS蛋白激活了小胶质细胞NLRP3炎性体。因此,抑制NLRP3可能是阻止小胶质细胞神经炎症和ALS疾病进展的潜在治疗方法。     

Protective role of retinoid X receptor in H9c2 cardiomyocytes from hypoxia/reoxygenation injury in rats

BACKGROUND:

Retinoid X receptor (RXR) plays a central role in the regulation of intracellular receptor signaling pathways. The activation of RXR has protective effect on H₂O₂-induced apoptosis of H9c2 ventricular cells in rats. But the protective effect and mechanism of activating RXR in cardiomyocytes against hypoxia/reoxygenation (H/R)-induced oxidative iniury are still unclear.

METHODS:

The model of H/R injury was established through hypoxia for 2 hours and reoxygenation for 4 hours in H9c2 cardiomyocytes of rats. 9-cis-retinoic acid (9-cis RA) was obtained as an RXR agonist, and HX531 as an RXR antagonist. Cultured cardiomyocytes were randomly divided into four groups: sham group, H/R group, H/R+9-cis RA -pretreated group (100 nmol/L 9-cis RA), and H/R+9-cis RA+HX531-pretreated group (2.5 μmol/L HX531). The cell viability was measured by MTT, apoptosis rate of cardiomyocytes by flow cytometry analysis, and mitochondrial membrane potential (ΔΨm) by JC-1 fluorescent probe, and protein expressions of Bcl-2, Bax and cleaved caspase-9 with Western blotting. All measurement data were expressed as mean±standard deviation, and analyzed using one-way ANOVA and the Dunnett test. Differences were considered significant when P was <0.05.

RESULTS:

Pretreatment with RXR agonist enhanced cell viability, reduced apoptosis ratio, and stabled ΔΨm. Dot blotting experiments showed that under H/R stress conditions, Bcl-2 protein level decreased, while Bax and cleaved caspase-9 were increased. 9-cis RA administration before H/R stress prevented these effects, but the protective effects of activating RXR on cardiomyocytes against H/R induced oxidative injury were abolished when pretreated with RXR pan-antagonist HX531.

CONCLUSION:

The activation of RXR has protective effects against H/R injury in H9c2 cardiomyocytes of rats through attenuating signaling pathway of mitochondria apoptosis.KEY WORDS: Retinoid X receptor, Cardiomyocytes, Apoptosis, Mitochondria, Hypoxia reoxygenation     

原钙黏附蛋白7在脂多糖诱导的肾小管上皮细胞焦亡中的作用

目的 探讨原钙黏附蛋白7（PCDH7）是否参与脂多糖（LPS）刺激引起的肾小管上皮细胞（HK-2）焦亡过程。方法 构建LPS刺激HK-2损伤模型,使用RT-PCR和蛋白免疫印迹法检测细胞中PCDH7、NLR家族Pyrin域蛋白3（NLRP3）、caspase-1、和IL-1β蛋白的表达变化。MTT法测定细胞活力,TUNEL法确定细胞焦亡率,ELISA法检测细胞分泌TNF-α、IL-6水平,全自动生化分析仪检测培养液中乳酸脱氢酶（LDH）含量。结果 与Control组比较,LPS组细胞生存率下降,细胞凋亡率升高,炎症因子TNF-α和IL-6水平升高,细胞损伤指标LDH含量明显升高,PCDH7的蛋白和mRNA表达明显下调,细胞焦亡指标NLRP3、caspase-1、IL-1β蛋白和mRNA表达明显上调（P均< 0.05）。结论 LPS通过下调PCDH7的表达,促进肾小管上皮细胞发生明显细胞焦亡损伤。     

Resveratrol ameliorates LPS-induced acute lung injury via NLRP3 inflammasome modulation

NLRP3 inflammasome plays a pivotal role in the development of acute lung injury (ALI), accelerating IL-1β and IL-18 release and inducing lung inflammation. Resveratrol, a natural phytoalexin, has anti-inflammatory properties via inhibition of oxidation, leukocyte priming, and production of inflammatory mediators. In this study, we aimed to investigate the effect of resveratrol on NLRP3 inflammasome in lipopolysaccharide-induced ALI. Mice were intratracheally instilled with 3 mg/kg lipopolysaccharide (LPS) to induce ALI. Resveratrol treatment alleviated the LPS-induced lung pathological damage, lung edema and neutrophil infiltration. In addition, resveratrol reversed the LPS-mediated elevation of IL-1β and IL-18 level in the BAL fluids. In lung tissue, resveratrol also inhibited the LPS-induced NLRP3, ASC, caspase-1 mRNA and protein expression, and NLRP3 inflammasome activation. Moreover, resveratrol administration not only suppressed the NF-κB p65 nuclear translocation, NF-κB activity and ROS production in the LPS-treated mice, but also inhibited the LPS-induced thioredoxin-interacting protein (TXNIP) protein expression and interaction of TXNIP-NLRP3 in lung tissue. Meanwhile, resveratrol obviously induced SIRT1 mRNA and protein expression in the LPS-challenged mice. Taken together, our study suggests that resveratrol protects against LPS-induced lung injury by NLRP3 inflammasome inhibition. These findings further suggest that resveratrol may be of great value in the treatment of ALI and a potential and an effective pharmacological agent for inflammasome-relevant diseases.     

Advances toward structure-based drug discovery for inflammasome targets

Inflammasome proteins play an important role in many diseases of high unmet need, making them attractive drug targets. However, drug discovery for inflammasome proteins has been challenging in part due to the difficulty in solving high-resolution structures using cryo-EM or crystallography. Recent advances in the structural biology of NLRP3 and NLRP1 have provided the first set of data that proves a promise for structure-based drug design for this important family of targets.

The innate immune system represents the first line of defense to invading foreign pathogens, being essential for efficient detection and clearance of the infection. It also plays a critical role in maintaining healthy tissue homeostasis, by detecting and removing damaged or dying cells, ensuring that organs maintain normal human physiological function throughout life. Following removal of pathogens or damaged cells, down-regulation of the innate immunity system is critical to ensure that unnecessary inflammation and tissue damage do not persist.Many proteins and pathways of the innate immune system have been validated as compelling drug targets by virtue of their causal role in monogenic auto-inflammatory diseases (Harapas et al., 2018), their activation and up-regulation in many common inflammatory (Li and Wu, 2021) and neurodegenerative diseases (Heneka et al., 2018), and the extensive validation in preclinical models of diseases. Inflammasome proteins compose one key group of innate immune mediators of diseases (Lamkanfi and Dixit, 2012), which mediate the activation of IL-1β, IL-18, and Gasdermin D and the promotion of a pro-inflammatory response and disease. Thus, inflammasome proteins represent compelling drug targets.Challenges of targeting inflammasome pathwaysWhile inflammasomes represent highly compelling drug targets, the pursuit of drug discovery for many of these targets has been challenging due to the absence of high-resolution protein structures. The development of highly potent and selective small-molecule drugs requires the optimization of the precise molecular interactions between drug candidates and the intended protein targets. While optimization of small-molecule drugs can be pursued empirically, by engineering random modifications of small molecules in an effort to find those compounds that will have improved affinity and selectivity, it is far more powerful to design small molecules based on an intimate and precise understanding of how the molecules interact within a binding pocket on the protein. This understanding enables rational design of small molecules, which often leads to best-in-class designs and to expedited processes relative to using empirical approaches.Recent advances in the structural biology of inflammasomesDue to the heterogeneous, highly aggregated nature of inflammasome proteins, it has been difficult to generate high-resolution structures that can enable structure-based drug design (SBDD). In recent years, technological improvements in cryo-electron microscopy (cryo-EM) including direct electron detectors and computer programs are revolutionizing its utility for high-resolution structure determination. Together with x-ray crystallography, cryo-EM has generated excitement recently in structure analysis of inflammasome proteins and complexes.NLRP3Of particular interest are the recent advances in the structural biology of NLRP3, as it represents a most compelling drug target and has elicited significant drug discovery and development efforts in academia as well as pharmaceutical and biotechnology companies. NLRP3 can be activated by numerous self-derived damage-associated molecular patterns (Swanson et al., 2019) and is a critical regulator of the innate immune response in many diseases. Due to the difficulty in expressing the monomeric form of NLRP3 and the challenge of solving high-resolution protein structures, much of the drug discovery effort, however, has been based on empirical approaches resulting in limited structural diversity of the chemical matter (El-Sharkawy et al., 2020).The first cryo-EM structure of NLRP3 was reported in 2019 as a complex with NEK7, the Ser/Thr kinase that acts as an essential scaffolding protein for NLRP3 activation (Sharif et al., 2019; see figure, panels A and B). This was accomplished with protein engineering that allowed for the expression of a stable form of monomeric NLRP3. While the resolution of the structure was not high enough to enable structure-based drug design, these data provided insights into the structural mechanism of NLRP3 activation. In an inactive state, NLRP3 adopts a closed conformation in its NACHT domain, preventing formation of an inflammasome assembly, and upon activation it may open up to allow for the formation of active NLRP3 inflammasome oligomers and the subsequent recruitment and activation of caspase-1. This cryo-EM study also suggested that not only the conformational change in NLRP3 is necessary, but also the leucine-rich repeat (LRR) domain-bound NEK7 bridges the gap between adjacent NLRP3 subunits to mediate the assembly of the higher order oligomeric complex.Structures of NLRP1 and NLRP3. (A) Domain architecture of NLRP3. NLRP3 is composed of an N-terminal pyrin domain (PYD); a central NACHT domain that is subdivided into NBD, helical domain 1 (HD1), winged helix domain (WHD), and helical domain 2 (HD2); and a C-terminal LRR domain. (B) Structure of monomeric NLRP3 in complex with NEK7 solved by cryo-EM (Protein Data Bank [PDB] ID: 6NPY). The structure is colored coded as in A by domains. (C) Crystal structure of NACHT domain of NLRP3 in complex with ADP and a small molecular inhibitor (PDB ID: 7ALV). The inhibitor locates at the multiple domain interface and locks protein in the inactive conformation. (D) Domain architecture of NLRP1 and DPP9. The autocatalytic FIIND is composed of ZU5 and UPA subdomains. (E) Cryo-EM structures of the ternary complex of NLRP1-NLRP1-DPP9 and the inhibition by VbP. In the ternary complex (left, PDB ID: 6X6A), two NLRP1 molecules bind to one DPP9 (green). The first NLRP1 has intact FIIND in which the ZU5 subdomain (blue) associates with the UPA subdomain (pink). The second NLRP1 has the UPA subdomain dissociated, and the N-terminal peptide of the UPA subdomain inserts into the DPP9 catalytic pocket (magenta). Vbp binding displaces the inserted N-terminal UPA peptide and reduces second UPA occupancy (PDB ID: 6X6C). (F) UPA-CARD released from DPP9 initiates inflammasome assembly.Recently, cryo-EM structures of full-length wild-type human and mouse NLRP3 as inactive oligomers were reported in two preprints (Hochheiser et al., 2021 Preprint; Andreeva et al., 2021 Preprint). These structures exhibit decamer to hexadecamer double-ring cage-like arrangement, and the decameric human NLRP3 cage was solved in complex with the selective and potent NLRP3 inhibitor CRID3/MCC950 (Hochheiser et al., 2021 Preprint). In this 4-Å decameric structure, the mechanism of action of CRID3 was demonstrated for the first time. The data revealed that CRID3 binds in a pocket only present in the closed conformation created by multiple subdomains of the NACHT domain (see figure, panel A), thereby stabilizing this structure and preventing activation. They not only confirmed previous biochemical and modeling studies suggesting a similar mechanism of action (El-Sharkawy et al., 2020), but also refuted previous data suggesting that the mechanism of inhibition was via competition with nucleotide.A crystal structure of the NACHT domain in complex with a CRID3-like small-molecule inhibitor at 2.8-Å resolution was also reported, which confirmed the binding of the inhibitor in a pocket formed by the four subdomains of the NACHT domain (Dekker et al., 2021; see figure, panel C). In an effort to help drive the discovery of differentiated chemical matter, we have recently successfully resolved a structure of monomeric NLRP3 complexed with a proprietary high affinity NLRP3 inhibitor (unpublished data). In this example we achieved a 2.6-Å resolution with a clear inhibitor bound pocket also formed by the subdomains of the NACHT domain. Thus, NLRP3 inhibitors, at least for the ones we have evidence for, act as an interdomain glue to lock the protein in an inactive conformation.NLRP1In addition to the recent advances on NLRP3, there have been significant advances in the structural biology for NLRP1. Like NLRP3, NLRP1 activation has been implicated in monogenic and complex pro-inflammatory diseases (Fenini et al., 2020). NLRP1 was the first inflammasome sensor to be identified, but only recently have we begun to elucidate the mechanism of activation of this protein. NLRP1 is unusual in that it contains a function-to-find domain (FIIND) that is auto-processed during protein synthesis to split into ZU5 and UPA subdomains, but the N- and C-terminal regions remain noncovalently bound (see figure, panel D). Through extensive recent studies, we now know that Anthrax lethal factor and viral proteases cleave an N-terminal region of NLRP1 to generate a neo-N-terminus, resulting in NLRP1 ubiquitination and N-terminal “functional” proteasomal degradation; this degradation leads to the release of the noncovalently associated C-terminal active fragment, which induce inflammasome assembly (Wang et al., 2021). Interestingly, the CARD8 protein, which also contains FIIND-CARD (caspase-1 recruitment domain) but lacks the nucleotide-binding domain (NBD)–LRR, was found to be able to form inflammasomes as well, and functional degradation can also explain the activation of the CARD8 inflammasome (Chui et al., 2020).NLRP1 activation is further regulated by DPP8 and DPP9, which are related intracellular prolyl dipeptidases, and inhibition of DPP8/9 by the small molecule Val-boroPro (VbP) or genetic knockouts leads to NLRP1 inflammasome activation (Wang et al., 2021). Further evidence uncovered that DPP8/9 directly interacts with the FIIND and mediates inhibition of the NLRP1 inflammasome activity. Recently, cryo-EM structures of NLRP1-DPP9 complex alone and in complex with the DPP8/9 inhibitor VbP have been reported (Hollingsworth et al., 2021; Huang et al., 2021). Surprisingly, the NLRP1-DPP9 forms a ternary complex comprised of DPP9, one intact FIIND of an autoinhibited NLRP1, and one C-terminal fragment (UPA-CARD) freed by N-terminal degradation. The N-terminal residues of UPA-CARD insert into the DPP9 active site, and VbP competes for this interaction (see figure, panels E and F). The binding of UPA-CARD to DPP9 requires full-length NLRP1, and the ternary complex could prevent UPA-CARD from self-oligomerization during homeostatic protein turnover (see figure, panel F). This work provides important insights into how the unique domain architecture and negative regulatory protein DPP8/9 participate in NLRP1 activation and may pave the way for the discovery of novel inhibitors of NLRP1 and a starting point to enable SBDD.Future prospectsThese recent advances in our structural understanding of the mechanism of activation and inhibition of these key inflammasome proteins offer tremendous insight into inflammasome signaling. Equally important, they provide initial templates from which to conduct rational-based drug design for these highly compelling targets. In the future, we are certain to see the fruits of these seminal pieces of work by assisting the development of inflammasome inhibitors using rational drug design approaches. We envision that these efforts will generate best-in-class molecules for the treatment of numerous diseases of high unmet medical need where inflammasomes are key pathogenic drivers.     

NLRP1在血液疾病中作用的研究进展

炎性复合体是存在于胞浆中的一组多蛋白复合体,它能够活化胱天蛋白酶（caspase）-1,后者介导IL（interleukin）-1β、IL-18和IL-33等促炎因子的成熟与释放.NALP1（NACHT leucine-rich-repeat protein 1）也称NLRP1,是最早被鉴定出来的具有明确配体的炎性复合体之一,它参与多种炎症反应和细胞凋亡的调节作用.此外,还有研究发现NLRP1在急性白血病的发生发展及诱导骨髓造血干细胞凋亡等血液系统疾病中也发挥着重要作用.本文将对NLRP1的结构、活化机制、调控及在造血系统中的作用进行综述.     

Oxidative metabolism enables Salmonella evasion of the NLRP3 inflammasome

Microbial infection triggers assembly of inflammasome complexes that promote caspase-1–dependent antimicrobial responses. Inflammasome assembly is mediated by members of the nucleotide binding domain leucine-rich repeat (NLR) protein family that respond to cytosolic bacterial products or disruption of cellular processes. Flagellin injected into host cells by invading Salmonella induces inflammasome activation through NLRC4, whereas NLRP3 is required for inflammasome activation in response to multiple stimuli, including microbial infection, tissue damage, and metabolic dysregulation, through mechanisms that remain poorly understood. During systemic infection, Salmonella avoids NLRC4 inflammasome activation by down-regulating flagellin expression. Macrophages exhibit delayed NLRP3 inflammasome activation after Salmonella infection, suggesting that Salmonella may evade or prevent the rapid activation of the NLRP3 inflammasome. We therefore screened a Salmonella Typhimurium transposon library to identify bacterial factors that limit NLRP3 inflammasome activation. Surprisingly, absence of the Salmonella TCA enzyme aconitase induced rapid NLRP3 inflammasome activation. This inflammasome activation correlated with elevated levels of bacterial citrate, and required mitochondrial reactive oxygen species and bacterial citrate synthase. Importantly, Salmonella lacking aconitase displayed NLRP3- and caspase-1/11–dependent attenuation of virulence, and induced elevated serum IL-18 in wild-type mice. Together, our data link Salmonella genes controlling oxidative metabolism to inflammasome activation and suggest that NLRP3 inflammasome evasion promotes systemic Salmonella virulence.Pattern recognition receptors (PRRs) that detect and respond to evolutionarily conserved microbial structures such as LPS or peptidoglycan, as well as pathogen-specific virulence activities, are critical for host immune defense (Medzhitov, 2007; Vance et al., 2009). To promote infection, microbial pathogens inject virulence factors into the cytosol of infected cells to disrupt or modulate critical host physiological processes (Cornelis, 2006). During this process, contamination of the target cell cytosol by microbial components triggers cytosolic PRRs of the nucleotide binding domain leucine-rich repeat (NLR) family (Lamkanfi and Dixit, 2009). Diverse NLRs respond to a variety of endogenous and exogenous signals associated with infection, tissue stress, or damage. For example, NLRC4 responds to microbial products such as bacterial flagellin or structurally related specialized secretion system components that are injected into the cytosol of infected cells during infection by bacterial pathogens including Pseudomonas, Legionella, and Salmonella spp. (Miao et al., 2006; Molofsky et al., 2006; Sutterwala et al., 2007). NLRs recruit pro–caspase-1 to multiprotein complexes termed inflammasomes, where pro–caspase-1 is processed and activated, leading to cleavage and secretion of caspase-1–dependent cytokines (Martinon et al., 2002, 2007), as well as pyroptosis, a caspase-1–dependent pro-inflammatory cell death (Bergsbaken et al., 2009).Inflammasome activation and subsequent production of caspase-1–dependent cytokines is important for both innate and adaptive antimicrobial responses (Mariathasan and Monack, 2007), as IL-1 family cytokines released upon inflammasome activation promote neutrophil migration to infected tissues and drive T_H17 and T_H1 responses against mucosal pathogens (Chung et al., 2009; Ichinohe et al., 2009). How pathogens evade inflammasome activation, and whether persistent bacterial pathogens evade or suppress inflammasome activation to establish or maintain persistence remains poorly understood.Salmonella enterica species cause a range of disease from severe gastroenteritis to persistent systemic infection (Bäumler et al., 1998). Salmonella enterica serovar Typhimurium (Stm) invades host cells by means of a type III secretion system (T3SS) encoded on Salmonella pathogenicity island I (SPI-1; Lee, 1996; Collazo and Galán, 1997). Salmonella subsequently replicates within a Salmonella-containing vacuole (SCV) that is established and maintained by the activity of a second T3SS, encoded on a second pathogenicity island, SPI-2 (Cirillo et al., 1998; Hensel et al., 1998). Intestinal inflammation during Stm infection is triggered by NLRC4-dependent responses to Stm flagellin, accompanied by caspase-1–dependent cytokine secretion and pyroptosis (Franchi et al., 2012). Activity of a SPI-1 effector protein, SopE, also contributes to SPI-1–dependent inflammasome activation in intestinal epithelial cells (Müller et al., 2009). Within the inflamed intestine, specialized adaptations allow Stm to resist mucosal antimicrobial defenses (Raffatellu et al., 2009; Winter et al., 2010; Thiennimitr et al., 2011). However, flagellin expression is down-regulated at systemic sites (Cummings et al., 2005, 2006), and enforced flagellin expression enhances NLRC4 activation and bacterial clearance, indicating that inflammasome activation in response to bacterial flagellin is detrimental for Stm replication during systemic infection (Miao et al., 2010a; Stewart et al., 2011).NLRP3 responds to a wide variety of structurally unrelated molecules and activities, including extracellular ATP, bacterial pore-forming proteins, bacterial nucleic acids, crystals, and unsaturated fatty acids (Kanneganti et al., 2006; Mariathasan et al., 2006; Martinon et al., 2006; Hornung et al., 2008; Wen et al., 2011). While ATP, crystals, and the Yersinia T3SS all induce rapid formation of an NLRP3 inflammasome that leads to caspase-1 activation within 1–2 h (Mariathasan et al., 2006; Martinon et al., 2006; Brodsky et al., 2010), Stm induces delayed activation of a noncanonical NLRP3 inflammasome 12–16 h after infection (Broz et al., 2010). This noncanonical NLRP3 inflammasome is independent of the activities of the SPI-1 T3SS and instead is regulated by caspase-11 and TLR4-dependent production of type I interferon (Broz et al., 2012; Gurung et al., 2012; Rathinam et al., 2012). We therefore hypothesized that Stm might evade or prevent rapid activation of a canonical NLRP3 inflammasome, and that this evasion might contribute to systemic bacterial virulence. Several bacterial and viral pathogens evade NLRP3 inflammasome activation (Taxman et al., 2010; Gregory et al., 2011), but whether Salmonella is capable of doing so is unknown.To identify potential negative regulators of NLRP3 inflammasome activation, we generated and screened a transposon library of flagellin-deficient Stm mutants for elevated inflammasome activation in NLRC4-deficient BM-derived macrophages (BMDMs). Sequencing of candidate hits identified acnB, the gene encoding the TCA cycle enzyme aconitase, which converts citrate to isocitrate, as well as several other genes that had been previously isolated in a screen for Salmonella genes that are required for persistent Salmonella infection in vivo (Lawley et al., 2006). Intriguingly, isocitrate lyase (encoded by aceA), which generates glyoxylate from isocitrate in the glyoxylate cycle, contributes to persistent but not acute infection by Salmonella as well as Mycobacterium tuberculosis (McKinney et al., 2000; Fang et al., 2005).To test the potential role of Salmonella TCA cycle metabolism in inflammasome modulation, we generated targeted deletions in acnB as well as genes encoding other TCA cycle enzymes. Notably, deletion of aconitase, isocitrate lyase, or isocitrate dehydrogenase (icdA), but not other TCA enzymes, induced rapid NLRP3-dependent inflammasome activation in Stm-infected macrophages, suggesting that activity of these enzymes limits NLRP3 inflammasome activation by intracellular Salmonella. Moreover, aconitase-deficient Salmonella exhibited a defect in acute systemic virulence after oral administration and were deficient in their ability to persist in a chronic infection. These findings define the first genes that mediate NLRP3 inflammasome evasion by Salmonella and suggest that inflammasome evasion contributes to persistence of bacterial pathogens. Our data further suggest that sensing of bacterial metabolites may provide an additional level of innate immune recognition, and that regulation of metabolite production by intracellular pathogens represents a pathogen immune evasion strategy.