POP1 might be recruiting its type‐Ia interface for NLRP3‐mediated PYD‐PYD interaction: Insights from MD simulation

Inflammasomes are multiprotein caspase‐activating complexes that enhance the maturation and release of proinflammatory cytokines (IL‐1β and IL‐18) in response to the invading pathogen and/or host‐derived cellular stress. These are assembled by the sensory proteins (viz NLRC4, NLRP1, NLRP3, and AIM‐2), adaptor protein (ASC), and effector molecule procaspase‐1. In NLRP3‐mediated inflammasome activation, ASC acts as a mediator between NLRP3 and procaspase‐1 for the transmission of signals. A series of homotypic protein‐protein interactions (NLRP3^PYD:ASC^PYD and ASC^CARD:CASP1^CARD) propagates the downstream signaling for the production of proinflammatory cytokines. Pyrin‐only protein 1 (POP1) is known to act as the regulator of inflammasome. It modulates the ASC‐mediated inflammasome assembly by interacting with pyrin domain (PYD) of ASC. However, despite similar electrostatic surface potential, the interaction of POP1 with NLRP3^PYD is obscured till date. Herein, to explore the possible PYD‐PYD interactions between NLRP3^PYD and POP1, a combined approach of protein‐protein docking and molecular dynamics simulation was adapted. The current study revealed that POP1's type‐Ia interface and type‐Ib interface of NLRP3^PYD might be crucial for 1:1 PYD‐PYD interaction. In addition to type‐I mode of interaction, we also observed type‐II and type‐III interaction modes in two different dynamically stable heterotrimeric complexes (POP1‐NLRP3‐NLRP3 and POP1‐NLRP3‐POP1). The inter‐residual/atomic distance calculation exposed several critical residues that possibly govern the said interaction, which need further investigation. Overall, the findings of this study will shed new light on hitherto concealed molecular mechanisms underlying NLRP3‐mediated inflammasome, which will have strong future therapeutic implications.     

The NLRP12 pyrin domain: structure, dynamics, and functional insights

The initial line of defense against infection is sustained by the innate immune system. Together, membrane-bound Toll-like receptors and cytosolic nucleotide-binding domain and leucine-rich repeat-containing receptors (NLR) play key roles in the innate immune response by detecting bacterial and viral invaders as well as endogenous stress signals. NLRs are multi-domain proteins with varying N-terminal effector domains that are responsible for regulating downstream signaling events. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP12 (NLRP12 PYD) determined using NMR spectroscopy. NLRP12 is a non-inflammasome NLR that has been implicated in the regulation of Toll-like receptor-dependent nuclear factor-κB activation. NLRP12 PYD adopts a typical six-helical bundle death domain fold. By direct comparison with other PYD structures, we identified hydrophobic residues that are essential for the stable fold of the NLRP PYD family. In addition, we report the first in vitro confirmed non-homotypic PYD interaction between NLRP12 PYD and the pro-apoptotic protein Fas-associated factor 1 (FAF-1), which links the innate immune system to apoptotic signaling. Interestingly, all residues that participate in this protein:protein interaction are confined to the α2-α3 surface, a region of NLRP12 PYD that differs most between currently reported NLRP PYD structures. Finally, we experimentally highlight a significant role for tryptophan 45 in the interaction between NLRP12 PYD and the FAF-1 UBA domain.     

Three-dimensional Structure of the NLRP7 Pyrin Domain: INSIGHT INTO PYRIN-PYRIN-MEDIATED EFFECTOR DOMAIN SIGNALING IN INNATE IMMUNITY*

The innate immune system provides an initial line of defense against infection. Nucleotide-binding domain- and leucine-rich repeat-containing protein (NLR or (NOD-like)) receptors play a critical role in the innate immune response by surveying the cytoplasm for traces of intracellular invaders and endogenous stress signals. NLRs themselves are multi-domain proteins. Their N-terminal effector domains (typically a pyrin or caspase activation and recruitment domain) are responsible for driving downstream signaling and initiating the formation of inflammasomes, multi-component complexes necessary for cytokine activation. However, the currently available structures of NLR effector domains have not yet revealed the mechanism of their differential modes of interaction. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP7 (NLRP7 PYD) obtained by NMR spectroscopy. The NLRP7 PYD adopts a six-α-helix bundle death domain fold. A comparison of conformational and dynamics features of the NLRP7 PYD with other PYDs showed distinct differences for helix α3 and loop α2-α3, which, in NLRP7, is stabilized by a strong hydrophobic cluster. Moreover, the NLRP7 and NLRP1 PYDs have different electrostatic surfaces. This is significant, because death domain signaling is driven by electrostatic contacts and stabilized by hydrophobic interactions. Thus, these results provide new insights into NLRP signaling and provide a first molecular understanding of inflammasome formation.     

Three-Dimensional Structure of Human NLRP10/PYNOD Pyrin Domain Reveals a Homotypic Interaction Site Distinct from Its Mouse Homologue

NLRPs (Nucleotide-binding domain, leucine-rich repeat and pyrin domain containing proteins) are a family of pattern-recognition receptors (PRRs) that sense intracellular microbial components and endogenous stress signals. NLRP10 (also known as PYNOD) is a unique NLRP member characterized by a lack of the putative ligand-binding leucine-rich repeat domain. Recently, human NLRP10 has been shown to inhibit the self-association of ASC into aggregates and ASC-mediated procaspase-1 processing. However, such activities are not found in mouse NLRP10. Here we report the solution structure and dynamics of human NLRP10 pyrin domain (PYD), whose helix H3 and loop H2–H3 adopt a conformation distinct from those of mouse NLRP10. Docking studies show that human and mouse NLRP10 PYDs may interact differently with ASC PYD. These results provide a possible structural explanation for the contrasting effect of NLRP10 on ASC aggregation in human cells versus mouse models. Finally, we also provide evidence that in human NLRP10 the PYD domain may not interact with the NOD domain to regulate its intrinsic nucleotide hydrolysis activity.     

Multiple Binding Sites on the Pyrin Domain of ASC Protein Allow Self-association and Interaction with NLRP3 Protein

A key process underlying an innate immune response to pathogens or cellular stress is activation of members of the NOD-like receptor family, such as NLRP3, to assemble caspase-1-activating inflammasome complexes. Activated caspase-1 processes proinflammatory cytokines into active forms that mediate inflammation. Activation of the NLRP3 inflammasome is also associated with common diseases including cardiovascular disease, diabetes, chronic kidney disease, and Alzheimer disease. However, the molecular details of NLRP3 inflammasome assembly are not established. The adaptor protein ASC plays a key role in inflammasome assembly. It is composed of an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain, which are protein interaction domains of the death fold superfamily. ASC interacts with NLRP3 via a homotypic PYD interaction and recruits procaspase-1 via a homotypic caspase recruitment domain interaction. Here we demonstrate that ASC PYD contains two distinct binding sites important for self-association and interaction with NLRP3 and the modulatory protein POP1. Modeling of the homodimeric ASC PYD complex formed via an asymmetric interaction using both sites resembles a type I interaction found in other death fold domain complexes. This interaction mode also permits assembly of ASC PYDs into filaments. Furthermore, a type I binding mode is likely conserved in interactions with NLRP3 and POP1, because residues critical for interaction of ASC PYD are conserved in these PYDs. We also demonstrate that ASC PYD can simultaneously self-associate and interact with NLRP3, rationalizing the model whereby ASC self-association upon recruitment to NLRP3 promotes clustering and activation of procaspase-1.     

Structure of the NLRP1 caspase recruitment domain suggests potential mechanisms for its association with procaspase‐1

The NLRP1 inflammasome responds to microbial challenges such as Bacillus anthracis infection and is implicated in autoimmune disease such as vitiligo. Human NLRP1 contains both an N‐terminal pyrin domain (PYD) and a C‐terminal caspase recruitment domain (CARD), with the latter being essential for its association with the downstream effector procaspase‐1. Here we report a 2.0 Å crystal structure of the human NLRP1 CARD as a fusion with the maltose‐binding protein. The structure reveals the six‐helix bundle fold of the NLRP1 CARD, typical of the death domain superfamily. The charge surface of the NLRP1 CARD structure and a procaspase‐1 CARD model suggests potential mechanisms for their association through electrostatic attraction. Proteins 2013; 81:1266–1270. © 2013 Wiley Periodicals, Inc.     

In vitro complex formation of human PYRIN domain-only protein 3 prevented by self-oligomerization of ASC PYD domain

The formation of inflammasome complexes contributes inactivation of inflammatory caspases viz caspase 1, which is generally considered essential for the innate response. Three proteins constituted this inflammasome complex, such as Nod-like receptors (NLRP or AIM2), ASC possessing caspase-recruiting domain, and caspase-1. The ASC proteins comprise two domains, the N-terminal PYD domain responsible for the interaction of various proteins, including PYD only protein 3 (POP3), and the CARD domain for association with other proteins. The PYRIN Domain-Only Protein POP3 negatively regulates responses to DNA virus infection by preventing the ALR inflammasome formation. POP3 directly interacts with ASC, therefore inhibiting ASC recruitment to AIM2-like receptors (ALRs). In the current study, we designed various constructs of the PYRIN Domain-Only Protein 3 (POP3) and ASC PYD domain to find the best-overexpressed construct for biochemical characterization as well as our complex studies. We cloned, purified, and characterized the PYD domain of pyrin only protein 3 and ASC PYD domain under physiological conditions. Our in vitro study clearly shows that the ASC PYD domain of corresponding amino acid 1–96 aa with ease self-oligomerization in physiological buffer conditions, and complex formation of POP3 PYD (1–83 aa) was inhibited by ASC PYD domain. Besides, we purified the PYD of POP3 protein in low and high salt conditions and different pH values for their biochemical characterization. Our results showed that POP3 formed a dimer under normal physiological conditions and was stable under normal buffer conditions; however, the purification in extremely low pH (pH5.0) conditions shows unstable behavior, the high salt conditions (500 mM NaCl) influence the protein aggregation. SDS PAGE arbitrated the homogeneity of the PYD domain of pyrin only protein 3 and ASC PYD domain of corresponding amino acids 1–83 and 1–96, respectively. Furthermore, our native PAGE shows the PYD domain of pyrin; only protein 3 did not form a complex with ASC PYD domain because of oligomerization mediated by the PYD domain.     

Single-Molecule Fluorescence Reveals the Oligomerization and Folding Steps Driving the Prion-like Behavior of ASC

Single-molecule fluorescence has the unique ability to quantify small oligomers and track conformational changes at a single-protein level. Here we tackled one of the most extreme protein behaviors, found recently in an inflammation pathway. Upon danger recognition in the cytosol, NLRP3 recruits its signaling adaptor, ASC. ASC start polymerizing in a prion-like manner and the system goes in “overdrive” by producing a single micron-sized “speck.” By precisely controlling protein expression levels in an in vitro translation system, we could trigger the polymerization of ASC and mimic formation of specks in the absence of inflammasome nucleators. We utilized single-molecule spectroscopy to fully characterize prion-like behaviors and self-propagation of ASC fibrils. We next used our controlled system to monitor the conformational changes of ASC upon fibrillation. Indeed, ASC consists of a PYD and CARD domains, separated by a flexible linker. Individually, both domains have been found to form fibrils, but the structure of the polymers formed by the full-length ASC proteins remains elusive. For the first time, using single-molecule Förster resonance energy transfer, we studied the relative positions of the CARD and PYD domains of full-length ASC. An unexpectedly large conformational change occurred upon ASC fibrillation, suggesting that the CARD domain folds back onto the PYD domain. However, contradicting current models, the “prion-like” conformer was not initiated by binding of ASC to the NLRP3 platform. Rather, using a new method, hybrid between Photon Counting Histogram and Number and Brightness analysis, we showed that NLRP3 forms hexamers with self-binding affinities around 300 nM. Overall our data suggest a new mechanism, where NLRP3 can initiate ASC polymerization simply by increasing the local concentration of ASC above a supercritical level.     

NLRP10蛋白的结构和自组装

NLRP10是NOD样受体(NOD-like receptor,NLR)蛋白家族的特殊成员,它具有N端热蛋白结构域(pyrin-like domain,PYD)和中间的核苷酸结合寡聚结构域(nucleotide-binding and oligomerization domain,NOD),却不具有亮氨酸富集重复结构域(leucine-rich repeat domain,LRR),这表明NLRP10可能并不直接参与病原微生物的识别,而是行使免疫调节的功能.研究表明,NLRP10能促进NOD1介导的免疫反应,也能抑制NLRP3炎症小体的激活.在参与免疫调控时,NLRP10以聚集体的形式与接头蛋白ASC结合,继而招募相应免疫信号通路中的其他成员.NLRP10蛋白在结构方面的研究很有限,目前仅有PYD结构域的相关报道.本文在尝试纯化全长人源NLRP10时,同时得到了PYD结构域缺失的稳定蛋白片段,电镜结果表明存在条形和环形两种形态的聚集体.通过序列分析和定点突变,我们找到了潜在的蛋白质降解位点,从而解决了纯化过程中蛋白质降解的问题,为后期的高分辨结构测定奠定了基础.     

Crystal Structure of the F27G AIM2 PYD Mutant and Similarities of Its Self-Association to DED/DED Interactions

Absent in melanoma 2 (AIM2) is a cytoplasmic double-stranded DNA sensor involved in innate immunity. It uses its C-terminal HIN domain for recognizing double-stranded DNA and its N-terminal pyrin domain (PYD) for eliciting downstream effects through recruitment and activation of apoptosis-associated Speck-like protein containing CARD (ASC). ASC in turn recruits caspase-1 and/or caspase-11 to form the AIM2 inflammasome. The activated caspases process proinflammatory cytokines IL-1β and IL-18 and induce the inflammatory form of cell death pyroptosis. Here we show that AIM PYD (AIM2^PYD) self-oligomerizes. We notice significant sequence homology of AIM2^PYD with the hydrophobic patches of death effector domain (DED)-containing proteins and confirm that mutations on these residues disrupt AIM2^PYD self-association. The crystal structure at 1.82 Å resolution of such a mutant, F27G of AIM2^PYD, shows the canonical six-helix (H1–H6) bundle fold in the death domain superfamily. In contrast to the wild-type AIM2^PYD structure crystallized in fusion with the large maltose-binding protein tag, the H2–H3 region of the AIM2^PYD F27G is well defined with low B-factors. Structural analysis shows that the conserved hydrophobic patches engage in a type I interaction that has been observed in DED/DED and other death domain superfamily interactions. While previous mutagenesis studies of PYDs point to the involvement of charged interactions, our results reveal the importance of hydrophobic interactions in the same interfaces. These centrally localized hydrophobic residues within fairly charged patches may form the hot spots in AIM2^PYD self-association and may represent a common mode of PYD/PYD interactions in general.     

Endothelial Acid Sphingomyelinase Promotes NLRP3 Inflammasome and Neointima Formation During Hypercholesterolemia

The NOD-like receptor pyrin domain 3 (NLRP3) inflammasome is activated during atherogenesis, but how this occurs is unclear. Here, we explored the mechanisms activating and regulating NLRP3 inflammasomes via the acid sphingomyelinase (ASM)-ceramide signaling pathway. As a neointima formation model, partial left carotid ligations were performed on endothelial cell (EC)-specific ASM transgene mice (Smpd1^trg/EC^cre) and their control littermates (Smpd1^trg/WT and WT/WT) fed on the Western diet (WD). We found neointima formation remarkably increased in Smpd1^trg/EC^cre mice over their control littermates. Next, we observed enhanced colocalization of NLRP3 versus adaptor protein ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) or caspase-1 in the carotid ECs of WD-treated Smpd1^trg/EC^cre mice but not in their control littermates. In addition, we used membrane raft (MR) marker flotillin-1 and found more aggregation of ASM and ceramide in the intima of Smpd1^trg/EC^cre mice than their control littermates. Moreover, we demonstrated by in situ dihydroethidium staining, carotid intimal superoxide levels were much higher in WD-treated Smpd1^trg/EC^cre mice than in their control littermates. Using ECs from Smpd1^trg/EC^cre and WT/WT mice, we showed ASM overexpression markedly enhanced 7-ketocholesterol (7-Ket)-induced increases in NLRP3 inflammasome formation, accompanied by enhanced caspase-1 activity and elevated interleukin-1β levels. These 7-Ket-induced increases were significantly attenuated by ASM inhibitor amitriptyline. Furthermore, we determined that increased MR clustering with NADPH oxidase subunits to produce superoxide contributes to 7-Ket-induced NLRP3 inflammasome activation via a thioredoxin-interacting protein-mediated controlling mechanism. We conclude that ceramide from ASM plays a critical role in NLRP3 inflammasome activation during hypercholesterolemia via MR redox signaling platforms to produce superoxide, which leads to TXNIP dissociation.     

1H, 13C and 15N resonance assignments of the pyrin domain from human PYNOD

PYNOD is a novel protein belonging to a large family of proteins containing the nucleotide-binding and oligomerization domain (NOD) involved in inflammation and apoptosis. Human PYNOD inhibits inflammatory response mediated by caspase-1 and apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). Here we report the ¹H, ¹³C and ¹⁵N resonance assignments and secondary structure identification of the pyrin domain (PYD) of human PYNOD as the first step towards elucidating the structural basis of the anti-inflammatory activity of PYNOD.     

Zinc depletion regulates the processing and secretion of IL-1β

Sterile inflammation contributes to many common and serious human diseases. The pro-inflammatory cytokine interleukin-1β (IL-1β) drives sterile inflammatory responses and is thus a very attractive therapeutic target. Activation of IL-1β in sterile diseases commonly requires an intracellular multi-protein complex called the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome. A number of disease-associated danger molecules are known to activate the NLRP3 inflammasome. We show here that depletion of zinc from macrophages, a paradigm for zinc deficiency, also activates the NLRP3 inflammasome and induces IL-1β secretion. Our data suggest that zinc depletion damages the integrity of lysosomes and that this event is important for NLRP3 activation. These data provide new mechanistic insight to how zinc deficiency contributes to inflammation and further unravel the mechanisms of NLRP3 inflammasome activation.     

Activation of CXCR7 promotes endothelial repair and reduces the carotid atherosclerotic lesions through inhibition of pyroptosis signaling pathways

Endothelial injuries, including cell pyroptosis, are ongoing inflammatory processes with key roles in atherosclerosis development. Our previous report showed that the chemokine CXCL12 and its receptor CXCR7 are associated with the proliferation and angiogenesis of endothelial cells. Nevertheless, the mechanism underlying these effects on atherosclerotic lesions, especially on endothelial dysfunction, remains unknown. Here, we demonstrated that CXCR7 was upregulated in human carotid atherosclerotic plaques, apolipoprotein E knockout (ApoE^?/?) mice fed with a high‐fat diet (HFD), and oxidized lipopolysaccharide‐treated (ox‐LDL) human umbilical vein endothelial cells (HUVECs). Further, the activation of CXCR7 reversed ox‐LDL‐induced HUVEC dysfunction, such as migration, tube formation, and cell pyroptosis; all of these protective effects were alleviated by inhibition of CXCR7. The NOD‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasomes were also elevated in human carotid atherosclerotic plaques, ApoE^?/? mice fed with HFD, and ox‐LDL‐injured HUVECs by regulation of caspase‐1 and interleukin (IL)‐1β expression. The activation of CXCR7 by TC14012 led to a decrease in atherosclerotic lesions in ApoE^?/? mice fed with HFD. TC14012 also inhibited the expression of the NLRP3 inflammasome signaling pathway in vivo. In conclusion, our study suggests that CXCR7 plays an important role in regulating NLRP3 inflammasome‐modulated pyroptosis in HUVECs, providing a potential novel therapy for atherosclerosis.     

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.     

Targeting the NLRP3 Inflammasome to Reduce Diet-Induced Metabolic Abnormalities in Mice

Although the molecular links underlying the causative relationship between chronic low-grade inflammation and insulin resistance are not completely understood, compelling evidence suggests a pivotal role of the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome. Here we tested the hypothesis that either a selective pharmacological inhibition or a genetic downregulation of the NLRP3 inflammasome results in reduction of the diet-induced metabolic alterations. Male C57/BL6 wild-type mice and NLRP3^−/− littermates were fed control diet or high-fat, high-fructose diet (HD). A subgroup of HD-fed wild-type mice was treated with the NLRP3 inflammasome inhibitor BAY 11-7082 (3 mg/kg intraperitoneally [IP]). HD feeding increased plasma and hepatic lipids and impaired glucose homeostasis and renal function. Renal and hepatic injury was associated with robust increases in profibrogenic markers, while only minimal fibrosis was recorded. None of these metabolic abnormalities were detected in HD-fed NLRP3^−/− mice, and they were dramatically reduced in HD-mice treated with the NLRP3 inflammasome inhibitor. BAY 11-7082 also attenuated the diet-induced increase in NLRP3 inflammasome expression, resulting in inhibition of caspase-1 activation and interleukin (IL)-1β and IL-18 production (in liver and kidney). Interestingly, BAY 11-7082, but not gene silencing, inhibited nuclear factor (NF)-κB nuclear translocation. Overall, these results demonstrate that the selective pharmacological modulation of the NLRP3 inflammasome attenuates the metabolic abnormalities and the related organ injury/dysfunction caused by chronic exposure to HD, with effects similar to those obtained by NLRP3 gene silencing.     

Plasticizer DBP Activates NLRP3 Inflammasome through the P2X7 Receptor in HepG2 and L02 Cells

Ditubyl phthalate (DBP), one of the most widely used plasticizers, can migrate out to contaminate our bodies and environment. A number of studies have showed that DBP is closely related to liver pathological changes and diseases. Inflammasomes are multiprotein complexes composed of procaspase and pattern recognition receptors such as Nucleotide oligomerization domain (NOD) like receptor family, pyrin domain containing 3 (NLRP3). Activation of NLRP3 inflammasome is implicated in the pathogeneses of liver damage. The aim of this study was to determine the effects of DBP on NLRP3 inflammasome. We found that DBP triggered the activation of NLRP3 inflammasome in hepatocyte cell lines. By using Ca‐074‐Me, N‐acetylcysteine and KN‐62, we observed that the P2X₇ receptor participated in the DBP‐induced activation of NLRP3 inflammasome. DBP could also trigger the ATP release. In conclusion, we demonstrated that DBP is one of the activator of NLRP3 inflammasome and may play an important role in liver damage.     

NLRP10 enhances Shigella-induced pro-inflammatory responses

Members of the NLR family evolved as intracellular sensors for bacterial and viral infection. However, our knowledge on the implication of most of the human NLR proteins in innate immune responses still remains fragmentary. Here we characterized the role of human NLRP10 in bacterial infection. Our data revealed that NLRP10 is a cytoplasmic localized protein that positively contributes to innate immune responses induced by the invasive bacterial pathogen Shigella flexneri. SiRNA-mediated knock-down studies showed that NLRP10 contributes to pro-inflammatory cytokine release triggered by Shigella in epithelial cells and primary dermal fibroblasts, by influencing p38 and NF-κB activation. This effect is dependent on the ATPase activity of NLRP10 and its PYD domain. Mechanistically, NLRP10 interacts with NOD1, a NLR that is pivotally involved in sensing of invasive microbes, and both proteins are recruited to the bacterial entry point at the plasma membrane. Moreover, NLRP10 physically interacts with downstream components of the NOD1 signalling pathway, such as RIP2, TAK1 and NEMO. Taken together, our data revealed a novel role of NLRP10 in innate immune responses towards bacterial infection and suggest that NLRP10 functions as a scaffold for the formation of the NOD1-Nodosome.     

Glucose regulates hypoxia-induced NLRP3 inflammasome activation in macrophages

Although the intimate linkage between hypoxia and inflammation is well known, the mechanism underlying this linkage has not been fully understood. Nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is an intracellular multiprotein complex that regulates interleukin-1β (IL-1β) secretion and pyroptosis, and is implicated in the pathogenesis of sterile inflammatory diseases. Here, we investigated the regulatory mechanism of NLRP3 inflammasome activation in response to hypoxia in macrophages. Severe hypoxia (0.1% O₂) induced the processing of pro-IL-1β, pro-caspase-1, and gasdermin D, as well as the release of IL-1β and lactate dehydrogenase in lipopolysaccharide (LPS)-primed murine macrophages, indicating that hypoxia induces NLRP3 inflammasome-driven inflammation and pyroptosis. NLRP3 deficiency and a specific caspase-1 blockade inhibited hypoxia-induced IL-1β release. Hypoxia-induced IL-1β release and cell death were augmented under glucose deprivation, and an addition of glucose in the media negatively regulated hypoxia-induced IL-1β release. Under hypoxia and glucose deprivation, hypoxia-induced glycolysis was not driven and subsequently, the intracellular adenosine triphosphates (ATPs) were depleted. Atomic absorption spectrometry analysis showed a reduction of intracellular K⁺ concentrations, indicating the K⁺ efflux occurring under hypoxia and glucose deprivation. Furthermore, hypoxia and glucose deprivation-induced IL-1β release was significantly prevented by inhibition of K⁺ efflux and K_ATP channel blockers. In vivo experiments further revealed that IL-1β production was increased in LPS-primed mice exposed to hypoxia (9.5% O₂), which was prevented by a deficiency of NLRP3, an apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1. Our results demonstrate that NLRP3 inflammasome can sense intracellular energy crisis as a danger signal induced by hypoxia and glucose deprivation, and provide new insights into the mechanism underlying hypoxia-induced inflammation.