NLRP3 phosphorylation in its LRR domain critically regulates inflammasome assembly.
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ABSTRACT:
SUBMITTER: Niu T
PROVIDER: S-EPMC8494922 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:NLRP3 is a cytosolic pattern recognition receptor that senses microbes and endogenous danger signals. Upon activation, NLRP3 forms an inflammasome with the adapter ASC, resulting in caspase-1 activation, release of proinflammatory cytokines and cell death. How NLRP3 activation is regulated by transcriptional and posttranslational mechanisms to prevent aberrant activation remains incompletely understood. Here, we identify three conserved phosphorylation sites in NLRP3 and demonstrate that NLRP3 activation is controlled by phosphorylation of its pyrin domain (PYD). Phosphomimetic residues in NLRP3 PYD abrogate inflammasome activation and structural modeling indicates that phosphorylation of the PYD regulates charge-charge interaction between two PYDs that are essential for NLRP3 activation. Phosphatase 2A (PP2A) inhibition or knock-down drastically reduces NLRP3 activation, showing that PP2A can license inflammasome assembly via dephosphorylating NLRP3 PYD. These results propose that the balance between kinases and phosphatases acting on the NLRP3 PYD is critical for NLRP3 activation.
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Project description:The NLRP3 inflammasome is a critical component of innate immunity that defends the host from microbial infections. However, its aberrant activation contributes to the pathogenesis of several inflammatory diseases. Activation of the NLRP3 inflammasome induces the secretion of proinflammatory cytokines IL-1β and IL-18 and pyroptotic cell death. NLRP3 contains a leucine-rich repeat (LRR) domain at its C terminus. Although posttranslational modifications in this LRR domain have been shown to regulate NLRP3 inflammasome activation, the role of the entire LRR domain in NLRP3 inflammasome activation remains controversial. Here, we generated mouse macrophages that express an endogenous NLRP3 mutant lacking the LRR domain. Deletion of the LRR domain diminished NLRP3 inflammasome activation in macrophages. Furthermore, using NLRP3-deficient macrophages that are reconstituted with NLRP3 mutants lacking the LRR domain, we found that deletion of the LRR domain inhibited NLRP3 inflammasome activation. Mechanistically, deletion of the LRR domain inhibited NLRP3 self-association, oligomerization, and interaction with the essential regulator NEK7. Our results demonstrate a critical role for the LRR domain in NLRP3 inflammasome activation.
| S-EPMC9763864 | biostudies-literature
Project description:The cytosolic pattern recognition receptor (PRR) NOD-like receptor family, pyrin domain containing 3 (NLRP3) senses a wide range of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Upon activation, NLRP3 triggers the assembly of inflammasome via the self-oligomerization and the recruitment of apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and pro-caspase-1, facilitating the robust immune responses including the secretion of proinflammatory cytokines and pyroptosis. The NLRP3 inflammasome must be well orchestrated to prevent the aberrant activations under physiological and pathological conditions, because uncontrolled activation of NLRP3 inflammasome is one of the major causes of a variety of autoimmune diseases and metabolic disorders. Therefore, understanding the molecular mechanisms for controlling NLRP3 inflammasome activation may provide novel strategies for the treatment of NLRP3-related diseases. Although NLRP3 inflammasome can be regulated at the transcriptional level, the post-translational modification (PTM) of NLRP3 as well as other inflammasome components has also been showed to be critical for the regulation of its activation. Several kinases and phosphatases have been shown to control NLRP3 inflammasome activation in response to either exogenous pathogen infections or endogenous molecules, such as bile acids. In this review, we summarize our current knowledge of phosphorylation patterns and their functional role in the regulation of NLRP3 inflammasome, and suggest interesting areas for future research.
| S-EPMC6186804 | biostudies-other
Project description:The NLRP3 inflammasome, a critical component of the innate immune system, induces caspase-1 activation and interleukin (IL)-1β maturation in response to microbial infection and cellular damage. However, aberrant activation of the NLRP3 inflammasome contributes to the pathogenesis of several inflammatory disorders, including cryopyrin-associated periodic syndromes, Alzheimer's disease, type 2 diabetes, and atherosclerosis. Here, we identify the receptor for activated protein C kinase 1 (RACK1) as a component of the NLRP3 complexes in macrophages. RACK1 interacts with NLRP3 and NEK7 but not ASC. Suppression of RACK1 expression abrogates caspase-1 activation and IL-1β release in response to NLRP3- but not NLRC4- or AIM2-activating stimuli. This RACK1 function is independent of its ribosomal binding activity. Mechanistically, RACK1 promotes the active conformation of NLRP3 induced by activating stimuli and subsequent inflammasome assembly. These results demonstrate that RACK1 is a critical mediator for NLRP3 inflammasome activation.
| S-EPMC7709964 | biostudies-literature
Project description:RAR-related orphan receptor γ (RORγ) is a nuclear receptor that plays an essential role in the development of T helper 17 (Th17) cells of the adaptive immune system. The NLRP3 inflammasome is a component of the innate immune system that processes interleukin (IL)-1β into a mature cytokine. Elevated activity of the NLRP3 inflammasome contributes to the progression of an array of inflammatory diseases. Bone marrow-derived macrophages (BMDMs) isolated from RORγ-null mice displayed reduced capacity to secrete IL-1β, and they also displayed a reduction in Nlrp3 and Il1b gene expression. Examination of the promoters of the Il1b and Nlrp3 genes revealed multiple putative ROR response elements (ROREs) that were occupied by RORγ. RORγ inverse agonists were effective inhibitors of the inflammasome. RORγ inverse agonists suppressed lipopolysaccharide (LPS)/ATP-stimulated IL-1β secretion and expression of Il1b and Nlrp3 in BMDMs. Additionally, the ability of the RORγ inverse agonists to suppress IL-1β secretion was lost in Nlrp3-null macrophages. The potential for targeting the NLRP3 inflammasome in vivo using RORγ inverse agonists was examined in two models: LPS-induced sepsis and fulminant hepatitis. Pharmacological inhibition of RORγ activity reduced plasma IL-1β as well as IL-1β production by peritoneal macrophages in a model of LPS-induced sepsis. Additionally, RORγ inverse agonists reduced mortality in an LPS/d-galactosamine-induced fulminant hepatitis mouse model. These results illustrate a major role for RORγ in regulation of innate immunity via modulation of NLRP3 inflammasome activity. Furthermore, these data suggest that inhibiting the NLRP3 inflammasome with RORγ inverse agonists may be an effective method to treat NLRP3-associated diseases.
| S-EPMC6322869 | biostudies-literature
Project description:BackgroundInterleukin-1? (IL-1?) is one of the most important cytokine secreted by activated microglia as it orchestrates the vicious cycle of inflammation by inducing the expression of various other pro-inflammatory cytokines along with its own production. Microglia-mediated IL-1? production is a tightly regulated mechanism which involves the activation of nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome pathway. Our previous study suggests the critical role of heat shock protein 60 (HSP60) in IL-1?-induced inflammation in microglia through TLR4-p38 MAPK axis. However, whether HSP60 regulates endogenous IL-1? production is not known. Therefore, to probe the underlying mechanism, we elucidate the role of HSP60 in endogenous IL-1? production.MethodsWe used in vitro (N9 murine microglial cells) and in vivo (BALB/c mouse) models for our study. HSP60 overexpression and knockdown experiment was done to elucidate the role of HSP60 in endogenous IL-1? production by microglia. Western blotting and quantitative real-time PCR was performed using N9 cells and BALB/c mice brain, to analyze various proteins and transcript levels. Reactive oxygen species levels and mitochondrial membrane depolarization in N9 cells were analyzed by flow cytometry. We also performed caspase-1 activity assay and enzyme-linked immunosorbent assay to assess caspase-1 activity and IL-1? production, respectively.ResultsHSP60 induces the phosphorylation and nuclear localization of NF-?B both in vitro and in vivo. It also induces perturbation in mitochondrial membrane potential and enhances reactive oxygen species (ROS) generation in microglia. HSP60 further activates NLRP3 inflammasome by elevating NLRP3 expression both at RNA and protein levels. Furthermore, HSP60 enhances caspase-1 activity and increases IL-1? secretion by microglia. Knockdown of HSP60 reduces the IL-1?-induced production of IL-1? both in vitro and in vivo. Also, we have shown for the first time that knockdown of HSP60 leads to decreased IL-1? production during Japanese encephalitis virus (JEV) infection, which eventually leads to decreased inflammation and increased survival of JEV-infected mice.ConclusionHSP60 mediates microglial IL-1? production by regulating NLRP3 inflammasome pathway and reduction of HSP60 leads to reduction of inflammation in JEV infection.
| S-EPMC5994257 | biostudies-literature
Project description:We previously reported that tumor inflammasomes play a key role in tumor control and act as favorable prognostic markers in nasopharyngeal carcinoma (NPC). Activated inflammasomes frequently form distinguishable specks and govern the cellular secretion of IL-1β. However, we know little about the biological and biochemical differences between cells with and without apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) speck formation. In this study, we used proteomic iTRAQ analysis to analyze the proteomes of NPC cells that differ in their ASC speck formation upon cisplatin treatment. We identified proteins that were differentially over-expressed in cells with specks, and found that they fell into two Gene ontology (GO) pathways: mitochondrial oxidative phosphorylation (OxPhos) and ubiquinone metabolism. We observed up-regulation of various components of the OxPhos machinery (including NDUFB3, NDUFB8 and ATP5B), and subsequently found that these changes lead to mitochondrial ROS (mtROS) production, which promotes the formation and activation of NLRP3 inflammasomes and subsequent pyroptosis. In NPC patients, better local recurrence-free survival was significantly associated with high-level expression of NDUFB8 (p = 0.037) and ATP5B (p = 0.029), as examined using immunohistochemistry. However, there were no significant associations between the expression of NDUFB8 and ATP5B with overall survival of NPC patients. Together, our results demonstrate that up-regulated mitochondrial OxPhos components are strongly associated with NLRP3 inflammasome activation in NPC. Our findings further suggest that high-level expression of OxPhos components could be markers for local recurrence and/or promising therapeutic targets in patients with NPC.
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Project description:The cytosolic pattern recognition receptor NLRP3 senses host-derived danger signals and certain microbe-derived products in both humans and rodents. NLRP3 activation assembles an inflammasome complex that contains the adapter proteins ASC and caspase-1, whose activation triggers the maturation and release of the proinflammatory cytokines IL-1β and IL-18. S5 phosphorylation of NLRP3 prevents its oligomerization and activation, whereas dephosphorylation of this residue by the phosphatase PP2A allows NLRP3 activation. However, the protein kinase that mediates NLRP3 S5 phosphorylation is unknown. In this study, we show that AKT associates with NLRP3 and phosphorylates it on S5, limiting NLRP3 oligomerization. This phosphorylation event also stabilizes NLRP3 by reducing its ubiquitination on lysine 496, which inhibits its proteasome-mediated degradation by the E3 ligase Trim31. Pharmacologic manipulation of AKT kinase activity reciprocally modulates NLRP3 inflammasome-mediated IL-1β production. Inhibition of AKT reduced IL-1β production following the i.p. injection of LPS into mice. We propose that AKT, Trim31, and PP2A together modulate NLRP3 protein levels and the tendency to oligomerize, thereby setting a tightly regulated threshold for NLRP3 activation.
| S-EPMC7541779 | biostudies-literature
Project description:Activation of the NLRP3 inflammasome and subsequent maturation of IL-1β have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis. We investigated the role of vimentin, a type III intermediate filament, in this process using three well-characterized murine models of ALI known to require NLRP3 inflammasome activation. We demonstrate that central pathophysiologic events in ALI (inflammation, IL-1β levels, endothelial and alveolar epithelial barrier permeability, remodelling and fibrosis) are attenuated in the lungs of Vim(-/-) mice challenged with LPS, bleomycin and asbestos. Bone marrow chimeric mice lacking vimentin have reduced IL-1β levels and attenuated lung injury and fibrosis following bleomycin exposure. Furthermore, decreased active caspase-1 and IL-1β levels are observed in vitro in Vim(-/-) and vimentin-knockdown macrophages. Importantly, we show direct protein-protein interaction between NLRP3 and vimentin. This study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the NLRP3 inflammasome.
| S-EPMC4358756 | biostudies-literature
Project description:The NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome is triggered by various stimuli. Whether the NLRP3 inflammasome is activated during the monocyte clearing of aged or damaged erythrocytes is unknown. This work aimed to determine whether the NLRP3 inflammasome is activated during the THP-1 cell engulfing of aged erythrocytes. In the study, THP-1 cells were treated with PMA and then coincubated with untreated red blood cells (RBCs), 42 °C-treated RBCs, immunoglobulin G (IgG) anti-D-sensitized RBCs, Rhnull/Rhmod RBC sample, hemoglobin, and RBC ghost. The activation of the NLRP3 inflammasome and production of some proinflammatory cytokines were determined using immunoblotting, cytometric bead array, and digital PCR. An NLRP3 inflammasome inhibitor was also used to evaluate the alteration of the NLRP3 activation and RBC clearance rate. The untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin induced the THP-1-cell-mediated activation of the NLRP3 inflammasome and the production of inflammatory cytokines. The RBC clearance rate exhibited a positive correlation with the expression of proinflammatory cytokines. The NLRP3 inflammasome inhibitor reduced the NLRP3 activation and RBC phagocytosis rate. The NLRP3 inflammasome was activated during the clearance of the aged erythrocytes through unopsonized and opsonized pathways. However, the mechanism of such phenomenon needs to be further elucidated. Such mechanism may provide new insight into the assessment of the safety of transfusing long-storage RBC based on cytokine levels.
| S-EPMC6061012 | biostudies-literature