Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3.
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ABSTRACT:
SUBMITTER: Kang S
PROVIDER: S-EPMC4480782 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background: Oral commensals contribute to microbe-host symbiosis in periodontal homeostasis, and Porphyromonas gingivalis (P. gingivalis) as the keystone pathogen critically accounts for the shift of symbiosis to dysbiosis and periodontal destruction. Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome-mediated interleukin-1β (IL-1β) is significantly involved in periodontal diseases, and notably P. gingivalis enables to modulate the induction and expression of NLRP3. Whereas, the exact mechanism by which NLRP3 inflammasome is regulated in response to commensal and pathogenic bacteria remains unclear. Methods: To examine the expression of IL-1β and NLRPs inflammasome in tissues with severe chronic periodontitis, and further investigate how Caspase-4-dependent non-canonical NLRP3 inflammasome pathways functioned during the interactions of Streptococcus mitis (S. mitis) and P. gingivalis with human THP-1 cells. Results: IL-1β and NLRP3, NLRP6, NLRP12, and absent in melanoma 2 (AIM2) inflammasomes are highly expressed in gingival tissues with severe chronic periodontitis. In human THP-1 cells, P. gingivalis activates the synthesis and secretion of IL-1β to higher levels than S. mitis. Importantly, NLRP3-, Caspase-1-, and Caspase-4-siRNA knockdown THP-1 cells treated with P. gingivalis exhibited a lower expression level of IL-1β as compared to the control cells. In addition, silencing of either CASP4 or CASP1 can lead to a concurrent or reciprocal decrease in the expression of the other. Of note, the IL-1β induction is not affected in the S. mitis-treated THP-1 cells with the silence of NLRP3, Caspase-1, and Caspase-4 genes. Conclusion: NLRP3/Caspase-4 and NLRP3/Caspase-1 dependent IL-1β production may crucially contribute to the dysregulated immuno-inflammatory response in periodontal pathogenesis.
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Project description:RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3-caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1? inflammatory responses independent of MLKL and necroptotic cell death.
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Project description:The assembly and activation of the inflammasomes are tightly regulated by post-translational modifications, including ubiquitin. Deubiquitinases (DUBs) counteract the addition of ubiquitin and are essential regulators of immune signalling pathways, including those acting on the inflammasome. How DUBs control the assembly and activation of inflammasomes is unclear. Here, we show that the DUBs USP7 and USP47 regulate inflammasome activation in macrophages. Chemical inhibition of USP7 and USP47 blocks inflammasome formation, independently of transcription, by preventing ASC oligomerisation and speck formation. We also provide evidence that the ubiquitination status of NLRP3 itself is altered by inhibition of USP7 and USP47. Interestingly, we found that the activity of USP7 and USP47 increased in response to inflammasome activators. Using CRISPR/Cas9 in the macrophage cell line THP-1, we show that inflammasome activation is reduced when both USP7 and USP47 are knocked down. Altogether, these data reveal a new post-transcriptional role for USP47 and USP7 in inflammation by regulating inflammasome activation and the release of the pro-inflammatory cytokines IL-1? and IL-18, and implicate dual USP7 and USP47 inhibitors as potential therapeutic agents for inflammatory disease.
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Project description:Caspase-11 detection of intracellular lipopolysaccharide (LPS) from invasive Gram-negative bacteria mediates noncanonical activation of the NLRP3 inflammasome. While avirulent bacteria do not invade the cytosol, their presence in tissues necessitates clearance and immune system mobilization. Despite sharing LPS, only live avirulent Gram-negative bacteria activate the NLRP3 inflammasome. Here, we found that bacterial mRNA, which signals bacterial viability, was required alongside LPS for noncanonical activation of the NLRP3 inflammasome in macrophages. Concurrent detection of bacterial RNA by NLRP3 and binding of LPS by pro-caspase-11 mediated a pro-caspase-11-NLRP3 interaction before caspase-11 activation and inflammasome assembly. LPS binding to pro-caspase-11 augmented bacterial mRNA-dependent assembly of the NLRP3 inflammasome, while bacterial viability and an assembled NLRP3 inflammasome were necessary for activation of LPS-bound pro-caspase-11. Thus, the pro-caspase-11-NLRP3 interaction nucleated a scaffold for their interdependent activation explaining their functional reciprocal exclusivity. Our findings inform new vaccine adjuvant combinations and sepsis therapy.
| S-EPMC9106893 | biostudies-literature
Project description:The assembly and activation of the inflammasomes are tightly regulated by post-translational modifications, including ubiquitin. Deubiquitinases counteract the addition of ubiquitin and are essential regulators of immune signalling pathways, including those acting on the inflammasome. How deubiquitinases control the assembly and activation of inflammasomes is unclear. Here we show that DUBs USP7 and USP47 regulate inflammasome activation in macrophages. Chemical inhibition of USP7 and USP47 blocks inflammasome formation, independently of transcription, by preventing ASC-oligomerisation and speck formation. We also provide evidence that the ubiquitination status of NLRP3 itself is altered by USP7 and USP47 inhibition. Interestingly, we found that the activity of USP7 and USP47 increased in response to inflammasome activators. Using CRISPR/Cas9 in the macrophage cell line THP-1, we show that inflammasome activation is reduced when both USP7 and USP47 are knocked down. Altogether these data reveal a new post-transcriptional role for USP47 and USP7 in inflammation by regulating inflammasome activation and release of the pro-inflammatory cytokines IL-1? and IL-18, and implicates dual USP7 and USP47 inhibitors as potential therapeutic agents for inflammatory disease.
| S-SCDT-EMBOR-2017-44766V1 | biostudies-other
Project description:Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.
| S-EPMC3708594 | biostudies-literature
Project description:The innate immune system acts as the first line of defense against infection. One key component of the innate immune response to gram-negative bacterial infections is inflammasome activation. The caspase-11 (CASP11)-nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is activated by cytosolic lipopolysaccharide, a gram-negative bacterial cell wall component, to trigger pyroptosis and host defense during infection. Although several cellular signaling pathways have been shown to regulate CASP11-NLRP3 inflammasome activation in response to lipopolysaccharide, the upstream molecules regulating CASP11 activation during infection with live pathogens remain unclear. Here, we report that the understudied caspase-6 (CASP6) contributes to the activation of the CASP11-NLRP3 inflammasome in response to infections with gram-negative bacteria. Using in vitro cellular systems with bone marrow-derived macrophages and 293T cells, we found that CASP6 can directly process CASP11 by cleaving at Asp59 and Asp285, the CASP11 auto-cleavage sites, which could contribute to the activation of CASP11 during gram-negative bacterial infection. Thus, the loss of CASP6 led to impaired CASP11-NLRP3 inflammasome activation in response to gram-negative bacteria. These results demonstrate that CASP6 potentiates activation of the CASP11-NLRP3 inflammasome to produce inflammatory cytokines during gram-negative bacterial infections.
| S-EPMC8633687 | biostudies-literature
Project description:Systemic infections with Gram-negative bacteria are characterized by high mortality rates due to the "sepsis syndrome," a widespread and uncontrolled inflammatory response. Though it is well recognized that the immune response during Gram-negative bacterial infection is initiated after the recognition of endotoxin by Toll-like receptor 4, the molecular mechanisms underlying the detrimental inflammatory response during Gram-negative bacteremia remain poorly defined. Here, we identify a TRIF pathway that licenses NLRP3 inflammasome activation by all Gram-negative bacteria. By engaging TRIF, Gram-negative bacteria activate caspase-11. TRIF activates caspase-11 via type I IFN signaling, an event that is both necessary and sufficient for caspase-11 induction and autoactivation. Caspase-11 subsequently synergizes with the assembled NLRP3 inflammasome to regulate caspase-1 activation and leads to caspase-1-independent cell death. These events occur specifically during infection with Gram-negative, but not Gram-positive, bacteria. The identification of TRIF as a regulator of caspase-11 underscores the importance of TLRs as master regulators of inflammasomes during Gram-negative bacterial infection.
| S-EPMC3660860 | biostudies-literature
Project description:Inflammasomes are caspase-1-activating multiprotein complexes. The mouse nucleotide-binding domain and leucine rich repeat pyrin containing 1b (NLRP1b) inflammasome was identified as the sensor of Bacillus anthracis lethal toxin (LT) in mouse macrophages from sensitive strains such as BALB/c. Upon exposure to LT, the NLRP1b inflammasome activates caspase-1 to produce mature IL-1β and induce pyroptosis. Both processes are believed to depend on autoproteolysed caspase-1. In contrast to human NLRP1, mouse NLRP1b lacks an N-terminal pyrin domain (PYD), indicating that the assembly of the NLRP1b inflammasome does not require the adaptor apoptosis-associated speck-like protein containing a CARD (ASC). LT-induced NLRP1b inflammasome activation was shown to be impaired upon inhibition of potassium efflux, which is known to play a major role in NLRP3 inflammasome formation and ASC dimerization. We investigated whether NLRP3 and/or ASC were required for caspase-1 activation upon LT stimulation in the BALB/c background. The NLRP1b inflammasome activation was assessed in both macrophages and dendritic cells lacking either ASC or NLRP3. Upon LT treatment, the absence of NLRP3 did not alter the NLRP1b inflammasome activity. Surprisingly, the absence of ASC resulted in IL-1β cleavage and pyroptosis, despite the absence of caspase-1 autoprocessing activity. By reconstituting caspase-1/caspase-11(-/-) cells with a noncleavable or catalytically inactive mutant version of caspase-1, we directly demonstrated that noncleavable caspase-1 is fully active in response to the NLRP1b activator LT, whereas it is nonfunctional in response to the NLRP3 activator nigericin. Taken together, these results establish variable requirements for caspase-1 cleavage depending on the pathogen and the responding NLR.
| S-EPMC4260594 | biostudies-literature
Project description:Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.
| S-EPMC5307433 | biostudies-other