Molecular characterization and functional study of the NLRP3 inflammasome genes in Tetraodon nigroviridis
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瞄准：为了检验 Nalp3 的激活， inflammasome 和它的下游的目标追随者 lipopolysaccharide (LPS ) 在肝导致了刺激。方法：Six-to-eight-week-old C57BL/6 食物喂的老鼠与 0.5 g/g bodyweight LPS intraperitoneally 被注射并且牺牲了 2， 4， 6， 18 或 24 h 以后。导致 LPS 的肝损坏被生物化学的试金证实检测丙氨酸 aminotransferase (中高音) 层次。决定在肝的 LPS 刺激是否导致了 inflammasome 的激活，即时量的聚合酶链反应被用来计算 Nalp3 inflammasome 的部件的 mRNA 表达式。连接酶的 immunosorbent 试金被用来决定 Nalp3 inflammasome 的几个下游的目标的蛋白质表示层次，包括 caspase-1 和 caspase-1， interleukin (IL )-1 和 IL-18 的二个 cytokine 目标。结果：我们发现那 LPS 注射处于由提高的中高音层次显示了的肝损坏结果。这与 mRNA 和 proinflammatory cytokine 肿瘤坏死因素(TNF ) 的蛋白质层次的重要增加被联系 - 在在浆液的 TNF 的肝，以及增加的层次。我们证明 LPS 刺激为 inflammasome 的所有受体部件在肝导致了 mRNA 层次的 upregulation，包括 Nalp3， Nalp1， pannexin-1 和适配器分子联系 apoptosis 像斑点， caspase 招募域域包含蛋白质。我们也为 caspase-1 发现 mRNA 和蛋白质的层次增加， inflammasome 的一个下游的目标。另外， LPS 挑战为 caspase-1， IL-1 和 IL-18 的二个 cytokine 目标在肝导致了 mRNA 和蛋白质的增加的层次。有趣地， pre-IL-1 和 pre-IL-18 的实质的基线表示在肝被发现。Inflammasome 和 caspase-1 激活被 IL-1 和 IL-18 的活跃形式的重要增加在 LPS 刺激以后显示。结论：我们的结果证明 Nalp3 inflammasome 是 upregulated 并且响应 LPS 刺激在肝激活。
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Interleukin 18
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Interleukin-1 beta (IL-1β) and its key regulator, the inflammasome, are suspected to play a role in the neuroinflammation observed in Alzheimer's disease (AD); no conclusive data are nevertheless available in AD patients. mRNA for inflammasome components (NLRP1, NLRP3, PYCARD, caspase 1, 5 and 8) and downstream effectors (IL-1β, IL-18) was up-regulated in severe and MILD AD. Monocytes co-expressing NLRP3 with caspase 1 or caspase 8 were significantly increased in severe AD alone, whereas those co-expressing NLRP1 and NLRP3 with PYCARD were augmented in both severe and MILD AD. Activation of the NLRP1 and NLRP3 inflammasomes in AD was confirmed by confocal microscopy proteins co-localization and by the significantly higher amounts of the pro-inflammatory cytokines IL-1β and IL-18 being produced by monocytes. In MCI, the expression of NLRP3, but not the one of PYCARD or caspase 1 was increased, indicating that functional inflammasomes are not assembled in these individuals: this was confirmed by lack of co-localization and of proinflammatory cytokines production. The activation of at least two different inflammasome complexes explains AD-associated neuroinflammation. Strategies targeting inflammasome activation could be useful in the therapy of AD.
NLRP1
AIM2
Proinflammatory cytokine
Interleukin 18
NALP3
Pyrin domain
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Synucleinopathies such as Parkinson's disease (PD) are hallmarked by α-synuclein (α-syn) pathology and neuroinflammation. This neuroinflammation involves activated microglia with increased secretion of interleukin-1β (IL-1β). The main driver of IL-1β secretion from microglia is the NLRP3 inflammasome. A critical link between microglial NLRP3 inflammasome activation and the progression of both α-syn pathology and dopaminergic neurodegeneration has been identified in various PD models in vivo. α-Syn is known to activate the microglial NLRP3 inflammasome in murine models, but its relationship to this inflammasome in human microglia has not been established. In this study, IL-1β secretion from primary mouse microglia induced by α-syn fibrils was dependent on NLRP3 inflammasome assembly and caspase-1 activity, as previously reported. We show that exposure of primary human microglia to α-syn fibrils also resulted in significant IL-1β secretion that was dependent on inflammasome assembly and involved the recruitment of caspase-1 protein to inflammasome scaffolds as visualized with superresolution microscopy. While canonical IL-1β secretion was clearly dependent on caspase-1 enzymatic activity, this activity was less clearly involved for α-syn-induced IL-1β secretion from human microglia. This work presents similarities between primary human and mouse microglia in the mechanisms of activation of the NLRP3 inflammasome by α-syn, but also highlights evidence to suggest that there may be a difference in the requirement for caspase-1 activity in IL-1β output. The data represent a novel characterization of PD-related NLRP3 inflammasome activation in primary human microglia and further implicate this mechanism in the pathology underlying PD.
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To the Editor We read the article of Osuka et al. (1) entitled “A Protective Role for Inflammasome Activation Following Injury” with great interest. However, we are concerned that the authors have not sufficiently ruled out the possibility that the major effects attributed to inflammasome inhibition were merely due to the solvent used. The authors describe inflammasome activation in burned mice 1 day after injury as revealed by caspase 1 activation and increased interleukin 1β (IL-1β) production. Interestingly, the data suggest that inhibiting caspase 1 activity—and thereby inhibiting inflammasome activation—with the Ac-YVAD-cmk peptide did not reduce inflammation as expected. On the contrary, it caused a significantly higher mortality and increased expression of the proinflammatory cytokines IL-6 and IL-33 as compared with untreated burned mice. The authors therefore conclude that inflammasome activation might have a protective role following severe injury. Inhibition of (pro)caspase 1 activity shifts the proportion between pro–caspase 1 and mature processed caspase 1 toward the pro form. Interestingly, a potential role of enzymatically inactive pro–caspase 1 in activating nuclear factor κB (NF-κB) via receptor-interacting protein 2 (RIP2) has been described (2, 3). Interleukin 6 transcription is regulated by an NF-κB–sensitive promotor. Hence, elevated IL-6 expression after inhibition of (pro)caspase 1 could indicate NF-κB activation and might be mediated by pro–caspase 1. The authors show this correlation for the first time in an in vivo model. According to this model, inflammasome formation could reduce the amount of pro–caspase 1 available for NF-κB activation via RIP2 and thereby downregulate this proinflammatory pathway (3). In the current study, (pro)caspase 1 activation was inhibited by injecting Ac-YVAD-cmk intraperitoneally. Ac-YVAD-cmk is a peptide inhibitor of (pro)caspase 1, which is typically dissolved in dimethyl sulfoxide (DMSO). Dimethyl sulfoxide is used in a variety of fields. For example, it is commonly applied as a cryoprotectant of cultured cells and as a solvent for hydrophobic compounds in biological studies. However, it is important to note that DMSO is biologically active (4). Although it can often be used safely as a drug vehicle there is a lot of evidence that it can have undesirable anti-inflammatory and/or proinflammatory, nonspecific, and toxic effects in vitro and in vivo (5). For instance, treatment with 1% DMSO activates NF-κB in THP-1 and HL-60 cells (6, 7), and DMSO increases lipopolysaccharide-induced IL-1β secretion in a dose dependent-manner in human peripheral blood mononuclear cells and mice (8). Furthermore, it induces neurophysiological changes in rats and leads to lymphocyte apoptosis in murine lymphoid organs (9, 10). These effects are undesirable and unpredictable when DMSO is used as solvent. Therefore, control experiments of the effects of DMSO itself always have to be performed. Unfortunately, in the study of Osuka et al., the control group was treated with saline solution. Thus, DMSO could have contributed to the enhanced expression of proinflammatory cytokines and the increased mortality in the Ac-YVAD-cmk–treated burned-mice group. Therefore, the effect of Ac-YVAD-cmk and the conclusions drawn by the authors remain unsettled. Stefan Winkler Michael C. Heyman Marcus Franke Anika Martin Joachim Roesler Angela Rösen-Wolff Department of Pediatrics University Medical Center Carl Gustav Carus Dresden, Germany
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Inflammasomes activate the protease caspase-1, which cleaves interleukin-1β and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1C284A, we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1C284A, we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis.
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The NLRP3 inflammasome responds to infection and tissue damage, and rapidly escalates the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. How the NLRP3 inflammasome is negatively regulated is poorly understood. Here we show that NLRP3 inflammasome activation is suppressed by sumoylation. NLRP3 is sumoylated by the SUMO E3-ligase MAPL, and stimulation-dependent NLRP3 desumoylation by the SUMO-specific proteases SENP6 and SENP7 promotes NLRP3 activation. Defective NLRP3 sumoylation, either by NLRP3 mutation of SUMO acceptor lysines or depletion of MAPL, results in enhanced caspase-1 activation and IL-1β release. Conversely, depletion of SENP7 suppresses NLRP3-dependent ASC oligomerisation, caspase-1 activation and IL-1β release. These data indicate that sumoylation of NLRP3 restrains inflammasome activation, and identify SUMO proteases as potential drug targets for the treatment of inflammatory diseases.
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Abstract The inflammasome pathway functions to regulate caspase‐1 activation in response to a broad range of stimuli. Caspase‐1 activation is required for the maturation of the pivotal pro‐inflammatory cytokines of the pro‐IL‐1β family. In addition, caspase‐1 activation leads to a certain type of cell death known as pyroptosis. Activation of the inflammasome has been shown to play a critical role in the recognition and containment of various microbial pathogens, including the intracellularly replicating Listeria monocytogenes ; however, the inflammasome pathways activated during L. monocytogenes infection are only poorly defined. Here, we demonstrate that L. monocytogenes activates both the NLRP3 and the AIM2 inflammasome, with a predominant involvement of the AIM2 inflammasome. In addition, L. monocytogenes ‐triggered cell death was diminished in the absence of both AIM2 and NLRP3, and is concomitant with increased intracellular replication of L. monocytogenes . Altogether, these data establish a role for DNA sensing through the AIM2 inflammasome in the detection of intracellularly replicating bacteria.
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Inflammasome activation culminates in activation of caspase‐1, which leads to the maturation and subsequent release of cytokines of the interleukin 1 (IL‐1) family and results in a particular form of cell death known as pyroptosis. In addition, in the murine system, a so‐called non‐canonical inflammasome involving caspase‐11 has been described that directly responds to cytosolic LPS. Here, we show that the human monocytic cell line THP1 activates the inflammasome in response to cytosolic LPS in a TLR4‐independent fashion. This response is mediated by caspase‐4 and accompanied by caspase‐1 activation, pyroptosis, and IL‐1β maturation. In addition to caspase‐4, efficient IL‐1β conversion upon intracellular LPS delivery relies on potassium efflux, NLRP3, ASC, and caspase‐1, indicating that although caspase‐4 activation alone is sufficient to induce pyroptosis, this process depends on the NLRP3 inflammasome activation to drive IL‐1β maturation. Altogether, this study provides evidence for the presence of a non‐canonical inflammasome in humans and its dependence on caspase‐4.
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