Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome

In a recent report, we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, C. albicans Sap2 and Sap6 potently induced IL‐1β, TNF‐α, and IL‐6 production. Here, we demonstrate that Sap2 and Sap6 proteins trigger IL‐1β and IL‐18 production through inflammasome activation. This occurs via NLRP3 and caspase‐1 activation, which cleaves pro‐IL‐1β into secreted bioactive IL‐1β, a cytokine that was induced by Saps in monocytes, in monocyte‐derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL‐1β. Inflammasome activation required Sap internalization via a clathrin‐dependent mechanism, intracellular induction of K⁺ efflux, and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS‐ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.     

Complement C5a potentiates uric acid crystal‐induced IL‐1β production

Anaphylatoxin C5a released upon complement activation is associated with both acute and chronic inflammations such as gout. The pathogenesis of gout was identified as uric acid crystal deposition in the joints that activates inflammasome, leading to IL‐1β release. However, little is known about the interaction between complement activation and monosodium urate/uric acid (MSU) crystal‐induced inflammasome activation or IL‐1β production. Here, we report that MSU crystal‐induced proinflammatory cytokines/chemokines in human whole blood is predominantly regulated by C5a through its interaction with C5a receptor. C5a induces pro‐IL‐1β and IL‐1β production in human primary monocytes, and potentiates MSU or cholesterol crystals in IL‐1β production. This potentiation is caspase‐1 dependent and requires intracellular Ca²⁺ mobilization, K⁺ efflux, and cathepsin B activity. Our results provide insight into the role of C5a as an endogenous priming signal that is required for the initiation of uric acid crystal‐induced IL‐1β production. C5a could potentially be a therapeutic target together with IL‐1β antagonists for the treatment of complement‐dependent and inflammasome‐associated diseases.     

Damage control: Management of cellular stress by the NLRP3 inflammasome

The NLRP3 inflammasome plays a critical role in regulating inflammatory and cell death pathways in response to a diverse array of stimuli. Activation of the NLRP3 inflammasome results in activation of the cysteine protease caspase‐1 and the subsequent processing and secretion of the proinflammatory cytokines IL‐1β and IL‐18. In this issue of the European Journal of Immunology, Licandro et al. [Eur. J. Immunol. 2013. 43, 2126–2137] show that the NLRP3 inflammasome contributes to oxidative DNA damage. In addition, activation of the NLRP3 inflammasome modulates a number of pathways involved in DNA damage repair, cell cycle, and apoptosis, suggesting a novel role for the NLRP3 inflammasome in DNA damage responses following cellular stress.     

Caspase‐4 mediates non‐canonical activation of the NLRP3 inflammasome in human myeloid cells

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.     

Inhibition of NLRP3 inflammasome: a new protective mechanism of cinnamaldehyde in endotoxin poisoning of mice

Context: Cinnamaldehyde (CA) has a protective effect in endotoxin poisoning of mice, but there is no direct evidence for the protective effect of CA through inhibition of NLRP3 inflammasome activation in endotoxin poisoning of mice.

Objective: We aimed to investigate the protective mechanism of CA in endotoxin poisoned mice through NLRP3 inflammasome.

Materials and methods: First, we evaluated the anti-inflammatory effect of CA in phorbol-12-myristate acetate–differentiated THP-1 cells through the NLRP3 inflammasome. Second, in a mouse model of lipopolysaccharide (LPS)-induced endotoxin poisoning, CA was administrated for 5 d (once a day) before the 15?mg/kg LPS challenge. Then, the levels of IL-1β in serum were measured, and the effect of CA on the NLRP3 inflammasome activation and the expression of cathepsin B and P2X7R proteins in lung were explored.

Results: In vitro, CA decreased the levels of p20, pro-IL-1β and IL-1β in cell culture supernatants, as well as the expression of NLRP3 and IL-1β mRNA in cells. In vivo, CA decreased IL-1β production in serum. Furthermore, CA suppressed LPS-induced NLRP3, p20, Pro-IL-1β, P2X7 receptor (P2X7R) and cathepsin B protein expression in lung, as well as the expression of NLRP3 and IL-1β mRNA.

Conclusions: CA has a protective effect in the endotoxin poisoned mice through the inhibition of NLRP3 inflammasome activation. Furthermore, CA suppresses the NLRP3 inflammasome activation by inhibiting the expression of cathepsin B and P2X7R protein expression. CA can be considered as a potential therapeutic candidate for diseases involved in endotoxin poisoning such as sepsis.     

Negative regulation of Nod‐like receptor protein 3 inflammasome activation by T cell Ig mucin‐3 protects against peritonitis

The Nod‐like receptor protein 3 (NLRP3) inflammasome plays roles in host defence against invading pathogens and in the development of autoimmune damage. Strict regulation of these responses is important to avoid detrimental effects. Here, we demonstrate that T cell Ig mucin‐3 (Tim‐3), an immune checkpoint inhibitor, inhibits NLRP3 inflammasome activation by damping basal and lipopolysaccharide‐induced nuclear factor‐κB‐mediated up‐regulation of NLRP3 and interleukin‐1β during the priming step and basal and ATP/lipopolysaccharide‐induced ATP production, K⁺ efflux, and reactive oxygen species production during the activation step. Residues Y256/Y263 in the C‐terminal region of Tim‐3 are required for these inhibitory effects on the NLRP3 inflammasome. In mice with alum‐induced peritonitis, blockade of Tim‐3 exacerbates peritonitis by overcoming the inhibitory effect of Tim‐3 on NLRP3 inflammasome activation, while transgenic expression of Tim‐3 attenuates inflammation by inhibiting NLRP3 inflammasome activation. Our results show that Tim‐3 is a critical negative regulator of NLRP3 inflammasome and provides a potential target for intervention of diseases with uncontrolled inflammasome activation.     

Potassium efflux fires the canon: Potassium efflux as a common trigger for canonical and noncanonical NLRP3 pathways

Murine caspase‐11 and its human orthologues, caspase‐4 and caspase‐5, activate an inflammatory response following cytoplasmic recognition of cell wall constituents from Gram‐negative bacteria, such as LPS. This inflammatory response involves pyroptotic cell death and the concomitant release of IL‐1α, as well as the production of IL‐1β and IL‐18 through the noncanonical NLR family, pyrin domain containing 3 (NLRP3) pathway. This commentary discusses three papers in this issue of the European Journal of Immunology that advance our understanding of the roles of caspase‐11, ‐4, and ‐5 in the noncanonical pathway. By utilizing the new gene editing technique, clustered regularly interspaced short palindromic repeats (CRISPR), as well as sensitive cell imaging techniques, these papers establish that cytoplasmic LPS‐dependent IL‐1β production requires the NLRP3 inflammasome and that its activation is dependent on K⁺ efflux, whereas IL‐1α release and pyroptotic cell death pathways are NLRP3‐independent. These findings expand on previous research implicating K⁺ efflux as the principal trigger for NLRP3 activation and suggest that canonical and noncanonical NLRP3 pathways are not as dissimilar as first thought.     

Mycoplasma pneumoniae lipids license TLR‐4 for activation of NLRP3 inflammasome and autophagy to evoke a proinflammatory response

Mycoplasma pneumoniae is an obligate pathogen that causes pneumonia, tracheobronchitis, pharyngitis and asthma in humans. It is well recognized that membrane lipoproteins are immunostimulants exerting as lipopolysaccharides (LPS) and play a crucial role in the pathogenesis of inflammatory responses upon M. pneumoniae infection. Here, we report that the M. pneumoniae‐derived lipids are another proinflammatory agents. Using an antibody‐neutralizing assay, RNA interference or specific inhibitors, we found that Toll‐like receptor 4 (TLR‐4) is essential for M. pneumoniae lipid‐induced tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β production. We also demonstrate that NLR family pyrin domain containing 3 inflammasome (NLRP3) inflammasome, autophagy and nuclear factor kappa B (NF‐κB)‐dependent pathways are critical for the secretion of proinflammatory cytokines, while inhibition of TLR‐4 significantly abrogates these events. Further characterization revealed that autophagy‐mediated inflammatory responses involved the activation of NF‐κB. In addition, the activation of NF‐κB promoted lipid‐induced autophagosome formation, as revealed by assays using pharmacological inhibitors, 3‐methyladenine (3‐MA) and Bay 11‐7082, or silencing of atg5 and beclin‐1. These findings suggest that, unlike the response to lipoprotein stimulation, the inflammation in response to M. pneumoniae lipids is mediated by the TLR‐4 pathway, which subsequently initiates the activation of NLRP3 inflammasome and formation of a positive feedback loop between autophagy and NF‐κB signalling cascade, ultimately promoting TNF‐α and Il‐1β production in macrophages.     

The protein kinase PKR is critical for LPS‐induced iNOS production but dispensable for inflammasome activation in macrophages

Inflammasomes are multi‐protein platforms that drive the activation of caspase‐1 leading to the processing and secretion of biologically active IL‐1β and IL‐18. Different inflammasomes including NOD‐like receptor (NLR) family pyrin domain‐containing 3 (NLRP3), NLR caspase‐recruitment domain‐containing 4 (NLRC4) and absent in melanoma 2 (AIM2) are activated and assembled in response to distinct microbial or endogenous stimuli. However, the mechanisms by which upstream stimuli trigger inflammasome activation remain poorly understood. Double‐stranded RNA‐activated protein kinase (PKR), a protein kinase activated by viral infection, has been recently shown to be required for the activation of the inflammasomes. Using macrophages from two different mouse strains deficient in PKR, we found that PKR is important for the induction of the inducible nitric oxide synthase (iNOS). However, PKR was dispensable for caspase‐1 activation, processing of pro‐IL‐1β/IL‐18 and secretion of IL‐1β induced by stimuli that trigger the activation of NLRP3, NLRC4 and AIM2. These results indicate that PKR is not required for inflammasome activation in macrophages.     

Histones trigger sterile inflammation by activating the NLRP3 inflammasome

Sterile cell death mediated inflammation is linked to several pathological disorders and involves danger recognition of intracellular molecules released by necrotic cells that activate different groups of innate pattern recognition receptors. Toll‐like receptors directly interact with their extrinsic or intrinsic agonists and induce multiple proinflammatory mediators. In contrast, the NLRP3 inflammasome is rather thought to represent a downstream element integrating various indirect stimuli into proteolytic cleavage of interleukin (IL)–1β and IL‐18. Here, we report that histones released from necrotic cells induce IL‐1β secretion in an NLRP3–ASC‐caspase‐1‐dependent manner. Genetic deletion of NLRP3 in mice significantly attenuated histone‐induced IL‐1β production and neutrophil recruitment. Furthermore, necrotic cells induced neutrophil recruitment, which was significantly reduced by histone‐neutralizing antibodies or depleting extracellular histones via enzymatic degradation. These results identify cytosolic uptake of necrotic cell‐derived histones as a triggering mechanism of sterile inflammation, which involves NLRP3 inflammasome activation and IL‐1β secretion via oxidative stress.     

Interleukin‐17A participates in podocyte injury by inducing IL‐1β secretion through ROS‐NLRP3 inflammasome‐caspase‐1 pathway

Studies show that the Th17/IL ‐17A axis plays an important role in the pathogenesis of kidney diseases. Previously, we also showed that IL ‐17A may play a role in the pathogenesis of primary nephrotic syndrome; however, the underlying mechanism(s) is unclear. The aim of this study was to explore the molecular mechanism of IL ‐17A‐inducing podocyte injury in vitro. In this study, the NLRP 3 inflammasome activation and the morphology of podocytes were detected by Western blot and immunofluorescence. The results showed that podocytes persistently expressed IL ‐17A receptor and that NLRP 3 inflammasome in these cells was activated upon exposure to IL ‐17A. Also, activity of caspase‐1 and secretion of IL ‐1β increased in the presence of IL ‐17A. In addition, IL ‐17A disrupted podocyte morphology by decreasing expression of podocin and increasing expression of desmin. Blockade of intracellular ROS or inhibition of caspase‐1 prevented activation of the NLRP 3 inflammasome, thereby restoring podocyte morphology. Taken together, the results suggest that IL ‐17A induces podocyte injury by activating the NLRP 3 inflammasome and IL ‐1β secretion and contributes to disruption of the kidney's filtration system.     

Effect of enterohaemorrhagic Escherichia coli O157:H7‐specific enterohaemolysin on interleukin‐1β production differs between human and mouse macrophages due to the different sensitivity of NLRP3 activation

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection in humans can cause acute haemorrhagic colitis and severe haemolytic uraemic syndrome. The role of enterohaemolysin (Ehx) in the pathogenesis of O157:H7‐mediated disease in humans remains undefined. Recent studies have revealed the importance of the inflammatory response in O157:H7 pathogenesis in humans. We previously reported that Ehx markedly induced interleukin‐1β (IL‐1β) production in human macrophages. Here, we investigated the disparity in Ehx‐induced IL‐1β production between human and mouse macrophages and explored the underlying mechanism regarding the activation of NOD‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasomes. In contrast to the effects on human differentiated THP‐1 cells and peripheral blood mononuclear cells, Ehx exerted no effect on IL‐1β production in mouse macrophages and splenocytes because of a disparity in pro‐IL‐1β cleavage into mature IL‐1β upon caspase‐1 activation. Additionally, Ehx significantly contributed to O157:H7‐induced ATP release from THP‐1 cells, which was not detected in mouse macrophages. Confocal microscopy demonstrated that Ehx was a key inducer of cathepsin B release in THP‐1 cells but not in mouse IC‐21 cells upon O157:H7 challenge. Inhibitor experiments indicated that O157:H7‐induced IL‐1β production was largely dependent upon caspase‐1 activation and partially dependent upon ATP signalling and cathepsin B release, which were both involved in NLRP3 activation. Moreover, inhibition of K⁺ efflux drastically diminished O157:H7‐induced IL‐1β production and cytotoxicity. The findings in this study may shed light on whether and how the Ehx contributes to the development of haemolytic uraemic syndrome in human O157:H7 infection.     

The purinergic 2X7 receptor participates in renal inflammation and injury induced by high‐fat diet: possible role of NLRP3 inflammasome activation

Renal disease associated with type 2 diabetes and the metabolic syndrome is characterized by a distinct inflammatory phenotype. The purinergic 2X₇ receptor (P2X₇R) and the nucleotide‐binding and oligomerization domain‐like receptor containing a pyrin domain 3 (NLRP3) inflammasome have been separately shown to play a role in two models of non‐metabolic chronic kidney disease. Moreover, the NLRP3 inflammasome has been implicated in chronic low‐grade sterile inflammation characterizing metabolic disorders, though the mechanism(s) involved in inflammasome activation under these conditions are still unknown. We investigated the role of P2X₇R (through activation of the NLRP3 inflammasome) in renal inflammation and injury induced by a high‐fat diet, an established model of the metabolic syndrome. On a high‐fat diet, mice lacking P2X₇R developed attenuated renal functional and structural alterations as well as reduced inflammation, fibrosis, and oxidative/carbonyl stress, as compared with wild‐type animals, in the absence of significant differences in metabolic parameters. This was associated with blunted up‐regulation of the NLRP3 inflammasome components NLRP3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), pro‐caspase 1, pro‐interleukin (IL)‐1β, and pro‐IL‐18, as well as reduced inflammasome activation, as evidenced by decreased formation of mature caspase 1, whereas mature IL‐1β and IL‐18 were not detected. Up‐regulated expression of NLRP3 and pro‐caspase 1, post‐translational processing of pro‐caspase‐1, and release of IL‐18 in response to lipopolysaccharide + 2′(3′)‐O‐(4‐benzoylbenzoyl)ATP were attenuated by P2X₇R silencing in cultured mouse podocytes. Protein and mRNA expression of P2X₇R, NLRP3, and ASC were also increased in kidneys from subjects with type 2 diabetes and the metabolic syndrome, showing histologically documented renal disease. These data provide evidence of a major role for the purinergic system, at least in part through activation of the NLRP3 inflammasome, in the process driving ‘metabolic’ renal inflammation and injury and identify P2X₇R and NLRP3 as novel therapeutic targets. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Caspase‐11 activates a canonical NLRP3 inflammasome by promoting K+ efflux

Recognition of microbe‐associated molecular patterns or endogenous danger signals by a subset of cytosolic PRRs results in the assembly of multiprotein signaling complexes, the so‐called inflammasomes. Canonical inflammasomes are assembled by NOD‐like receptor (NLR) or PYHIN family members and activate caspase‐1, which promotes the induction of pyroptosis and the release of mature interleukin‐1β/‐18. Recently, a noncanonical inflammasome pathway was discovered that results in caspase‐11 activation in response to bacterial lipopolysaccharide (LPS) in the cytosol. Interestingly, caspase‐11 induces pyroptosis by itself, but requires NLRP3, the inflammasome adapter ASC, and caspase‐1 to promote cytokine secretion. Here, we have studied the mechanism by which caspase‐11 controls IL‐1β secretion. Investigating NLRP3/ASC complex formation, we find that caspase‐11 functions upstream of a canonical NLRP3 inflammasome. The activation of NLRP3 by caspase‐11 during LPS transfection is a cell‐intrinsic process and is independent of the release of danger signals. Furthermore, we show that active caspase‐11 leads to a drop of intracellular potassium levels, which is necessary to activate NLRP3. Our study, therefore, sheds new light on the mechanism of noncanonical inflammasome signaling.     

Molecular Mechanism of NLRP3 Inflammasome Activation

The inflammasome is an intracellular multimolecular complex that controls caspase-1 activity in the innate immune system. NLRP3, a member of the NLR family of cytosolic pattern recognition receptors, along with the adaptor protein ASC, mediates caspase-1 activation via assembly of the inflammasome in response to various pathogen-derived factors as well as danger-associated molecules. The active NLRP3 inflammasome drives innate immune response towards invading pathogens and cellular damage, and regulates adaptive immune response. Here, we review identified agonists of the NLRP3 inflammasome and the molecular mechanism by which they induce NLRP3 inflammasome activation. Three signaling pathways involving potassium efflux, generation of reactive oxygen species, and cathepsin B release are discussed.     

Inhibition of the NLRP3 inflammasome by HSP90 inhibitors

Excessive and dysregulated inflammation is known to contribute to disease progression. HSP90 is an intracellular chaperone known to regulate inflammatory processes including the NLRP3 inflammasome and secretion of the pro‐inflammatory cytokine interleukin(IL)‐1β. Here, primarily using an in vitro inflammasome ASC speck assay, and an in vivo model of murine peritonitis, we tested the utility of HSP90 inhibitors as anti‐inflammatory molecules. We report that the HSP90 inhibitor EC144 effectively inhibited inflammatory processes including priming and activation of NLRP3 in vitro and in vivo. A specific inhibitor of the β HSP90 isoform was ineffective suggesting the importance of the α isoform in inflammatory signalling. EC144 inhibited IL‐1β and IL‐6 in vivo when administered orally, and was brain‐penetrant. These data suggest that HSP90 inhibitors may be useful for targeting inflammation in diverse diseases that are worsened by the presence of inflammation.