Impaired NLRP3 inflammasome activity during fetal development regulates IL‐1β production in human monocytes

Interleukin‐1β (IL‐1β) production is impaired in cord blood monocytes. However, the mechanism underlying this developmental attenuation remains unclear. Here, we analyzed the extent of variability within the Toll‐like receptor (TLR)/NLRP3 inflammasome pathways in human neonates. We show that immature low CD14 expressing/CD16^pos monocytes predominate before 33 weeks of gestation, and that these cells lack production of the pro‐IL‐1β precursor protein upon LPS stimulation. In contrast, high levels of pro‐IL‐1β are produced within high CD14 expressing monocytes, although these cells are unable to secrete mature IL‐1β. The lack of secreted IL‐1β in these monocytes parallels a reduction of NLRP3 induction following TLR stimulation resulting in a lack of caspase‐1 activity before 29 weeks of gestation, whereas expression of the apoptosis‐associated speck‐like protein containing a CARD and function of the P2×7 receptor are preserved. Our analyses also reveal a strong inhibitory effect of placental infection on LPS/ATP‐induced caspase‐1 activity in cord blood monocytes. Lastly, secretion of IL‐1β in preterm neonates is restored to adult levels during the neonatal period, indicating rapid maturation of these responses after birth. Collectively, our data highlight important developmental mechanisms regulating IL‐1β responses early in gestation, in part due to a downregulation of TLR‐mediated NLRP3 expression. Such mechanisms may serve to limit potentially damaging inflammatory responses in a developing fetus.     

NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL‐1β secretion but not in inflammasome activation

Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin‐1 beta (IL‐1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL‐1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL‐1β processing in human neutrophils is dependent on caspase‐1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase‐1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL‐1β; whereas ROS negatively controlled caspase‐1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL‐1β out of the cell, a role never previously described. The complex ROS‐mediated regulation of neutrophil IL‐1β secretion might constitute a physiological mechanism to control IL‐1β‐dependent inflammatory processes where neutrophils play a crucial role.     

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.     

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.     

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.     

High‐density lipoprotein reduces inflammation from cholesterol crystals by inhibiting inflammasome activation

Interleukin‐1β (IL‐1β), a potent pro‐inflammatory cytokine, has been implicated in many diseases, including atherosclerosis. Activation of IL‐1β is controlled by a multi‐protein complex, the inflammasome. The exact initiating event in atherosclerosis is unknown, but recent work has demonstrated that cholesterol crystals (CC) may promote atherosclerosis development by activation of the inflammasome. High‐density lipoprotein (HDL) has consistently been shown to be anti‐atherogenic and to have anti‐inflammatory effects, but its mechanism of action is unclear. We demonstrate here that HDL is able to suppress IL‐1β secretion in response to cholesterol crystals in THP‐1 cells and in human‐monocyte‐derived macrophages. HDL is able to blunt inflammatory monocyte cell recruitment in vivo following intraperitoneal CC injection in mice. HDL appears to modulate inflammasome activation in several ways. It reduces the loss of lysosomal membrane integrity following the phagocytosis of CC, but the major mechanism for the suppression of inflammasome activation by HDL is decreased expression of pro‐IL‐1β and NLRP3, and reducing caspase‐1 activation. In summary, we have described a novel anti‐inflammatory effect of HDL, namely its ability to suppress inflammasome activation by CC by modulating the expression of several key components of the inflammasome.     

Brucella abortus nitric oxide metabolite regulates inflammasome activation and IL‐1β secretion in murine macrophages

NLRP3 inflammasome is a protein complex crucial to caspase‐1 activation and IL‐1β and IL‐18 maturation. This receptor participates in innate immune responses to different pathogens, including the bacteria of genus Brucella. Our group recently demonstrated that Brucella abortus‐induced IL‐1β secretion involves NLRP3 inflammasome and it is partially dependent on mitochondrial ROS production. However, other factors could be involved, such as P2X7‐dependent potassium efflux, membrane destabilization, and cathepsin release. Moreover, there is increasing evidence that nitric oxide acts as a modulator of NLRP3 inflammasome. The aim of this study was to unravel the mechanism of NLRP3 inflammasome activation induced by B. abortus, as well as the involvement of bacterial nitric oxide (NO) as a modulator of this inflammasome pathway. We demonstrated that NO produced by B. abortus can be used by the bacteria to modulate IL‐1β secretion in infected murine macrophages. Additionally, our results suggest that B. abortus‐induced IL‐1β secretion depends on a P2X7‐independent potassium efflux, lysosomal acidification, cathepsin release, mechanisms clearly associated to NLRP3 inflammasome. In summary, our results help to elucidate the molecular mechanisms of NLRP3 activation and regulation during an intracellular bacterial infection.     

Type I NKT‐cell‐mediated TNF‐α is a positive regulator of NLRP3 inflammasome priming

The NLRP3 inflammasome plays a crucial role in the innate immune response to pathogens and exogenous or endogenous danger signals. Its activity must be precisely and tightly regulated to generate tailored immune responses. However, the immune cell subsets and cytokines controlling NLRP3 inflammasome activity are still poorly understood. Here, we have shown a link between NKT‐cell‐mediated TNF‐α and NLRP3 inflammasome activity. The NLRP3 inflammasome in APCs was critical to potentiate NKT‐cell‐mediated immune responses, since C57BL/6 NLRP3 inflammasome‐deficient mice exhibited reduced responsiveness to α‐galactosylceramide. Importantly, NKT cells were found to act as regulators of NLRP3 inflammasome signaling, as NKT‐cell‐derived TNF‐α was required for optimal IL‐1β and IL‐18 production by myeloid cells in response to α‐galactosylceramide, by acting on the NLRP3 inflammasome priming step. Thus, NKT cells play a role in the positive regulation of NLRP3 inflammasome priming by mediating the production of TNF‐α, thus demonstrating another means by which NKT cells control early inflammation.     

Inflammasome activation in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE)

The aptly named inflammasomes are powerful signaling complexes that sense inflammatory signals under a myriad of conditions, including those from infections and endogenous sources. The inflammasomes promote inflammation by maturation and release of the pro‐inflammatory cytokines, IL‐1β and IL‐18. Several inflammasomes have been identified so far, but this review focuses mainly on the NLRP3 inflammasome. By still ill‐defined activation mechanisms, a sensor molecule, NLRP3 (NACHT, LRR and PYD domains‐containing protein 3), responds to danger signals and rapidly recruits ASC (apoptosis‐associated speck‐like protein containing a CARD) and pro‐caspase‐1 to form a large oligomeric signaling platform—the inflammasome. Involvement of the NLRP3 inflammasome in infections, metabolic disorders, autoinflammation, and autoimmunity, underscores its position as a central player in sensing microbial and damage signals and coordinating pro‐inflammatory immune responses. Indeed, evidence in patients with multiple sclerosis (MS) suggests inflammasome activation occurs during disease. Experiments with the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), specifically describe the NLRP3 inflammasome as critical and necessary to disease development. This review discusses recent studies in EAE and MS which describe associations of inflammasome activation with promotion of T cell pathogenicity, infiltration of cells into the central nervous system (CNS) and direct neurodegeneration during EAE and MS.     

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.     

NLRP3 inflammasome: A likely target for the treatment of allergic diseases

Allergic diseases, such as asthma, rhinitis, dermatitis, conjunctivitis, and anaphylaxis, have recently become a global public health concern. According to previous studies, the NLRP3 inflammasome is a multi‐protein complex known to be associated with many inflammatory conditions. In response to allergens or allergen/damage‐associated molecular signals, NLRP3 changes its conformation to allow the assembly of the NLRP3 inflammasome complex and activates caspase‐1, which is an evolutionarily conserved enzyme that proteolytically cleaves other proteins, such as the precursors of the inflammatory cytokines IL‐1β and IL‐18. Subsequently, active caspase‐1 cleaves pro‐IL‐1 and pro‐IL‐18. Recently, accumulating human and mouse experimental evidence has demonstrated that the NLRP3 inflammasome, IL‐1β, and IL‐18 are critically involved in the development of allergic diseases. Furthermore, the application of specific NLRP3 inflammasome inhibitors has been demonstrated in animal models. Therefore, these inhibitors may represent potential therapeutic methods for the management of clinical allergic disorders. This review summarizes findings related to the NLRP3 inflammasome and its related factors and concludes that specific NLRP3 inflammasome inhibitors may be potential therapeutic agents for allergic diseases.     

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.     

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.     

The NLRP3 inflammasome: role in airway inflammation

Asthma is characterized by airway inflammation, airway hyperresponsiveness and airway remodelling. Uncontrolled airway inflammation or repeated asthma exacerbations can lead to airway remodelling, which cannot be reversed by current pharmacological treatment, and consequently lead to decline in lung function. Thus, it is critical to understand airway inflammation in asthma and infectious exacerbation. The inflammasome has emerged as playing a key role in innate immunity and inflammation. Upon ligand sensing, inflammasome components assemble and self‐oligomerize, leading to caspase‐1 activation and maturation of pro‐IL‐1β and pro‐IL‐18 into bioactive cytokines. These bioactive cytokines then play a pivotal role in the initiation and amplification of inflammatory processes. In addition to facilitating the proteolytic activation of IL‐1β and IL‐18, inflammasomes also participate in cell death through caspase‐1‐mediated pyroptosis. In this review, we describe the structure and function of the inflammasome and provide an overview of our current understanding of role of the inflammasome in airway inflammation. We focus on nucleotide‐binding domain and leucine‐rich repeat protein 3 (NLRP3) inflammasome as it is the best‐characterized subtype shown expressed in airway and considered to play a key role in chronic airway diseases such as asthma.     

The murine neutrophil NLRP3 inflammasome is activated by soluble but not particulate or crystalline agonists

Neutrophils express pattern recognition receptors (PRRs) and regulate immune responses via PRR‐dependent cytokine production. An emerging theme is that neutrophil PRRs often exhibit cell type‐specific adaptations in their signalling pathways. This prompted us to examine inflammasome signalling by the PRR NLRP3 in murine neutrophils, in comparison to well‐established NLRP3 signalling pathways in macrophages. Here, we demonstrate that while murine neutrophils can indeed signal via the NLRP3 inflammasome, neutrophil NLRP3 selectively responds to soluble agonists but not to the particulate/crystalline agonists that trigger NLRP3 activation in macrophages via phagolysosomal rupture. In keeping with this, alum did not trigger IL‐1β production from human PMN, and the lysosomotropic peptide Leu‐Leu‐OMe stimulated only weak NLRP3‐dependent IL‐1β production from murine neutrophils, suggesting that lysosomal rupture is not a strong stimulus for NLRP3 activation in neutrophils. We validated our in vitro findings for poor neutrophil NLRP3 responses to particles in vivo, where we demonstrated that neutrophils do not significantly contribute to alum‐induced IL‐1β production in mice. In all, our studies highlight that myeloid cell identity and the nature of the danger signal can strongly influence signalling by a single PRR, thus shaping the nature of the resultant immune response.     

A novel “complement–metabolism–inflammasome axis” as a key regulator of immune cell effector function

The inflammasomes are intracellular multiprotein complexes that induce and regulate the generation of the key pro‐inflammatory cytokines IL‐1β and IL‐18 in response to infectious microbes and cellular stress. The activation of inflammasomes involves several upstream signals including classic pattern or danger recognition systems such as the TLRs. Recently, however, the activation of complement receptors, such as the anaphylatoxin C3a and C5a receptors and the complement regulator CD46, in conjunction with the sensing of cell metabolic changes, for instance increased amino acid influx and glycolysis (via mTORC1), have emerged as additional critical activators of the inflammasome. This review summarizes recent advances in our knowledge about complement‐mediated inflammasome activation, with a specific focus on a novel “complement – metabolism – NLRP3 inflammasome axis.”     

Signaling by ROS drives inflammasome activation

Inflammasomes are innate immune signaling pathways that sense pathogens and injury to direct the proteolytic maturation of inflammatory cytokines such as IL‐1β and IL‐18. Among inflammasomes, the NLRP3/NALP3 inflammasome is the most studied. However, little is known on the molecular mechanisms that mediate its assembly and activation. Recent findings suggest that ROS are produced by NLRP3/NALP3 activators and are essential secondary messengers signaling NLRP3/NALP3 inflammasome activation.