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.     

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.     

Pannexin‐1‐dependent caspase‐1 activation and secretion of IL‐1β is regulated by zinc

Inflammatory processes induced by IL‐1β are critical for host defence responses, but are also implicated in disease. Zinc deficiency is a common consequence of, or contributor to, human inflammatory disease. However, the molecular mechanisms through which zinc contributes to inflammatory disease remain largely unknown. We report here that zinc metabolism regulates caspase‐1 activation and IL‐1β secretion. One of the endogenous mediators of IL‐1β secretion is adenosine triphosphate, acting via the P2X7‐receptor and caspase‐1 activation in cells primed with an inflammatory stimulus such as LPS. We show that this process is selectively abolished by a brief pre‐treatment with the zinc chelator N,N,N′,N′‐tetrakis‐(2‐pyridylmethyl) ethylene diamine (TPEN). These effects on IL‐1β secretion were independent of rapid changes in free zinc within the cell, not a direct effect on caspase‐1 activity, and upstream of caspase‐1 activation. TPEN did however inhibit the activity of pannexin‐1, a hemi‐channel critical for adenosine triphosphate and nigericin‐induced IL‐1β release. These data provide new insights into the mechanisms of caspase‐1 activation and how zinc metabolism contributes to inflammatory mechanisms.     

Cytotoxins of the human pathogen Aeromonas hydrophila trigger,via the NLRP3 inflammasome,caspase‐1 activation in macrophages

Aeromonas hydrophila is a Gram‐negative pathogen that causes serious infectious disease in humans. A. hydrophila induces apoptosis in infected macrophages, but the host proinflammatory responses triggered by macrophage death are largely unknown. Here, we demonstrate that the infection of mouse macrophages with A. hydrophila triggers the activation of caspase‐1 and release of IL‐1β. Caspase‐1 activation was abrogated in macrophages deficient in Nod‐like receptor family, pyrin domain containing 3 (NLRP3) and apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), but not NLR family, CARD domain containing 4 (NLRC4). The activation of the NLRP3 inflammasome was mediated by three cytotoxins (aerolysin, hemolysin and multifunctional repeat‐in‐toxin) produced by A. hydrophila. Our results indicated that the NLRP3 inflammasome senses A. hydrophila infection through the action of bacterial cytotoxins.     

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.     

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.     

The Cag pathogenicity island and interaction between TLR2/NOD2 and NLRP3 regulate IL‐1β production in Helicobacter pylori infected dendritic cells

Helicobacter pylori colonization of the stomach affects about half of the world population and is associated with the development of gastritis, ulcers, and cancer. Polymorphisms in the IL1B gene are linked to an increased risk of H. pylori associated cancer, but the bacterial and host factors that regulate interleukin (IL)‐1β production in response to H. pylori infection remain unknown. Using murine BM‐derived DCs, we show that the bacterial virulence factors cytotoxin‐associated genes pathogenicity island and CagL, but not vacuolating cytotoxin A or CagA, regulate the induction of pro‐IL‐1β and the production of mature IL‐1β in response to H. pylori infection. We further show that the host receptors, Toll‐like receptor 2 (TLR2) and nucleotide‐binding oligomerization domain 2 (NOD2), but not NOD1, are required for induction of pro‐IL‐1β and NOD‐like receptor pyrin domain containing 3 (NLRP3) in H. pylori infected DCs. In contrast, NLRP3 and the adaptor ASC were essential for the activation of caspase‐1, processing of pro‐IL‐1β into IL‐1β, and IL‐1β secretion. Finally, we show that mice deficient in caspase‐1, IL‐1β, and IL‐1 receptor, but not NLRP3, are impaired in the clearance of CagA‐positive H. pylori from the stomach when compared with WT mice. These studies identify bacterial cag pathogenicity island and the cooperative interaction among host innate receptors TLR2, NOD2, and NLRP3 as important regulators of IL‐1β production in H. pylori infected DCs.     

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.     

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.     

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.     

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.     

Listeria monocytogenes is sensed by the NLRP3 and AIM2 inflammasome

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.     

IL‐1β activation in response to Staphylococcus aureus lung infection requires inflammasome‐dependent and independent mechanisms

Maintaining balanced levels of IL‐1β is extremely important to avoid host tissue damage during infection. Our goal was to understand the mechanisms behind the reduced pathology and decreased bacterial burdens in Ifnlr1^?/? mice during lung infection with Staphylococcus aureus. Intranasal infection of Ifnlr1^?/? mice with S. aureus led to significantly improved bacterial clearance, survival and decrease of proinflammatory cytokines in the airway including IL‐1β. Ifnlr1^?/? mice treated with recombinant IL‐1β displayed increased bacterial burdens in the airway and lung. IL‐1β levels in neutrophils from Ifnlr1^?/? infected mice lungs were decreased when compared to neutrophils from WT mice. Mice lacking NLRP3 and caspase‐1 had reduced IL‐1β levels 4 h after infection, due to reductions or absence of active caspase‐1 respectively, but levels at 24 h were comparable to WT infected mice. Ifnlr1^?/? infected mice had decreases in both active caspase‐1 and neutrophil elastase indicating an important role for the neutrophil serine protease in IL‐1β processing. By inhibiting neutrophil elastase, we were able to decrease IL‐1β levels by 39% in Nlrp3^?/? infected mice when compared to WT mice. These results highlight the crucial role of both proteases in IL‐1β processing, via inflammasome‐dependent and ‐independent mechanisms.     

NLRP3 inflammasome mediates interleukin‐1β production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice

Acinetobacter baumannii is a multi‐drug resistant, Gram‐negative bacteria and infection with this organism is one of the major causes of mortality in intensive care units. Inflammasomes are multiprotein oligomers that include caspase‐1, and their activation is required for maturation of interleukin‐1β (IL‐1β). Inflammasome signalling is involved in host defences against various microbial infections, but the precise mechanism by which A. baumannii activates inflammasomes and the roles of relevant signals in host defence against pulmonary A. baumannii infection are unknown. Our results showed that NLRP3, ASC and caspase‐1, but not NLRC4, are required for A. baumannii‐induced production of IL‐1β in macrophages. An inhibitor assay revealed that various pathways, including P2X7R, K⁺ efflux, reactive oxygen species production and release of cathepsins, are involved in IL‐1β production in macrophages in response to A. baumannii. Interleukin‐1β production in bronchoalveolar lavage (BAL) fluid was impaired in NLRP3‐deficient and caspase‐1/11‐deficient mice infected with A. baumannii, compared with that in wild‐type (WT) mice. However, the bacterial loads in BAL fluid and lungs were comparable between WT and NLRP3‐deficient or caspase‐1/11‐deficient mice. The severity of lung pathology was reduced in NLRP3‐ deficient, caspase‐1/11‐ deficient and IL‐1‐receptor‐deficient mice, although the recruitment of immune cells and production of inflammatory cytokines and chemokines were not altered in these mice. These findings indicate that A. baumannii leads to the activation of NLRP3 inflammasome, which mediates IL‐1β production and lung pathology.     

Caspase‐11: The driving factor for noncanonical inflammasomes

Inflammasomes are large multiprotein platforms that mediate the processing of caspase‐1, which in turn promotes the maturation and release of IL‐1β and IL‐18 in response to microbial and danger signals. While the canonical pathway of inflammasome activation has been known for some time, a novel mechanism of noncanonical inflammasome activation mediated by caspase‐11 was more recently identified. This pathway engages caspase‐11 to trigger both caspase‐1‐dependent and ‐independent production of the inflammatory cytokines IL‐1β, IL‐18, and IL‐1α, as well as to promote pyroptosis, a form of genetically programmed cell death that is associated with the release of such cytokines. In this review, we gather together studies on both the mechanisms and implications of caspase‐11‐mediated noncanonical inflammasome activation, and discuss the emerging importance of this pathway in regulating host defense against intracellular bacterial pathogens.     

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.     

Caspase‐11 non‐canonical inflammasome: a critical sensor of intracellular lipopolysaccharide in macrophage‐mediated inflammatory responses

Inflammatory responses mediated by macrophages are part of the innate immune system, whose role is to protect against invading pathogens. Lipopolysaccharide (LPS) found in the outer membrane of Gram‐negative bacteria stimulates an inflammatory response by macrophages. During the inflammatory response, extracellular LPS is recognized by Toll‐like receptor 4, one of the pattern recognition receptors that activates inflammatory signalling pathways and leads to the production of inflammatory mediators. The innate immune response is also triggered by intracellular inflammasomes, and inflammasome activation induces pyroptosis and the secretion of pro‐inflammatory cytokines such as interleukin‐1β (IL‐1β) and IL‐18 by macrophages. Cysteine‐aspartic protease (caspase)‐11 and the human orthologues caspase‐4/caspase‐5 were recently identified as components of the ‘non‐canonical inflammasome’ that senses intracellular LPS derived from Gram‐negative bacteria during macrophage‐mediated inflammatory responses. Direct recognition of intracellular LPS facilitates the rapid oligomerization of caspase‐11/4/5, which results in pyroptosis and the secretion of IL‐1β and IL‐18. LPS is released into the cytoplasm from Gram‐negative bacterium‐containing vacuoles by small interferon‐inducible guanylate‐binding proteins encoded on chromosome 3 (GBP^chr3)‐mediated lysis of the vacuoles. In vivo studies have clearly shown that caspase‐11^−/− mice are more resistant to endotoxic septic shock by excessive LPS challenge. Given the evidence, activation of caspase‐11 non‐canonical inflammasomes by intracellular LPS is distinct from canonical inflammasome activation and provides a new paradigm in macrophage‐mediated inflammatory responses.     

The adaptor ASC exacerbates lethal Listeria monocytogenes infection by mediating IL‐18 production in an inflammasome‐dependent and ‐independent manner

Listeria monocytogenes induces the formation of inflammasomes and subsequent caspase‐1 activation, and the adaptor apoptosis‐associated speck‐like protein containing a CARD (ASC) is crucial for this response. However, the role of ASC in L. monocytogenes infection in vivo is unclear. In this study, we demonstrate that ASC has a detrimental effect on host defense against L. monocytogenes infection at a lethal dose (10⁶ CFU), but not at a sublethal dose (10³ CFU). During lethal L. monocytogenes infection, serum levels of IL‐18 and IL‐10 were markedly elevated in WT mice, but not in ASC KO mice. IL‐18 KO mice were more resistant to lethal L. monocytogenes infection than WT mice and had lower levels of serum IL‐10. Furthermore, blockade of IL‐10 receptor resulted in a reduction in bacterial counts, suggesting that ASC and IL‐18 might exacerbate L. monocytogenes infection through induction of IL‐10. We noticed that maturation of IL‐18 during lethal infection was partially independent of caspase‐1, but was critically dependent on ASC. ASC was required for the elevation of serum neutrophil serine protease activity, which correlated with caspase‐1‐independent IL‐18 maturation and IL‐10 production. Collectively, these results suggest that ASC plays a detrimental role in lethal L. monocytogenes infection through IL‐18 production in an inflammasome‐dependent and ‐independent manner.