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.     

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.     

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.     

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.     

NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase‐4 and caspase‐5

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase‐4 and caspase‐5. When activated, these trigger pyroptotic cell death and caspase‐1‐dependent IL‐1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase‐4/5‐dependent IL‐1β production elicited by transfected LPS. Given that both caspase‐4 and caspase‐5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase‐4 and caspase‐5 were genetically deleted either individually or together. We found that the deletion of caspase‐4 suppressed cell death and IL‐1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase‐5 did not confer protection against transfected LPS, cell death and IL‐1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase‐4 and caspase‐5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL‐1β maturation, downstream of cytoplasmic LPS detection by caspase‐4/5. We also show that both caspase‐4 and caspase‐5 are functionally important for appropriate responses to intracellular Gram‐negative bacteria.     

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.     

Propionibacterium acnes activates caspase‐1 in human neutrophils

Propionibacterium acnes is a Gram‐positive, slow‐growing, anaerobic bacillus, predominantly found as a commensal on the skin and mucous membranes of adults. It is, however, also considered an opportunistic pathogen; mostly associated with acne vulgaris, but rarely also with severe infections such as infective endocarditis, prosthetic joint infections, and deep sternal wound infections following cardiothoracic surgery. In addition, P. acnes has recently been found in high frequency in prostate tissue from patients with prostatitis and prostate cancer. The NOD‐like receptors (NLR) act as intracellular sensors of microbial components, and a number of various bacteria have been found to induce assembling and activation of NLR‐inflammasomes; leading to a pro‐inflammatory response. The inflammasome‐mediated formation of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β) and IL‐18 involves the auto‐proteolytic maturation of caspase‐1. This study investigated if P. acnes activates inflammasomes. Propionibacterium acnes isolates (n = 29) with diverse origin were used as stimuli for peripheral leukocytes obtained from blood donors (BDs). The activity of inflammasomes was determined by measuring caspase‐1 by flow cytometry and cytokine production by ELISA. A significant amount of caspase‐1 was found in neutrophils upon P. acnes stimulation, whereas only a modest activation was seen in monocytes. The activation was mainly produced by components of the bacterial cell and no exo‐products, because heat‐killed and live bacteria caused high activation of caspase‐1 as well as cytokine production, whereas the bacterial supernatant elicited minor effect. The response among different BDs varied significantly, almost fivefold. In addition, P. acnes of various origins showed considerable variation, however, the commensal isolates showed a stronger response compared with the invasive. In conclusion, although regarded as a harmless commensal of the skin, P. acnes strongly activates the inflammasome of human peripheral neutrophils.     

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.     

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.     

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 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 contributes to host protection during Sporothrix schenckii infection

Sporotrichosis is a mycosis caused by fungi from the Sporothrix schenckii species complex, whose prototypical member is Sporothrix schenckii sensu stricto. Pattern recognition receptors (PRRs) recognize and respond to pathogen‐associated molecular patterns (PAMPs) and shape the following adaptive immune response. A family of PRRs most frequently associated with fungal recognition is the nucleotide‐binding oligomerization domain‐like receptor (NLR). After PAMP recognition, NLR family pyrin domain‐containing 3 (NLRP3) binds to apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) and caspase‐1 to form the NLRP3 inflammasome. When activated, this complex promotes the maturation of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β) and IL‐18 and cell death through pyroptosis. In this study, we aimed to evaluate the importance of the NLRP3 inflammasome in the outcome of S. schenckii infection using the following three different knockout (KO) mice: NLRP3^−/−, ASC^−/− and caspase‐1^−/−. All KO mice were more susceptible to infection than the wild‐type, suggesting that NLRP3‐triggered responses contribute to host protection during S. schenckii infection. Furthermore, the NLRP3 inflammasome appeared to be critical for the ex vivo release of IL‐1β, IL‐18 and IL‐17 but not interferon‐γ. Additionally, a role for the inflammasome in shaping the adaptive immune response was suggested by the lower frequencies of type 17 helper T (Th17) cells and Th1/Th17 but not Th1 cells in S. schenckii‐infected KO mice. Overall, our results indicate that the NLRP3 inflammasome links the innate recognition of S. schenckii to the adaptive immune response, so contributing to protection against this infection.     

Inflammasomes in host response to protozoan parasites

Inflammasomes are multimeric complexes of proteins that are assembled in the host cell cytoplasm in response to specific stress signals or contamination of the cytoplasm by microbial molecules. The canonical inflammasomes are composed of at least three main components: an inflammatory caspase (caspase-1, caspase-11), an adapter molecule (such as ASC), and a sensor protein (such as NLRP1, NLRP3, NLRP12, NAIP1, NAIP2, NAIP5, or AIM2). The sensor molecule determines the inflammasome specificity by detecting specific microbial products or cell stress signals. Upon activation, these molecular platforms facilitate restriction of microbial replication and trigger an inflammatory form of cell death called pyroptosis, thus accounting for the genesis of inflammatory processes. Inflammasome activation has been widely reported in response to pathogenic bacteria. However, recent reports have highlighted the important role of the inflammasomes in the host response to the pathogenesis of infections caused by intracellular protozoan parasites. Herein, we review the activation and specific roles of inflammasomes in recognition and host responses to intracellular protozoan parasites such as Trypanosoma cruzi, Toxoplasma gondii, Plasmodium spp., and Leishmania spp.     

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.     

Pyroptosis of resident macrophages differentially orchestrates inflammatory responses to Staphylococcus aureus in resistant and susceptible mice

The relationship between Staphylococcus aureus and innate immunity is highly complex and requires further investigation to be deciphered. i.p. challenge of C57BL/6 and DBA/2 mice, resistant and susceptible to the infection, respectively, resulted in different patterns of cytokine production and neutrophil recruitment. Staphylococcus aureus infection induced macrophage pyroptosis, an inflammasome‐dependent cell death program, whose rates significantly differed between C57BL/6 and DBA/2 mice. Fast rate pyroptosis of C57BL/6 macrophages released high levels of IL‐1β but limited the synthesis of other cytokines such as TNF‐α, IL‐6, CXCL1, and CXCL2. Conversely, the extended survival of DBA/2 macrophages allowed substantial production of these NF‐κB‐related cytokines. Phenotyping of resting macrophages in different mouse strains revealed differential predisposition toward specific macrophage phenotypes that modulate S. aureus‐mediated inflammasome activation. Treatment of DBA/2 susceptible mice with inflammasome inducers (i.e. nigericin and ATP) artificially increased pyroptosis and lowered the levels of NF‐κB‐related inflammatory cytokines, but restored IL‐1β to levels similar to those in C57BL/6 mice. Collectively, this study promotes the concept that, in association with host genetics, the basal phenotype of resident macrophages influences the early inflammatory response and possibly participates in S. aureus infection outcome via the inflammasome pathway and subsequent pyroptosis.     

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.     

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.     

IL‐1β, IL‐18, and eicosanoids promote neutrophil recruitment to pore‐induced intracellular traps following pyroptosis

Inflammasomes activate caspase‐1, initiating a lytic form of programmed cell death termed pyroptosis, which is an important innate immune defense mechanism against intracellular infections. We recently demonstrated in a mouse infection model of pyroptosis that instead of releasing bacteria into the extracellular space, bacteria remain trapped within the pyroptotic cell corpse, termed the pore‐induced intracellular trap (PIT). This trapping mediates efferocytosis of the PIT and associated bacteria by neutrophils; bacteria are subsequently killed via neutrophil ROS. Using this pyroptosis model, we now show that the pro‐inflammatory cytokines IL‐1β and IL‐18 and inflammatory lipid mediators termed eicosanoids are required for effective clearance of bacteria downstream of pyroptosis. We further show that IL‐1β, IL‐18, and eicosanoids affect this in part by mediating neutrophil recruitment to the PIT. This is in addition to our prior findings that complement is also important to attract neutrophils. Thus, the PIT initiates a robust and coordinated innate immune response involving multiple mediators that attract neutrophils to efferocytose the PIT and its entrapped bacteria.