Activation of the NLRP3 inflammasome by Mycobacterium tuberculosis is uncoupled from susceptibility to active tuberculosis

As a hallmark of tuberculosis (TB), Mycobacterium tuberculosis (MTB) induces granulomatous lung lesions and systemic inflammatory responses during active disease. Molecular regulation of inflammation is associated with inflammasome assembly. We determined the extent to which MTB triggers inflammasome activation and how this impacts on the severity of TB in a mouse model. MTB stimulated release of mature IL-1β in macrophages while attenuated M. bovis BCG failed to do so. Tubercle bacilli specifically activated the NLRP3 inflammasome and this propensity was strictly controlled by the virulence-associated RD1 locus of MTB. However, Nlrp3-deficient mice controlled pulmonary TB, a feature correlated with NLRP3-independent production of IL-1β in infected lungs. Our studies demonstrate that MTB activates the NLRP3 inflammasome in macrophages in an ESX-1-dependent manner. However, during TB, MTB promotes NLRP3- and caspase-1-independent IL-1β release in myeloid cells recruited to lung parenchyma and thus overcomes NLRP3 deficiency in vivo in experimental models.     

The NLRP3 inflammasome: A sensor of immune danger signals

The innate immune system senses danger signals via evolutionary conserved receptors. The nucleotide-binding domain leucine-rich repeat containing receptor (NLR) family is a group of intracellular receptors that drive a wide variety of inflammatory responses. A number of the NLR family members can form inflammasomes, which are multiprotein complexes that can activate caspase-1 and ultimately lead to the processing and secretion of interleukin (IL)-1β, IL-18 and IL-33. One of the best-studied members of the NLR family is NLRP3 for which a number of divergent activators have recently been described. These and other studies examining the NLRP3 inflammasome will be discussed in this review.     

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.     

Critical functions of priming and lysosomal damage for NLRP3 activation

Inflammasomes are cytosolic multi‐protein complexes that form in response to infectious or injurious challenges. Inflammasomes control the activity of caspase‐1, which is essential for the maturation and release of IL‐1β family cytokines. The NLRP1, IPAF and AIM2 inflammasomes recognize specific substances, while the NLRP3 inflammasome responds to many structurally and chemically diverse triggers. Here, we discuss the critical roles of priming and lysosomal damage in NLRP3 inflammasome activation.     

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.     

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.     

The NLRP3 inflammasome,a target for therapy in diverse disease states

A role for NLRP3 inflammasome in recurrent and chronic inflammation was initially described in a group of rare autoinflammatory conditions, termed cryopyrin‐associated periodic syndrome. Subsequently, inflammasomes have been implicated in the pathology of many common diseases, including cancer, gout and diabetes. Despite diverse pathologies, the central role of the inflammasome in innate defences and tumour elimination suggests common therapeutic approaches to reduce inflammation where appropriate.     

How location and cellular signaling combine to activate the NLRP3 inflammasome

NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) is a cytosolic innate immune sensor of cellular stress signals, triggered by infection and sterile inflammation. Upon detection of an activating stimulus, NLRP3 transitions from an inactive homo-oligomeric multimer into an active multimeric inflammasome, which promotes the helical oligomeric assembly of the adaptor molecule ASC. ASC oligomers provide a platform for caspase-1 activation, leading to the proteolytic cleavage and activation of proinflammatory cytokines in the IL-1 family and gasdermin D, which can induce a lytic form of cell death. Recent studies investigating both the cellular requirement for NLRP3 activation and the structure of NLRP3 have revealed the complex regulation of NLRP3 and the multiple steps involved in its activation. This review presents a perspective on the biochemical and cellular processes controlling the assembly of the NLRP3 inflammasome with particular emphasis on structural regulation and the role of organelles. We also highlight the latest research on metabolic control of this inflammatory pathway and discuss promising clinical targets for intervention.     

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.     

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.     

Expression and regulation of the NALP3 inflammasome complex in periodontal diseases

Periodontitis is an infectious process characterized by inflammation affecting the supporting structures of the teeth. Porphyromonas gingivalis is a major oral bacterial species implicated in the pathogenesis of periodontitis. Processing of interleukin (IL)‐1 family cytokines is regulated by an intracellular innate immune response system, known as the NALP3 [nacht domain‐, leucine‐rich repeat‐, and pyrin domain (PYD)‐containing protein 3] inflammasome complex. The aim of the present study was to investigate by quantitative real‐time polymerase chain reaction (PCR) the mRNA expression of NALP3, its effector molecule apoptosis associated speck‐like protein (ASC), its putative antagonist NLRP2 (NLR family, PYD‐containing protein 2), IL‐1β and IL‐18 (i) in gingival tissues from patients with gingivitis (n = 10), chronic periodontitis (n = 18), generalized aggressive periodontitis (n = 20), as well as in healthy subjects (n = 20), (ii) in vitro in a human monocytic cell line (Mono‐Mac‐6), in response to P. gingivalis challenge for 6 h. The clinical data indicate that NALP3 and NLRP2, but not ASC, are expressed at significantly higher levels in the three forms of inflammatory periodontal disease compared to health. Furthermore, a positive correlation was revealed between NALP3 and IL‐1β or IL‐18 expression levels in these tissues. The in vitro data demonstrate that P. gingivalis deregulates the NALP3 inflammasome complex in Mono‐Mac‐6 cells by enhancing NALP3 and down‐regulating NLRP2 and ASC expression. In conclusion, this study reveals a role for the NALP3 inflammasome complex in inflammatory periodontal disease, and provides a mechanistic insight to the host immune responses involved in the pathogenesis of the disease by demonstrating the modulation of this cytokine‐signalling pathway by bacterial challenge.     

Expression and function of the NALP3 inflammasome in rheumatoid synovium

The NACHT, LRR and PYD domains containing protein (NALP3) inflammasome is a key regulator of interleukin‐1β (IL‐1β) secretion. As there is strong evidence for a pro‐inflammatory role of IL‐1β in rheumatoid arthritis (RA) and in murine models of arthritis, we explored the expression of the different components of the NALP3 inflammasome as well as other nucleotide oligomerization domain (NOD)‐like receptors (NLRs) in synovium obtained from patients with RA. The expression of NLRs was also studied in fibroblast lines derived from joint tissue. By immunohistology, NALP3 and apoptosis‐associated speck‐like protein containing a CARD domain (ASC) were expressed in myeloid and endothelial cells and B cells. T cells expressed ASC but lacked NALP3. In synovial fibroblast lines, NALP3 expression was not detected at the RNA and protein levels and stimulation with known NALP3 agonists failed to induce IL‐1β secretion. Interestingly, we were unable to distinguish RA from osteoarthritis synovial samples on the basis of their basal level of RNA expression of known NLR proteins, though RA samples contained higher levels of caspase‐1 assayed by enzyme‐linked immunsorbent assay. These results indicate that myeloid and endothelial cells are the principal sources of inflammasome‐mediated IL‐1β production in the synovium, and that synovial fibroblasts are unable to activate caspase‐1 because they lack NALP3. The NALP3 inflammasome activity does not account for the difference in level of inflammation between RA and osteoarthritis.     

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.     

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.     

Inflammasomes as microbial sensors

Members of the Nod‐like receptor family and the adaptor ASC assemble into multiprotein platforms, termed inflammasomes, to mediate the activation of caspase‐1 and subsequent secretion of IL‐1β and IL‐18. Recent studies have identified microbial and endogenous molecules as well as possible mechanisms involved in inflammasome activation.     

The Nlrp3 inflammasome,IL‐1β, and neutrophil recruitment are required for susceptibility to a nonhealing strain of Leishmania major in C57BL/6 mice

Infection of C57BL/6 mice with most Leishmania major strains results in a healing lesion and clearance of parasites from the skin. Infection of C57BL/6 mice with the L. major Seidman strain (LmSd), isolated from a patient with chronic lesions, despite eliciting a strong Th1 response, results in a nonhealing lesion, poor parasite clearance, and complete destruction of the ear dermis. We show here that in comparison to a healing strain, LmSd elicited early upregulation of IL‐1β mRNA and IL‐1β‐producing dermal cells and prominent neutrophil recruitment to the infected skin. Mice deficient in Nlrp3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain, or caspase‐1/11, or lacking IL‐1β or IL‐1 receptor signaling, developed healing lesions and cleared LmSd from the infection site. Mice resistant to LmSd had a stronger antigen‐specific Th1 response. The possibility that IL‐1β might act through neutrophil recruitment to locally suppress immunity was supported by the healing observed in neutropenic Genista mice. Secretion of mature IL‐1β by LmSd‐infected macrophages in vitro was dependent on activation of the Nlrp3 inflammasome and caspase‐1. These data reveal that Nlrp3 inflammasome‐dependent IL‐1β, associated with localized neutrophil recruitment, plays a crucial role in the development of a nonhealing form of cutaneous leishmaniasis in conventionally resistant mice.