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.     

Uric acid induces trophoblast IL-1β production via the inflammasome: implications for the pathogenesis of preeclampsia

Citation
Mulla MJ, Myrtolli K, Potter J, Boeras C, Kavathas PB, Sfakianaki AK, Tadesse S, Norwitz ER, Guller S, Abrahams VM. Uric acid induces trophoblast IL‐1β production via the inflammasome: implications for the pathogenesis of preeclampsia. Am J Reprod Immunol 2010; 65: 542–548 Problem  Preeclampsia is associated with hyperuricemia, which correlates with the disease severity. Levels of circulating uric acid increase before the clinical manifestations, suggesting that they may be causally related. Uric acid, or monosodium urate (MSU), activates the Nod‐like receptor, Nalp3, leading to inflammasome activation and IL‐1β processing. Because preeclampsia is associated with placental immune/inflammatory dysregulation, we sought to determine in the trophoblast, the presence of the Nalp3 inflammasome, and the effect of MSU on its activation. Method of study  Isolated first‐ and third‐trimester trophoblasts were assessed for expression of the inflammasome components, Nalp1, Nalp3, and ASC. First‐trimester trophoblast cells were incubated with or without MSU, and after which, IL‐1β secretion and processing and caspase‐1 activation were determined. Results  Trophoblast cells expressed Nalp1, Nalp3, and ASC under basal conditions. Following incubation with MSU, first‐trimester trophoblast IL‐1β secretion was upregulated. This correlated with increased expression levels of active IL‐1β and active caspase‐1. ASC knockdown reduced MSU‐induced IL‐1β secretion. Conclusion  These findings demonstrate that uric acid activates the inflammasome in the trophoblast, leading to IL‐1β production. This may provide a novel mechanism for the induction of inflammation at the maternal–fetal interface leading to placental dysfunction and adverse pregnancy outcome, including preeclampsia.     

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.     

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.     

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.     

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.     

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.     

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.     

Distinct expression of interleukin (IL)‐36α, β and γ, their antagonist IL‐36Ra and IL‐38 in psoriasis,rheumatoid arthritis and Crohn's disease

Interleukin (IL)‐36α, IL‐36β and IL‐36γ are expressed highly in skin and are involved in the pathogenesis of psoriasis, while the antagonists IL‐36Ra or IL‐38, another potential IL‐36 inhibitor, limit uncontrolled inflammation. The expression and role of IL‐36 cytokines in rheumatoid arthritis (RA) and Crohn's disease (CD) is currently debated. Here, we observed that during imiquimod‐induced mouse skin inflammation and in human psoriasis, expression of IL‐36α, γ and IL‐36Ra, but not IL‐36β and IL‐38 mRNA, was induced and correlated with IL‐1β and T helper type 17 (Th17) cytokines (IL‐17A, IL‐22, IL‐23, CCL20). In mice with collagen‐induced arthritis and in the synovium of patients with RA, IL‐36α, β, γ, IL‐36Ra and IL‐38 were all elevated and correlated with IL‐1β, CCL3, CCL4 and macrophage colony‐stimulating factor (M‐CSF), but not with Th17 cytokines. In the colon of mice with dextran sulphate sodium‐induced colitis and in patients with CD, only IL‐36α, γ and IL‐38 were induced at relatively low levels and correlated with IL‐1β and IL‐17A. We suggest that only a minor subgroup of patients with RA (17–29%) or CD (25%) had an elevated IL‐36 agonists/antagonists ratio, versus 93% of patients with psoriasis. By immunohistochemistry, IL‐36 cytokines were produced by various cell types in skin, synovium and colonic mucosa such as keratinocytes, CD68⁺ macrophages, dendritic/Langerhans cells and CD79α⁺ plasma cells. In primary cultures of monocytes or inflammatory macrophages (M1), IL‐36β and IL‐36Ra were produced constitutively, but IL‐36α, γ and IL‐38 were produced after lipopolysaccharide stimulation. These distinct expression profiles may help to explain why only subgroups of RA and CD patients have a potentially elevated IL‐36 agonists/antagonists ratio.     

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.     

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.     

Neurodegeneration and NLRP3 inflammasome expression in the anterior thalamus of SOD1(G93A) ALS mice

Nowadays, amyotrophic lateral sclerosis (ALS) is considered as a multisystem disorder, characterized by a primary degeneration of motor neurons as well as neuropathological changes in non‐motor regions. Neurodegeneration in subcortical areas, such as the thalamus, are believed to contribute to cognitive and behavioral abnormalities in ALS patients. In the present study, we investigated neurodegenerative changes including neuronal loss and glia pathology in the anterodorsal thalamic nucleus (AD) of SOD1(G93A) mice, a widely used animal model for ALS. We detected massive dendrite swelling and neuronal loss in SOD1(G93A) animals, which was accompanied by a mild gliosis. Furthermore, misfolded SOD1 protein and autophagy markers were accumulating in the AD. Since innate immunity and activation inflammasomes seem to play a crucial role in ALS, we examined protein expression of Nod‐like receptor protein 3 (NLRP3), apoptosis‐associated speck‐like protein containing a caspase‐1 recruitment domain (ASC) and the cytokine interleukin 1 beta (IL1β) in AD glial cells and neurons. NLRP3 and ASC were significantly up‐regulated in the AD of SOD1(G93A) mice. Finally, co‐localization studies revealed expression of NLRP3, ASC and IL1β in neurons. Our study yielded two main findings: (i) neurodegenerative changes already occur at an early symptomatic stage in the AD and (ii) increased inflammasome expression may contribute to neuronal cell death. In conclusion, neurodegeneration in the anterior thalamus may critically account for cognitive changes in ALS pathology.     

Experimental cerebral malaria progresses independently of the Nlrp3 inflammasome

Cerebral malaria is the most severe complication of Plasmodium falciparum infection in humans and the pathogenesis is still unclear. Using the P. berghei ANKA infection model of mice, we investigated a potential involvement of Nlrp3 and the inflammasome in the pathogenesis of cerebral malaria. Nlrp3 mRNA expression was upregulated in brain endothelial cells after exposure to P. berghei ANKA. Although β‐hematin, a synthetic compound of the parasites heme polymer hemozoin, induced the release of IL‐1β in macrophages through Nlrp3, we did not obtain evidence for a role of IL‐1β in vivo. Nlrp3 knock‐out mice displayed a delayed onset of cerebral malaria; however, mice deficient in caspase‐1, the adaptor protein ASC or the IL‐1 receptor succumbed as WT mice. These results indicate that the role of Nlrp3 in experimental cerebral malaria is independent of the inflammasome and the IL‐1 receptor pathway.     

The role of high‐mobility group box protein 1 in collagen antibody‐induced arthritis is dependent on vascular endothelial growth factor

High‐mobility group box 1 (HMGB1) has been implicated in angiogenesis and rheumatoid arthritis (RA). The aim of this study was to define more clearly the role of HMGB1 in the synovial angiogenesis and pathogenesis of an immune model of arthritis. BALB/c mice were injected with monoclonal anti‐collagen antibody cocktail followed by lipopolysaccharide to induce arthritis. HMGB1 and vascular endothelial growth factor (VEGF) were over‐expressed in the areas of the synovium where more inflammation and neoangiogenesis were present. The selective blockade of HMGB1 or VEGF resulted alternatively in a lower severity of arthritis evaluated by the arthritis index. Furthermore, exogenous HMGB1 administration caused a worsening of arthritis, associated with VEGF up‐regulation and increased synovial angiogenesis. The selective inhibition of VEGF also resulted in no induction of arthritis in mice receiving exogenous HMGB1. Cytokine enzyme‐linked immunosorbent assay (ELISA) analyses performed on peripheral blood and synovial fluid demonstrated a significant reduction of interleukin (IL)?1β, IL‐6 and tumour necrosis factor (TNF)‐α in mice where HMGB1 and VEGF pathways were blocked. Interestingly, the selective blockade of HMGB1 and VEGF resulted in an increase of the peripheral IL‐17A concentration. The development of arthritis mediated by HMGB1 and the synovial angiogenesis can be blocked by inhibiting the VEGF activity. The proinflammatory and proangiogenic cytokine IL‐17A was increased when HMGB1 is inhibited, but the synovial angiogenesis was nevertheless reduced in this model of arthritis. Taken together, these findings shed new light on the role of this nuclear protein in the pathogenesis of arthritis in an RA‐like model.