Mycobacterium tuberculosis inhibits the NLRP3 inflammasome activation via its phosphokinase PknF

Mycobacterium tuberculosis (Mtb) has evolved to evade host innate immunity by interfering with macrophage functions. Interleukin-1β (IL-1β) is secreted by macrophages after the activation of the inflammasome complex and is crucial for host defense against Mtb infections. We have previously shown that Mtb is able to inhibit activation of the AIM2 inflammasome and subsequent pyroptosis. Here we show that Mtb is also able to inhibit host cell NLRP3 inflammasome activation and pyroptosis. We identified the serine/threonine kinase PknF as one protein of Mtb involved in the NLRP3 inflammasome inhibition, since the pknF deletion mutant of Mtb induces increased production of IL-1β in bone marrow-derived macrophages (BMDMs). The increased production of IL-1β was dependent on NLRP3, the adaptor protein ASC and the protease caspase-1, as revealed by studies performed in gene-deficient BMDMs. Additionally, infection of BMDMs with the pknF deletion mutant resulted in increased pyroptosis, while the IL-6 production remained unchanged compared to Mtb-infected cells, suggesting that the mutant did not affect the priming step of inflammasome activation. In contrast, the activation step was affected since potassium efflux, chloride efflux and the generation of reactive oxygen species played a significant role in inflammasome activation and subsequent pyroptosis mediated by the Mtb pknF mutant strain. In conclusion, we reveal here that the serine/threonine kinase PknF of Mtb plays an important role in innate immune evasion through inhibition of the NLRP3 inflammasome.     

Measles virus V protein inhibits NLRP3 inflammasome-mediated interleukin-1β secretion

Inflammasomes are cytosolic protein complexes that stimulate the activation of caspase-1, which in turn induces the secretion of the inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. Recent studies have indicated that the inflammasome known as the NOD-like-receptor-family, pyrin domain-containing 3 (NLRP3) inflammasome recognizes several RNA viruses, including the influenza and encephalomyocarditis viruses, whereas the retinoic acid-inducible gene I (RIG-I) inflammasome may detect vesicular stomatitis virus. We demonstrate that measles virus (MV) infection induces caspase-1-dependent IL-1β secretion in the human macrophage-like cell line THP-1. Gene knockdown experiments indicated that IL-1β secretion in MV-infected THP-1 cells was mediated by the NLRP3 inflammasome but not the RIG-I inflammasome. MV produces the nonstructural V protein, which has been shown to antagonize host innate immune responses. The recombinant MV lacking the V protein induced more IL-1β than the parental virus. THP-1 cells stably expressing the V protein suppressed NLRP3 inflammasome-mediated IL-1β secretion. Furthermore, coimmunoprecipitation assays revealed that the V protein interacts with NLRP3 through its carboxyl-terminal domain. NLRP3 was located in cytoplasmic granular structures in THP-1 cells stably expressing the V protein, but upon inflammasome activation, NLRP3 was redistributed to the perinuclear region, where it colocalized with the V protein. These results indicate that the V protein of MV suppresses NLRP3 inflammasome-mediated IL-1β secretion by directly or indirectly interacting with NLRP3.     

Mycobacterium abscessus activates the NLRP3 inflammasome via Dectin-1-Syk and p62/SQSTM1

Numerous atypical mycobacteria, including Mycobacterium abscessus (Mabc), cause nontuberculous mycobacterial infections, which present a serious public health threat. Inflammasome activation is involved in host defense and the pathogenesis of autoimmune diseases. However, inflammasome activation has not been widely characterized in human macrophages infected with atypical mycobacteria. Here, we demonstrate that Mabc robustly activates the nucleotide binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via dectin-1/Syk-dependent signaling and the cytoplasmic scaffold protein p62/SQSTM1 (p62) in human macrophages. Both dectin-1 and Toll-like receptor 2 (TLR2) were required for Mabc-induced mRNA expression of pro-interleukin (IL)-1β, cathelicidin human cationic antimicrobial protein-18/LL-37 and β-defensin 4 (DEFB4). Dectin-1-dependent Syk signaling, but not that of MyD88, led to the activation of caspase-1 and secretion of IL-1β through the activation of an NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) inflammasome. Additionally, potassium efflux was required for Mabc-induced NLRP3/ASC inflammasome activation. Furthermore, Mabc-induced p62 expression was critically involved in NLRP3 inflammasome activation in human macrophages. Finally, NLRP3/ASC was critical for the inflammasome in antimicrobial responses to Mabc infection. Taken together, these data demonstrate the induction mechanism of the NLRP3/ASC inflammasome and its role in innate immunity to Mabc infection.     

The NLRP3 inflammasome: activation and regulation

The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome is a cytoplasmic supramolecular complex that is activated in response to cellular perturbations triggered by infection and sterile injury. Assembly of the NLRP3 inflammasome leads to activation of caspase-1, which induces the maturation and release of interleukin-1β (IL-1β) and IL-18, as well as cleavage of gasdermin D (GSDMD), which promotes a lytic form of cell death. Production of IL-1β via NLRP3 can contribute to the pathogenesis of inflammatory disease, whereas aberrant IL-1β secretion through inherited NLRP3 mutations causes autoinflammatory disorders. In this review, we discuss recent developments in the structure of the NLRP3 inflammasome, and the cellular processes and signaling events controlling its assembly and activation.     

Activation of an NLRP3 inflammasome restricts Mycobacterium kansasii infection

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1β secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1β derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii.     

Celastrol ameliorates Propionibacterium acnes/LPS-induced liver damage and MSU-induced gouty arthritis via inhibiting K63 deubiquitination of NLRP3

BackgroundCelastrol, a pentacyclic triterpenoid quinonemethide isolated from several spp. of Celastraceae family, exhibits anti-inflammatory activities in a variety of diseases including arthritis.PurposeThis study aims to investigate whether the inhibition of NLRP3 inflammasome is engaged in the anti-inflammatory activities of celastrol and delineate the underlying mechanism.MethodsThe influence of celastrol on NLRP3 inflammasome activation was firstly studied in lipopolysaccharide (LPS)-primed mouse bone marrow-derived macrophages (BMDMs) and phorbol 12-myristate 13-acetate (PMA)-primed THP-1 cells treated with nigericin. Reconstituted inflammasome was also established by co-transfecting NLRP3, ASC, pro-caspase-1 and pro-IL-1β in HEK293T cells. The changes of inflammasome components including NLRP3, ASC, pro-caspase-1/caspase-1 and pro-IL-1β/IL-1β were examined by enzyme-linked immunosorbent assay (ELISA), western blotting and immunofluorescence. Furthermore, Propionibacterium acnes (P. acnes)/LPS-induced liver injury and monosodium urate (MSU)-induced gouty arthritis in mice were employed in vivo to validate the inhibitory effect of celastrol on NLRP3 inflammasome.ResultsCelastrol significantly suppressed the cleavage of pro-caspase-1 and pro-IL-1β, while not affecting the protein expressions of NLRP3, ASC, pro-caspase-1 and pro-IL-1β in THP-1 cells, BMDMs and HEK293T cells. Celastrol suppressed NLRP3 inflammasome activation and alleviated P. acnes/LPS-induced liver damage and MSU-induced gouty arthritis. Mechanism study revealed that celastrol could interdict K63 deubiquitination of NLRP3, which may concern interaction of celastrol and BRCA1/BRCA2-containing complex subunit 3 (BRCC3), and thereby prohibited the formation of NLRP3, ASC and pro-caspase-1 complex to block the generation of mature IL-1β.ConclusionCelastrol suppresses NLRP3 inflammasome activation in P. acnes/LPS-induced liver damage and MSU-induced gouty arthritis via inhibiting K63 deubiquitination of NLRP3, which presents a novel insight into inhibition of celastrol on NLRP3 inflammasome and provides more evidences for its application in the therapy of inflammation-related diseases.     

Participation of c-FLIP in NLRP3 and AIM2 inflammasome activation

Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage survival. Recent studies have revealed a selective role of caspase-8 in noncanonical IL-1β production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP_L is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1β. Hemizygotic deletion of c-FLIP impaired ATP- and monosodium uric acid (MSU)-induced IL-1β production in macrophages primed through Toll-like receptors (TLRs). Decreased IL-1β expression was attributed to a reduced activation of caspase-1 in c-FLIP hemizygotic cells. In contrast, the production of TNF-α was not affected by downregulation in c-FLIP. c-FLIP_L interacted with NLRP3 or procaspase-1. c-FLIP is required for the full NLRP3 inflammasome assembly and NLRP3 mitochondrial localization, and c-FLIP is associated with NLRP3 inflammasome. c-FLIP downregulation also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic-, FasL-, or Dectin-1-induced IL-1β generation that is caspase-8-mediated. Our results demonstrate a prominent role of c-FLIP_L in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by over-activation of inflammasomes.     

Endogenous and Uric Acid-Induced Activation of NLRP3 Inflammasome in Pregnant Women with Preeclampsia

Preeclampsia (PE) is a specific syndrome of pregnancy, characterized by hypertension and proteinuria. This pathology is associated with hyperuricemia and elevated serum levels of inflammatory cytokines. Uric acid crystals may activate an intracellular complex called inflammasome, which is important for processing and release of inflammatory cytokines. This study investigated the state of monocyte activation, both endogenous and stimulated with monosodium urate (MSU), by gene expression of NLRP1 and NLRP3 receptors as well as their association with inflammatory cytokines expression. Monocytes were obtained from peripheral blood of 23 preeclamptic pregnant women, 23 normotensive pregnant women (NT) and 23 healthy non-pregnant women (NP). Inflammasome activation was evaluated by the gene expression of NLRP1, NLRP3, caspase-1, IL-1β, IL-18 and TNF-α by RT-qPCR in unstimulated monocytes (endogenous expression), or after cell stimulation with MSU (stimulated expression). The concentration of cytokines was assessed by ELISA. In preeclamptic pregnant women, gene expression of NLRP1, NLRP3, caspase-1, IL-1β and TNF-α by monocytes stimulated or not with MSU was significantly higher than in NT and NP groups. Stimulation of monocytes from preeclamptic and non-pregnant women with MSU induced increased gene expression of NLRP3, caspase-1 and TNF-α in relation to the endogenous expression in these groups, while this was not observed in the NT group. The cytokine determination showed that monocytes from women with PE produced higher endogenous levels of IL-1β, IL-18 and TNF-α compared to the other groups, while the stimulus with MSU led to higher production of these cytokines in preeclamptic group than in the NT group. In conclusion, the results showed increased basal gene expression of NLRP1 and NLRP3 receptors in monocytes from PE group. These cells stimulation with MSU demonstrates that uric acid plays a role in NLRP3 inflammasome activation, suggesting the participation of this inflammatory complex in the pathogenesis of preeclampsia.     

MicroRNA-133a-1 regulates inflammasome activation through uncoupling protein-2

Inflammasomes are multimeric protein complexes involved in the processing of IL-1β through Caspase-1 cleavage. NLRP3 is the most widely studied inflammasome, which has been shown to respond to a large number of both endogenous and exogenous stimuli. Although studies have begun to define basic pathways for the activation of inflammasome and have been instrumental in identifying therapeutics for inflammasome related disorders; understanding the inflammasome activation at the molecular level is still incomplete. Recent functional studies indicate that microRNAs (miRs) regulate molecular pathways and can lead to diseased states when hampered or overexpressed. Mechanisms involving the miRNA regulatory network in the activation of inflammasome and IL-1β processing is yet unknown. This report investigates the involvement of miR-133a-1 in the activation of inflammasome (NLRP3) and IL-1β production. miR-133a-1 is known to target the mitochondrial uncoupling protein 2 (UCP2). The role of UCP2 in inflammasome activation has remained elusive. To understand the role of miR-133a-1 in regulating inflammasome activation, we either overexpressed or suppressed miR-133a-1 in differentiated THP1 cells that express the NLRP3 inflammasome. Levels of Caspase-1 and IL-1β were analyzed by Western blot analysis. For the first time, we showed that overexpression of miR-133a-1 increases Caspase-1 p10 and IL-1β p17 cleavage, concurrently suppressing mitochondrial uncoupling protein 2 (UCP2). Surprisingly, our results demonstrated that miR-133A-1 controls inflammasome activation without affecting the basal expression of the individual inflammasome components NLRP3 and ASC or its immediate downstream targets proIL-1β and pro-Caspase-1. To confirm the involvement of UCP2 in the regulation of inflammasome activation, Caspase-1 p10 and IL-1β p17 cleavage in UCP2 of overexpressed and silenced THP1 cells were studied. Suppression of UCP2 by siRNA enhanced the inflammasome activity stimulated by H₂O₂ and, conversely, overexpression of UCP2 decreased the inflammasome activation. Collectively, these studies suggest that miR-133a-1 suppresses inflammasome activation via the suppression of UCP2.     

Baeckein E suppressed NLRP3 inflammasome activation through inhibiting both the priming and assembly procedure: Implications for gout therapy

BackgroundBaeckein E (BF-2) was isolated from the aerial parts of Baeckea frutescens L., which has a long history of use in traditional medicine in Southeast Asia to treat inflammatory disease.PurposeBF-2 was identified to have inhibitory activity on nucleotide oligomerization domain (NOD)-like receptor protein-3 inflammasome (NLRP3) activation. This study aimed to investigate the related signaling cascade of BF-2 in both lipopolysaccharides (LPS)/ATP induced pyroptosis in J774A.1 macrophages and its application in a mouse model of gout induced by monosodium urate crystal (MSU).MethodsThe effect of BF-2 on NLRP3 inflammasome activation and gouty arthritis was studied in J774A.1 macrophages and male C57BL/6 mice. The J774A.1 macrophages were primed with LPS and stained by propidium iodide (PI) for cell pyroptosis detection. A gout mouse model was established by subcutaneous injection of MSU crystals into the hind paw of C57BL/6 mice. Mice were then randomly divided into different groups. The concentrations of IL-1β and IL-18 in both J774A.1 macrophage and gout mouse model were analyzed by ELISA. The NLRP3 inflammasome related protein expression was detected by western blot analysis. The inhibitory effects of BF-2 on NLRP3 inflammasome assembly were analyzed by immunoprecipitation assay. The roles of BF-2 in mitochondrial damage were imaged by Mito Tracker Green and Mito Tracker Red probes. The inhibitory effects of BF-2 on ROS production were imaged by DCF (2′,7′-dichlorofluorescein diacetate) probe.ResultsThe results demonstrated BF-2 could significantly suppress the cell pyroptosis and IL-1β secretion in macrophages. Furthermore, BF-2 significantly inhibited NLRP3 inflammasome activation and reduced ankle swelling in the gout mouse model. In detail, it alleviated mitochondrial damage mediated oxidative stress and inhibited the assembly of NLRP3 inflammasome by affecting the binding of pro-Caspase 1 and ASC. Moreover, BF-2 blocked NLRP3 activation by inhibiting the MAPK/NF-κB signaling pathways.ConclusionsResults demonstrated BF-2 inhibited NLRP3 inflammasome activation in both LPS primed macrophages and mouse model of gout through blocking MAPK/NF-κB signaling pathway and mitochondrial damage mediated oxidative stress. This study strongly suggests BF-2 could be a promising drug candidate against inflammatory diseases associated with NLRP3 inflammasome activation.     

TLR2/MyD88/NF-κB pathway, reactive oxygen species, potassium efflux activates NLRP3/ASC inflammasome during respiratory syncytial virus infection

Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although "controlled" inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1β (IL-1β) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 to yield mature IL-1β that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1β release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1β secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1β production during RSV infection. Further studies illustrated that prior to inflammasome formation; the "first signal" constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection. Following expression of these genes, two "second signals" are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K(+)) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1β release during RSV infection.     

Vascular endothelial cells senescence is associated with NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation via reactive oxygen species (ROS)/thioredoxin-interacting protein (TXNIP) pathway

Endothelial dysfunction caused by endothelial cells senescence and chronic inflammation is tightly linked to the development of cardiovascular diseases. NLRP3 (NOD-like receptor family pyrin domain-containing3) inflammasome plays a central role in inflammatory response that is associated with diverse inflammatory diseases. This study explores the effects and possible mechanisms of NLRP3 inflammasome in endothelial cells senescence. Results show an increment of pro-inflammatory cytokine interleukin (IL) −1β secretion and caspase-1 activation during the senescence of endothelial cells induced by bleomycin. Moreover, secreted IL-1β promoted endothelial cells senescence through up-regulation of p53/p21 protein expression. NLRP3 inflammasome was found to mediate IL-1β secretion through the production of ROS (reactive oxygen species) during the senescence of endothelial cells. Furthermore, the association of TXNIP (thioredoxin-interacting protein) with NLRP3 induced by ROS promoted NLRP3 inflammasome activation in senescent endothelial cells. In addition, the expressions of NLRP3 inflammasome related genes, ASC (apoptosis associated speck-like protein containing a CARD), TXNIP, cleaved caspase-1 and IL-1β, were also increased in vitro and in vivo studies. These findings indicate that endothelial senescence could be mediated through ROS and NLRP3 inflammasome signaling pathways, suggesting a potential target for the prevention of endothelial senescence-related cardiovascular diseases.     

CD36 promotes NLRP3 inflammasome activation via the mtROS pathway in renal tubular epithelial cells of diabetic kidneys

Tubulointerstitial inflammation plays a key role in the pathogenesis of diabetic nephropathy (DN). Interleukin-1β (IL-1β) is the key proinflammatory cytokine associated with tubulointerstitial inflammation. The NLRP3 inflammasome regulates IL-1β activation and secretion. Reactive oxygen species (ROS) represents the main mediator of NLRP3 inflammasome activation. We previously reported that CD36, a class B scavenger receptor, mediates ROS production in DN. Here, we determined whether CD36 is involved in NLRP3 inflammasome activation and explored the underlying mechanisms. We observed that high glucose induced-NLRP3 inflammasome activation mediate IL-1β secretion, caspase-1 activation, and apoptosis in HK-2 cells. In addition, the levels of CD36, NLRP3, and IL-1β expression (protein and mRNA) were all significantly increased under high glucose conditions. CD36 knockdown resulted in decreased NLRP3 activation and IL-1β secretion. CD36 knockdown or the addition of MitoTempo significantly inhibited ROS production in HK-2 cells. CD36 overexpression enhanced NLRP3 activation, which was reduced by MitoTempo. High glucose levels induced a change in the metabolism of HK-2 cells from fatty acid oxidation (FAO) to glycolysis, which promoted mitochondrial ROS (mtROS) production after 72 h. CD36 knockdown increased the level of AMP-activated protein kinase (AMPK) activity and mitochondrial FAO, which was accompanied by the inhibition of NLRP3 and IL-1β. The in vivo experimental results indicate that an inhibition of CD36 could protect diabetic db/db mice from tubulointerstitial inflammation and tubular epithelial cell apoptosis. CD36 mediates mtROS production and NLRP3 inflammasome activation in db/db mice. CD36 inhibition upregulated the level of FAO-related enzymes and AMPK activity in db/db mice. These results suggest that NLRP3 inflammasome activation is mediated by CD36 in renal tubular epithelial cells in DN, which suppresses mitochondrial FAO and stimulates mtROS production.Subject terms: Biochemistry, Cell biology     

Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1

Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.