Attenuated NLRP3 Inflammasome Activation Research Articles

Sirtuin 3-induced macrophage autophagy in regulating NLRP3 inflammasome activation

Defective autophagy of monocytes or macrophages might result in NLRP3 inflammasome activation and cause vascular metabolic inflammation. However, the mechanism underlying the initiation of the autophagy response to hyperlipidaemia remains unclear. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is sensitive to the metabolic status and mediates adaptation responses. In this study, we investigated the role of SIRT3-mediated autophagy in regulating NLRP3 inflammasome activation. We determined that the inhibition of autophagy and the activation of the NLRP3 inflammasome were concomitant with reduced SIRT3 levels both in peripheral blood monocytes from obese humans and in palmitate-treated THP-1 cells. Furthermore, we demonstrated that SIRT3 could form a molecular complex with ATG5, while SIRT3 overexpression altered the acetylation of endogenous ATG5. ATG5 acetylation inhibited autophagosome maturation and induced NLRP3 inflammasome activation. In parallel, SIRT3 overexpression in THP-1 cells decreased the palmitate-induced generation of mitochondrial reactive oxygen species, restored autophagy, and attenuated NLRP3 inflammasome activation. The incubation of human aortic endothelial cells (HAECs) with macrophage-conditioned medium (MCM) induced HAEC expression of vascular cell adhesion molecule-1, intercellular adhesion molecule 1, α-smooth muscle actin, and collagen-1. The effect of MCM could be reversed by the addition of neutralizing anti-IL-1β antibody or the overexpression of SIRT3. Consistent with this, en face analyses displayed a marked increase in α-SMC-positive endothelial cells in SIRT3−/− mice with acute hyperlipidaemia. Taken together, these findings revealed that SIRT3-deficient macrophages displayed impaired autophagy and accelerated NLRP3 inflammasome activation and endothelial dysfunction.

MicroRNA-132 Negatively Regulates Palmitate-Induced NLRP3 Inflammasome Activation through FOXO3 Down-Regulation in THP-1 Cells.

Saturated fatty acids were proposed to activate the NLRP3 inflammasome, a molecular platform that mediates the processing of interleukin (IL)-1β and IL-18. However, the mechanisms underlying the miRNA-mediated regulation of palmitate (PA)-induced inflammasome activation are unclear. We examined the role of miR-132 in PA-induced NLRP3 inflammasome activation in THP-1 cells. To understand the regulatory role of miR-132 in inflammasome activation, we either overexpressed or suppressed miR-132 in THP-1 cells that expressed the NLRP3 inflammasome in response to stimulation by PA. We analyzed the mRNA and protein levels of NLRP3, caspase-1 p10, IL-18, and IL-1β; caspase-1 activity; and IL-1β secretion. The presence of PA activated the NLRP3 inflammasome and increased miR-132 expression. Overexpression of miR-132 reduced caspase-1 p10, IL-18, and IL-1β, while the suppression of miR-132 enhanced inflammasome activation. In addition, miR-132 regulated the mRNA and protein expression of FOXO3, which is a potential target of miR-132 in these cells. FOXO3 suppression by small interfering RNA decreased NLRP3 inflammasome activity stimulated by PA. Knockdown of FOXO3 attenuated NLRP3 inflammasome activation by the miR-132 inhibitor. Based on these findings, we conclude that miR-132 negatively regulates PA-induced NLRP3 inflammasome activation through FOXO3 down-regulation in THP-1 cells.

BOT-4-one attenuates NLRP3 inflammasome activation: NLRP3 alkylation leading to the regulation of its ATPase activity and ubiquitination

The ATPase activity of NLRP3 has pivotal role in inflammasome activation and is recognized as a good target for the development of the NLRP3 inflammasome-specific inhibitor. However, signals in the vicinity of the ATPase activity of NLRP3 have not been fully elucidated. Here, we demonstrate NLRP3 inflammasome-specific action of a benzoxathiole derivative, BOT-4-one. BOT-4-one exhibited an inhibition of NLRP3 inflammasome activation, which was attributable to its alkylating capability to NLRP3. In particular, the NLRP3 alkylation by BOT-4-one led to an impaired ATPase activity of NLRP3, thereby obstructing the assembly of the NLRP3 inflammasome. Additionally, we found that NLRP3 alkylators, including BOT-4-one, enhance the ubiquitination level of NLRP3, which might also contribute to the inhibition of NLRP3 inflammasome activation. Finally, BOT-4-one appeared to be superior to other known NLRP3 alkylators in inhibiting the functionality of the NLRP3 inflammasome and its resulting anti-inflammatory activity was confirmed in vivo using a monosodium urate-induced peritonitis mouse model. Collectively, the results suggest that NLRP3 alkylators function by inhibiting ATPase activity and increasing the ubiquitination level of NLRP3, and BOT-4-one could be the type of NLRP3 inhibitor that may be potentially useful for the novel development of a therapeutic agent in controlling NLRP3 inflammasome-related diseases.

Induction of heme oxygenas-1 attenuates NLRP3 inflammasome activation in lipopolysaccharide-induced mastitis in mice

Mastitis is one of most prevalent production disease in dairy herds worldwide, and is responsible for enormous economic losses. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme, which is involved in the response to oxidative stress and inflammatory response. The purpose of this study was to detect the protective effect of HO-1 on LPS-induced mastitis in mice. BALB/c mice were pretreated with hemin (HO-1 inducer) and zinc protoporphyrin (ZnPP; HO-1 inhibitor) at 2h before LPS stimulation. The results showed that the mammary gland damage, production of inflammatory cytokines IL-1β, and MPO activity in mammary gland tissues were significantly reduced after pretreated with hemin compared with the group of LPS stimulation only. However, ZnPP reversed the effects of hemin. Furthermore, we found that the levels of ROS and NLRP3 inflammasome were increased after LPS stimulation. The increases were inhibited by hemin and the inhibition of hemin on ROS production and NLRP3 inflammasome activation were blocked by ZnPP. In addition, the results showed that hemin reduced the expression of thioredoxin-interacting protein (TXNIP) induced by LPS, and ZnPP attenuated these changes. In conclusion, the results suggested that overproduction of HO-1 may inhibit the activation of NLRP3 inflammasome and the expression of TXNIP. Induction of HO-1 may be served as a promising method against mastitis induced by LPS.

Melatonin alleviates inflammasome-induced pyroptosis through inhibiting NF-κB/GSDMD signal in mice adipose tissue.

Pyroptosis is a proinflammatory form of cell death that is associated with pathogenesis of many chronic inflammatory diseases. Melatonin is substantially reported to possess anti-inflammatory properties by inhibiting inflammasome activation. However, the effects of melatonin on inflammasome-induced pyroptosis in adipocytes remain elusive. Here, we demonstrated that melatonin alleviated lipopolysaccharides (LPS)-induced inflammation and NLRP3 inflammasome formation in mice adipose tissue. The NLRP3 inflammasome-mediated pyroptosis was also inhibited by melatonin in adipocytes. Further analysis revealed that gasdermin D (GSDMD), the key executioner of pyroptosis, was the target for melatonin inhibition of adipocyte pyroptosis. Importantly, we determined that nuclear factor κB (NF-κB) signal was required for the GSDMD-mediated pyroptosis in adipocytes. We also confirmed that melatonin alleviated adipocyte pyroptosis by transcriptional suppression of GSDMD. Moreover, GSDMD physically interacted with interferon regulatory factor 7 (IRF7) and subsequently formed a complex to promote adipocyte pyroptosis. Melatonin also attenuated NLRP3 inflammasome activation and pyroptosis, which was induced by LPS or obesity. In summary, our results demonstrate that melatonin alleviates inflammasome-induced pyroptosis by blocking NF-κB/GSDMD signal in mice adipose tissue. Our data reveal a novel function of melatonin on adipocyte pyroptosis, suggesting a new potential therapy for melatonin to prevent and treat obesity caused systemic inflammatory response.

Involvement of c-Abl Kinase in Microglial Activation of NLRP3 Inflammasome and Impairment in Autolysosomal System

A growing body of evidence suggests that excessive microglial activation and pesticide exposure may be linked to the etiology of PD; however, the mechanisms involved remain elusive. Emerging evidence indicates that intracellular inflammasome complex namely NLRP3 complex is involved in the recognition and execution of host inflammatory response. Thus, in the present study, we investigated the hypothesis that NLRP3 inflammasome activation is linked to rotenone (ROT)-induced microglial activation which is dependent upon a priming stimulus by a pathogen-associated molecular pattern (PAMP) or damage associated molecular pattern (DAMP), respectively. Herein using both BV2 cells and primary microglial cells, we show that LPS priming and subsequent ROT stimulation enhanced NLRP3 inflammasome activation, c-Abl and PKCδ activation, mitochondrial dysfunction, NF-κB activation, and autophagic markers, while TFEB levels were decreased dramatically. Mechanistic studies revealed c-Abl acts as a proximal signal that exacerbated the activation of the afore mentioned markers. Intriguingly, siRNA-mediated depletion or pharmacological inhibition of c-Abl via dasatinib abrogated LPS and ROT-induced microglial activation response via attenuation of NLRP3 inflammasome activation, mitochondrial oxidative stress, and ALS dysfunction. Moreover, mitoTEMPO, a mitochondrial antioxidant, attenuated NLRP3 inflammasome activation effects via blockade of c-Abl and PKCδ activation. In LPS treated mice, dasatinib attenuated NLRP3 inflammasome activation, c-Abl and PKCδ activation; and sickness behavior. Together our findings identify an exaggerated ROS/c-Abl/NLRP3 signaling axis in the heightened microglial activation response evidenced in LPS-primed ROT-stimulated microglial cells and suggest that targeting c-Abl-regulated NLRP3 inflammasome signaling offers a novel therapeutic strategy for PD treatment. Graphical Abstract ᅟ.

Abstract 96: Melatonin Prevents Atherosclerosis via Regulating NLRP3 Inflammasome: The Role of Sirt3 Signaling

Introduction: NLRP3 inflammasome mediated inflammatory factors secretion is critically involved in atherosclerosis (AS). Melatonin has anti-inflammatory properties. However, it is unknown whether melatonin is beneficial in AS. Hypothesis: Melatonin plays a beneficial role in AS by decreasing NLRP3 inflammasome activation in macrophages. Methods: AS model was induced with high fat diet in apoE -/- mice. Plaque stability was examined with historical staining. In vitro study was performed in ox-LDL treated RAW264.7 cells. NLRP3 inflammasome activation, inflammatory factors secretion, mitochondrial ROS generation, autophagy, mitophagy indexes and potential signaling pathways were investigated. Results: Historical staining results showed that melatonin treatment markedly alleviated AS plaque progression. Despite of unchanged protein expression, Sirt 3 activity was elevated in plaque tissue in melatonin treated mice. Melatonin attenuated NLRP3 inflammasome activation and inflammatory factors secretion in ox-LDL treated macrophages, while this protective effect was abolished by Sirt3-siRNA. Mitochondrial ROS (mitoROS), which was an inducer for NLRP3 inflammasome, was reduced by melatonin through the elimination of damaged mitochondria (mitophagy). Similar with Sirt3-siRNA, autophagy inhibitor 3-MA also abolished the effects of melatonin on mitoROS clearance, indicating the crucial role of autophagy and mitophagy in melatonin caused NLRP3 inactivation. Furthermore, melatonin protected against AS via Sirt3/FoxO3/Parkin signaling pathway. Conclusions: Melatonin prevented atherosclerotic progression. Melatonin reduced mitochondrial ROS through the activation of autophagy and mitophagy, thereby attenuating NLRP3 inflammasome activation in macrophages. Moreover, the protective effect of melatonin was mediated by Sirt3/FoxO3/Parkin signaling pathway. Our study provides insight into a new therapeutic target for AS.

A novel mechanism of diabetic vascular endothelial dysfunction: Hypoadiponectinemia-induced NLRP3 inflammasome activation

It has been well documented that hypoadiponectinemia is associated with impaired endothelium-dependent vasodilation. However, the exact molecular mechanism which mediates this process has not been fully described. The current study aimed to investigate the role of hypoadiponectinemia-induced NLRP3 inflammasome activation in diabetic vascular endothelial dysfunction and its molecular mechanism. Male adult adiponectin knockout mice and wild type mice were fed with a high fat diet to establish a type 2 diabetic mellitus model. In addition, human umbilical vein endothelial cells (HUVECs) were cultured and subjected to high glucose/high fat (HG/HF). The NLRP3 inflammasome activation was increased in type 2 diabetic mice and treatment of diabetic aortic segments with MCC950, a potent selective inhibitor of NLRP3 inflammasome ex vivo improved endothelial-dependent vasorelaxation. NLRP3 inflammasome activation and vascular endothelial injury were significantly increased in APN-KO mice compared with WT mice in diabetes and MCC950 decreased diabetic vascular endothelial dysfunction to comparable levels in APN-KO mice and WT mice. Adiponectin could decrease NLRP3 inflammasome activation and attenuate endothelial cell injury, which was abolished by NLRP3 inflammasome overexpression. Inhibition of peroxynitrite formation preferentially attenuated NLRP3 inflammasome activation in APN-KO diabetic mice. The current study demonstrated for the first time that hypoadiponectinemia-induced NLRP3 inflammasome activation was a novel mechanism of diabetic vascular endothelial dysfunction.

Nonsaponin fractions of Korean Red Ginseng extracts prime activation of NLRP3 inflammasome

BackgroundKorean Red Ginseng extracts (RGE) have been suggested as effective immune modulators, and we reported that ginsenosides possess anti-inflammasome properties. However, the properties of nonsaponin components of RGE have not been well studied. MethodsTo assess the roles of nonsaponin fractions (NS) in NLRP3 inflammasome activation, we treated murine macrophages with or without first or second inflammasome activation signals with RGE, NS, or saponin fractions (SF). The first signal was nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated transcription of pro-interleukin (IL)-1β and NLRP3 while the second signal triggered assembly of inflammasome components, leading to IL-1β maturation. In addition, we examined the role of NS in IL-6 production and IL-1β maturation in mice. ResultsNS induced IL-1β and NLRP3 transcription via toll-like receptor 4 signaling, whereas SF blocked expression. During the second signal, SF attenuated NLRP3 inflammasome activation while NS did not. Further, NS-injected mice presented increased IL-1β maturation and IL-6 production. ConclusionSF and NS of RGE play differential roles in the NLRP3 inflammasome activation. Hence, RGE can be suggested as an NLRP3 inflammasome modulator.

Non-transcriptional regulation of NLRP3 inflammasome signaling by IL-4.

Th2 cytokine IL-4 has been previously shown to suppress the production of proinflammatory cytokines in monocytes. However, the underlying molecular mechanism by which IL-4 signaling antagonizes proinflammatory responses is poorly characterized. In particular, whether IL-4 can modulate inflammasome signaling remains unknown. Here, we provide evidence that IL-4 suppresses NLRP3-dependent caspase-1 activation and the subsequent IL-1β secretion but does not inhibit absent in melanoma 2 (AIM2)- or NLRC4 (NOD-like receptor family, CARD domain-containing 4)-dependent caspase-1 activation in THP-1 and mouse bone marrow-derived macrophages. Upon lipopolysaccharide (LPS) or LPS/ATP stimulation, IL-4 markedly inhibited the assembly of NLRP3 inflammasome, including NLRP3-dependent ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) oligomerization, NLRP3-ASC interaction and NLRP3 speck-like oligomeric structure formation. The negative regulation of NLRP3 inflammasome by IL-4 was not due to the impaired mRNA or protein production of NLRP3 and proinflammatory cytokines. Supporting this observation, IL-4 attenuated NLRP3 inflammasome activation even in reconstituted NLRP3-expressing macrophages in which NLRP3 expression is not transcriptionally regulated by TLR-NF-κB signaling. Furthermore, the IL-4-mediated suppression of NLRP3 inflammasome was independent of STAT6-dependent transcription and mitochondrial reactive oxygen species (ROS). Instead, IL-4 inhibited subcellular redistribution of NLRP3 into mitochondria and microtubule polymerization upon NLRP3-activating stimulation. Our results collectively suggest that IL-4 could suppress NLRP3 inflammasome activation in a transcription-independent manner, thus providing an endogenous regulatory machinery to prevent excessive inflammasome activation.

Methylsulfonylmethane inhibits NLRP3 inflammasome activation

Methylsulfonylmethane (MSM) is an organosulfur compound and the health benefits associated with MSM include inflammation. Although MSM has been shown to have various physiological effects, no study has yet focused on inflammasome activation. The inflammasome is a multiprotein complex that serves as a platform for caspase 1-dependent proteolytic maturation and secretion of interleukin-1β (IL-1β). In this study, we tested the effect of MSM on inflammasome activation using mouse and human macrophages. In our results, MSM significantly attenuated NLRP3 inflammasome activation in lipopolysaccharide-primed macrophages, although it had no effect on NLCR4 or AIM2 inflammasome activation. Extracts of MSM-enriched vegetables presented the same inhibitory effect on NLRP3 inflammasome activation as MSM. MSM also attenuated the transcriptional expression of IL-1α, IL-1β, IL-6, and NLRP3. Taken together, these results show that MSM has anti-inflammatory characteristics, interrupts NLRP3 inflammasome activation, and inhibits pro-cytokine expression. We further confirmed the intracellular mechanism of MSM in relation to NLRP3 inflammasome activation, followed by comparison with that of DMSO. Both chemicals showed a synergic effect on anti-NLRP3 activation and attenuated production of mitochondrial reactive oxygen species (ROS). Thus, MSM is a selective inhibitor of NLRP3 inflammasome activation and can be developed as a supplement to control several metabolic disorders.

Dimethyl sulfoxide inhibits NLRP3 inflammasome activation

Dimethyl sulfoxide (DMSO) is an amphipathic molecule that is commonly/widely used as a solvent for biological compounds. In addition, DMSO has been studied as a medication for the treatment of inflammation, cystitis, and arthritis. Based on the anti-inflammatory characteristics of DMSO, we elucidated the effects of DMSO on activation of inflammasomes, which are cytoplasmic multi-protein complexes that mediate the maturation of interleukin (IL)-1β by activating caspase-1 (Casp1). In the present study, we prove that DMSO attenuated IL-1β maturation, Casp1 activity, and ASC pyroptosome formation via NLRP3 inflammasome activators. Further, NLRC4 and AIM2 inflammasome activity were not affected, suggesting that DMSO is a selective inhibitor of the NLRP3 inflammasomes. The anti-inflammatory effect of DMSO was further confirmed in animal, LPS-endotoxin sepsis and inflammatory bowel disease models. In addition, DMSO inhibited LPS-mediating IL-1s transcription. Taken together, DMSO shows anti-inflammatory characteristics, attenuates NLRP3 inflammasome activation, and mediates inhibition of IL-1s transcription.

Contribution of Reactive Oxygen Species to NLRP3 Inflammasome Activation in Glomeruli of Mice with Hyperhomocysteinemia

Our previous studies have shown the importance of NADPH oxidase‐derived reactive oxygen species (ROS) in hyperhomocysteinemia (hHcys)‐induced glomerular injury. Using SOD mimetic TEMPOL, catalase, and uric acid to reduce superoxide (O2•−), hydrogen peroxide (H2O2), and peroxynitrite (ONOO−), we have recently shown that these ROS are involved in hHcys‐induced inflammasome activation in podocytes. The present study was designed to further test whether in vivo scavenging of O2•−and H2O2 attenuates NLRP3 inflammasome activation and glomerular injury in mice with hHcys. Uninephrectomized C57BL/6J wild type mice were fed either normal chow (ND) or a folate free (FF) diet for 4 weeks to produce hHcys. Confocal microscopy showed that hHcys increased the colocalization of inflammasome proteins NLRP3 with ASC or caspase‐1 in the glomeruli compared to ND fed mice. Inflammasome formation mainly occurred in podocytes as shown by colocalization of NLRP3 with podocyte marker podocin. In vivo scavenging of O2•− by TEMPOL and removal of H2O2 by catalase substantially inhibited NLRP3 inflammasome formation in glomeruli of hHcys mice. Correspondingly, TEMPOL and catalase treatment inhibited hHcys‐induced caspase‐1 activation, respectively by 25.9% and 33.8%, and decreased IL‐1β production by 38.8% and 35.7%. ESR analysis demonstrated that TEMPOL produced a 44.3% decrease of renal O2•−production, while catalase produced a 42.4% decrease. Importantly, reduction of both O2•−and H2O2 was found to protect glomerular function and structure, preventing hHcys‐induced proteinuria, albuminuria and glomerular morphological pathology. Together, these results indicate that endogenously produced O2•− and H2O2 contribute to NLRP3 inflammasome formation and activation in vivo at early stage hHcys and that inhibition of these ROS may prevent NLRP3 inflammasome activation and subsequent glomerular injury (supported by NIH grants DK54927 and 1F31AG043289).