Increased NLRP3 Inflammasome Activation Research Articles

Co-Treatment with Phlorotannin and Extracellular Vesicles from Ecklonia cava Inhibits UV-Induced Melanogenesis.

Hyperpigmentation due to ultraviolet (UV)-induced melanogenesis causes various esthetic problems. Phlorotannin (PT) and extracellular vesicles (EVs) derived from various plants suppress melanogenesis pathways. We used UV-exposed keratinocytes and animal skin to determine if co-treatment with PT and EVs from Ecklonia cava (EVE) could inhibit melanogenesis by reducing UV-induced oxidative stress and the expression of the thioredoxin-interacting protein (TXNIP)/nucleotide-binding oligomerization domain-like receptor family pyrin domain containing the 3 (NLRP3)/interleukin-18 (IL-18) pathway, which are upstream signals of the microphthalmia-associated transcription factor. UV exposure increased oxidative stress in keratinocytes and animal skin, as evaluated by 8-OHdG expression, and this effect was reduced by co-treatment with PT and EVE. UV also increased binding between NLRP3 and TXNIP, which increased NLRP3 inflammasome activation and IL-18 secretion, and this effect was reduced by co-treatment with PT and EVE in keratinocytes and animal skin. In melanocytes, conditioned media (CM) from UV-exposed keratinocytes increased the expression of melanogenesis-related pathways; however, these effects were reduced with CM from UV-exposed keratinocytes treated with PT and EVE. Similarly, PT and EVE treatment reduced melanogenesis-related signals, melanin content, and increased basement membrane (BM) components in UV-exposed animal skin. Thus, co-treatment with PT and EVE reduced melanogenesis and restored the BM structure by reducing oxidative stress and TXNIP/NLRP3/IL-18 pathway expression.

Altered vacuole membrane protein 1 (VMP1) expression is associated with increased NLRP3 inflammasome activation and mitochondrial dysfunction.

Altered expression of vacuole membrane protein 1 (VMP1) has recently been observed in the context of multiple sclerosis and Parkinson's disease (PD). However, how changes in VMP1 expression may impact pathogenesis has not been explored. This study aimed to characterize how altered VMP1 expression affects NLRP3 inflammasome activation and mitochondrial function. VMP1 expression was depleted in a monocytic cell line using CRISPR-Cas9. The effect of VMP1 on NLRP3 inflammasome activation was examined by stimulating cells with LPS and ATP or α-synuclein fibrils. Inflammasome activation was determined by caspase-1 activation using both a FLICA assay and a biosensor as well as by the release of proinflammatory molecules measured by ELISA. RNA-sequencing was utilized to define global gene expression changes resulting from VMP1 deletion. SERCA activity and mitochondrial function were investigated using various fluorescence microscopy-based approaches including a novel method that assesses the function of individual mitochondria in a cell. Here, we report that genetic deletion of VMP1 from a monocytic cell line resulted in increased NLRP3 inflammasome activation and release of proinflammatory molecules. Examination of the VMP1-dependent changes in these cells revealed that VMP1 deficiency led to decreased SERCA activity and increased intracellular [Ca2+]. We also observed calcium overload in mitochondria in VMP1 depleted cells, which was associated with mitochondrial dysfunction and release of mitochondrial DNA into the cytoplasm and the extracellular environment. Collectively, these studies reveal VMP1 as a negative regulator of inflammatory responses, and we postulate that decreased expression of VMP1 can aggravate the inflammatory sequelae associated with neurodegenerative diseases like PD.

Lysophosphatidic Acid Induces Podocyte Pyroptosis in Diabetic Nephropathy by an Increase of Egr1 Expression via Downregulation of EzH2.

Podocyte damage and renal inflammation are the main features and pathogenesis of diabetic nephropathy (DN). Inhibition of lysophosphatidic acid (LPA) receptor 1 (LPAR1) suppresses glomerular inflammation and improves DN. Herein, we investigated LPA-induced podocyte damage and its underlying mechanisms in DN. We investigated the effects of AM095, a specific LPAR1 inhibitor, on podocytes from streptozotocin (STZ)-induced diabetic mice. E11 cells were treated with LPA in the presence or absence of AM095, and the expression of NLRP3 inflammasome factors and pyroptosis were measured. A chromatin immunoprecipitation assay and Western blotting were performed to elucidate underlying molecular mechanisms. Gene knockdown by transfecting small interfering RNA was used to determine the role of the transcription factor Egr1 (early growth response protein 1) and histone methyltransferase EzH2 (Enhancer of Zeste Homolog 2) in LPA-induced podocyte injury. AM095 administration inhibited podocyte loss, NLRP3 inflammasome factor expression, and cell death in STZ-induced diabetic mice. In E11 cells, LPA increased NLRP3 inflammasome activation and pyroptosis via LPAR1. Egr1 mediated NLRP3 inflammasome activation and pyroptosis in LPA-treated E11 cells. LPA decreased H3K27me3 enrichment at the Egr1 promoter in E11 cells by downregulating EzH2 expression. EzH2 knockdown further increased LPA-induced Egr1 expression. In podocytes from STZ-induced diabetic mice, AM095 suppressed Egr1 expression increase and EzH2/H3K27me3 expression reduction. Collectively, these results demonstrate that LPA induces NLRP3 inflammasome activation by downregulating EzH2/H3K27me3 and upregulating Egr1 expression, resulting in podocyte damage and pyroptosis, which may be a potential mechanism of DN progression.

Increased NLRP3 Inflammasome Activation and Pyroptosis in Patients With Multiple Sclerosis With Fingolimod Treatment Failure.

Inflammasomes are involved in the pathogenesis of different neuroimmune and neurodegenerative diseases, including multiple sclerosis (MS). In a previous study by our group, the nucleotide-binding oligomerization domain, leucine-rich repeat receptor and pyrin-domain-containing 3 (NLRP3) inflammasome was reported to be associated with the response to interferon-beta in MS. Based on recent data showing the potential for the oral therapy fingolimod to inhibit NLRP3 inflammasome activation, here we investigated whether fingolimod could also be implicated in the response to this therapy in patients with MS. NLRP3 gene expression levels were measured by real-time PCR in peripheral blood mononuclear cells at baseline and after 3, 6, and 12 months in a cohort of patients with MS treated with fingolimod (N = 23), dimethyl fumarate (N = 21), and teriflunomide (N = 21) and classified into responders and nonresponders to the treatment according to clinical and radiologic criteria. In a subgroup of fingolimod responders and nonresponders, the percentage of monocytes with an oligomer of ASC was determined by flow cytometry, and the levels of interleukin (IL)-1β, IL-18, IL-6, tumor necrosis factor (TNF)α, and galectin-3 were quantified by ELISA. NLPR3 expression levels were significantly increased in fingolimod nonresponders after 3 (p = 0.03) and 6 months (p = 0.008) of treatment compared with the baseline but remained similar in responders at all time points. These changes were not observed in nonresponders to the other oral therapies tested. The formation of an oligomer of ASC in monocytes after lipopolysaccharide and adenosine 5'-triphosphate stimulation was significantly decreased in responders (p = 0.006) but increased in nonresponders (p = 0.0003) after 6 months of fingolimod treatment compared with the baseline. Proinflammatory cytokine release from stimulated peripheral blood mononuclear cells was comparable between responders and nonresponders, but galectin-3 levels on cell supernatants, as a marker of cell damage, were significantly increased in fingolimod nonresponders (p = 0.02). The differential effect of fingolimod on the formation of an inflammasome-triggered ASC oligomer in monocytes between responders and nonresponders could be used as a response biomarker after 6 months of fingolimod treatment and suggests that fingolimod may exert their beneficial effects by reducing inflammasome signaling in a subset of patients with MS.

YAP1 protects against PM2.5-induced lung toxicity by suppressing pyroptosis and ferroptosis

Pollution from fine particulate matter (PM2.5) has become a major threat to public health and has been related to lung toxicity. One of the key regulators of the Hippo signaling system, Yes-associated protein 1 (YAP1), is speculated to play a role in ferroptosis development. Here, we focused on investigating the function of YAP1 in pyroptosis and ferroptosis, aiming to explore its therapeutic potential in PM2.5-induced lung toxicity. PM2.5-induced lung toxicity was induced in Wild-type WT and conditional YAP1-knockout mice, and lung epithelial cells were stimulatd by PM2.5 in vitro. We used western blot, transmission electron microscopy, and fluorescence microscopy to investigate pyroptosis- and ferroptosis-related characteristics. We found that PM2.5 leads to lung toxicity using mechanisms involving pyroptosis and ferroptosis. YAP1 knockdown impeded pyroptosis, ferroptosis, and PM2.5-induced lung damage, as shown by increased histopathology, higher levels of proinflammatory cytokines, GSDMD protein, lipid peroxidation, and iron accumulation, as well as increased NLRP3 inflammasome activation and decreased SLC7A11 expression. YAP1 silencing consistently promoted NLRP3 inflammasome activation and reduced SLC7A11 levels, aggravating PM2.5-induced cellular damage. In contrast, YAP1-overexpressing cells inhibited NLRP3 inflammasome activation and increased SLC7A11 levels, preventing pyroptosis and ferroptosis. Overall, our data suggest that YAP1 ameliorates PM2.5-induced lung injury by inhibiting NLRP3-mediated pyroptosis and SL7A11-dependent ferroptosis.

Inactivation of mitochondrial pyruvate carrier promotes NLRP3 inflammasome activation and gout development via metabolic reprogramming.

The nucleotide-binding and oligomerization domain, leucine-rich repeats, and pyrin domain-containing protein 3 (NLRP3) inflammasome plays a crucial role in innate immunity and is involved in the pathogenesis of autoinflammatory diseases. Glycolysis regulates NLRP3 inflammasome activation in macrophages. However, how lactic acid fermentation and pyruvate oxidation controlled by the mitochondrial pyruvate carrier (MPC) affect NLRP3 inflammasome activation and autoinflammatory disease remains elusive. We found that the inactivation of MPC with genetic depletion or pharmacological inhibitors, MSDC-0160 or pioglitazone, increased NLRP3 inflammasome activation and IL-1β secretion in macrophages. Glycolytic reprogramming induced by MPC inhibition skewed mitochondrial ATP-associated oxygen consumption into cytosolic lactate production, which enhanced NLRP3 inflammasome activation in response to monosodium urate (MSU) crystals. As pioglitazone is an insulin sens MSDC-itizer used for diabetes, its MPC inhibitory effect in diabetic individuals was investigated. The results showed that MPC inhibition exacerbated MSU-induced peritonitis in diabetic mice and increased the risk of gout in patients with diabetes. Altogether, we found that glycolysis controlled by MPC regulated NLRP3 inflammasome activation and gout development. Accordingly, prescriptions for medications targeting MPC should consider the increased risk of NLRP3-related autoinflammatory diseases.

Recombinant protein diannexin prevents preeclampsia-like symptoms in a pregnant mouse model via reducing the release of microparticles.

Preeclampsia (PE) is characterized by placenta-mediated pregnancy complication. The only effective treatment for PE is the delivery of the placenta. However, this treatment may cause preterm birth and neonatal death. Therefore, preventing PE is needed. The mechanism of PE involves abnormal placentation, which leads to the release of anti-angiogenic and inflammatory mediators into maternal circulation. These mediators contribute to systemic vascular dysfunction, inflammatory responses, and excessive thrombin generation. Microparticles (MPs) are reportedly involved in PE by promoting the thromboinflammatory response. This study describes a strategy to prevent PE by reducing MP release using the recombinant protein, diannexin. Results showed that the patients with PE had elevated MP number and procoagulant activity and increased NLRP3 inflammasome activation. Additionally, diannexin remarkably reduced the release of MPs from activated cells by binding to phosphatidylserine exposed on the surface of activated cells. Moreover, in vivo results showed that diannexin could prevent PE-like symptoms by decreasing MPs and NLRP3 inflammasome activation in pregnant mice. Furthermore, diannexin effectively inhibited trophoblast cell activation and NLRP3 inflammasome activation in vitro. These findings suggested that diannexin inhibited MP release and might be an effective therapeutic strategy for preventing PE.

Abstract P355: Involvement Of Nlrp3 Inflammasome In Vascular Injury-induced Neointimal Formation And PDGF-BB Stimulated Monocyte Retention And Pyroptosis In Arterial Smooth Muscle Cells

Intimal hyperplasia due to pathological vascular remodeling is a common feature of cardiovascular diseases such as atherosclerosis and restenosis after angioplasty. Nlrp3 inflammasome has been reported to trigger endothelial injury leading to pathological vascular remodeling in metabolic diseases. This study aimed to elucidate whether the Nlrp3 inflammasome in smooth muscle cells (SMCs) per se is involved in their phenotypic regulation and thereby contributes to pathological vascular remodeling. The ligation injury-induced neointimal hyperplasia model was established in hypercholesterolemic mice. Mice received a single intravenous injection of AAV encoding mutant mPCSK9 (rAAV8-D377Y-mPCSK9). Then mice received partial ligation surgery in left carotid artery (LCA) and consumed a high fat, high cholesterol diet for 4 weeks. The neointimal formation and various markers related to synthetic phenotype and Nlrp3 inflammasome pathways were assessed in ligated LCA. The collateral un-ligated right carotid artery (RCA) served as controls. Our data revealed that ligation injury induced neointimal formation in Nlrp3 +/+ mice, which was markedly attenuated in Nlrp3 -/- mice (intima area: Nlrp3 +/+ 15.97±4.07 μm 2 vs. Nlrp3 -/- 4.31±1.27 μm 2 ). Ligation injury drastically increased Nlrp3 inflammasome activation, monocyte adhesion, pyroptosis, and synthetic phenotype trans-differentiation in LCAs of Nlrp3 +/+ mice. These changes were largely limited in the SMC-enriched neointimal region and abrogated in Nlrp3 -/- mice. PDGF-BB, a ligation injury-associated factor promoting SMC synthetic phenotype transition, activated Nlrp3 inflammasomes in cultured SMCs. Inhibition of inflammasome-dependent caspase-1 by Ac-YVAD-cmk attenuated PDGF-BB-induced SMC-monocyte interaction and pyroptosis, whereas proliferation and migration of SMCs were unaffected. Together, these results suggest that in arterial SMCs, activation of Nlrp3 inflammasomes and subsequent events including induction of pyroptosis and adhesion of monocytes are essential for ligation injury-induced neointimal formation. These results provide new insight into the mechanisms of Nlrp3 inflammasome action in SMCs in pathological vascular remodeling in hypercholesterolemia.

The Ala134Thr variant in TMEM176B exerts a beneficial role in colorectal cancer prognosis by increasing NLRP3 inflammasome activation.

TMEM176B was recently described as a negative modulator of Nlrp3 inflammasome activation in mice. In the mouse model, the inhibition of TMEM176B leads to an increased anti-tumoral activity which is dependent on Nlrp3. Since we have recently shown that single nucleotide variants (SNPs) in inflammasome genes, including NLRP3, significantly affect colorectal cancer (CRC) prognosis, we proposed to investigate here the association between genetic variants in TMEM176B and CRC prognosis. Considering that, up to now, no genetic study analyzing this gene in humans exists, we selected possible functional SNPs and genotyped them in a cohort of CRC patients submitted to surgery and followed up for more than 10years. Genotype-guided assays were realized to evaluate the effect of the variant on NLRP3 inflammasome activation. Gene expression from The Cancer Genome Atlas (TCGA) cohort was analyzed to valid possible prognostic and predictive features. We identified the Ala134Thr variant (rs2072443) in TMEM176B as a protective factor for CRC prognosis. This SNP is associated with decreased gene expression and with an increased activation of NLRP3 inflammasome, at least in monocytes and dendritic cells. Furthermore, low TMEM176B expression is associated with higher overall survival. Altogether, these findings supported the role of TMEM176B in NLRP3 inflammasome biology and for the first time demonstrated the genetic association between rs2072443 and CRC in humans.

IBD-associated G protein-coupled receptor 65 variant compromises signalling and impairs key functions involved in inflammation

Background and aimsInflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these. MethodsWe used HEK 293 cells stably expressing GPR65 and deficient for either Gαs, Gαq/11 or Gα12/13, to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation. ResultsWe confirmed that GPR65 signals via the Gαs pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1β secretion in response to an inflammatory stimulus. ConclusionsWe demonstrate that GPR65 exerts its effects through Gαs- and Gα12/13-mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies.

Dopamine signaling modulates microglial NLRP3 inflammasome activation: implications for Parkinson\u2019s disease

BackgroundParkinson’s disease (PD) is characterized by the loss of nigral dopaminergic neurons leading to impaired striatal dopamine signaling, α-synuclein- (α-syn-) rich inclusions, and neuroinflammation. Degenerating neurons are surrounded by activated microglia with increased secretion of interleukin-1β (IL-1β), driven largely by the NLRP3 inflammasome. A critical role for microglial NLRP3 inflammasome activation in the progression of both dopaminergic neurodegeneration and α-syn pathology has been demonstrated in parkinsonism mouse models. Fibrillar α-syn activates this inflammasome in mouse and human macrophages, and we have shown previously that the same holds true for primary human microglia. Dopamine blocks microglial NLRP3 inflammasome activation in the MPTP model, but its effects in this framework, highly relevant to PD, remain unexplored in primary human microglia and in other in vivo parkinsonism models.MethodsBiochemical techniques including quantification of IL-1β secretion and confocal microscopy were employed to gain insight into dopamine signaling-mediated inhibition of the NLRP3 inflammasome mechanism in primary human microglia and the SYN120 transgenic mouse model. Dopamine and related metabolites were applied to human microglia together with various inflammasome activating stimuli. The involvement of the receptors through which these catecholamines were predicted to act were assessed with agonists in both species.ResultsWe show in primary human microglia that dopamine, l-DOPA, and high extracellular K+, but not norepinephrine and epinephrine, block canonical, non-canonical, and α-syn-mediated NLRP3 inflammasome-driven IL-1β secretion. This suggests that dopamine acts as an inflammasome inhibitor in human microglia. Accordingly, we provide evidence that dopamine exerts its inhibitory effect through dopamine receptor D1 and D2 (DRD1 and DRD2) signaling. We also show that aged mice transgenic for human C-terminally truncated (1–120) α-syn (SYN120 tg mice) display increased NLRP3 inflammasome activation in comparison to WT mice that is diminished upon DRD1 agonism.ConclusionsDopamine inhibits canonical, non-canonical, and α-syn-mediated activation of the NLRP3 inflammasome in primary human microglia, as does high extracellular K+. We suggest that dopamine serves as an endogenous repressor of the K+ efflux-dependent microglial NLRP3 inflammasome activation that contributes to dopaminergic neurodegeneration in PD, and that this reciprocation may account for the specific vulnerability of these neurons to disease pathology.

Quinolinic Acid, a Tryptophan Metabolite of the Skin Microbiota, Negatively Regulates NLRP3 Inflammasome through AhR in Psoriasis

Psoriasis is a chronic inflammatory skin disease whose pathogenesis involves skin microbiota dysbiosis. Multiple studies have revealed the changes in microbiota abundances between psoriatic lesions and healthy skin. However, the metabolic pathways of skin microbiota (especially tryptophan metabolism, which is closely related to immunosuppression) are far less understood. In this study, we first detected the major microbial metabolites of tryptophan on the skin surfaces, finding that the quinolinic acid was significantly lower in the lesional skin of patients with psoriasis than in that of healthy subjects and correlated negatively with the severity of psoriasis. Invitro and invivo, applying quinolinic acid significantly alleviated skin inflammation in an AhR-dependent manner, resulting in inhibition of the NLRP3 inflammasome activation. Furthermore, in mice with imiquimod-induced psoriasis-like dermatitis, topical application of Ahr-targeted small interfering RNA substantially exacerbated the disease severity, with increased NLRP3 inflammasome activation. Collectively, our data suggest that quinolinic acid, a skin microbiota-derived metabolite, negatively regulates aryl hydrocarbon receptor-NLRP3 inflammasome signaling activation in patients with psoriasis, providing an insight into the correlation between microbiota metabolism and the host skin in individuals with psoriasis.

TFEB signaling attenuates NLRP3-driven inflammatory responses in severe asthma.

NLRP3-driven inflammatory responses by circulating and lung-resident monocytes are critical drivers of asthma pathogenesis. Autophagy restrains NLRP3-induced monocyte activation in asthma models. Yet, the effects of autophagy and its master regulator, transcription factor EB (TFEB), on monocyte responses in human asthma remain unexplored. Here, we investigated whether activation of autophagy and TFEB signaling suppress inflammatory monocyte responses in asthmatic individuals. Peripheral blood CD14+ monocytes from asthmatic patients (n=83) and healthy controls (n=46) were stimulated with LPS/ATP to induce NLRP3 activation with or without the autophagy inducer, rapamycin. ASC specks, caspase-1 activation, IL-1β and IL-18 levels, mitochondrial function, ROS release, and mTORC1 signaling were examined. Autophagy was evaluated by LC3 puncta formation, p62/SQSTM1 degradation and TFEB activation. In a severe asthma (SA) model, we investigated the role of NLRP3 signaling using Nlrp3-/- mice and/or MCC950 administration, and the effects of TFEB activation using myeloid-specific TFEB-overexpressing mice or administration of the TFEB activator, trehalose. We observed increased NLRP3 inflammasome activation, concomitant with impaired autophagy in circulating monocytes that correlated with asthma severity. SA patients also exhibited mitochondrial dysfunction and ROS accumulation. Autophagy failed to inhibit NLRP3-driven monocyte responses, due to defective TFEB activation and excessive mTORC1 signaling. NLRP3 blockade restrained inflammatory cytokine release and linked airway disease. TFEB activation restored impaired autophagy, attenuated NLRP3-driven pulmonary inflammation, and ameliorated SA phenotype. Our studies uncover a crucial role for TFEB-mediated reprogramming of monocyte inflammatory responses, raising the prospect that this pathway can be therapeutically harnessed for the management of SA.

Modulation of the NLRP3 inflammasome by Sars-CoV-2 Envelope protein

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1β and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1β, levels of IL-1β and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.

Cardiomyocyte NLRP3 Contributes to the Cardiac Inflammatory Response

Despite extensive clinical and basic science research, cardiovascular diseases continue to be the leading cause of death worldwide. In response to pathological insults, the heart attempts to adapt and normalize cardiac function by initiating a compensatory response that drives wound healing and remodeling of the myocardium. The immune system and its inflammatory mediators are currently recognized as active participants in cardiac disease progression and the development of heart failure. We have previously demonstrated that the cardiomyocyte is the initial site of proinflammatory gene transcription following transverse aortic constriction (TAC) and that this occurs through signal transduction by activated Ca2+/Calmodulin-Dependent Kinase II (CaMKIIδ). Activation of cardiomyocyte CaMKIIδ transduces cardiac cellular stress signals into inflammatory gene expression via canonical NFκβ signaling and the NLRP3 inflammasome. The NLRP3 inflammasome is rapidly primed and activated in cardiomyocytes following TAC as indicated by caspase-1 activation and generation of proinflammatory cytokines IL-1β and IL-18. Interleukin-1β is regarded as a potent mediator of sterile inflammation often associated with neutrophilic infiltration, and its biological activity is tightly controlled by multiple levels of regulation including the requirement for proteolytic cleavage of the full-length transcript to its biologically active form by active caspase-1 recruited to the NLRP3 inflammasome complex. To investigate the role of the NLRP3 inflammasome in cardiomyocytes, we have generated mice with constitutive NLRP3 (designated as FCAS after the human mutation in familial cold autoinflammatory syndrome) expression in cardiomyocytes. This was achieved by administration of cardiomyocyte-specific adeno-virus associated virus serotype 9 (AAV9) expressing cre recombinase into mice floxed with the mutant FCAS gene. At the conclusion of 3 weeks after virus administration, a low dose of 2mg/kg lipopolysaccharide (LPS) was administered as a priming event for the transcription of inflammasome signaling components. At the conclusion of 6 hours after LPS injection, blood was collected for measurements of systemic inflammation, and whole hearts were collected for analysis NLRP3 priming (gene expression) and NLRP3 activation using qPCR and measuring caspase1 enzymatic activity. Inflammasome priming, as measured by an increase in Il-18 and caspase-1, were increased in FCAS LPS animals compared to wild type control LPS animals, although we did not observe differences in Il-1β or NLRP3 gene expression. Whole ventricular tissue from these mice were probed for activated NLRP3 via the measurement of caspase-1 enzymatic activity AAV9-Cre administered mice with floxed FCAS gene increased NLRP3 inflammasome activation compared to virus injection into wild type mice, but there were no measured differences between FCAS LPS and wild type control LPS mice. Blood from FCAS LPS mice had significantly increased serum Il-1β levels compared to control LPS mice suggesting that cardiomyocyte-derived inflammasome products can contribute to appreciable levels of inflammation.

MicroRNA-10 negatively regulates inflammation in diabetic kidney via targeting activation of the NLRP3 inflammasome

NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome activation has emerged as a central mediator of kidney inflammation in diabetic kidney disease (DKD). However, the mechanism underlying this activation in DKD remains poorly defined. In this study, we found that kidney-enriched microRNA-10a and -10b (miR-10a/b), predominantly expressed in podocytes and tubular epithelial cells, were downregulated in kidney from diabetic mice and patients with DKD. High glucose decreased miR-10a/b expression invitro in an osmolarity-independent manner. miR-10a/b functioned as negative regulators of the NLRP3 inflammasome through targeting the 3'untranslated region of NLRP3 mRNA, inhibiting assembly of the NLRP3 inflammasome and decreasing caspase-1-dependent release of pro-inflammatory cytokines. Delivery of miR-10a/b into kidney prevented NLRP3 inflammasome activation and renal inflammation, and it reduced albuminuria in streptozotocin (STZ)-treated mice, whereas knocking down miR-10a/b increased NLRP3 inflammasome activation. Restoration of miR-10a/b expression in established DKD ameliorated kidney inflammation and mitigated albuminuria in both db/db and STZ-treated mice. These results suggest a novel intervention strategy for inhibiting kidney inflammation in DKD by targeting the NLRP3 inflammasome.

NLRP3 inflammasome mediated pyroptosis is involved in cadmium exposure-induced neuroinflammation through the IL-1β/IkB-α-NF-κB-NLRP3 feedback loop in swine

Cadmium (Cd) chloride, as widely distributed toxic environmental pollutants by using in industry, severely imperils animal and human health. Pyroptosis is a Cas1-dependent pro-inflammatory programmed cell death and involves in various types of diseases. Nevertheless, the mechanism of pyroptosis and Cd-induced neurotoxicity remains obscure. To investigate the specific molecular mechanisms of Cd-induced neurotoxicity, 10 weaned piglets were randomly divided into 2 groups treated with 0 and 20 mg/kg CdCl2 in the diet for 40 days. The levels of pyroptosis, mitochondrial and inflammation-related genes were validated by qRT-PCR and WB in vivo. Our results revealed that Cd caused cerebral histopathology lesions, inducing cerebral pyroptosis and the mass generation of inflammatory cytokines, as indicated by the increased NLRP3 inflammasome activation (NLRP3, Cas1 and ASC) and the upregulation of inflammation factors IL-2, IL-6, IL-7 and inhibition of IL-10. Subsequently, further research indicated that Cd triggered pyroptosis via activating the TRAF6-IkB-α-NF-κB pathway, which interfered with the phosphorylation and ubiquitination of IkB-α. Furthermore, Cd caused mitochondrial dysfunction and fragmentation by inhibiting the AMPK-PGC-1α-NRF1/2 signaling pathway and reduced the expression of mitochondrial-related regulatory factors OPA1, TFAM and mtDNA, resulting in the increase of NLRP3 inflammasome. Besides, we found eight hub genes (IKK, IKB-α, NLRP3, TRAF6, NF-κB, AMPK, TNFα and PGC-1α), mainly related to the interaction between the NF-κB pathway and NLRP3 inflammasome. Overall, these results demonstrated that Cd could promote the IL-1β/IkB-α-NF-κB-NLRP3 inflammasome activation positive feedback loop to result in neuroinflammation in swine, which provided new insights in understanding Cd-induced toxicity.

Overexpression of Macrophage-Inducible C-Type Lectin Mincle Aggravates Proinflammatory Responses to Streptococcus pneumoniae with Fatal Outcome in Mice.

Macrophage-inducible C-type lectin (Mincle)-dependent sensing of pathogens triggers proinflammatory immune responses in professional phagocytes that contribute to protecting the host against pathogen invasion. In this study, we examined whether overexpression of Mincle that is designed to improve early pathogen sensing by professional phagocytes would improve lung-protective immunity against Streptococcus pneumoniae in mice. Proteomic profiling of alveolar macrophages of Mincle transgenic (tg) mice stimulated with the Mincle-specific pneumococcal ligand glucosyl-diacylglycerol (Glc-DAG) revealed increased Nlrp3 inflammasome activation and downstream IL-1β cytokine release that was not observed in Glc-DAG-stimulated Mincle knockout or Nlrp3 knockout macrophages. Along this line, Mincle tg mice also responded with a stronger Nlrp3 expression and early proinflammatory cytokine release after challenge with S. pneumoniae, ultimately leading to fatal pneumonia in the Mincle tg mice. Importantly, Nlrp3 inhibitor treatment of Mincle tg mice significantly mitigated the observed hyperinflammatory response to pneumococcal challenge. Together, we show that overexpression of the pattern recognition receptor Mincle triggers increased Glc-DAG-dependent Nlrp3 inflammasome activation in professional phagocytes leading to fatal pneumococcal pneumonia in mice that is amenable to Nlrp3 inhibitor treatment. These data show that ectopic expression of the Mincle receptor confers increased susceptibility rather than resistance to S. pneumoniae in mice, thus highlighting the importance of an inducible Mincle receptor expression in response to microbial challenge.

The deubiquitinase STAMBP modulates cytokine secretion through the NLRP3 inflammasome

The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a multimeric, cytoplasmic, protein complex that regulates maturation and secretion of interleukin (IL)-1β, a potent pro-inflammatory cytokine. Critical to host defense against pathogens, IL-1β amplifies early innate immune responses by activating transcription of numerous other cytokines and chemokines. Excessive IL-1β is associated with poor outcomes in inflammatory illnesses, such as sepsis and the acute respiratory distress syndrome (ARDS). Tight regulation of this signaling axis is vital, but little is known about mechanisms to limit excessive inflammasome activity. Here we identify the deubiquitinase STAM-binding protein (STAMBP) as a negative regulator of the NLRP3 inflammasome. In monocytes, knockout of STAMBP by CRISPR/Cas9 gene editing increased expression of numerous cytokines and chemokines in response to Toll-like receptor (TLR) agonists or bacterial lipopolysaccharide (LPS). This exaggerated inflammatory response was dependent on IL-1β signaling, and STAMBP knockout directly increased release of IL-1β with TLR ligation. While STAMBP does not modulate NLRP3 protein abundance, cellular depletion of the deubiquitinase increased NLRP3 K63 chain polyubiquitination resulting in increased NLRP3 inflammasome activation. These findings describe a unique mechanism of non-degradative ubiquitination of NLRP3 by STAMBP to limit excessive inflammasome activation and to reduce injurious IL-1β signaling.

Metformin protects against myocardial ischemia-reperfusion injury and cell pyroptosis via AMPK/NLRP3 inflammasome pathway.

Ischemia/reperfusion (I/R) injury is a life-threatening vascular emergency following myocardial infarction. Our previous study showed cardioprotective effects of metformin against myocardial I/R injury. In this study, we further examined the involvement of AMPK mediated activation of NLRP3 inflammasome in this cardioprotective effect of metformin. Myocardial I/R injury was simulated in a rat heart Langendorff model and neonatal rat ventricle myocytes (NRVMs) were subjected to hypoxi/reoxygenation (H/R) to establish an in vitro model. Outcome measures included myocardial infarct size, hemodynamic monitoring, myocardial tissue injury, myocardial apoptotic index and the inflammatory response. myocardial infarct size and cardiac enzyme activities. First, we found that metformin postconditioning can not only significantly alleviated myocardial infarct size, attenuated cell apoptosis, and inhibited myocardial fibrosis. Furthermore, metformin activated phosphorylated AMPK, decreased pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β, and decreased NLRP3 inflammasome activation. In isolated NRVMs metformin increased cellular viability, decreased LDH activity and inhibited cellular apoptosis and inflammation. Importantly, inhibition of AMPK phosphorylation by Compound C (CC) resulted in decreased survival of cardiomyocytes mainly by inducing the release of inflammatory cytokines and increasing NLRP3 inflammasome activation. Finally, in vitro studies revealed that the NLRP3 activator nigericin abolished the anti-inflammatory effects of metformin in NRVMs, but it had little effect on AMPK phosphorylation. Collectively, our study confirmed that metformin exerts cardioprotective effects by regulating myocardial I/R injury-induced inflammatory response, which was largely dependent on the enhancement of the AMPK pathway, thereby suppressing NLRP3 inflammasome activation.