SLC25A3 negatively regulates NLRP3 inflammasome activation by restricting the function of NLRP3.

The NACHT, leucine-rich repeat, and pyrin domains-containing protein 3 (collectively known as NLRP3) inflammasome activation plays a critical role in innate immune and pathogenic microorganism infections. However, excessive activation of NLRP3 inflammasome will lead to cellular inflammation and tissue damage, and naturally it must be precisely controlled in the host. Here, we discovered that solute carrier family 25 member 3 (SLC25A3), a mitochondrial phosphate carrier protein, plays an important role in negatively regulating NLRP3 inflammasome activation. We found that SLC25A3 could interact with NLRP3, overexpression of SLC25A3 and knockdown of SLC25A3 could regulate NLRP3 inflammasome activation, and the interaction of NLRP3 and SLC25A3 is significantly boosted in the mitochondria when the NLRP3 inflammasome is activated. Our detailed investigation demonstrated that the interaction between NLRP3 and SLC25A3 disrupted the interaction of NLRP3-NEK7, promoted ubiquitination of NLRP3, and negatively regulated NLRP3 inflammasome activation. Thus, these findings uncovered a new regulatory mechanism of NLRP3 inflammasome activation, which provides a new perspective for the therapy of NLRP3 inflammasome-associated inflammatory diseases.

Desloratadine alleviates ALS-like pathology in hSOD1G93A mice via targeting 5HTR2A on activated spinal astrocytes.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with progressive loss of motor neurons in the spinal cord, cerebral cortex and brain stem. ALS is characterized by gradual muscle atrophy and dyskinesia. The limited knowledge on the pathology of ALS has impeded the development of therapeutics for the disease. Previous studies have shown that autophagy and astrocyte-mediated neuroinflammation are involved in the pathogenesis of ALS, while 5HTR2A participates in the early stage of astrocyte activation, and 5HTR2A antagonism may suppress astrocyte activation. In this study, we evaluated the therapeutic effects of desloratadine (DLT), a selective 5HTR2A antagonist, in human SOD1G93A (hSOD1G93A) ALS model mice, and elucidated the underlying mechanisms. HSOD1G93A mice were administered DLT (20 mg·kg-1·d-1, i.g.) from the age of 8 weeks for 10 weeks or until death. ALS onset time and lifespan were determined using rotarod and righting reflex tests, respectively. We found that astrocyte activation accompanying with serotonin receptor 2 A (5HTR2A) upregulation in the spinal cord was tightly associated with ALS-like pathology, which was effectively attenuated by DLT administration. We showed that DLT administration significantly delayed ALS symptom onset time, prolonged lifespan and ameliorated movement disorders, gastrocnemius injury and spinal motor neuronal loss in hSOD1G93A mice. Spinal cord-specific knockdown of 5HTR2A by intrathecal injection of adeno-associated virus9 (AAV9)-si-5Htr2a also ameliorated ALS pathology in hSOD1G93A mice, and occluded the therapeutic effects of DLT administration. Furthermore, we demonstrated that DLT administration promoted autophagy to reduce mutant hSOD1 levels through 5HTR2A/cAMP/AMPK pathway, suppressed oxidative stress through 5HTR2A/cAMP/AMPK/Nrf2-HO-1/NQO-1 pathway, and inhibited astrocyte neuroinflammation through 5HTR2A/cAMP/AMPK/NF-κB/NLRP3 pathway in the spinal cord of hSOD1G93A mice. In summary, 5HTR2A antagonism shows promise as a therapeutic strategy for ALS, highlighting the potential of DLT in the treatment of the disease. DLT as a 5HTR2A antagonist effectively promoted autophagy to reduce mutant hSOD1 level through 5HTR2A/cAMP/AMPK pathway, suppressed oxidative stress through 5HTR2A/cAMP/AMPK/Nrf2-HO-1/NQO-1 pathway, and inhibited astrocytic neuroinflammation through 5HTR2A/cAMP/AMPK/NF-κB/NLRP3 pathway in the spinal cord of hSOD1G93A mice.

A positive feedback cycle between the alarmin S100A8/A9 and NLRP3 inflammasome-GSDMD signalling reinforces the innate immune response in Candida albicans keratitis.

OBJECTIVE: Fungal keratitis is a severe sight-threatening ocular infection, without effective treatment strategies available now. Calprotectin S100A8/A9 has recently attracted great attention as a critical alarmin modulating the innate immune response against microbial challenges. However, the unique role of S100A8/A9 in fungal keratitis is poorly understood. METHODS: Experimental fungal keratitis was established in wild-type and gene knockout (TLR4-/- and GSDMD-/-) mice by infecting mouse corneas with Candida albicans. The degree of mouse cornea injuries was evaluated by clinical scoring. To interrogate the molecular mechanism in vitro, macrophage RAW264.7 cell line was challenged with Candida albicans or recombinant S100A8/A9 protein. Label-free quantitative proteomics, quantitative real-time PCR, Western blotting, and immunohistochemistry were conducted in this research. RESULTS: Herein, we characterized the proteome of mouse corneas infected with Candida albicans and found that S100A8/A9 was robustly expressed at the early stage of the disease. S100A8/A9 significantly enhanced disease progression by promoting NLRP3 inflammasome activation and Caspase-1 maturation, accompanied by increased accumulation of macrophages in infected corneas. In response to Candida albicans infection, toll-like receptor 4 (TLR4) sensed extracellular S100A8/A9 and acted as a bridge between S100A8/A9 and NLRP3 inflammasome activation in mouse corneas. Furthermore, the deletion of TLR4 resulted in noticeable improvement in fungal keratitis. Remarkably, NLRP3/GSDMD-mediated macrophage pyroptosis in turn facilitates S100A8/A9 secretion during Candida albicans keratitis, thus forming a positive feedback cycle that amplifies the proinflammatory response in corneas. CONCLUSIONS: The present study is the first to reveal the critical roles of the alarmin S100A8/A9 in the immunopathology of Candida albicans keratitis, highlighting a promising approach for therapeutic intervention in the future.

Nanocellulose Length Determines the Differential Cytotoxic Effects and Inflammatory Responses in Macrophages and Hepatocytes.

Nanocellulose including cellulose nanocrystal (CNC) and cellulose nanofiber (CNF) has attracted much attention due to its exceptional mechanical, chemical, and rheological properties. Although considered biocompatible, recent reports have demonstrated nanocellulose can be hazardous, including serving as drug carriers that accumulate in the liver. However, the nanocellulose effects on liver cells, including Kupffer cells (KCs) and hepatocytes are unclear. Here, the toxicity of nanocellulose with different lengths is compared, including the shorter CNCs (CNC-1, CNC-2, and CNC-3) and longer CNF (CNF-1 and CNF-2), to liver cells. While all CNCs triggered significant cytotoxicity in KCs and only CNC-2 induced toxicity to hepatocytes, CNFs failed to induce significant cytotoxicity due to their minimal cellular uptake. The phagocytosis of CNCs by KCs induced mitochondria ROS generation, caspase-3/7 activation, and apoptotic cell death as well as lysosomal damage, cathepsin B release, NLRP3 inflammasome and caspase-1 activation, and IL-1ß production. The cellular uptake of CNC-2 by hepatocytes is through clathrin-mediated endocytosis, and it induced the caspase-3/7-mediated apoptosis. CNC-2 shows the highest levels of uptake and cytotoxicity among CNCs. These results demonstrate the length-dependent mechanisms of toxicity on liver cells in a cell type-dependent fashion, providing information to safely use nanocellulose for biomedical applications.

A mini-review on ion fluxes that regulate NLRP3 inflammasome activation.

The activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome can be induced by a wide spectrum of activators. This is unlikely achieved by the binding of different activators directly to the NLRP3 protein itself, as the activators found so far show different forms of chemical structures. Previous studies have shown that these activators can induce potassium ion (K+) and chloride ion (Cl-) efflux, calcium (Ca2+) and other ion mobilization, mitochondrial dysfunction, and lysosomal disruption, all of which are believed to cause NLRP3 inflammasome activation; how these events are induced by the activators and how they coordinate with each other in inducing the NLRP3 inflammasome activation are not fully understood. Increasing evidence suggests that the coordinated change of intracellular ion concentrations may be a common mechanism for the NLRP3 activation by different activators. In this mini-review, we present a brief summary of the current knowledge about how different ionic flows (including K+, sodium ion, Ca2+, magnesium ion, manganese ion, zinc ion, iron ion, and Cl-) are involved in regulating the NLRP3 inflammasome activation in macrophages.

NLRP3-associated autoinflammatory diseases: Phenotypic and molecular characteristics of germline versus somatic mutations.

BACKGROUND: NLRP3-associated autoinflammatory diseases (NLRP3-AIDs) include conditions of various severities, due to germline or somatic mosaic NLRP3 mutations. OBJECTIVE: To identify mosaic- versus germline-specific NLRP3 mutations' characteristics, we reinterpreted all the mutations reported in NLRP3-AIDs and performed an in-depth study of 3 novel patients. METHODS: The pathogenicity of all reported mosaic/germline mutations was reassessed according to international recommendations and their location on the NLRP3 3-dimensional structure. Deep-targeted sequencing and NLRP3-inflammasome-activation assays were used to identify the disease-causing mutation in 3 patients. RESULTS: We identified, in 3 patients, mosaic mutations affecting the same NLRP3 amino acid (Glu569). This residue belongs to 1 of the 2 mosaic mutational hot spots that face each other in the core of the NLRP3 ATPase domain. The review of the 90 NLRP3 mutations identified in 277 patients revealed that those hot spots account for 68.5% of patients (37 of 54) with mosaic mutations. Glu569 is affected in 22% of the patients (12 of 54) with mosaic mutations and in 0.4% of patients (1 of 223) with germline mutations. Only 8 of 90 mutations were found in mosaic and germinal states. All of the germline mutations were associated with a severe phenotype. These data suggest that mutations found only in mosaic state could be incompatible with life if present in germinal state. None of the 5 most frequent germline mutations was identified in mosaic state. Mutations found only in germinal state could, therefore, be asymptomatic in mosaic state. CONCLUSIONS: The phenotypic spectrum of NLRP3-AIDs appears to be related to the germinal/mosaic status and localization of the underlying mutations.

NLRP3 Inflammasome Activation Controls Vascular Smooth Muscle Cells Phenotypic Switch in Atherosclerosis.

(1) Background: Monocytes and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome orchestrate lipid-driven amplification of vascular inflammation promoting the disruption of the fibrous cap. The components of the NLRP3 inflammasome are expressed in macrophages and foam cells within human carotid atherosclerotic plaques and VSMCs in hypertension. Whether monocytes and NLRP3 inflammasome activation are direct triggers of VSMC phenotypic switch and plaque disruption need to be investigated. (2) Methods: The direct effect of oxLDL-activated monocytes in VSMCs co-cultured system was demonstrated via flow cytometry, qPCR, ELISA, caspase 1, and pyroptosis assay. Aortic roots of VSMCs lineage tracing mice fed normal or high cholesterol diet and human atherosclerotic plaques were used for immunofluorescence quantification of NLRP3 inflammasome activation/VSMCs phenotypic switch. (3) Results: OxLDL-activated monocytes reduced α-SMA, SM22α, Oct-4, and upregulation of KLF-4 and macrophage markers MAC2, F4/80 and CD68 expression as well as caspase 1 activation, IL-1ß secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1ß in phenotypically modified VSMCs was detected in the aortic roots of VSMCs lineage tracing mice fed high cholesterol diet and in human atherosclerotic plaques from carotid artery disease patients who experienced a stroke. (4) Conclusions: Taken together, these results provide evidence that monocyte promote VSMC phenotypic switch through VSMC NLRP3 inflammasome activation with a likely detrimental role in atherosclerotic plaque stability in human atherosclerosis.

Mechanistic Differences in Cell Death Responses to Metal-Based Engineered Nanomaterials in Kupffer Cells and Hepatocytes.

The mononuclear phagocyte system in the liver is a frequent target for nanoparticles (NPs). A toxicological profiling of metal-based NPs is performed in Kupffer cell (KC) and hepatocyte cell lines. Sixteen NPs are provided by the Nanomaterial Health Implications Research Consortium of the National Institute of Environmental Health Sciences to study the toxicological effects in KUP5 (KC) and Hepa 1-6 cells. Five NPs (Ag, CuO, ZnO, SiO2 , and V2 O5 ) exhibit cytotoxicity in both cell types, while SiO2 and V2 O5 induce IL-1ß production in KC. Ag, CuO, and ZnO induced caspase 3 generated apoptosis in both cell types is accompanied by ion shedding and generation of mitochondrial reactive oxygen species (ROS) in both cell types. However, the cell death response to SiO2 in KC differs by inducing pyroptosis as a result of potassium efflux, caspase 1 activation, NLRP3 inflammasome assembly, IL-1ß release, and cleavage of gasdermin-D. This releases pore-performing peptide fragments responsible for pyroptotic cell swelling. Interestingly, although V2 O5 induces IL-1ß release and delays caspase 1 activation by vanadium ion interference in membrane Na+ /K+ adenosine triphosphate (ATP)ase activity, the major cell death mechanism in KC (and Hepa 1-6) is caspase 3 mediated apoptosis. These findings improve the understanding of the mechanisms of metal-based engineered nanomaterial (ENM) toxicity in liver cells toward comprehensive safety evaluation.

[Triptolide inhibits NLRP3 inflammasome activation and ameliorates podocyte epithelial-mesenchymal transition induced by high glucose].

The aim of this paper was to explore the effects of triptolide( TP),the effective component of Tripterygium wilfordii on improving podocyte epithelial-mesenchymal transition( EMT) induced by high glucose( HG),based on the regulative mechanisms of Nod-like receptor protein 3( NLRP 3) inflammasome in the kidney of diabetic kidney disease( DKD). The immortalized podocytes of mice in vitro were divided into the normal( N) group,the HG( HG) group,the low dose of TP( L-TP) group,the high dose of TP( HTP) group and the mannitol( MNT) group,and treated by the different measures,respectively. More specifically,the podocytes in each group were separately treated by D-glucose( DG,5 mmol·L~(-1)) or HG( 30 mmol·L~(-1)) or HG( 30 mmol·L~(-1)) + TP( 5 µg·L~(-1))or HG( 30 mmol·L~(-1)) + TP( 10 µg·L~(-1)) or DG( 5 mmol·L~(-1)) + MNT( 24. 5 mmol·L~(-1)). After the treatment of HG or TP at 24,48 and 72 h,firstly,the activation of podocyte proliferation was investigated. Secondly,the protein expression levels of the epithelial markers in podocytes such as nephrin and ZO-1,the mesenchymal markers such as collagen Ⅰ and fibronectin( FN) were detected,respectively. Finally,the protein expression levels of NLRP3 and apoptosis-associated speck-like protein( ASC) as the key signaling molecules of NLRP3 inflammasome activation,as well as the downstream effector proteins including caspase-1,interleutin( IL)-1ß and IL-18 were examined,severally. The results indicated that,for the cultured podocytes in vitro,HG could cause the low protein expression levels of nephrin and ZO-1,induce the high protein expression levels of collagen Ⅰ and FN and trigger podocyte EMT. Also HG could cause the high protein expression levels of NLRP3,ASC,caspase-1,IL-1ß and IL-18 and induce NLRP3 inflammasome activation. On the other hand,the co-treatment of TP( L-TP or H-TP) and HG for podocytes could recover the protein expression levels of nephrin and ZO-1,inhibit the protein expression levels of collagen Ⅰ and FN and ameliorate podocyte EMT. Also the co-treatment of TP( L-TP or H-TP) and HG could down-regulate the protein expression levels of NLRP3 and ASC,inhibit NLRP3 inflammasome activation and reduce the protein expression levels of the downstream effector molecules including caspase-1,IL-1ß and IL-18. On the whole,HG could activate NLRP3 inflammasome and induce podocyte EMT in vitro. TP at the appropriate dose range could inhibit NLRP3 inflammasome activation and ameliorate podocyte EMT,which may be one of the critical molecular mechanisms of TP protecting againstpodocyte inflammatory injury in DKD.

Interleukin-17A participates in podocyte injury by inducing IL-1ß 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 NLRP3 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 NLRP3 inflammasome in these cells was activated upon exposure to IL-17A. Also, activity of caspase-1 and secretion of IL-1ß 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 NLRP3 inflammasome, thereby restoring podocyte morphology. Taken together, the results suggest that IL-17A induces podocyte injury by activating the NLRP3 inflammasome and IL-1ß secretion and contributes to disruption of the kidney's filtration system.

Lysine-containing cationic liposomes activate the NLRP3 inflammasome: Effect of a spacer between the head group and the hydrophobic moieties of the lipids.

Cationic lipids containing lysine head groups and ditetradecyl, dihexadecyl or dioctadecyl glutamate hydrophobic moieties with/without propyl, pentyl or heptyl spacers were applied for the preparation of cationic liposomes using a simple bath type-sonicator. The size distribution, zeta potential, cellular internalization, and cytotoxicity of the liposomes were characterized, and the innate immune stimulation, e.g., the NLRP3 inflammasome activation of human macrophages and THP-1 cells, was evaluated by the detection of IL-1ß release. Comparatively, L3C14 and L5C14 liposomes, made from the lipids bearing lysine head groups, ditetradecyl hydrophobic chains and propyl or pentyl spacers, respectively, were the most potent to activate the NLRP3 inflammasome. The possible mechanism includes endocytosis of the cationic liposomes and subsequent lysosome rupture without significant inducement of reactive oxygen species production. In summary, we first disclosed the structural effect of cationic liposomes on the NLRP3 inflammasome activation, which gives an insight into the application of nanoparticles for improved immune response.

Aloe-emodin alleviates cerebral ischemia-reperfusion injury by regulating microglial polarization and pyroptosis through inhibition of NLRP3 inflammasome activation.

BACKGROUND: Microglial activation plays a crucial role in injury and repair after cerebral ischemia, and microglial pyroptosis exacerbates ischemic injury. NOD-like receptor protein 3 (NLRP3) inflammasome activation has an important role in microglial polarization and pyroptosis. Aloe-emodin (AE) is a natural anthraquinone compound originated from rhubarb and aloe. It exerts antioxidative and anti-apoptotic effects during cerebral ischemia/reperfusion (I/R) injury. However, whether AE affects microglial polarization, pyroptosis, and NLRP3 inflammasome activation remains unknown. PURPOSE: This study aimed to explore the effects of AE on microglial polarization, pyroptosis, and NLRP3 inflammasome activation in the cerebral infarction area after I/R. METHODS: The transient middle cerebral artery occlusion (tMCAO) and oxygen-glucose deprivation/re-oxygenation (OGD/R) methods were used to create cerebral I/R models in vivo and in vitro, respectively. Neurological scores and triphenyl tetrazolium chloride and Nissl staining were used to assess the neuroprotective effects of AE. Immunofluorescence staining, quantitative polymerase chain reaction and western blot were applied to detect NLRP3 inflammasome activation and microglial polarization and pyroptosis levels after tMCAO or OGD/R. Cell viability and levels of interleukin (IL)-18 and IL-1ß were measured. Finally, MCC950 (an NLRP3-specific inhibitor) was used to evaluate whether AE affected microglial polarization and pyroptosis by regulating the activation of the NLRP3 inflammasome. RESULTS: AE improved neurological function scores and reduced the infarct area, brain edema rate, and Nissl-positive cell rate following I/R injury. It also showed a protective effect on BV-2 cells after OGD/R. AE inhibited microglial pyroptosis and induced M1 to M2 phenotype transformation and suppressed microglial NLRP3 inflammasome activation after tMCAO or OGD/R. The combined administration of AE and MCC950 had a synergistic effect on the inhibition of tMCAO- or OGD/R-induced NLRP3 inflammasome activation, which subsequently suppressed microglial pyroptosis and induced microglial phenotype transformation. CONCLUSION: AE exerts neuroprotective effects by regulating microglial polarization and pyroptosis through the inhibition of NLRP3 inflammasome activation after tMCAO or OGD/R. These findings provide new evidence of the molecular mechanisms underlying the neuroprotective effects of AE and may support the exploration of novel therapeutic strategies for cerebral ischemia.

Potassium ion efflux induces exaggerated mitochondrial damage and non-pyroptotic necrosis when energy metabolism is blocked.

Damage-associated molecular patterns (DAMPs) such as extracellular ATP and nigericin (a bacterial toxin) not only act as potassium ion (K+) efflux inducers to activate NLRP3 inflammasome, leading to pyroptosis, but also induce cell death independently of NLRP3 expression. However, the roles of energy metabolism in determining NLRP3-dependent pyroptosis and -independent necrosis upon K+ efflux are incompletely understood. Here we established cellular models by pharmacological blockade of energy metabolism, followed by stimulation with a K+ efflux inducer (ATP or nigericin). Two energy metabolic inhibitors, namely CPI-613 that targets α-ketoglutarate dehydrogenase and pyruvate dehydrogenase (a rate-limiting enzyme) and 2-deoxy-d-glucose (2-DG) that targets hexokinase, are recruited in this study, and Nlrp3 gene knockout macrophages were used. Our data showed that CPI-613 and 2-DG dose-dependently inhibited NLRP3 inflammasome activation, but profoundly increased cell death in the presence of ATP or nigericin. The cell death was K+ efflux-induced but NLRP3-independent, which was associated with abrupt reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential, and oligomerization of mitochondrial proteins, all indicating mitochondrial damage. Notably, the cell death induced by K+ efflux and blockade of energy metabolism was distinct from pyroptosis, apoptosis, necroptosis or ferroptosis. Furthermore, fructose 1,6-bisphosphate, a high-energy intermediate of glycolysis, significantly suppressed CPI-613+nigericin-induced mitochondrial damage and cell death. Collectively, our data show that energy deficiency diverts NLRP3 inflammasome activation-dependent pyroptosis to Nlrp3-independent necrosis upon K+ efflux inducers, which can be dampened by high-energy intermediate, highlighting a critical role of energy metabolism in cell survival and death under inflammatory conditions.

Air pollution induces pyroptosis of human monocytes through activation of inflammasomes and Caspase-3-dependent pathways.

According to the World Health Organization (WHO), air pollution is one of the most serious threats for our planet. Despite a growing public awareness of the harmful effects of air pollution on human health, the specific influence of particulate matter (PM) on human immune cells remains poorly understood. In this study, we investigated the effect of PM on peripheral blood monocytes in vitro. Monocytes from healthy donors (HD) were exposed to two types of PM: NIST (SRM 1648a, standard urban particulate matter from the US National Institute for Standards and Technology) and LAP (SRM 1648a with the organic fraction removed). The exposure to PM-induced mitochondrial ROS production followed by the decrease of mitochondrial membrane potential and activation of apoptotic protease activating factor 1 (Apaf-1), Caspase-9, and Caspase-3, leading to the cleavage of Gasdermin E (GSDME), and initiation of pyroptosis. Further analysis showed a simultaneous PM-dependent activation of inflammasomes, including NLRP3 (nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3) and Caspase-1, followed by cleavage of Gasdermin D (GSDMD) and secretion of IL-1ß. These observations suggest that PM-treated monocytes die by pyroptosis activated by two parallel signaling pathways, related to the inorganic and organic PM components. The release of IL-1ß and expression of danger-associated molecular patterns (DAMPs) by pyroptotic cells further activated the remnant viable monocytes to produce inflammatory cytokines (TNF-α, IL-6, IL-8) and protected them from death induced by the second challenge with PM.In summary, our report shows that PM exposure significantly impacts monocyte function and induces their death by pyroptosis. Our observations indicate that the composition of PM plays a crucial role in this process-the inorganic fraction of PM is responsible for the induction of the Caspase-3-dependent pyroptotic pathway. At the same time, the canonical inflammasome path is activated by the organic components of PM, including LPS (Lipopolysaccharide/endotoxin). PM-induced pyroptosis of human monocytes. Particulate matter (PM) treatment affects monocytes viability already after 15 min of their exposure to NIST or LAP in vitro. The remnant viable monocytes in response to danger-associated molecular patterns (DAMPs) release pro-inflammatory cytokines and activate Th1 and Th17 cells. The mechanism of PM-induced cell death includes the increase of reactive oxygen species (ROS) production followed by collapse of mitochondrial membrane potential (ΔΨm), activation of Apaf-1, Caspase-9 and Caspase-3, leading to activation of Caspase-3-dependent pyroptotic pathway, where Caspase-3 cleaves Gasdermin E (GSDME) to produce a N-terminal fragment responsible for the switch from apoptosis to pyroptosis. At the same time, PM activates the canonical inflammasome pathway, where activated Caspase-1 cleaves the cytosolic Gasdermin D (GSDMD) to produce N-terminal domain allowing IL-1ß secretion. As a result, PM-treated monocytes die by pyroptosis activated by two parallel pathways-Caspase-3-dependent pathway related to the inorganic fraction of PM and the canonical inflammasome pathway dependent on the organic components of PM.

Oleanolic Acid Provides Neuroprotection against Ischemic Stroke through the Inhibition of Microglial Activation and NLRP3 Inflammasome Activation.

Oleanolic acid (OA), a natural pentacyclic triterpenoid, has been reported to exert protective effects against several neurological diseases through its anti-oxidative and anti-inflammatory activities. The goal of the present study was to evaluate the therapeutic potential of OA against acute and chronic brain injuries after ischemic stroke using a mouse model of transient middle cerebral artery occlusion (tMCAO, MCAO/reperfusion). OA administration immediately after reperfusion significantly attenuated acute brain injuries including brain infarction, functional neurological deficits, and neuronal apoptosis. Moreover, delayed administration of OA (at 3 h after reperfusion) attenuated brain infarction and improved functional neurological deficits during the acute phase. Such neuroprotective effects were associated with attenuation of microglial activation and lipid peroxidation in the injured brain after the tMCAO challenge. OA also attenuated NLRP3 inflammasome activation in activated microglia during the acute phase. In addition, daily administration of OA for 7 days starting from either immediately after reperfusion or 1 day after reperfusion significantly improved functional neurological deficits and attenuated brain tissue loss up to 21 days after the tMCAO challenge; these findings supported therapeutic effects of OA against ischemic stroke-induced chronic brain injury. Together, these findings showed that OA exerted neuroprotective effects against both acute and chronic brain injuries after tMCAO challenge, suggesting that OA is a potential therapeutic agent to treat ischemic stroke.

Treadmill exercise training alleviates diabetes-induced depressive-like behavior and cognitive impairment by improving hippocampal CA1 neurons injury in db/db mice.

Patients with diabetes mellitus (DM) have an increased risk of diabetic encephalopathy symptoms such as depressive-like behaviour and cognitive impairment. Exercise is an effective strategy for preventing and treating DM and diabetic complications. The aim of this study is to investigate the effects and potential mechanisms of treadmill exercise training on diabetes-induced depressive-like behavior and cognitive impairment in db/db mice. In this study, the mice were divided into three groups (n = 10 per group) as follows: healthy-sedentary (db/m), diabetes-sedentary (db/db), and diabetes-treadmill exercise training (db/db-TET). The db/db-TET mice were performed five days per week at a speed of 8 m/min for 60 min/day for 8 weeks, following which body weight, fasting blood glucose, insulin resistance, behavioral, synaptic ultrastructure, oxidative stress, apoptotic signaling, and inflammatory responses were evaluated. As a result, treadmill exercise training significantly decreased body weight and fasting blood glucose levels, increased insulin sensitivity, protected synaptic ultrastructure, reduced depression-like behavior, and improved learning and memory deficits in db/db mice. In addition, treadmill exercise training significantly suppressed NOX2-mediated oxidative stress, resulting in a decrease in NOX2-dependent ROS generation in the db/db mouse hippocampus CA1 region. Reduced ROS generation prevented the apoptotic signaling pathway and NLRP3 inflammasome activation, thereby ameliorating hippocampus neuronal damage. In summary, the results indicated that treadmill exercise training significantly ameliorates hippocampus injury by suppressing oxidative stress-induced apoptosis and NLRP3 inflammasome activation, consequently ameliorating diabetes-induced depressive-like behavior and cognitive impairment in db/db mice.

NLRP3 Inflammasome Activation: A Therapeutic Target for Cerebral Ischemia-Reperfusion Injury.

Millions of patients are suffering from ischemic stroke, it is urgent to figure out the pathogenesis of cerebral ischemia-reperfusion (I/R) injury in order to find an effective cure. After I/R injury, pro-inflammatory cytokines especially interleukin-1ß (IL-1ß) upregulates in ischemic brain cells, such as microglia and neuron. To ameliorate the inflammation after cerebral I/R injury, nucleotide-binding oligomerization domain (NOD), leucine-rich repeat (LRR), and pyrin domain-containing protein 3 (NLRP3) inflammasome is well-investigated. NLRP3 inflammasomes are complicated protein complexes that are activated by endogenous and exogenous danger signals to participate in the inflammatory response. The assembly and activation of the NLRP3 inflammasome lead to the caspase-1-dependent release of pro-inflammatory cytokines, such as interleukin (IL)-1ß and IL-18. Furthermore, pyroptosis is a pro-inflammatory cell death that occurs in a dependent manner on NLRP3 inflammasomes after cerebral I/R injury. In this review, we summarized the assembly and activation of NLRP3 inflammasome; moreover, we also concluded the pivotal role of NLRP3 inflammasome and inhibitors, targeting the NLRP3 inflammasome in cerebral I/R injury.

An inter-laboratory comparison of an NLRP3 inflammasome activation assay and dendritic cell maturation assay using a nanostructured lipid carrier and a polymeric nanomedicine, as exemplars.

Nanoparticles including nanomedicines are known to be recognised by and interact with the immune system. As these interactions may result in adverse effects, for safety evaluation, the presence of such interactions needs to be investigated. Nanomedicines in particular should not unintendedly interact with the immune system, since patient's exposure is not minimised as in the case of 'environmental' nanoparticles, and repeated exposure may be required. NLRP3 inflammasome activation and dendritic cell (DC) maturation are two types of immune mechanisms known to be affected by nanoparticles including nanomedicines. NLRP3 inflammasome activation results in production of the pro-inflammatory cytokines IL-1ß and IL-18, as well as a specific type of cell death, pyroptosis. Moreover, chronic NLRP3 inflammasome activation has been related to several chronic diseases. Upon maturation, DC activate primary T cells; interference with this process may result in inappropriate activation and skewing of the adaptive immune response. Here, we evaluated the effect of two nanomedicines, representing nanostructured lipid carriers and polymers, on these two assays. Moreover, with a view to possible future standardisation and regulatory application, these assays were subject to an inter-laboratory comparison study using common SOPs. One laboratory performed three independent NLRP3 inflammasome activation experiments, while the other performed a single experiment. Two laboratories each performed three independent DC maturation experiments. While the nanostructured lipid carrier only showed marginal effects, the polymers showed major cytotoxicity. No evidence for inflammasome activation or DC maturation was demonstrated. Intra- and inter-laboratory comparison showed clearly reproducible results.

NLRP3 inflammasome activation contributes to the pathogenesis of cardiocytes aging.

OBJECTIVE: The NOD-like receptor protein 3 (NOD-like receptor protein 3, NLRP3) inflammasome is associated with many physiological processes related to aging. We investigated whether NLRP3 inflammasome activation contributes to the pathogenesis of cardiocytes aging dissected the underlying mechanism. METHODS: H9c2 cells were treated with different concentrations of D-galactose (D-gal, 0, 2, 10 and 50 g/L) for 24 hours. The cytochemical staining, flow cytometry and fluorescence microscope analysis were employed to detect the ß-galactosidase (ß-gal) activity. Western blot analysis was used to detect the age-associated proteins (P53, P21) and NLRP3 inflammasome proteins [NLRP3, apoptosis-associated speck-like protein (ASC)]. Confocal fluorescent images were applied to capture the colocalization of NLRP3 and caspase-1. Intracellular reactive oxygen species (ROS) was measured using 2'7'-dichlorodihydrofluorescein diacetate (DCFH-DA) by flow cytometry and visualized using a fluorescence microscope. The IL-1ß, IL-18 and lactate dehydrogenase (LDH) release were also detected. RESULTS: D-gal induced-H9c2 cells caused cardiocytes' aging changes (ß-gal staining, CellEvent™ Senescence Green staining, P53, P21) in a concentration-dependent manner. NLRP3 inflammasomes were activated, IL-1ß, IL-18 and LDH release and ROS generation were increased in the cardiocytes aging progress. When MCC950 inhibited NLRP3 inflammasomes, it attenuated the cardiocytes aging, yet the ROS generation was similar. Inhibition of ROS by NAC attenuated cardiocytes aging and inhibited the NLRP3 inflammasome activation at the same time. NLRP3 inflammasome activation by nigericin-induced cardiocytes cells aging progress. CONCLUSIONS: NLRP3 inflammasome activation contributes to the pathogenesis of cardiocytes aging, and ROS generation may serve as a potential mechanism by which NLRP3 inflammasome is activated.

Native and oxidised lipoproteins negatively regulate the serum amyloid A-induced NLRP3 inflammasome activation in human macrophages.

OBJECTIVES: The NLRP3 inflammasome plays a key role in arterial wall inflammation. In this study, we elucidated the role of serum lipoproteins in the regulation of NLRP3 inflammasome activation by serum amyloid A (SAA) and other inflammasome activators. METHODS: The effect of lipoproteins on the NLRP3 inflammasome activation was studied in primary human macrophages and THP-1 macrophages. The effect of oxidised low-density lipoprotein (LDL) was examined in an in vivo mouse model of SAA-induced peritoneal inflammation. RESULTS: Native and oxidised high-density lipoproteins (HDL3) and LDLs inhibited the interaction of SAA with TLR4. HDL3 and LDL inhibited the secretion of interleukin (IL)-1ß and tumor necrosis factor by reducing their transcription. Oxidised forms of these lipoproteins reduced the secretion of mature IL-1ß also by inhibiting the activation of NLRP3 inflammasome induced by SAA, ATP, nigericin and monosodium urate crystals. Specifically, oxidised LDL was found to inhibit the inflammasome complex formation. No cellular uptake of lipoproteins was required, nor intact lipoprotein particles for the inhibitory effect, as the lipid fraction of oxidised LDL was sufficient. The inhibition of NLRP3 inflammasome activation by oxidised LDL was partially dependent on autophagy. Finally, oxidised LDL inhibited the SAA-induced peritoneal inflammation and IL-1ß secretion in vivo. CONCLUSIONS: These findings reveal that both HDL3 and LDL inhibit the proinflammatory activity of SAA and this inhibition is further enhanced by lipoprotein oxidation. Thus, lipoproteins possess major anti-inflammatory functions that hinder the NLRP3 inflammasome-activating signals, particularly those exerted by SAA, which has important implications in the pathogenesis of cardiovascular diseases.