Activation Of NOD-like Receptor 3 Inflammasome Research Articles

HIF‑1α promotes NLRP3 inflammasome activation in bleomycin‑induced acute lung injury.

The inflammatory response is one of the most important factors in the occurrence and development of acute lung injury (ALI). Hypoxia‑inducible factor‑1α (HIF‑1α) and the NOD‑like receptor 3 (NLRP3) inflammasome have been demonstrated to serve an important role in the pathogenesis of ALI. The objective of the present study was to investigate whether HIF‑1α could regulate activation of the NLRP3 inflammasome and its potential function and specific mechanism in bleomycin (BLM)‑induced ALI. Activation of the NLRP3 inflammasome and secretion of IL‑1β were detected following silencing of HIF‑1α or NF‑κB, respectively, in BLM‑treated A549 and RLE‑6TN cells. The results demonstrated that the NLRP3 inflammasome could be activated after BLM treatment. HIF‑1α and NF‑κB expression significantly increased in the BLM group. The levels of NF‑κB‑ and NLRP3 inflammasome‑associated proteins, including NLRP3, apoptosis‑associated speck‑like protein containing CARD and caspase‑1, markedly decreased after treating A549 and RLE‑6TN cells with HIF‑1α small interfering RNA. Activation of the NLRP3 inflammasome was also inhibited after silencing NF‑κB. Furthermore, the levels of IL‑1β markedly decreased in the cellular culture supernatants following inhibition of HIF‑1α and NF‑κB. Therefore, the present study indicated that HIF‑1α could modulate the activation of the NLRP3 inflammasome and the secretion of IL‑1β through NF‑κB signaling in BLM‑induced ALI. The current results improve understanding of the mechanism of ALI and may provide new ideas for identifying therapeutic targets of ALI.

GEN-27 exhibits anti-inflammatory effects by suppressing the activation of NLRP3 inflammasome and NF-κB pathway.

Prolonged inflammation and deregulated cytokine production are associated with diversified inflammatory diseases. Genistein (GEN), the active and predominant isoflavonoid in dietary soybean, possesses anti-inflammatory activity. Our study aimed to assess the anti-inflammatory effects of GEN-27, a derivative of GEN, as well as explore the potential molecular mechanisms using lipopolysaccharide (LPS)-induced RAW264.7 cells. In our study, we demonstrated that GEN-27 administration (1, 5, or 10 μM) dose-dependently inhibited nitrite and nitric oxide (NO) levels in LPS-stimulated RAW264.7 cells. Also, GEN-27 suppressed the release of LPS-induced pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-18. Moreover, GEN-27 attenuated LPS-induced inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) expressions at messenger RNA and protein levels, and reversed the promoter activity of iNOS in RAW264.7 cells. Mechanistically, GEN-27 abated LPS-induced reactive oxygen species production, as well as mitigated LPS-induced increase of caspase 1 activity and the protein levels of NOD-like receptor 3 (NLRP3), anti-apoptosis-associated speck-like protein-containing a CRAD (ASC), and caspase 1 in RAW264.7 cells in a dose-dependent manner. Similarly, GEN-27 dose-dependently weakened adenosine triphosphate-induced NLRP3 and IL-1β in RAW264.7 cells. In addition, GEN-27 treatment significantly suppressed LPS-induced phosphorylation of nuclear factor-κB (NF-κB) p65 and alleviated LPS-induced increase of transcriptional activity of NF-κB in RAW264.7 cells. In summary, these results revealed that GEN-27 exhibited anti-inflammatory effects by suppressing the activation of NLRP3 inflammasome and NF-κB pathway, suggesting that GEN-27 may be served as a promising therapeutic agent for the prevention and therapy of inflammatory-associated diseases.

Protective Effect of Ethyl Pyruvate against Myocardial Ischemia Reperfusion Injury through Regulations of ROS-Related NLRP3 Inflammasome Activation.

Emerging evidence indicates the pronounced role of inflammasome activation linked to reactive oxygen species (ROS) in the sterile inflammatory response triggered by ischemia/reperfusion (I/R) injury. Ethyl pyruvate (EP) is an antioxidant and conveys myocardial protection against I/R injury, while the exact mechanisms remain elusive. We aimed to investigate the effect of EP on myocardial I/R injury through mechanisms related to ROS and inflammasome regulation. The rats were randomly assigned to four groups: (1) sham, (2) I/R-control (IRC), (3) EP-pretreatment + I/R, and (4) I/R + EP-posttreatment. I/R was induced by a 30 min ligation of the left anterior descending artery followed by 4 h of reperfusion. EP (50 mg/kg) was administered intraperitoneally at 1 h before ischemia (pretreatment) or upon reperfusion (posttreatment). Both pre- and post-EP treatment resulted in significant reductions in myocardial infarct size (by 34% and 31%, respectively) and neutrophil infiltration. I/R-induced myocardial expressions of NADPH oxidase-4, carnitine palmitoyltransferase 1A, and thioredoxin-interacting protein (TXNIP) were mitigated by EP. EP treatment was associated with diminished inflammasome activation (NOD-like receptor 3 (NLRP3), apoptosis-associated speck-like protein, and caspase-1) and interleukin-1β induced by I/R. I/R-induced phosphorylation of ERK and p38 were also mitigated with EP treatments. In H9c2 cells, hypoxia-induced TXNIP and NLRP3 expressions were inhibited by EP and to a lesser degree by U0126 (MEK inhibitor) and SB203580 (p38 inhibitor) as well. EP's downstream protective mechanisms in myocardial I/R injury would include mitigation of ROS-mediated NLRP3 inflammasome upregulation and its associated pathways, partly via inhibition of hypoxia-induced phosphorylation of ERK and p38.

Role of Pyroptosis in Cardiovascular Diseases and its Therapeutic Implications.

Pyroptotic cell death or pyroptosis is characterized by caspase-1-dependent formation of plasma membrane pores, leading to the release of pro-inflammatory cytokines and cell lysis. Pyroptosis tightly controls the inflammatory responses and coordinates antimicrobial host defenses by releasing pro-inflammatory cellular contents, such as interleukin (IL)-1β and IL-18, and consequently expands or sustains inflammation. It is recognized as an important innate immune effector mechanism against intracellular pathogens. The induction of pyroptosis is closely associated with the activation of the NOD-like receptor 3 (NLRP3) inflammasome which has been linked to key cardiovascular risk factors including hyperlipidemia, diabetes, hypertension, obesity, and hyperhomocysteinemia. Emerging evidence has indicated pyroptosis as an important trigger and endogenous regulator of cardiovascular inflammation. Thus, pyroptosis may play an important role in the pathogenesis of cardiovascular diseases. Design of therapeutic strategies targeting the activation of NLRP3 inflammasome and pyroptosis holds promise for the treatment of cardiovascular diseases.

Acinetobacter baumannii outer membrane protein 34 elicits NLRP3 inflammasome activation via mitochondria-derived reactive oxygen species in RAW264.7 macrophages

Acinetobacter baumannii (A. baumannii) is a Gram-negative bacterium, which acts as an opportunistic pathogen and causes hospital-acquired pneumonia and bacteremia by infecting the alveoli of epithelial cells and macrophages. Evidence reveals that A. baumannii outer membrane protein 34 (Omp34) elicits cellular immune responses and inflammation. The innate immunity NOD-like receptor 3 (NLRP3) inflammasome exerts critical function against pneumonia caused by A. baumannii infection, however, the role of Omp34 in the activation of the NLRP3 inflammasome and its corresponding regulatory mechanism are not clearly elucidated. The present study aimed to investigate whether Omp34 elicited NLRP3 inflammasome activation through the mitochondria-derived reactive oxygen species (ROS). Our results showed that Omp34 triggered cell pyroptosis by up-regulating the expression of NLRP3 inflammasome-associated proteins and IL-1β release in a time- and dose-dependent manner. Omp34 induced the expression of caspase-1-p10 and IL-1β, which was significantly attenuated by NLRP3 gene silencing in RAW264.7 mouse macrophage cells. Additionally, Omp34 stimulated RAW264.7 mitochondria to generate ROS, while the ROS scavenger Mito-TEMPO inhibited the Omp34-triggered expression of NLRP3 inflammasome-associated proteins and IL-1β synthesis. The above findings indicate that mitochondria-derived ROS play an important role in the process of NLRP3 inflammasome activation. In summary, our study demonstrates that the A. baumannii pathogen pattern recognition receptor Omp34 activates NLRP3 inflammasome via mitochondria-derived ROS in RAW264.7 cells. Accordingly, down-regulating the mitochondria-derived ROS prevents the severe infection consequences caused by A. baumannii-induced NLRP3 inflammasome hyper-activation.

Role of NLRP3 inflammasome in pyroptosis during myocardial ischemia-reperfusion in diabetic rats

Objective To evaluate the role of NOD-like receptor 3 (NLRP3) inflammasome in pyroptosis during myocardial ischemia-reperfusion (I/R) in diabetic rats. Methods SPF male Sprague-Dawley rats, aged 6-8 weeks, weighing 210-230 g, were studied.Type 1 diabetes mellitus (DM) was induced by intraperitoneal injection of 1% streptozotocin 60 mg/kg.Forty-two rats with type 1 DM were divided into 4 groups using a random number table method: sham operation group (DS group, n=6), myocardial I/R group (DI/R group, n=12), myocardial I/R plus caspase-1 inhibitor Ac-YVAD-CMK group (DI/R+ Ac group, n=12) and myocardial I/R plus NLRP3 inhibitor BAY11-7082 group (DI/R+ BAY group, n=12). Eighteen healthy adult male Sprague-Dawley rats were selected and randomly divided into sham operation group (group S, n=6) and myocardial I/R group (group I/R, n=12). Myocardial I/R was induced by 30 min occlusion of the left anterior descending branch of the coronary artery followed by 120 min reperfusion at 8 weeks after successful establishment of the type 1 DM model.Ac-YVAD-CMK 1.5 mg/kg and BAY11-7082 5 mg/kg were intraperitoneally injected at 10 min before reperfusion in DI/R+ Ac group and DI/R+ BAY group, respectively.At the end of reperfusion, the myocardial infarct size was measured using TTC staining, the activities of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in serum were detected by enzyme-linked immunosorbent assay, the ultrastructure of myocardium was observed by transmission electron microscopy, and the expression of NLRP3, pro-caspase-1, caspase-1, apoptosis-associated speck-like protein containing CARD (ASC) and interleukin-1 (IL-1β) in myocardial tissues was detected by Western blot. Results Compared with group S or group DS, the activities of serum CK-MB and LDH were significantly increased, and the expression of NLRP3, pro-caspase-1, caspase-1, ASC and IL-1β was down-regulated in I/R and DI/R groups (P<0.05). Compared with group DI/R, the percentage of myocardial infarct size and activities of serum CK-MB and LDH were significantly decreased, and the expression of NLRP3, pro-caspase-1, caspase-1, ASC and IL-1β was down-regulated in group I/R, the percentage of myocardial infarct size and activities of serum CK-MB and LDH were significantly decreased, and the expression of pro-caspase-1, ASC and IL-1β was down-regulated in group DI/R+ Ac, and the percentage of myocardial infarct size and activities of serum CK-MB and LDH were significantly decreased, and the expression of NLRP3, pro-caspase-1, caspase-1, ASC and IL-1β was down-regulated (P<0.05), and the pathological changes of myocardium were significantly attenuated in group DI/R+ BAY. Conclusion The mechanism of pyroptosis during myocardial I/R may be related to the activation of NLRP3 inflammasome in diabetic rats. Key words: Diabetes mellitus; Myocardial reperfusion injury; Pyroptosis; NOD-like receptor protein 3

Inhibitory Effects of Toll-Like Receptor 4, NLRP3 Inflammasome, and Interleukin-1β on White Adipocyte Browning.

Adipose tissue expansion is accompanied by infiltration and accumulation of pro-inflammatory macrophages, which links obesity to pathologic conditions such as type 2 diabetes. However, little is known regarding the role of pro-inflammatory adipose tissue remodeling in the thermogenic activation of brown/beige fat. Here, we investigated the effect of pattern recognition receptors (PRR) activation in macrophages, especially the toll-like receptor 4 (TLR4) and Nod-like receptor 3 (NLRP3), on white adipocyte browning. We report that TLR4 activation by lipopolysaccharide repressed white adipocyte browning in response to β3-adrenergic receptor activation and caused ROS production and mitochondrial dysfunction, while genetic deletion of TLR4 protected mitochondrial function and thermogenesis. In addition, activation of NLRP3 inflammasome in macrophages attenuated UCP1 induction and mitochondrial respiration in cultures of primary adipocytes, while the absence of NLRP3 protected UCP1 in adipocytes. The effect of NLRP3 inflammasome activation on browning was mediated by IL-1β signaling, as blocking IL-1 receptor in adipocytes protected thermogenesis. We also report that IL-1β interferes with thermogenesis via oxidative stress stimulation and mitochondrial dysfunction as we observed a statistically significant increase in ROS production, decrease in SOD enzyme activity, and increase in mitochondrial depolarization in adipocytes treated with IL-1β. Collectively, we demonstrated that inflammatory response to obesity, such as TLR4 and NLRP3 inflammasome activation as well as IL-1β secretion, attenuates β3-adrenoreceptor-induced beige adipocyte formation via oxidative stress and mitochondrial dysfunction. Our findings provide insights into targeting innate inflammatory system for enhancement of the adaptive thermogenesis against obesity.

Baicalein ameliorates TNBS-induced colitis by suppressing TLR4/MyD88 signaling cascade and NLRP3 inflammasome activation in mice

Baicalein (5,6,7-trihydroxyflavone), a predominant bioactive component isolated from the root of Scutellaria baicalensis Georgi, has established potent anti-inflammatory activity via multi-targeted mechanisms. However, little is known about the effect of baicalein on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, which shares pathology related to human Crohn’s disease (CD). The present study demonstrated that baicalein alleviated the severity of TNBS-induced colitis in mice by decreasing the activity of myeloperoxidase (MPO) and the expression of pro-inflammatory mediators. The decline in the activation of nuclear factor-kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) correlated with a decrease in the expression of mucosal toll-like receptor 4 (TLR4) and its adaptor myeloid differentiation factor 88 (MyD88). In vitro, baicalein down-regulated the TLR4/MyD88 signaling cascades (NF-κB and MAPKs) in lipopolysaccharide (LPS)-stimulated macrophages. At the upstream level, baicalein bound to the hydrophobic region of the myeloid differentiation protein-2 (MD-2) pocket and inhibited the formation of the LPS-induced MD-2/TLR4 complex. Furthermore, baicalein reduced NOD-like receptor 3 (NLRP3) inflammasome activation and downstream interleukin-1β expression in a dose-dependent manner. Our study provided evidence for the first time that baicalein attenuated TNBS-induced colitis, at least in part, via inhibition of TLR4/MyD88 signaling cascade as well as inactivation of NLRP3 inflammasome.

Triggering of inflammasome by impaired autophagy in response to acute experimental Parkinson's disease: involvement of the PI3K/Akt/mTOR pathway.

Several lines of evidence suggest that the inflammasome activation is involved in the progression of neurodegenerative diseases. However, the relation between Parkinson’s disease (PD) and the inflammasome is still unclear. This study was designed to assess the involvement of inflammasome in acute experimental PD. Specifically, acute PD was induced in C57BL/6 mice by an injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). At seven days from MPTP induction, mice were euthanized and the midbrains were sampled to carry out immunohistochemical evaluations and western blot analysis. Our results show the activation of Nod-like receptor-3 inflammasome in acute MPTP mice, as suggested by the increase of nuclear factor-κB expression, which represents the first signal for inflammasome induction. The Nod-like receptor-3 assembly induces the activation of caspase-1, which in turn activates interleukin-1β and interleukin-18 production, as confirmed by our evaluations. A dysregulation of autophagy system was also found in acute MPTP mice by looking at the expression of Beclin-1, LC-3, and Bcl-2, chosen as markers of autophagy. Thus, in an effort to identify the molecular mechanism underlying the well-known crosstalk between autophagy and the inflammasome, we evaluated the involvement of the phosphoinositide-3 kinase/protein kinase-B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, which plays a key role in autophagy. Our results showed a clear upregulation of this signaling after MPTP induction. Taken together, our findings suggest that the triggering of inflammasome could be linked to impaired autophagy because of aberrant upstream activation of the PI3K/Akt/mTOR pathway. Finally, our results propose the inflammasome as a new potential therapeutic target in the management of PD.

Gamma Tocotrienol Suppresses NLRP3 Inflammasome by Dual Mechanism of A20‐ mediated Priming Inhibition and AMPK/autophagy Axis Activation

Nod‐like receptor 3 (NLRP3) inflammasome is the intracellular innate immunity sensor that processes and releases IL‐1β in response to microbial invasion or metabolic danger signals such as saturated fatty acids and free cholesterol. Aberrant activation of NLRP3 is intimately associated with numerous chronic inflammatory diseases. However, little information is available as to whether NLRP3 inflammasome is regulated by dietary factors. The aim of this study is to investigate whether gamma‐tocotrienol (γT3), an unsaturated form of vitamin E, downregulates NLRP3 inflammasome activation, and to determine the molecular mechanisms. Pretreatment of γT3 (0.5–10 mM) significantly suppressed activation of NLRP3 inflammasome reporter, caspase‐1 cleavage, and IL‐1β and IL‐18 secretion in J774 macrophages as well as bone marrow‐derived murine macrophages (BMDM). To further determine the mechanisms, we investigate the role of γT3 on NFκB activation and AMPK/autophagy axis activation. γT3 significantly attenuated proinflammatory gene expressions of IL‐1β, IL‐18 and NLRP3, and induced A20 expression, an ubiquitin‐editing enzyme to interrupt NFκB signaling. Induction of A20 concurred with 1) the decrease of p‐IKKα/β, p‐ERK, and p‐IκBα; 2) the reappearance of IκBα, and 3) the disappearance of TRAF6, suggesting a negative feedback of A20 on TRAF6/IKK/NF‐κB signaling. Moreover, silencing of A20 markedly dampened the inflammasome inhibitory effects of γT3. Intriguingly, γT3 treatment was also associated with AMPK/autophagy axis activation shown through LC3II accumulation and increased levels of AMPK phosphorylation. The inhibition of AMPK with Compound C completely reversed γT3's inhibitory role on NLRP3 inflammasome and caspase‐1 cleavage. Taken together, we demonstrated that γT3 suppresses NLRP3 inflammasome by two mechanisms, 1) inhibition of TRAF6/NFκB axis activation through induction of A20, and 2) activation of AMP kinase/autophagy thereby inhibiting inflammasome assembly.Support or Funding InformationAmerican Heart Association

The role of Resveratrol-induced mitophagy/autophagy in peritoneal mesothelial cells inflammatory injury via NLRP3 inflammasome activation triggered by mitochondrial ROS

It has been suggested that continuous exposure of peritoneal mesothelial cells (PMCs) to high glucose-containing peritoneal dialysis (PD) solutions may result in peritoneal inflammatory injury and impairment of local peritoneal host defence. Here, we investigated the effect of glucose-based PD solutions on mitochondrial reactive oxygen species (ROS) and nod-like receptor 3 (NLRP3) inflammasome activation in human PMCs (HPMCs). Exposure of HPMCs to high glucose-based PD solutions resulted in ROS production, which can trigger NLRP3 activation, leading to IL-1β secretion. Additionally, resveratrol (RSV) treatment induced mitophagy/autophagy via adenosine monophosphate-activated protein kinase (AMPK) activation. Increased mitochondrial ROS concentrations and IL-1β upregulation were confirmed following inhibition (siRNA against Beclin1 and ATG5 or autophagy inhibitor 3MA), but not induction (RSV), of mitophagy/autophagy. Furthermore, we observed that ATG5 and Beclin1 downregulation sensitised cells to IL-1β release induced by MSU or nigericin, which is an NLRP3 inflammasome activator. RSV treatment attenuated this effect. Taken together, this study may provide a potential therapeutic strategy for peritoneal inflammatory injury via NLRP3 inflammasome activation triggered by mitochondrial ROS.

Suppression of NLRP3 inflammasome by γ-tocotrienol ameliorates type 2 diabetes

The Nod-like receptor 3 (NLRP3) inflammasome is an intracellular sensor that sets off the innate immune system in response to microbial-derived and endogenous metabolic danger signals. We previously reported that γ-tocotrienol (γT3) attenuated adipose tissue inflammation and insulin resistance in diet-induced obesity, but the underlying mechanism remained elusive. Here, we investigated the effects of γT3 on NLRP3 inflammasome activation and attendant consequences on type 2 diabetes. γT3 repressed inflammasome activation, caspase-1 cleavage, and interleukin (IL) 1β secretion in murine macrophages, implicating the inhibition of NLRP3 inflammasome in the anti-inflammatory and antipyroptotic properties of γT3. Furthermore, supplementation of leptin-receptor KO mice with γT3 attenuated immune cell infiltration into adipose tissue, decreased circulating IL-18 levels, preserved pancreatic β-cells, and improved insulin sensitivity. Mechanistically, γT3 regulated the NLRP3 inflammasome via a two-pronged mechanism: 1) the induction of A20/TNF-α interacting protein 3 leading to the inhibition of the TNF receptor-associated factor 6/nuclear factor κB pathway and 2) the activation of AMP-activated protein kinase/autophagy axis leading to the attenuation of caspase-1 cleavage. Collectively, we demonstrated, for the first time, that γT3 inhibits the NLRP3 inflammasome thereby delaying the progression of type 2 diabetes. This study also provides an insight into the novel therapeutic values of γT3 for treating NLRP3 inflammasome-associated chronic diseases.

Lycium barbarum polysaccharide attenuates alcoholic cellular injury through TXNIP-NLRP3 inflammasome pathway

Lycium barbarum has been used as a traditional Chinese medicine to nourish liver, kidneys and the eyes. However, the underlying mechanisms of its hepatic-protective properties remain uncertain. In this study, we aimed to investigate whether thioredoxin-interacting protein (TXNIP) and NOD-like receptor 3 (NLRP3) inflammasome mediated the attenuation of ethanol-induced hepatic injury by Lycium barbarum polysaccharide (LBP). Rat normal hepatocyte line BRL-3A was pre-treated with LBP prior to ethanol incubation. Hepatic damages, including apoptosis, inflammation, and oxidative stress, were measured. Then the inhibition of endogenous TXNIP expression was achieved by using its specific siRNA to test its possible involvement in the injury attenuation. We found that 50μg/ml LBP pre-treatment significantly alleviated 24-h ethanol exposure-induced overexpression of TXNIP, increased cellular apoptosis, secretion of inflammatory cytokines, activation of NLRP3 inflammasome, production of ROS, and reduced antioxidant enzyme expression. Silence of TXNIP suppressed the activated NLRP3 inflammasome, increased oxidative stress and worsened apoptosis in the cells. Further addition of LBP did not influence the effects of TXNIP inhibition on the cells. In conclusion, inhibition of hepatic TXNIP by LBP contributes to the reduction of cellular apoptosis, oxidative stress and NLRP3 inflammasome-mediated inflammation.

Defective mitophagy driven by dysregulation of rheb and KIF5B contributes to mitochondrial reactive oxygen species (ROS)-induced nod-like receptor 3 (NLRP3) dependent proinflammatory response and aggravates lipotoxicity

High-fat diet (HFD) and inflammation are the key contributors to insulin resistance and type 2 diabetes (T2D). Previous study shows fatty acid-induced accumulation of damaged, reactive oxygen species (ROS)-generating mitochondria, and this in turn activates the NLRP3 inflammasome interference with insulin signaling. Our previous research shows NLRP3 inflammasome activation signal originates from defects in autophagy. Yet how the fatty acid related to mitophagy alteration leads to the activation of NLRP3-ASC inflammasome has not been considered. Here we demonstrated that palmitate (PA) induced mitophagy deficiency, leading to damaged mitochondrion as characterized by mito-ROS production and loss of membrane potential. Antioxidant APDC or Ca2+ signaling inhibitor Nifedipine blocked PA-induced NLRP3 inflammasome activation. Further, we provided evidences that PA reduced the expression of Ras homolog enriched in brain (Rheb) and disrupted Rheb recruitment to the mitochondrial outer membrane. In addition, sustained PA caused disassociation of kinesin family member 5B (KIF5B) from binding with mitochondria via Ca2+-dependent effects. Disruption of Rheb and KIF5B interaction with mitochondria blocked mitochondrial degradation along with IL-1β dependent insulin resistance, which was majorly attenuated by Rheb/KIF5B overexpression. In a consequence, defective mitophagy led to the accumulation of damaged-ROS-generating mitochondria, down pathway of NLRP3-ASC-Caspase 1 activation, and subsequently, insulin resistance. These findings provide insights into the association of inflammation, mitophagy and T2D.

Quercetin and allopurinol reduce liver thioredoxin‐interacting protein to alleviate inflammation and lipid accumulation in diabetic rats

Thioredoxin-interacting protein (TXNIP), a regulator of cellular oxidative stress, has been associated with activation of NOD-like receptor 3 (NLRP3) inflammasome, inflammation and lipid metabolism, suggesting it has a role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) in diabetes. In this study we investigated whether TXNIP is involved in type 1 diabetes-associated NAFLD and whether antioxidants, quercetin and allopurinol, alleviate NAFLD by targeting TXNIP. Diabetes was induced in male Sprague-Dawley rats by a single i.p. injection of 55 mg · kg⁻¹ streptozotocin. Quercetin and allopurinol were given p.o. to diabetic rats for 7 weeks. Hepatic function, oxidative stress, inflammation and lipid levels were determined. Rat BRL-3A and human HepG2 cells were exposed to high glucose (30 mM) in the presence and absence of antioxidants, TXNIP siRNA transfection or caspase-1 inhibitor, Ac-YVAD-CMK. Quercetin and allopurinol significantly inhibited the TXNIP overexpression, activation of NLRP3 inflammasome, down-regulation of PPARα and up-regulation of sterol regulatory element binding protein-1c (SREBP-1c), SREBP-2, fatty acid synthase and liver X receptor α, as well as elevation of ROS and IL-1β in diabetic rat liver. These effects were confirmed in hepatocytes in vitro and it was further shown that TXNIP down-regulation contributed to the suppression of NLRP3 inflammasome activation, inflammation and changes in PPARα and SREBPs. Inhibition of hepatic TXNIP by quercetin and allopurinol contributes to the reduction in liver inflammation and lipid accumulation under hyperglycaemic conditions. The targeting of hepatic TXNIP by quercetin and allopurinol may have therapeutic implications for prevention of type 1 diabetes-associated NAFLD.

ATP-P2X4 signaling mediates NLRP3 inflammasome activation: A novel pathway of diabetic nephropathy

Tubulointerstitial inflammation plays a key role in the development of diabetic nephropathy (DN). Cytokines in the IL-1 family are the key pro-inflammatory cytokines of tubulointerstitial inflammation. Extracellular ATP can cause P2X receptors to activate the NOD-like receptor 3 (NLRP3) inflammasome and cause IL-1β and IL-18 maturation and release. We investigated the role of ATP-P2X4 signaling in NLRP3 inflammasome activation and renal interstitial inflammation characteristic of DN. Ex vivo studies, P2X4 showed increased expression in renal tubule epithelial cells in patients with nephropathy due to type 2 diabetes compared to those in the control group. Linear correlation analysis shows that P2X4 expression was positively related with urine IL-1β and IL-18 levels. Moreover, P2X4 expression was co-localized with NLRP3, IL-1β, and IL-18 expression. In vitro culture experiments showed NLRP3 protein expression, cleavage of caspase-1 and IL-1β, and release of IL-1β, IL-18 and ATP in HK-2 cells significantly increased after high glucose stimulation. However, apyrase, which consumes extracellular ATP, completely blocked the changes caused by high glucose. The P2 receptor antagonist suramin, P2X receptor antagonist TNP-ATP, P2X4 selective antagonist 5-BDBD, and P2X4 gene silencing attenuated NLRP3 expression, cleavage of caspase-1 and IL-1β, and release of IL-1β and IL-18 induced by high glucose. Taken together, these results suggest that ATP-P2X4 signaling mediates high glucose-induced activation of the NLRP3 inflammasome, regulates IL-1 family cytokine secretion, and causes the development of tubulointerstitial inflammation in DN.

Critical role of the inflammasome during central nervous system Staphylococcus aureus infection (114.1)

Abstract Staphylococcus aureus (S. aureus) infection in the central nervous system (CNS) can lead to abscess formation typified by widespread inflammation and necrosis. A key cytokine driving inflammation during this process is IL-1β, which is processed into its active form by the inflammasome. Our recent studies demonstrated that the NOD-like receptor 3 (NLRP3) inflammasome and its adapter protein ASC are required for IL-1β secretion by microglia, the resident immune cells of the CNS. To understand the role of NLRP3 inflammasome activation during brain abscess formation, C57BL/6 wild type (WT), ASC knockout (KO), and NLRP3 KO mice received intracerebral infections with 1000 cfu S. aureus. Interestingly, ASC KO mice succumbed to infection within 24 h, yet bacterial burdens were identical to WT animals. Inflammatory mediators including CXCL1, CXCL10, CCL2, and IL-6 were significantly reduced in ASC KO mice, suggesting that ASC loss may impact processes extending beyond IL-1β cleavage. To extend the survival of ASC KO mice, animals were infected with a 1-log lower inoculum, revealing significant reductions in neutrophil and CD4+ T cell infiltrates compared to WT animals. In contrast, NLRP3 loss had minimal impact on brain abscess development. Collectively, these results indicate that ASC is involved in diverse pathways affecting inflammatory mediator expression and immune cell infiltration into the CNS, which may occur independently of its classical inflammasome actions.

Allopurinol, quercetin and rutin ameliorate renal NLRP3 inflammasome activation and lipid accumulation in fructose-fed rats

The NOD-like receptor 3 (NLRP3) inflammasome-mediated inflammation is recently recognized in the development of renal injury. However, the mechanisms of the inflammasome-mediated inflammation and lipid accumulation in renal injury and the actions of lowing urate agents remain unclear. The present study used fructose to induce hyperuricemia and dyslipidemia, which caused renal NLRP3 inflammasome activation characterized by over-expression of the NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1, resulting in overproduction of interleukin (IL)-1β and IL-18, as well as IL-6 and tumor necrosis factor α (TNF-α) in rats. The elevated levels of these pro-inflammatory cytokines impaired renal janus-activated kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3)/peroxisome proliferator-activated receptor α (PAPR-α), and insulin receptor (IR)/insulin receptor substrate 1 (IRS1)/protein kinase B (Akt)/extracellular signal-regulated kinase1/2 (ERK1/2) signaling pathways with over-expression of suppressor of cytokine signaling 3 (SOCS3), exacerbating renal lipid accumulation and injury in fructose-fed rats. The restoration of fructose-induced hyperuricemia and dyslipidemia by the treatment of allopurinol, quercetin and rutin blocked the NLRP3 inflammasome activation to improve the signaling impairments and reduce lipid accumulation in the kidney of rats. These results suggest that the activation of renal NLRP3 inflammasome may play an important role in the link among renal inflammation, JAK2/STAT3/PAPR-α and IR/IRS1/Akt/ERK1/2 signaling impairment, and lipid accumulation driven by fructose. The NLRP3 inflammasome may be the target mediating the improvement of urate-lowering agents allopurinol, quercetin and rutin on fructose-induced renal lipid accumulation and injury.