The role of NLRP3-CASP1 in inflammasome-mediated neuroinflammation and autophagy dysfunction in manganese-induced,hippocampal-dependent impairment of learning and memory ability

Central nervous system (CNS) inflammation and autophagy dysfunction are known to be involved in the pathology of neurodegenerative diseases. Manganese (Mn), a neurotoxic metal, has the potential to induce microglia-mediated neuroinflammation as well as autophagy dysfunction. NLRP3 (NLR family, pyrin domain containing 3)- CASP1 (caspase 1) inflammasome-mediated neuroinflammation in microglia has specific relevance to neurological diseases. However, the mechanism driving these phenomena remains poorly understood. We demonstrate that Mn activates the NLRP3-CASP1 inflammasome pathway in the hippocampus of mice and BV2 cells by triggering autophagy-lysosomal dysfunction. The autophagy-lysosomal dysfunction is induced by lysosomal damage caused by excessive Mn accumulation, damaging the structure and normal function of these organelles. Additionally, we show that the release of lysosomal CTSB (cathepsin B) plays an important role in Mn-induced NLRP3-CASP1 inflammasome activation, and that the increased autophagosomes in the cytoplasm are not the main cause of NLRP3-CASP1 inflammasome activation. The accumulation of proinflammatory cytokines, such as IL1B (interleukin 1 β) and IL18 (interleukin 18), as well as the dysfunctional autophagy pathway may damage hippocampal neuronal cells, thus leading to hippocampal-dependent impairment in learning and memory, which is associated with the pathogenesis of Alzheimer disease (AD).     

NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice

While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the age‐dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD⁺ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan.     

牛病毒性腹泻病毒Erns蛋白激活NOD样受体热蛋白结构域相关蛋白3炎症小体诱发细胞焦亡的研究

【目的】牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV)是引起牛病毒性腹泻-黏膜病的关键病毒。BVDV的结构蛋白Erns可在病毒感染的初期削弱宿主的免疫防御,引发牛群炎症反应。核苷酸寡聚化结构域样受体(nucleotide-binding oligomerization domain, NOD)热蛋白结构域相关蛋白3 (NLRP3)炎症小体是NOD样受体(NOD-like receptor, NLRs)家族重要成员,调控炎症性疾病的发生发展,同时激活的NLRP3炎症小体能够引起宿主细胞焦亡,进而诱发级联放大的炎症反应。但BVDV Erns蛋白在BVDV感染诱发炎症反应的分子机制尚不清楚。【方法】为进一步探索Erns蛋白对BVDV感染激活NLRP3炎症小体诱发细胞焦亡的影响,构建了BVDV Erns蛋白的真核表达质粒pCMV-HA-Erns,过表达BVDV Erns蛋白,检测BVDV感染细胞中NLRP3炎症小体组分[半胱氨酸蛋白酶(caspase-1)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein, ASC)和NLRP3]、IL-1β的mRNA转录水平和蛋白表达水平,以及细胞死亡调节蛋白(gasdermin D, GSDMD)的基因表达和蛋白剪切情况,并通过扫描电镜观察牛睾丸(bovine testis, BT)细胞膜成孔及BT细胞内容物释放情况,以分析Erns蛋白诱导BT细胞产生细胞焦亡。【结果】Erns蛋白能够显著引起NLRP3炎症小体活化进而激活caspase-1,活化的caspase-1一方面切割GSDMD,形成有活性的GSDMD-N端并在BT细胞膜形成孔洞,释放内容物,诱导BT细胞发生细胞焦亡;另一方面活化的caspase-1切割pro-IL-1β,形成有活性的IL-1β,并释放到BT细胞外,引起BT细胞上清中IL-1β水平上升。【结论】系统解析了BVDV Erns蛋白激活NLRP3炎症小体介导细胞焦亡的产生,对疫苗及治疗药物的研制具有重要指导意义。     

Palmitoylation prevents sustained inflammation by limiting NLRP3 inflammasome activation through chaperone-mediated autophagy

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Autophagy induced by AXL receptor tyrosine kinase alleviates acute liver injury via inhibition of NLRP3 inflammasome activation in mice

Severe hepatic inflammation is a common cause of acute or chronic liver disease. Macrophages are one of the key mediators which regulate the progress of hepatic inflammation. Increasing evidence shows that the TAM (TYRO3, AXL and MERTK) family of RTKs (receptor tyrosine kinases), which is expressed in macrophages, alleviates inflammatory responses through a negative feedback loop. However, the functional contribution of each TAM family member to the progression of hepatic inflammation remains elusive. In this study, we explore the role of individual TAM family proteins during autophagy induction and evaluate their contribution to hepatic inflammation. Among the TAM family of RTKs, AXL (AXL receptor tyrosine kinase) only induces autophagy in macrophages after interaction with its ligand, GAS6 (growth arrest specific 6). Based on our results, autophosphorylation of 2 tyrosine residues (Tyr815 and Tyr860) in the cytoplasmic domain of AXL in mice is required for autophagy induction and AXL-mediated autophagy induction is dependent on MAPK (mitogen-activated protein kinase)14 activity. Furthermore, induction of AXL-mediated autophagy prevents CASP1 (caspase 1)-dependent IL1B (interleukin 1, β) and IL18 (interleukin 18) maturation by inhibiting NLRP3 (NLR family, pyrin domain containing 3) inflammasome activation. In agreement with these observations, axl^?/? mice show more severe symptoms than do wild-type (Axl^+/+) mice following acute hepatic injury induced by administration of lipopolysaccharide (LPS) or carbon tetrachloride (CCl₄). Hence, GAS6-AXL signaling-mediated autophagy induction in murine macrophages ameliorates hepatic inflammatory responses by inhibiting NLRP3 inflammasome activation.     

Negative regulation of NLRP3 inflammasome signaling

Inflammasomes are multiprotein complexes that serve as a platform for caspase-1 activation and interleukin-1β (IL-1β) maturation as well as pyroptosis. Though a number of inflammasomes have been described, the NLRP3 inflammasome is the most extensively studied. NLRP3 inflammasome is triggered by a variety of stimuli, including infection, tissue damage and metabolic dysregulation, and then activated through an integrated cellular signal. Many regulatory mechanisms have been identifi ed to attenuate NLRP3 inflammasome signaling at multiple steps. Here, we review the developments in the negative regulation of NLRP3 inflammasome that protect host from inflammatory damage.     

Cellular localization of NLRP3 inflammasome

Infl ammasome is a large protein complex activated upon cellular stress or microbial infection, which triggers maturation of pro-inflammatory cytokines interleukin-1β and interleukin-18 through caspase-1 activation. Nod-like receptor family protein 3 (NLRP3) is the most characterized infl ammasome activated by various stimuli. However, the mechanism of its activation is unclear and its exact cellular localization is still unknown. We examined the potential co-localization of NLRP3 infl ammasome with mitochondria and seven other organelles under adenosine triphosphate, nigericin or monosodium urate stimulation in mouse peritoneal macrophages using confocal microscopy approach. Our results revealed that the activated endogenous apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome forms in the cytoplasm and co-localizes with NLRP3 and caspase-1, but not with any of the organelles screened. This study indicates that the ASC pyroptosome universally localizes within the cytoplasm rather than with any specifi c organelles.     

Purple sweet potato color attenuated NLRP3 inflammasome by inducing autophagy to delay endothelial senescence

Autophagy is a vital negative factor regulating cellular senescence. Purple sweet potato color (PSPC), one type of flavonoid, has been demonstrated to suppress endothelial senescence and restore endothelial function in diabetic mice by inhibiting the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome. However, the roles of autophagy in the inflammatory response during endothelial senescence are unknown. Here, we found that PSPC augmented autophagy to restrict high-glucose-induced premature endothelial senescence. In addition, PSPC administration impaired endothelium aging in diabetic mice by increasing autophagy. Inhibition of autophagy accelerated endothelial senescence, while enhancement of autophagy delayed senescence. Moreover, deactivation of the NLRP3 inflammasome triggered by PSPC was autophagy-dependent. Autophagy receptor microtubule-associated protein 1 light chain 3 and p62 interacted with the inflammasome component NLRP3, suggesting that autophagosomes target the NLRP3 inflammasome and deliver it to the lysosome for degradation. Altogether, PSPC amplified cellular autophagy, subsequently attenuated NLRP3 inflammasome activity and finally delayed endothelial senescence to ameliorate cardiovascular complication. These results suggest a potential therapeutic target in senescence-related cardiovascular diseases.     

Immunoproteasome modulates NLRP3 inflammasome‐mediated neuroinflammation under cerebral ischaemia and reperfusion conditions

Compelling evidence showed that both nucleotide‐binding oligomerization domain‐like receptor family, pyrin domain‐containing protein 3 (NLRP3) inflammasomes and the immunoproteasome participate in neuroinflammatory responses in cerebral ischaemia injury. Moreover, inhibition of either NLRP3 inflammasomes or the immunoproteasome attenuates both neuroinflammation and neurological deterioration during ischaemic stroke. However, the underlying mechanism between the immunoproteasome and NLRP3 inflammasomes under ischaemic stroke conditions remains to be established. In this study, using both in vitro and in vivo ischaemic models, we demonstrated that the immunoproteasome inhibition reduced the expressions of NLRP3 inflammasome‐associated proteins, including NLRP3, apoptosis‐associated speck‐like protein (ASC), caspase‐1 and mature cytokines (interleukin [IL]‐1β and IL‐18). It also downregulated the levels of nuclear factor (NF)‐κB and pyroptotic‐ and apoptotic‐related proteins, and improved cell viability. In addition, inhibition of NF‐κB by the small molecule inhibitor Bay‐11‐7082 led to lower levels of NLRP3 inflammasomes and cleaved caspase‐1 proteins in BV2 cells after oxygen‐glucose deprivation and reoxygenation. Together, these findings suggest that the immunoproteasome may be responsible for inducing the expression and activation of NLRP3 inflammasomes via the NF‐κB pathway. Therapeutic interventions that target activation of the immunoproteasome/NF‐κB/NLRP3 inflammasome pathway may provide novel prospects for the future treatment of ischaemic stroke.     

Mechanistic insight into the attenuation of gouty inflammation by Taiwanese green propolis via inhibition of the NLRP3 inflammasome

Dysregulation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including gouty arthritis. Activation of the NLRP3 inflammasome requires priming and activation signals: the priming signal controls the expression of NLRP3 and interleukin (IL)-1β precursor (proIL-1β), while the activation signal leads to the assembly of the NLRP3 inflammasome and to caspase-1 activation. Here, we reported the effects of the alcoholic extract of Taiwanese green propolis (TGP) on the NLRP3 inflammasome in vitro and in vivo. TGP inhibited proIL-1β expression by reducing nuclear factor kappa B activation and reactive oxygen species (ROS) production in lipopolysaccharide-activated macrophages. Additionally, TGP also suppressed the activation signal by reducing mitochondrial damage, ROS production, lysosomal rupture, c-Jun N-terminal kinases 1/2 phosphorylation and apoptosis-associated speck-like protein oligomerization. Furthermore, we found that TGP inhibited the NLRP3 inflammasome partially via autophagy induction. In the in vivo mouse model of uric acid crystal-induced peritonitis, TGP attenuated the peritoneal recruitment of neutrophils, and the levels of IL-1β, active caspase-1, IL-6 and monocyte chemoattractant protein-1 in lavage fluids. As a proof of principle, in this study, we purified a known compound, propolin G, from TGP and identified this compound as a potential inhibitor of the NLRP3 inflammasome. Our results indicated that TGP might be useful for ameliorating gouty inflammation via inhibition of the NLRP3 inflammasome.     

NLRP3 inflammasome negatively regulates podocyte autophagy in diabetic nephropathy

Inflammasome mechanisms are recognized as a key pathophysiology of diabetic nephropathy (DN). The nucleotide-oligomerization domain-like receptor 3 (NLRP3) inflammasome has attracted the most attention. Autophagy as a conserved intracellular catabolic pathway plays essential roles in the maintenance of podocytes. Although autophagy was involved in preventing excessive inflammatory responses in kidney diseases, a clear understanding of the regulation of NLRP3 inflammasome on autophagy in glomerular damage in DN is still lacking. In this study, we focused on the effect of the activation of NLRP3 inflammasome on the suppression of podocyte autophagy and aimed to investigate the role of autophagy in podocyte injury in DN. Podocyte autophagy has been confirmed to be inhibited in high-fat diet/streptozotocin (HFD/STZ)-induced DN mice, and NLRP3 has been found to be upregulated in both mice and human DN biopsies and in vitro. Activation of NLRP3 inflammasome exacerbated podocyte autophagy and reduced podocyte nephrin expression, while silencing of NLRP3 efficiently restored podocyte autophagy and ameliorated podocyte injury induced by high glucose. The results showed that NLRP3 was a negative regulator of autophagy and suggested that restoration of podocyte autophagy by inactivation of NLRP3 under high glucose could reduce podocyte injury. Proper modification of autophagy and inflammasome has the potential to benefit the kidney in DN.     

Bacterial infection reinforces host metabolic flux from arginine to spermine for NLRP3 inflammasome evasion

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Biological functions of NLRP3 inflammasome: A therapeutic target in inflammatory bowel disease

Cases of inflammatory bowel disease (IBD), a debilitating intestinal disorder with complex pathological mechanisms, have been increasing in recent years, straining the capacity of healthcare systems. Thus, novel therapeutic targets and innovative agents must be developed. Notably, the NLRP3 inflammasome is upregulated in patients with IBD and/or in animal experimental models. As an innate immune supramolecular assembly, the NLRP3 inflammasome is persistently activated during the pathogenesis of IBD by multiple stimuli. Moreover, this protein complex regulates pro-inflammatory cytokines. Thus, targeting this multiprotein oligomer may offer a feasible way to relieve IBD symptoms and improve clinical outcomes. The mechanisms by which the NLRP3 inflammasome is activated, its role in IBD pathogenesis, and the drugs administered to target this protein complex are reviewed herein. This review establishes that the use of inflammasome-targeting drugs are effective for IBD treatment. Moreover, this review suggests that the value and potential of naturally sourced or derived medicines for IBD treatment must be recognized and appreciated.     

ER stress activates the NLRP3 inflammasome via an UPR-independent pathway

Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1β. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.     

Verapamil inhibits early acute liver failure through suppressing the NLRP3 inflammasome pathway

Acute liver failure (ALF) is a rare disease characterized by the sudden onset of serious hepatic injury, as manifested by a profound liver dysfunction and hepatic encephalopathy in patients without prior liver disease. In this paper, we aim to investigate whether verapamil, an antagonist of TXNIP, inhibits early ALF through suppressing the NLRP3 inflammasome pathway. Firstly, an ALF mouse model was induced by lipopolysaccharide (LPS)/D-galactosamine (GalN) treatment. The optimal concentration of verapamil in treating early ALF mice was determined followed by investigation on its mechanism in LPS/GalN-induced liver injury. Western blot analysis and co-immunoprecipitation were performed to determine the activation of the TXNIP/NLRP3 inflammasome pathway. Subsequently, overexpression of NLRP3 in mouse liver was induced by transfection with AAV-NRLP3 in vivo and in vitro to identity whether verapamil inhibited early ALF through suppressing the activation of NLRP3 inflammasome. We found that ALF was induced by LPS/GalN in mice but was alleviated by verapamil through a mechanism that correlated with suppression of the NLRP3 inflammasome pathway. Oxidative stress and inflammatory response were induced by intraperitoneal injection of LPS/GalN, but alleviated with injection of verapamil. Overexpression of NLRP3 via AAV in mouse liver in vivo and in vitro reduced the therapeutic effect of verapamil on LPS/GalN-induced ALF. Taken together, the TXNIP antagonist verapamil could inhibit activation of the NLRP3 inflammasome, inflammatory responses and oxidative stress to alleviate LPS/GalN-induced ALF.