Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome

Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen that mainly infects immunocompromised individuals such as AIDS patients. Although cell surface receptors for recognition of C. neoformans have been studies intensively, cytoplasmic recognition of this pathogen remains unclear. As an important detector of pathogen infection, inflammasome can sense and get activated by infection of various pathogens, including pathogenic fungi such as Candida albicans and Aspergillus fumigatus. Our present study showed that acapsular C. neoformans (cap59Δ) activated the NLRP3-, but not AIM2-nor NLRC4- inflammasome. During this process, viability of the fungus was required. Moreover, our in vivo results showed that during the pulmonary infection of cap59Δ, immune cell infiltration into the lung and effective clearance of the fungus were both dependent on the presence of NLRP3 inflammasome. In summary, our data suggest that the capsule of C. neoformans prevents recognition of the fungus by host NLRP3 inflammasome and indicate that manipulation of inflammasome activity maybe a novel approach to control C. neoformans infection.     

Adenovirus membrane penetration activates the NLRP3 inflammasome

Adenovirus type 5 (Ad5) infection of macrophages results in rapid secretion of interleukin-1β (IL-1β) and is dependent on the inflammasome components NLRP3 and ASC and the catalytic activity of caspase-1. Using lentivirus-expressed short hairpin RNA (shRNA) and competitive inhibitors, we show that Ad-induced IL-1β release is dependent upon Toll-like receptor 9 (TLR9) sensing of the Ad5 double-stranded DNA (dsDNA) genome in human cell lines and primary monocyte-derived macrophages but not in mouse macrophages. Additionally, a temperature-sensitive mutant of Ad5 unable to penetrate endosomal membranes, ts1, is unable to induce IL-1β release in TLR2-primed THP-1 cells, suggesting that penetration of endosomal membranes is required for IL-1β release. Disruption of lysosomal membranes and the release of cathepsin B into the cytoplasm are required for Ad-induced NLRP3 activation. Ad5 cell entry also induces reactive oxygen species (ROS) production, and inhibitors of ROS prevent Ad-induced IL-1β release. Ad5 activation of NLRP3 also induces necrotic cell death, resulting in the release of the proinflammatory molecule HMGB1. This work further defines the mechanisms of virally induced inflammasome activation.     

Serum amyloid A activates the NLRP3 inflammasome and promotes Th17 allergic asthma in mice

IL-1β is a cytokine critical to several inflammatory diseases in which pathogenic Th17 responses are implicated. Activation of the NLRP3 inflammasome by microbial and environmental stimuli can enable the caspase-1-dependent processing and secretion of IL-1β. The acute-phase protein serum amyloid A (SAA) is highly induced during inflammatory responses, wherein it participates in systemic modulation of innate and adaptive immune responses. Elevated levels of IL-1β, SAA, and IL-17 are present in subjects with severe allergic asthma, yet the mechanistic relationship among these mediators has yet to be identified. In this study, we demonstrate that Saa3 is expressed in the lungs of mice exposed to several mixed Th2/Th17-polarizing allergic sensitization regimens. SAA instillation into the lungs elicits robust TLR2-, MyD88-, and IL-1-dependent pulmonary neutrophilic inflammation. Furthermore, SAA drives production of IL-1α, IL-1β, IL-6, IL-23, and PGE(2), causes dendritic cell (DC) maturation, and requires TLR2, MyD88, and the NLRP3 inflammasome for secretion of IL-1β by DCs and macrophages. CD4(+) T cells polyclonally stimulated in the presence of conditioned media from SAA-exposed DCs produced IL-17, and the capacity of polyclonally stimulated splenocytes to secrete IL-17 is dependent upon IL-1, TLR2, and the NLRP3 inflammasome. Additionally, in a model of allergic airway inflammation, administration of SAA to the lungs functions as an adjuvant to sensitize mice to inhaled OVA, resulting in leukocyte influx after Ag challenge and a predominance of IL-17 production from restimulated splenocytes that is dependent upon IL-1R signaling.     

TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3^?/?) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1β (IL-1β), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3^?/? mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.     

Atherosclerosis in ApoE-deficient mice progresses independently of the NLRP3 inflammasome

The interleukin-1 (IL-1) family of cytokines has been implicated in the pathogenesis of atherosclerosis in previous studies. The NLRP3 inflammasome has recently emerged as a pivotal regulator of IL-1β maturation and secretion by macrophages. Little is currently known about a possible role for the NLRP3 inflammasome in atherosclerosis progression in vivo. We generated ApoE−/− Nlrp3−/−, ApoE−/− Asc−/− and ApoE−/− caspase-1−/− double-deficient mice, fed them a high-fat diet for 11 weeks and subsequently assessed atherosclerosis progression and plaque phenotype. No differences in atherosclerosis progression, infiltration of plaques by macrophages, nor plaque stability and phenotype across the genotypes studied were found. Our results demonstrate that the NLRP3 inflammasome is not critically implicated in atherosclerosis progression in the ApoE mouse model.     

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.     

U1-small nuclear ribonucleoprotein activates the NLRP3 inflammasome in human monocytes

The NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is a caspase-1-containing cytosolic protein complex that is essential for processing and secretion of IL-1β. The U1-small nuclear ribonucleoprotein (U1-snRNP) that includes U1-small nuclear RNA is a highly conserved intranuclear molecular complex involved in splicing pre-mRNA. Abs against this self nuclear molecule are characteristically found in autoimmune diseases like systemic lupus erythematosus, suggesting a potential role of U1-snRNP in autoimmunity. Although endogenous DNA and microbial nucleic acids are known to activate the inflammasomes, it is unknown whether endogenous RNA-containing U1-snRNP could activate this molecular complex. In this study, we show that U1-snRNP activates the NLRP3 inflammasome in CD14(+) human monocytes dependently of anti-U1-snRNP Abs, leading to IL-1β production. Reactive oxygen species and K(+) efflux were responsible for this activation. Knocking down the NLRP3 or inhibiting caspase-1 or TLR7/8 pathway decreased IL-1β production from monocytes treated with U1-snRNP in the presence of anti-U1-snRNP Abs. Our findings indicate that endogenous RNA-containing U1-snRNP could be a signal that activates the NLRP3 inflammasome in autoimmune diseases like systemic lupus erythematosus where anti-U1-snRNP Abs are present.     

Human pathogenic fungus Trichophytonschoenleinii activates the NLRP3 inflammasome

The fungus Trichophyton schoenleinii (T. schoenleinii) is the causative agent of Trichophytosis and Tinea favosa of the scalp in certain regions of Eurasia and Africa. Human innate immune system plays an important role in combating with various pathogens including fungi. The inflammasome is one of the most critical arms of host innate immunity, which is a protein complex controlling maturation of IL-1β. To clarify whether T. schoenleinii is able to activate the inflammasome, we analyzed human monocytic cell line THP-1 for IL-1β production upon infection with T. schoenleinii strain isolated from Tinea favosa patients, and rapid IL-1β secretion from THP-1 cells was observed. Moreover, applying competitive inhibitors and gene specific silencing with shRNA, we found that T. schoenleinii induced IL-1β secretion, ASC pyroptosome formation as well as caspase-1 activation were all dependent on NLRP3. Cathepsin B activity, ROS production and K⁺ efflux were required for the inflammasome activation by T. schoenleinii. Our data thus reveal that the NLRP3 inflammasome plays an important role in host defense against T. schoenleinii, and suggest that manipulating NLRP3 signaling can be a novel approach for control of diseases caused by T. schoenleinii infection.     

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。     

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.     

Mycobacterium abscessus activates the NLRP3 inflammasome via Dectin-1-Syk and p62/SQSTM1

Numerous atypical mycobacteria, including Mycobacterium abscessus (Mabc), cause nontuberculous mycobacterial infections, which present a serious public health threat. Inflammasome activation is involved in host defense and the pathogenesis of autoimmune diseases. However, inflammasome activation has not been widely characterized in human macrophages infected with atypical mycobacteria. Here, we demonstrate that Mabc robustly activates the nucleotide binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via dectin-1/Syk-dependent signaling and the cytoplasmic scaffold protein p62/SQSTM1 (p62) in human macrophages. Both dectin-1 and Toll-like receptor 2 (TLR2) were required for Mabc-induced mRNA expression of pro-interleukin (IL)-1β, cathelicidin human cationic antimicrobial protein-18/LL-37 and β-defensin 4 (DEFB4). Dectin-1-dependent Syk signaling, but not that of MyD88, led to the activation of caspase-1 and secretion of IL-1β through the activation of an NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) inflammasome. Additionally, potassium efflux was required for Mabc-induced NLRP3/ASC inflammasome activation. Furthermore, Mabc-induced p62 expression was critically involved in NLRP3 inflammasome activation in human macrophages. Finally, NLRP3/ASC was critical for the inflammasome in antimicrobial responses to Mabc infection. Taken together, these data demonstrate the induction mechanism of the NLRP3/ASC inflammasome and its role in innate immunity to Mabc infection.     

Lysosomal destabilization activates the NLRP3 inflammasome in human umbilical vein endothelial cells (HUVECs)

Inflammation is a crucial component in the pathogenesis of many vascular diseases, such as atherosclerosis and diabetes. Inflammasomes are intracellular signalling complexes whose activation promotes inflammation. Nucleotide-binding domain and Leucine-rich repeat Receptor containing a Pyrin domain 3 (NLRP3) is a pattern recognition receptor (PRR) forming the best-known inflammasome. Disturbances in NLRP3 have been associated with multiple diseases. The purpose of this study was to explore the lysosomal destabilization-related NLRP3 inflammasome signaling pathway in human endothelial cells. In order to prime and activate NLRP3, human umbilical vein cells (HUVECs) were exposed to TNF-α and the lysosomal destructive agent Leusine-Leusine-O-Methylesther (Leu-Leu-OMe), respectively. A caspase-1 inhibitor was used to block caspase-1’s enzymatic function and an interleukin 1 receptor antagonist (IL-1RA) to prevent any possible secondary effects of IL-1β. Leu-Leu-OMe increased the expression of NLRP3, IL-1β, and IL-18 in HUVECs. Exposure to Leu-Leu-OMe significantly promoted the production of IL-6 and IL-8 in primed HUVECs; this effect was prevented by the pre-treatment of cells with an IL-1RA. Our results suggest that lysosomal destabilization activates the NLRP3 inflammasome pathway that promotes the production of IL-6 and IL-8 in an autocrine manner in HUVEC cells.     

Berberine ameliorates aGVHD by gut microbiota remodelling,TLR4 signalling suppression and colonic barrier repairment for NLRP3 inflammasome inhibition

Berberine (BBR), an isoquinoline alkaloid, is used to treat gastrointestinal disorders as an herbal medicine in China. The aim of this study was to investigate the anti‐inflammatory activities of BBR in a mouse model with acute graft‐versus‐host disease (aGVHD). Mice were intravenously injected with bone marrow cells from donors combined with splenocytes to develop aGVHD. The body weight, survival rate and clinical scores were monitored. Then the levels of inflammatory cytokines, histological changes (lung, liver and colon), colonic mucosal barrier and gut microbiota were analysed. Moreover, the toll‐like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (Myd88)/nuclear factor‐κB signalling pathway, NLRP3 inflammasome and its cytokines’ expressions were determined. The results showed that the gavage of BBR lessened GVHD‐induced weight loss, high mortality and clinical scores, inhibited inflammation and target organs damages and prevented GVHD‐indued colonic barrier damage. Additionally, BBR modulated gut microbiota, suppressed the activation of the TLR4 signaling pathway and inhibited NLRP3 inflammasome and its cytokine release. This study indicated that BBR might be a potential therapy for aGVHD through NLRP3 inflammasome inhibition.     

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.     

The NLRP3 inflammasome: activation and regulation

The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome is a cytoplasmic supramolecular complex that is activated in response to cellular perturbations triggered by infection and sterile injury. Assembly of the NLRP3 inflammasome leads to activation of caspase-1, which induces the maturation and release of interleukin-1β (IL-1β) and IL-18, as well as cleavage of gasdermin D (GSDMD), which promotes a lytic form of cell death. Production of IL-1β via NLRP3 can contribute to the pathogenesis of inflammatory disease, whereas aberrant IL-1β secretion through inherited NLRP3 mutations causes autoinflammatory disorders. In this review, we discuss recent developments in the structure of the NLRP3 inflammasome, and the cellular processes and signaling events controlling its assembly and activation.     

Celastrol specifically inhibits the activation of NLRP3 inflammasome

正Dear Editor,Inflammasomes are vital components of the innate immune system that survey microbial infections or sterile challenges in the cytosol(de Zoete et al.,2014).A typical inflammasome is composed of a sensor protein such as NOD like receptor(NLR)or AIM2 like receptor(ALR),the adaptor     

Serum amyloid A activates the NLRP3 inflammasome via P2X7 receptor and a cathepsin B-sensitive pathway

Serum amyloid A (SAA) is an acute-phase protein, the serum levels of which can increase up to 1000-fold during inflammation. SAA has a pathogenic role in amyloid A-type amyloidosis, and increased serum levels of SAA correlate with the risk for cardiovascular diseases. IL-1β is a key proinflammatory cytokine, and its secretion is strictly controlled by the inflammasomes. We studied the role of SAA in the regulation of IL-1β production and activation of the inflammasome cascade in human and mouse macrophages, as well as in THP-1 cells. SAA could provide a signal for the induction of pro-IL-1β expression and for inflammasome activation, resulting in secretion of mature IL-1β. Blocking TLR2 and TLR4 attenuated SAA-induced expression of IL1B, whereas inhibition of caspase-1 and the ATP receptor P2X(7) abrogated the release of mature IL-1β. NLRP3 inflammasome consists of the NLRP3 receptor and the adaptor protein apoptosis-associated speck-like protein containing CARD (a caspase-recruitment domain) (ASC). SAA-mediated IL-1β secretion was markedly reduced in ASC(-/-) macrophages, and silencing NLRP3 decreased IL-1β secretion, confirming NLRP3 as the SAA-responsive inflammasome. Inflammasome activation was dependent on cathepsin B activity, but it was not associated with lysosomal destabilization. SAA also induced secretion of cathepsin B and ASC. In conclusion, SAA can induce the expression of pro-IL-1β and activation of the NLRP3 inflammasome via P2X(7) receptor and a cathepsin B-sensitive pathway. Thus, during systemic inflammation, SAA may promote the production of IL-1β in tissues. Furthermore, the SAA-induced secretion of active cathepsin B may lead to extracellular processing of SAA and, thus, potentially to the development of amyloid A amyloidosis.     

Dexmedetomidine alleviates lipopolysaccharide-induced acute kidney injury by inhibiting the NLRP3 inflammasome activation via regulating the TLR4/NOX4/NF-κB pathway

Dexmedetomidine (DEX) prevents kidney damage caused by sepsis, but the mechanism of this effect remains unclear. In this study, the protective molecular mechanism of DEX in lipopolysaccharide (LPS)-induced acute kidney injury was investigated and its potential pharmacological targets from the perspective of inhibiting oxidative stress damage and the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activation. Intraperitoneal injection of DEX (30 μg/kg) significantly improved LPS (10 mg/kg) induced renal pathological damage and renal dysfunction. DEX also ameliorated oxidative stress damage by reducing the contents of reactive oxygen species, malondialdehyde and hydrogen peroxide, and increasing the level of glutathione, as well as the activity of superoxide dismutase and catalase. In addition, DEX prevented nuclear factor-kappa B (NF-κB) activation and I-kappa B (IκB) phosphorylation, as well as the expressions of NLRP3 inflammasome-associated protein and downstream IL-18 and IL-1β. The messengerRNA (mRNA) and protein expressions of toll-like receptor 4 (TLR4), NADPH oxidase-4 (NOX4), NF-κB, and NLRP3 were also significantly reduced by DEX. Their expressions were further evaluated by immunohistochemistry, yielding results were consistent with the results of mRNA and protein detection. Interestingly, the protective effects of DEX were reversed by atipamezole-an alpha 2 adrenal receptor (α₂AR) inhibitor, whereas idazoxan-an imidazoline receptor (IR) inhibitor failed to reverse this change. In conclusion, DEX attenuated LPS-induced AKI by inhibiting oxidative stress damage and NLRP3 inflammasome activation via regulating the TLR4/NOX4/NF-κB pathway, mainly acting on the α₂AR rather than IR.