Heterologous Expression and Auto-Activation of Human Pro-Inflammatory Caspase-1 in Saccharomyces cerevisiae and Comparison to Caspase-8.

Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of caspase-8. Overexpression of both caspases in the heterologous model led to their activation and caused mitochondrial hyperpolarization, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and caspase-8 was more aggressive than caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of caspase-1 enhanced its activity, whereas the deletion of the death effector domain (DED) of caspase-8 diminished it. We show that caspase-1 is able to efficiently process its target gasdermin D (GSDMD) when co-expressed in yeast. In sum, we propose that S. cerevisiae provides a manageable tool to explore caspase-1 activity and structure-function relationships.

A phenotypic high-content, high-throughput screen identifies inhibitors of NLRP3 inflammasome activation.

Inhibition of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome has recently emerged as a promising therapeutic target for several inflammatory diseases. After priming and activation by inflammation triggers, NLRP3 forms a complex with apoptosis-associated speck-like protein containing a CARD domain (ASC) followed by formation of the active inflammasome. Identification of inhibitors of NLRP3 activation requires a well-validated primary high-throughput assay followed by the deployment of a screening cascade of assays enabling studies of structure-activity relationship, compound selectivity and efficacy in disease models. We optimized a NLRP3-dependent fluorescent tagged ASC speck formation assay in murine immortalized bone marrow-derived macrophages and utilized it to screen a compound library of 81,000 small molecules. Our high-content screening assay yielded robust assay metrics and identified a number of inhibitors of NLRP3-dependent ASC speck formation, including compounds targeting HSP90, JAK and IKK-ß. Additional assays to investigate inflammasome priming or activation, NLRP3 downstream effectors such as caspase-1, IL-1ß and pyroptosis form the basis of a screening cascade to identify NLRP3 inflammasome inhibitors in drug discovery programs.

Subventricular zone-derived extracellular vesicles promote functional recovery in rat model of spinal cord injury by inhibition of NLRP3 inflammasome complex formation.

Spinal cord injury (SCI) is the destruction of spinal cord motor and sensory resulted from an attack on the spinal cord, which can cause significant physiological damage. The inflammasome is a multiprotein oligomer resulting in inflammation; the NLRP3 inflammasome composed of NLRP3, apoptosis-associated speck-like protein (ASC), procaspase-1, and cleavage of procaspase-1 into caspase-1 initiates the inflammatory response. Subventricular Zone (SVZ) is the origin of neural stem/progenitor cells (NS/PCs) in the adult brain. Extracellular vesicles (EVs) are tiny lipid membrane bilayer vesicles secreted by different types of cells playing an important role in cell-cell communications. The aim of this study was to investigate the effect of intrathecal transplantation of EVs on the NLRP3 inflammasome formation in SCI rats. Male wistar rats were divided into three groups as following: laminectotomy group, SCI group, and EVs group. EVs was isolated from SVZ, and characterized by western blot and DLS, and then injected into the SCI rats. Real-time PCR and western blot were carried out for gene expression and protein level of NLRP3, ASC, and Caspase-1. H&E and cresyl violet staining were performed for histological analyses, as well as BBB test for motor function. The results indicated high level in mRNA and protein level in SCI group in comparison with laminectomy (p < 0.001), and injection of EVs showed a significant reduction in the mRNA and protein levels in EVs group compared to SCI (p < 0.001). H&E and cresyl violet staining showed recovery in neural cells of spinal cord tissue in EVs group in comparison with SCI group. BBB test showed the promotion of motor function in EVs group compared to SCI in 14 days (p < 0.05). We concluded that the injection of EVs could recover the motor function in rats with SCI and rescue the neural cells of spinal cord tissue by suppressing the formation of the NLRP3 inflammasome complex.

Impairment of DHA synthesis alters the expression of neuronal plasticity markers and the brain inflammatory status in mice.

Docosahexaenoic acid (DHA) is a ω-3 fatty acid typically obtained from the diet or endogenously synthesized through the action of elongases (ELOVLs) and desaturases. DHA is a key central nervous system constituent and the precursor of several molecules that regulate the resolution of inflammation. In the present study, we questioned whether the impaired synthesis of DHA affected neural plasticity and inflammatory status in the adult brain. To address this question, we investigated neural and inflammatory markers from mice deficient for ELOVL2 (Elovl2-/- ), the key enzyme in DHA synthesis. From our findings, Elovl2-/- mice showed an altered expression of markers involved in synaptic plasticity, learning, and memory formation such as Egr-1, Arc1, and BDNF specifically in the cerebral cortex, impacting behavioral functions only marginally. In parallel, we also found that DHA-deficient mice were characterized by an increased expression of pro-inflammatory molecules, namely TNF, IL-1ß, iNOS, caspase-1 as well as the activation and morphologic changes of microglia in the absence of any brain injury or disease. Reintroducing DHA in the diet of Elovl2-/- mice reversed such alterations in brain plasticity and inflammation. Hence, impairment of systemic DHA synthesis can modify the brain inflammatory and neural plasticity status, supporting the view that DHA is an essential fatty acid with an important role in keeping inflammation within its physiologic boundary and in shaping neuronal functions in the central nervous system.

Ginkgolide B ameliorates NLRP3 inflammasome activation after hypoxic-ischemic brain injury in the neonatal male rat.

INTRODUCTION: Perinatal hypoxic-ischemic (HI) insult is an important cause of brain injury in neonates. The development of novel treatment strategies for neonates with HI brain injury is urgently needed. Ginkgolide B (GB) is a main component of Ginkgo biloba extracts with a long history of use in traditional Chinese medicine. However, it is unknown whether GB could play a protective role in hypoxic stress in immature animals. METHODS: Using neonatal hypoxic-ischemic (HI) brain injury model of rat pups, neurological score, infarct size, and brain edema were evaluated after HI injury. The activation of microglia and the production of IL-1ß and IL-18 were detected by immunohistochemistry and ELISA, respectively. A priming signal (NF-κB P65) and an activation signal (Caspase-1) of NLRP3 inflammasome activation were detected by western blot analyses. RESULTS: GB administrated 30 min prior to ischemia induction can improve neurological disorder, reduce infarct volume and alleviate cerebral edema. Compared with the HI groups, GB inhibited the activation of microglia and decreased the production of IL-1ß and IL-18 in neocortex. Furthermore, GB reduced NLRP3 expression mainly in microglia, and significantly inhibited the expression of Caspase-1 and the nuclear translocation of NF-κB P65, preventing NLRP3 inflammasome activation. CONCLUSIONS: GB ameliorates hypoxic-ischemic brain injury in the neonatal male rat via inhibiting NLRP3 inflammasome activation.

The involvement and mechanism of febuxostat in non-alcoholic fatty liver disease cells.

It has been proved that hyperuricemia is associated with non-alcoholic fatty liver disease (NAFLD). The xanthine oxidase (XO) inhibitor, febuxostat, decreases free fatty acids-induced fat accumulation in HFDT-fed mice. Here, it is shown that febuxostat attenuates fat accumulation and reactive oxygen species (ROS) in HepG2 cells. It was further found that the underlying mechanism is related to the reduction in expression of NLRP3/caspase-1/IL-18/IL-1beta and improved insulin resistance (IR). This finding highlights the possible molecular pathways involving NLRP3 activation for management of ROS and insulin IR. In conclusion, febuxostat may be a promising potential treatment for patients with NAFLD.

Cellular differentiation of non-transformed intestinal epithelial cells is regulated by Lactobacillus rhamnosus and L. casei strains.

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.

BMSCs-derived miR-223-containing exosomes contribute to liver protection in experimental autoimmune hepatitis.

Autoimmune hepatitis is a chronic inflammatory disease in the liver with potential to the development of liver fibrosis. Recent evidences suggest that bone marrow derived mesenchymal stem cells (BMSCs) may exert its therapeutic activity through exosomes. Moreover, miR-223 is highly expressed in BMSCs and plays an important role in autoimmune diseases. Therefore, in this study, hepatoprotective role of BMSCs and miR-223 was investigated in both mice and hepatocytes. Liver antigen S100 was used to establish autoimmune hepatitis model in mice while LPS and ATP were used to establish cell injury model in hepatocyte. Before the experiments, BMSCs were infected with pre-miR-223 and transfected with miR-223 inhibitor respectively. Exosomes from bone marrow stem cells were isolated by ultracentrifugation. Liver injury was evaluated by serum levels of ALT and AST as well as liver histology. Inflammation and cell death were examined by inflammatory cytokines and lactase dehydrogenase respectively. Both BMSCs-exo and BMSCs-exomiR-223(+) significantly reversed either S100 or LPS/ATP induced injury in mice and hepatocytes. Meanwhile, the expressions of cytokines, NLRP3 and caspase-1 were also downregulated by BMSCs-exo and BMSCs-exomiR-223(+) at both protein and mRNA levels in mice and hepatocytes. Moreover, BMSCs-exomiR-223(-) reverses the effects of BMSCs-exo and BMSCs-exomiR-223(+) in mouse AIH and in hepatocytes. In conclusion, bone marrow stem cell derived exosomes can protect liver injury in an experimental model of autoimmune hepatitis and the mechanism could be related to exosomal miR-223 regulation of NLRP3 and caspase-1.

MD-2 regulates LPS-induced NLRP3 inflammasome activation and IL-1beta secretion by a MyD88/NF-κB-dependent pathway in alveolar macrophages cell line.

Myeloid differentiation protein 2 (MD-2) is required in the recognition of lipopolysaccharide (LPS) by toll-like receptor 4 (TLR4), and participates in LPS-induced alveolar macrophage (AM) inflammation during acute lung injury (ALI). Activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome aggravates inflammation in LPS-induced ALI. However, there is currently little known about the relationship between MD-2 signaling and the NLRP3 inflammasome. This study showed that NLRP3 expression, IL-1beta (IL-1ß) secretion, and pyroptosis were up-regulated after LPS stimulation in the NR8383 AM cell-line. MD-2 gene knock-down reduced LPS-induced mRNA and protein expression of NLRP3 and IL-1ß secretion in NR8383 cells, and inhibited the MyD88/NF-κB signaling pathway. Conversely, over-expression of MD-2 not only heightened NLRP3, MyD88, and NF-κB p65 protein expression, it also aggravated the LPS-induced inflammatory response. Furthermore, the NF-κB inhibitor SN50 had a beneficial role in decreasing NLRP3 and caspase-1 mRNA and protein expression. The observations suggest that MD-2 helps to regulate LPS-induced NLRP3 inflammasome activation and the inflammatory response in NR8383 cells, and likely does so by affecting MyD88/NF-κB signaling.

Upregulation of NLRP3 inflammasome components in Mooren's ulcer.

PURPOSE: Mooren's ulcer (MU) is a peripheral corneal ulceration of presumed autoimmune etiology. NLRP3 inflammasome has been shown to be involved in a variety of autoimmune and auto-inflammatory diseases. However, the role of NLRP3 inflammasome in MU has not been investigated. Here, we evaluate the expression of NLRP3 inflammasome and its downstream inflammatory factors in human MU. METHODS: Conjunctival biopsy specimens were obtained from seven patients with MU and six healthy donors. The removed conjunctivas were histopathologically evaluated for NLRP3 inflammasome component expression using antibodies directed against NLRP3, Caspase-1 (CASP1), and Interleukin-1ß (IL-1ß). Quantitative real-time PCR was used to measure the mRNA expression of NLRP3 and IL-1ß, and the protein expressions of NLRP3, pro-CASP1, CASP1, and IL-1ß were detected by Western blotting. RESULTS: NLRP3 and IL-1ß mRNA expression showed higher levels in the MU group than in healthy controls. Western-blot and immunofluorescence analysis also showed that basal expression of NLRP3 inflammasome components (NLRP3, CAPS1, and IL-1ß) was elevated in patients with MU compared with healthy controls. Most importantly, we found that the cleavaged form of CASP1 and IL-1ß was significantly increased in MU patients compared with healthy donors, which indicates that the upregulation of NLRP3 inflammasome was probably responsible for the enhanced IL-1ß production in MU patients. CONCLUSIONS: This study demonstrated that the expression of the NLRP3-CASP1-IL-1ß signaling pathway was markedly increased in the conjunctival lesions of patients with MU, suggesting the involvement of NLRP3 inflammasome in the onset and development of the inflammation in MU.

The protease inhibitor cystatin C down-regulates the release of IL-ß and TNF-α in lipopolysaccharide activated monocytes.

Human cystatin C, a member of the cysteine proteinase-inhibitory family, is produced by all nucleated cells and has important roles in regulating natural immunity. Nematode homologs to human cystatin C have been shown to have anti-inflammatory effects on monocytes and to reduce colitis in mice. In Crohn's disease, pathogenic activated monocytes help drive inflammatory processes via the release of proinflammatory cytokines and chemokines. In particular, tumor necrosis factor-α-producing inflammatory monocytes have a central role in the intestinal inflammation in patients with Crohn's disease. We investigated the potential of human cystatin C to regulate pathogenic activated monocytes and its potential as an Immunomodulator in Crohn's disease. We found that cystatin C significantly decreased the lipopolysaccharide-stimulated release and expression of interleukin-1ß and tumor necrosis factor-α in monocyte and peripheral blood mononuclear cell cultures from healthy donors, whereas interleukin-6 and interleukin-8 levels were unchanged. A similar reduction of interleukin-1ß and tumor necrosis factor-α was also seen in peripheral blood mononuclear cell cultures from patients with Crohn's disease, and in particular, tumor necrosis factor-α was reduced in supernatants from lamina propria cell cultures from patients with Crohn's disease. Further investigation revealed that cystatin C was internalized by monocytes via an active endocytic process, decreased phosphorylation of the mitogen-activated protein kinase pathway extracellular signal-regulated kinase-1/2, and altered surface marker expression. The ability of cystatin C to modulate the cytokine expression of monocytes, together with its protease-inhibitory function, indicates that modulation of the local cystatin C expression could be an option in future Crohn's disease therapy.

Hydrogen sulfide diminishes the levels of thymic stromal lymphopoietin in activated mast cells.

Bamboo salt (BS) is a Korean traditional type of salt and has been reported to have therapeutic effects on allergic inflammation. Thymic stromal lymphopoietin (TSLP) aggravates inflammation in the pathogenesis of allergic reactions, such as allergic rhinitis (AR). To confirm an active compound of BS, we investigated the effect of sulfur, a compound of BS, on the levels of TSLP in a human mast cell line, HMC-1 cells and a mouse model of AR using hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaSH). We treated NaSH or BS in HMC-1 cells and activated the HMC-1 cells with phorbol myristate acetate and calcium ionophore A23187 (PMACI). ELISA for the production measurement of TSLP, PCR for the mRNA expression measurement of TSLP, and western blot analysis for the expression measurement of upstream mediators were performed. Mice were treated with NaSH and sensitized with ovalbumin (OVA). The levels of TSLP were measured in serum and nasal mucosa tissue in an OVA-induced AR mouse model. NaSH or BS diminished the production and mRNA expression of TSLP as well as interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the PMACI-activated HMC-1 cells. NaSH or BS diminished the level of intracellular calcium in the PMACI-activated HMC-1 cells. NaSH or BS reduced the expression and activity of caspase-1 in the PMACI-activated HMC-1 cells. And NaSH or BS inhibited the expression of receptor interacting protein-2 and the phosphorylation of extracellular signal-regulated kinase in the PMACI-activated HMC-1 cells. The translocation of NF-κB into the nucleus as well as the phosphorylation and degradation of IκBα in the cytoplasm were diminished by NaSH or BS in the PMACI-activated HMC-1 cells. Furthermore, NaSH inhibited the production of TSLP, IL-6, and IL-8 in TNF-α-activated HMC-1 cells. Finally, the administration of NaSH showed a decrease in number of rubs on mice with OVA-induced AR. And the levels of immunoglobulin E and TSLP in the serum and the level of TSLP in the nasal mucosa tissue of the OVA-induced AR mice were reduced by NaSH. In conclusion, these findings show that H2S, as an active compound of BS is a potential agent to cure allergic inflammation.

Dysregulation of the NLRP3 inflammasome complex and related cytokines in patients with multiple myeloma.

OBJECTIVE: The NLRP3 inflammasome complex, an important regulatory factor of inflammation and cell apoptosis, has attracted considerable attention in the development of tumor. Here, we analyzed the expression and clinical significance of NLRP3 inflammasome complex and related cytokines in patients with multiple myeloma (MM). METHODS: Peripheral blood and bone marrow of 38 newly diagnosed myeloma patients and 25 age- and gender-matched healthy people were studied. NLRP3 and caspase-1 were analyzed using quantitative real-time polymerase chain reaction and Western blot and IL-1beta, IL-18, RANKL, and OPG were evaluated by enzyme-linked immunosorbent assay. RESULTS: We showed that aberrant NLRP3 and caspase-1 expression were observed in MM and down-regulated compared with the healthy people. We further demonstrated that NLRP3 mRNA was negatively correlated with beta2-microglobulin and plasma cell percentage in MM. The downstream cytokines IL-18 and sRANKL/OPG in MM patients were higher than that in control group. Moreover, the lower mRNA levels of NLRP3 and caspase-1 were shown to be positively correlated with IL-1beta in newly diagnosed MM patients. CONCLUSIONS: This study demonstrated that dysregulated expression of NLRP3-caspase-1-IL-1beta axis was observed in patients with MM, suggesting they might be involved in the pathogenesis of MM.

Pseudomonas aeruginosa Triggers Macrophage Autophagy To Escape Intracellular Killing by Activation of the NLRP3 Inflammasome.

Assembly of the inflammasome has recently been identified to be a critical event in the initiation of inflammation. However, its role in bacterial killing remains unclear. Our study demonstrates that Pseudomonas aeruginosa infection induces the assembly of the NLRP3 inflammasome and the sequential secretion of caspase1 and interleukin-1ß (IL-1ß) in human macrophages. More importantly, activation of the NLRP3 inflammasome reduces the killing of P. aeruginosa in human macrophages, without affecting the generation of antimicrobial peptides, reactive oxygen species, and nitric oxide. In addition, our results demonstrate that P. aeruginosa infection increases the amount of the LC3-II protein and triggers the formation of autophagosomes in human macrophages. The P. aeruginosa-induced autophagy was enhanced by overexpression of NLRP3, ASC, or caspase1 but was reduced by knockdown of these core molecules of the NLRP3 inflammasome. Treatment with IL-1ß enhanced autophagy in human macrophages. More importantly, IL-1ß decreased the macrophage-mediated killing of P. aeruginosa, whereas knockdown of ATG7 or Beclin1 restored the IL-1ß-mediated suppression of bacterial killing. Collectively, our study explores a novel mechanism employed by P. aeruginosa to escape from phagocyte killing and may provide a better understanding of the interaction between P. aeruginosa and host immune cells, including macrophages.

Activation of NLRP3 inflammasome in human middle ear cholesteatoma and chronic otitis media.

CONCLUSIONS: The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome plays an important role in the pathogenesis of middle ear diseases. Modulation of inflammasome-mediated inflammation may be a novel therapeutic strategy for cholesteatoma and chronic otitis media. OBJECTIVE: NLRP3 inflammasome is a critical molecule mediating interleukin (IL)-1ß responses. However, the expression of NLRP3 in the pathogenesis of cholesteatoma and chronic otitis media has not been fully examined. This study sought to assess the expression of NLRP3, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain and a pyrin domain), and caspase-1 in middle ear tissues in patients with cholesteatoma or chronic otitis media. METHODS: Middle ear tissue samples were obtained from patients with cholesteatoma or chronic otitis media. Control middle ear samples were collected during cochlear implant surgery of patients without middle ear inflammation. The expression of NLRP3, ASC, and caspase-1 were examined by reverse transcription polymerase chain reaction (RT-PCR) assay and immunohistochemical study. RESULTS: The levels of mRNA of NLRP3, ASC, and caspase-1 were significantly elevated in cholesteatoma and chronic otitis media as compared with that of normal controls. The proteins of NLRP3, ASC, and caspase-1 were observed in infiltrating inflammatory cells in cholesteatoma and chronic otitis media.

Excess aldosterone is a critical danger signal for inflammasome activation in the development of renal fibrosis in mice.

High levels of aldosterone impair renal function by activating proinflammatory and profibrotic pathways. However, the molecular mechanism underlying aldosterone-induced inflammation and fibrosis is unknown. Inflammasome activation contributes to chronic kidney disease. We hypothesized that aldosterone induces renal tubulointerstitial inflammation and fibrosis by activating the inflammasome. Infusing wild-type mice with aldosterone (0.25 mg/kg/d) caused tubulointerstitial damage, increased expression of inflammasome components, caspase 1 activation, and overproduction of IL-1ß and IL-18. These changes were suppressed by eplerenone treatment (100 mg/kg/d) in wild-type mice or in mice deficient in apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC). Caspase 1-positive and F4/80-positive cells colocalized in the interstitium. Bone marrow transplantation using ASC-deficient mice indicated that inflammasome activation in macrophages mediated aldosterone-induced renal fibrosis. IL-18 was detected in culture supernatants of macrophages treated with aldosterone, and mitochondria-derived reactive oxygen species activated the inflammasome in these macrophages. Our results indicate that exposure of macrophages to high levels of aldosterone resulted in the activation of inflammasomes via the mitochondria-derived reactive oxygen species. Thus, inflammasome activation in macrophages may serve as a new therapeutic target for chronic kidney disease.

Toll-like receptor 2 and NLRP3 cooperate to recognize a functional bacterial amyloid, curli.

Amyloids are proteins with cross-ß-sheet structure that contribute to pathology and inflammation in complex human diseases, including Alzheimer's disease, Parkinson's disease, type II diabetes, and secondary amyloidosis. Bacteria also produce amyloids as a component of their extracellular matrix during biofilm formation. Recently, several human amyloids were shown to activate the NLRP3 inflammasome, leading to the activation of caspase 1 and production of interleukin 1ß (IL-1ß). In this study, we investigated the activation of the NLRP3 inflammasome by bacterial amyloids using curli fibers, produced by Salmonella enterica serovar Typhimurium and Escherichia coli. Here, we show that curli fibers activate the NLRP3 inflammasome, leading to the production of IL-1ß via caspase 1 activation. Investigation of the underlying mechanism revealed that activation of Toll-like receptor 2 (TLR2) by curli fibers is critical in the generation of IL-1ß. Interestingly, activation of the NLRP3 inflammasome by curli fibers or by amyloid ß of Alzheimer's disease does not cause cell death in macrophages. Overall, these data identify a cross talk between TLR2 and NLRP3 in response to the bacterial amyloid curli and generation of IL-1ß as a product of this interaction.

Absent in melanoma 2 (AIM2) expressed in human dental pulp mediates IL-1ß secretion in response to cytoplasmic DNA.

The inflammasome has been determined to play an important role in inflammatory diseases in recent years. Absent in melanoma 2 (AIM2), an inflammasome that recognizes cytoplasmic DNA, has recently been identified as a critical regulator of immune responses. In this study, we explored whether AIM2 was expressed in human dental pulp and defined the role of AIM2 in regulating interleukin (IL)-1ß secretion. We demonstrated that AIM2 was only detected in the odontoblast layer of healthy dental pulp, whereas strong expression was observed in inflamed dental pulp. Stimulation with interferon gamma (IFN-γ) and cytoplasmic DNA significantly activated the AIM2 inflammasome and increased IL-1ß secretion in human dental pulp cells (HDPCs) in a time- and dose-dependent manner. Moreover, the knockdown of AIM2 downregulated both cleaved-caspase-1 expression and IL-1ß release in HDPCs. These results suggest that AIM2 expressed in human dental pulp plays an important role in the immune defense by activating the inflammasome signaling pathway.

Transcriptomic analysis unveils correlations between regulative apoptotic caspases and genes of cholesterol homeostasis in human brain.

Regulative circuits controlling expression of genes involved in the same biological processes are frequently interconnected. These circuits operate to coordinate the expression of multiple genes and also to compensate dysfunctions in specific elements of the network. Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Silencing of caspase-2 expression in cultured glioblastoma cells allows the up-regulation of a limited number of genes, among which some are related to cholesterol homeostasis. Lysosomal Acid Lipase A (LIPA) was up-regulated in two different cell lines in response to caspase-2 down-regulation and cells silenced for caspase-2 exhibit reduced cholesterol staining in the lipid droplets. We expanded this observation by large-scale analysis of mRNA expression. All caspases were analyzed in terms of co-expression in comparison with 166 genes involved in cholesterol homeostasis. In the brain, hierarchical clustering has revealed that the expression of regulative apoptotic caspases (CASP2, CASP8 CASP9, CASP10) and of the inflammatory CASP1 is linked to several genes involved in cholesterol homeostasis. These correlations resulted in altered GBM (Glioblastoma Multiforme), in particular for CASP1. We have also demonstrated that these correlations are tissue specific being reduced (CASP9 and CASP10) or different (CASP2) in the liver. For some caspases (CASP1, CASP6 and CASP7) these correlations could be related to brain aging.

Losartan inhibits LPS + ATP-induced IL-1beta secretion from mouse primary macrophages by suppressing NALP3 inflammasome.

OBJECTIVES: IL-1beta is a potent proinflammatory, pro-fibrogenetic and pro-athrosclerosis cytokine which has been shown to play an important role in an expanding number of noninfectious, chronic inflammatory conditions including cardiovascular disease, renal fibrosis, rheumatoid arthritis and even type 2 diabetes. Losartan is an angiotensin II receptor antagonist widely used for the treatment of hypertension, diabetic nephropathy and congestive heart failure. In this study, we attempted to clarify whether losartan has an inhibitory effect on IL-1beta. To further elucidate the molecular mechanism underlying the anti-IL-1beta property of losartan, we studied the LPS+ATP-induced activation of NALP3 inflammasome which controls the muturation and secretion of IL-1beta. METHODS: LPS and ATP were used to stimulate the release of IL-1beta from thioglycollate-elicited macrophages from BALB/c mice. The production of IL-1beta was evaluated by ELISA assay and NALP3, caspase-1, IL-beta mRNA levels were determined by reverse transcription-polymerase chain reaction. RESULTS: In cultured thioglycollate-elicited macrophages, we observed that LPS + ATP greatly enhanced IL-1 beta secretion (6938.00 +/- 83.45; P < 0.05) and the mRNA levels of NALP3, caspase-1 which are two main components of NALP3 inflammasome (60.88 +/- 8.28; 1.31 +/- 0.04, P < 0.05 for both). The macrophages co-cultured with losartan showed low production of IL-1beta (3907.50 +/- 143.61; P < 0.05) and low production of NALP3, caspase-1mRNA (29.82 +/- 6.92; 1.12 +/- 0.05, P < 0.05 for both). Losartan did not reduce IL-1beta mRNA(P > 0.05). CONCLUSIONS: Our results show that the NALP3 inflammasome is up-regulated and activated in the mouse macrophage in response to LPS + ATP stimulation. Losartan is able to suppress the LPS + ATP-induced production of IL-1beta protein. In addition, this effectmay be partially mediated by suppressing NALP3 inflammasome activation.