Pterostilbene inhibits amyloid-ß-induced neuroinflammation in a microglia cell line by inactivating the NLRP3/caspase-1 inflammasome pathway.

Neuroinflammation has been known as an important pathogenetic contributor of Alzheimer's disease (AD). Pterostilbene is a natural compound which has neuroprotective activity. However, the effect of pterostilbene on amyloid-ß (Aß)-induced neuroinflammation has not been clarified. The aim of the present study was to investigate the effect of pterostilbene on Aß-induced neuroinflammation in microglia. The results indicated that pterostilbene attenuated Aß1-42 -induced cytotoxicity of BV-2 cells. Aß1-42 induced NO production and iNOS mRNA and protein expression, while pterostilbene inhibited the induction. The expression and secretion levels of IL-6, IL-1ß, and TNF-α were enhanced by Aß1-42 treatment, whereas pterostilbene decreased them. Aß1-42 activated NLRP3/caspase-1 inflammasome, which was inactivated by pterostilbene. In addition, the inhibitor of caspase-1 Z-YVAD-FMK attenuated the Aß1-42 -induced neuroinflammation in BV-2 cells. In conclusion, pterostilbene attenuated the neuroinflammatory response induced by Aß1-42 in microglia through inhibiting the NLRP3/caspase-1 inflammasome pathway, indicating that pterostilbene might be an effective therapy for AD.

Laquinimod decreases Bax expression and reduces caspase-6 activation in neurons.

Laquinimod is an immunomodulatory compound that has shown neuroprotective benefits in clinical trials for multiple sclerosis. Laquinimod ameliorates both white and gray matter damage in human patients, and prevents axonal degeneration in animal models of multiple sclerosis. Axonal damage and white matter loss are a common feature shared between different neurodegenerative diseases. Caspase-6 activation plays an important role in axonal degeneration on the molecular level. Increased activity of caspase-6 has been demonstrated in brain tissue from presymptomatic Huntington disease mutation carriers, and it is an early marker of axonal dysfunction. Since laquinimod is currently undergoing a clinical trial in Huntington disease (LEGATO-HD, clinicaltrials.gov ID: NCT02215616), we set out to evaluate its impact on neuronal caspase-6 activation. We find that laquinimod ameliorates DNA-damage induced activation of caspase-6 in primary neuronal cultures. This is an indirect effect that is not mediated by direct inhibition of the enzyme. The investigation of potential caspase-6 activating mechanisms revealed that laquinimod reduces the expression of Bax, a pro-apoptotic molecule that causes mitochondrial cytochrome c release and caspase activation. Bax expression is furthermore increased in striatal tissues from the YAC128 mouse model of HD in an age-dependent manner. Our results demonstrate that laquinimod can directly downregulate neuronal apoptosis pathways relevant for axonal degeneration in addition to its known effects on astrocytes and microglia in the CNS. It targets a pathway that is relevant for the pathogenesis of HD, supporting the hypothesis that laquinimod may provide clinical benefit.

Heterogeneity of subordination of the IL-18/IFN-γ axis to caspase-1 among patients with Crohn's disease.

In Crohn's disease (CD), hierarchical architecture of the inflammatory network, including subordination of IL-18, an IFN-γ-inducing cytokine, to the inflammasome, have remained undeciphered. Heterogeneity among patients of such a subordination cannot be evaluated by animal models, monofactorial in their etiology and homogenous in disease progression. To address these issues, we set up an ex vivo model of inflamed mucosa explant cultures from patients with active long-standing CD. Th1 cytokine production, especially IFN-γ and IL-18, was assessed in relation with inflammation intensity. Subordination of the Th1 response to caspase-1, effector of the inflammasome, was determined in explant cultures subjected to pharmacological inhibition of caspase-1 by YVAD. We showed a correlation between secreted IFN-γ/IL-18 levels, and caspase-1 activation, with inflammation intensity of intestinal CD mucosa explants. Inhibition of caspase-1 activation using the specific inhibitor YVAD identified a homogenous non responder group featuring a caspase-1-independent IL-18/IFN-γ response, and a heterogenous responder group, in which both IL-18 and IFN-γ responses were caspase-1-dependent, with a 40-70% range of inhibition by YVAD. These findings bring out the concept of heterogeneity of subordination of the Th1 response to inflammasome activation among CD patients. This ex vivo model should have therapeutic relevance in allowing to determine eligibility of CD patients for new targeted therapies.

Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation.

Dengue is the most frequent hemorrhagic viral disease and re-emergent infection in the world. Although thrombocytopenia is characteristically observed in mild and severe forms of dengue, the role of platelet activation in dengue pathogenesis has not been fully elucidated. We hypothesize that platelets have major roles in inflammatory amplification and increased vascular permeability during severe forms of dengue. Here we investigate interleukin (IL)-1ß synthesis, processing, and secretion in platelets during dengue virus (DV) infection and potential contribution of these events to endothelial permeability during infection. We observed increased expression of IL-1ß in platelets and platelet-derived microparticles from patients with dengue or after platelet exposure to DV in vitro. We demonstrated that DV infection leads to assembly of nucleotide-binding domain leucine rich repeat containing protein (NLRP3) inflammasomes, activation of caspase-1, and caspase-1-dependent IL-1ß secretion. Our findings also indicate that platelet-derived IL-1ß is chiefly released in microparticles through mechanisms dependent on mitochondrial reactive oxygen species-triggered NLRP3 inflammasomes. Inflammasome activation and platelet shedding of IL-1ß-rich microparticles correlated with signs of increased vascular permeability. Moreover, microparticles from DV-stimulated platelets induced enhanced permeability in vitro in an IL-1-dependent manner. Our findings provide new evidence that platelets contribute to increased vascular permeability in DV infection by inflammasome-dependent release of IL-1ß.

Differential regulation of high glucose-induced glyceraldehyde-3-phosphate dehydrogenase nuclear accumulation in Müller cells by IL-1beta and IL-6.

PURPOSE: This study determined the role of the proinflammatory cytokines known to be elevated in the diabetic retina, namely IL-1beta, TNFalpha, and IL-6, in a high glucose-induced nuclear accumulation of GAPDH in retinal Müller cells, an event considered crucial for the induction of cell death. METHODS: With use of the transformed rat Müller cell line (rMC-1) and isolated human Müller cells (HMCs), the authors examined the effect of high glucose (25 mM), IL-1beta, TNFalpha, IL-6, and high glucose (25 mM) plus inhibitors of the caspase-1/IL-1beta signaling pathway on GAPDH nuclear accumulation, which was evaluated by immunofluorescence analysis. RESULTS: High glucose induced IL-1beta, weak IL-6, and no TNFalpha production by rMC-1 and HMCs. IL-1beta (1-10 ng/mL) significantly increased GAPDH nuclear accumulation in Müller cells in a concentration-dependent manner within 24 hours. Further, high glucose-induced GAPDH nuclear accumulation in Müller cells was mediated by IL-1beta. Inhibition of the IL-1 receptor using an IL-1 receptor antagonist (IL-1ra; 50 ng/mL) or inhibition of IL-1beta production using a specific caspase-1 inhibitor (YVAD-fmk; 100 microM) significantly decreased high glucose-induced GAPDH nuclear accumulation. In contrast, IL-6 (2 ng/mL) had a strong protective effect attenuating high glucose and IL-1beta-induced GAPDH nuclear accumulation in Müller cells. TNFalpha (1-10 ng/mL) did not have any effect on GAPDH nuclear accumulation. CONCLUSIONS: These results revealed a novel mechanism for high glucose-induced GAPDH nuclear accumulation in Müller cells through production and autocrine stimulation by IL-1beta. The protective role of IL-6 in high glucose- and IL-1beta-induced toxicity indicates that changes in the balance of these cytokines might contribute to cellular damage mediated by elevated glucose levels.

Simvastatin induces interleukin-18 production in human peripheral blood mononuclear cells.

The effects of statins on immune response depend on the inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase and leukocyte function-associated antigen (LFA)-1, which is a ligand of intercellular adhesion molecule (ICAM)-1. Simvastatin, an HMG-CoA reductase inhibitor with mild inhibition of LFA-1, induced the production of interleukin (IL)-18, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in human peripheral blood mononuclear cells (PBMC). The IL-18 production is located upstream of the cytokine cascade activated by simvastatin. Moreover, simvastatin concentration-dependently inhibited the expression of ICAM-1 and induced the expression of CD40 on monocytes. In the presence of IL-18, simvastatin suppressed the expression of ICAM-1 and CD40 as well as the production of IL-12, TNF-alpha and IFN-gamma in PBMC, contributing to the anti-inflammatory effect of simvastatin. The effects of simvastatin were abolished by the addition of the product of the HMG-CoA reductase, mevalonate, indicating the involvement of HMG-CoA reductase in the action of simvastatin.

Differential effect of LFA703, pravastatin, and fluvastatin on production of IL-18 and expression of ICAM-1 and CD40 in human monocytes.

A novel, proinflammatory cytokine, interleukin (IL)-18 production was detected in the medium of human monocytes treated with 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitors, pravastatin, and fluvastatin (0.1 and 1 muM) but not with the statin-derived lymphocyte function-associated antigen-1 (LFA-1) inhibitor LFA703, which did not inhibit HMG-CoA reductase. Pravastatin and fluvastatin also induced the production of IL-18, tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in human peripheral blood mononuclear cells (PBMC) in contrast to LFA703. IL-18 production by PBMC is located upstream of the cytokine cascade activated by these statins. The IL-18-induced cytokine production was demonstrated to be dependent on adhesion molecule expression on monocytes. In the absence and presence of lower concentrations (0.1 and 1 ng/ml) of IL-18, pravastatin and fluvastatin inhibited the expression of intercellular adhesion molecule (ICAM)-1 and induced the expression of CD40, whereas LFA703 had no effect. In the presence of higher concentrations (5, 10, and 100 ng/ml) of IL-18, pravastatin, fluvastatin, and LFA703 similarly inhibited the expression of ICAM-1 and CD40 as well as the production of IL-12, TNF-alpha, and IFN-gamma in PBMC. The effects of pravastatin and fluvastatin but not LFA703 were abolished by the addition of mevalonate, indicating the involvement of HMG-CoA reductase in the action of pravastatin and fluvastatin. Thus, the effects of LFA703 were distinct from those of pravastatin and fluvastatin in the presence of lower concentrations of IL-18. It was concluded that LFA703 has the inhibitory effect on an IL-18-initiated immune response without any activation on monocytes.

alpha1-Adrenergic receptor antagonists induce production of IL-18 and expression of ICAM-1 and CD40 in human monocytes.

The activation of T cells plays a role in antitumor response. Monocytes activate T cells by inducing the cell-to-cell interaction that involves the engagement of adhesion molecules with their ligands, and the production of IL-18. The authors examined the effect of the quinazoline-based alpha1-adrenergic receptor antagonists bunazosin, doxazosin, prazosin, and terazosin on the expression of adhesion molecules such as ICAM-1, B7.1, B7.2, CD40, and CD40L on monocytes isolated from human peripheral blood mononuclear cells. Doxazosin, prazosin, and terazosin induced the expression of ICAM-1 and CD40 but had no effect on the expression of B7.1, B7.2, and CD40L. Moreover, IL-18 was detected in the medium of incubated monocytes treated with doxazosin, prazosin, and terazosin. Bunazosin did not affect adhesion molecule expression and IL-18 production, suggesting that the chemical structure of quinazoline might not be related to the effect of doxazosin, prazosin, and terazosin. Although caspase-1 inhibitor completely abolished the production of IL-18, anti-IL-18 mAb and caspase-1 inhibitor partially inhibited the increase in ICAM-1 and CD40 expression induced by doxazosin, prazosin, and terazosin. Doxazosin, prazosin, and terazosin can induce monocyte activation with a specific pattern of expression of adhesion molecules and IL-18 production, and this may lead to T-cell activation through the cell-to-cell interaction. The activation of T cells induced by the increase of the expression of ICAM-1 and CD40 and the production of IL-18 may be involved in the anti-cancer effects of doxazosin, prazosin, and terazosin.

PS-341, a novel proteasome inhibitor, induces Bcl-2 phosphorylation and cleavage in association with G2-M phase arrest and apoptosis.

Treatment with the proteasome inhibitor, PS-341 resulted in concentration- and time-dependent effects on Bcl-2 phosphorylation and cleavage in H460 cells that coincided with the PS-341-induced G2-M phase arrest. The observed Bcl-2 cleavage paralleled the degree of PS-341-induced apoptosis but was detected to a similar extent with comparable concentrations of two other proteasome inhibitors (MG-132 and PSI). Calpain inhibitors, ALLM and ALLN, and the caspase inhibitors, Z-VAD and AC-YVAD did not induce BcI-2 phosphorylation and cleavage. Exposure to PS-341 resulted in an additional Mr 25,000 cleavage fragment of Bcl-2, whereas only a Mr 23,000 fragment was observed with other anticancer agents. The formation of the Mr 25,000 fragment was not prevented by caspase inhibitors unlike the Mr 23,000 fragment, which suggests mediation by a caspase-independent pathway. Cell fractionation studies revealed that the Bcl-2 cleaved fragments localize within membrane structures and was an early event (at approximately 12 h, posttreatment), and before the observed cleavage of poly(ADP-ribose) polymerase (PARP), beta-catenin, and DNA fragmentation (at approximately 36 h posttreatment). The Mr 23,000 Bcl-2 cleavage product was inhibited by the pan-caspase inhibitor and the inhibitors of capase-3, -8, -9; but the PARP cleavage was prevented only by the pan-caspase and caspase-3 inhibitors, which suggests that the Mr 23,000 Bcl-2 cleavage occurred at both the initiation and execution stages of apoptosis. The inhibition of the ubiquitin/proteasome pathway by PS-341 leads, at an early stage of apoptosis, to Bcl-2 phosphorylation and a unique proteolytic cleavage product, which are associated with G2-M phase arrest and the induction of apoptosis.

Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor.

There is evidence in the literature that serine (Ser) proteases, like caspases, are activated during apoptosis. Little is known, however, about individual Ser proteases and the mechanism of their activation. In the present study, we employed a new type of cell permeant reagent to detect activation of chymotrypsin-like proteases in human leukemic HL-60 cells induced to undergo apoptosis. The reagent, 5(6)-carboxyfluoresceinyl-L-phenylalanyl-chloromethyl ketone (FFCK), is a fluorochrome-labeled analog of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), the inhibitor known to specifically and covalently bind to the active center of chymotrypsin-like enzymes. In cultures treated with the DNA topoisomerase I inhibitor, camptothecin (CPT), or tumor necrosis factor (TNFalpha), populations of cells appeared that had the capability to bind FFCK. Most FFCK-binding cells were identified by fluorescence microscopy and laser scanning cytometry (LSC) as the cells undergoing apoptosis. Frequency of cells binding FFCK strongly correlated with frequency of cells having activated caspases (r=0.98 in CPT-treated, and r=0.99 in TNFalpha-treated cultures). The observed induction of FFCK binding we interpret as representing the activation of a chymotrypsin-like apoptotic Ser protease(s). Pretreatment of cells with the poly-caspase inhibitor, Z-VAD-FMK, prevented the activation of these Ser protease(s). Pretreatment with TPCK, however, had a less pronounced, although distinct and reproducible suppressive effect, on caspase activation. The data, thus, suggest that activation of caspases is an upstream event required for activation of Ser protease(s). Activation of the latter, however, appears to additionally amplify, in a cascade-like mode, caspases activation. Differential color fluorochrome-labeling allowed us to discriminate, within the same cells, between the activation of active caspases and Ser protease(s). Despite a certain degree of co-localization, the inter- and intra-cellular pattern of caspase- vs. Ser-protease(s) was different. Our approach makes it possible to simultaneously monitor activation of caspases and Ser proteases in the same live cells that are induced to apoptosis.

Multiple caspases are involved in beta-amyloid-induced neuronal apoptosis.

beta-amyloid peptide (Abeta) has been implicated in the pathogenesis of Alzheimer disease and has been reported to induce apoptotic death in cell culture. Cysteine proteases, a family of enzymes known as caspases, mediate cell death in many models of apoptosis. Multiple caspases have been implicated in Abeta toxicity; these reports are conflicting. We show that treatment of cerebellar granule cells (CGC) with Abeta25-35 causes apoptosis associated with increased activity of caspases-2, -3 and -6. Selective inhibition of each of these three caspases provides significant protection against Abeta-mediated apoptosis. In contrast, no change in caspase-1 activity was seen after Abeta25-35 application, nor was inhibition of caspase-1 neuroprotective. Similar to CGC, cortical neuronal cultures treated with Abeta25-35 demonstrate increased caspase-3 activity but not caspase-1 activity. Furthermore, significant neuroprotection is elicited by selective inhibition of caspase-3 in cortical neurons administered Abeta25-35, whereas selective caspase-1 inhibition has no effect. Taken together, these findings indicate that multiple executioner caspases may be involved in neuronal apoptosis induced by Abeta.

Cell-specific caspase expression by different neuronal phenotypes in transient retinal ischemia.

Emerging evidence supports an important role for caspases in neuronal death following ischemia-reperfusion injury. This study assessed whether cell specific caspases participate in neuronal degeneration and whether caspase inhibition provides neuroprotection following transient retinal ischemia. We utilized a model of transient global retinal ischemia. The spatial and temporal pattern of the active forms of caspase 1, 2 and 3 expression was determined in retinal neurons following ischemic injury. Double-labeling with cell-specific markers identified which cells were expressing different caspases. In separate experiments, animals received various caspase inhibitors before the induction of ischemia. Sixty minutes of ischemia resulted in a delayed, selective neuronal death of the inner retinal layers at 7 days. Expression of caspase 1 was not detected at any time point. Maximal expression of caspase 2 was found at 24 h primarily in the inner nuclear and ganglion cell layers of the retina and localized to ganglion and amacrine neurons. Caspase 3 also peaked at 24 h in both the inner nuclear and outer nuclear layers and was predominantly expressed in photoreceptor cells and to a lesser extent in amacrine neurons. The pan caspase inhibitor, Boc-aspartyl fmk, or an antisense oligonucleotide inhibitor of caspase 2 led to significant histopathologic and functional improvement (electroretinogram) at 7 days. No protection was found with the caspase 1 selective inhibitor, Y-vad fmk. These observations suggest that ischemia-reperfusion injury activates different caspases depending on the neuronal phenotype in the retina and caspase inhibition leads to both histologic preservation and functional improvement. Caspases 2 and 3 may act in parallel in amacrine neurons following ischemia-reperfusion. These results in the retina may shed light on differential caspase specificity in global cerebral ischemia.

Anti-inflammatory activity of diazomethyl ketone and chloromethyl ketone analogs prepared from N-tosyl amino acids.

A series of diazomethyl ketone and chloromethyl ketone analogs prepared from N-tosyl amino acids was shown to have anti-inflammatory activity in mice at 20 mg/kg and in rats at 10 mg/kg. N-Tosyl-L-alanine and N-tosyl-beta-alanine chloromethyl ketones demonstrated the most potent anti-inflammatory activity. The writhing reflex also was inhibited at 20 mg/kg in mice. In the tail flick test, N-tosyl-D,L-alanine and N-tosyl-D,L-isoleucine chloromethyl ketones demonstrated the highest increase in time. Toxicity studies indicated good therapeutic indexes for most of these agents.