Release Of IL-1beta Research Articles

Ragweed pollen activates inflammasome in epidermal keratinocytes and release IL-1beta and IL-18

Pollen is an important trigger for allergic rhinitis, conjunctivitis, and asthma, and is an exacerbating factor for atopic dermatitis (AD). Pollen of white birch and short ragweed contains not only serine protease activity but also cysteine protease activity like house dust mite (HDM) allergens. HDM allergens are important factors for the onset and exacerbation of AD. Previously, we have demonstrated cysteine protease of HDM allergens directly activate NLRP3 inflammasome in human keratinocyte and release mature IL-1beta and IL-18.

Abstract 447: Activation of IL-1β-Producing Inflammasomes Triggered by RNA Receptor RIG-I in Mouse Endothelial Cells

Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase and recently identified as a cytosolic RNA receptor in mammalian cells. The role of RIG-I in the regulation of vascular function under physiological and pathological conditions is unknown. The present study tested whether RIG-I activation triggers inflammasome formation, turning on inflammation in mouse endothelial cells (EOMA cell line). By real time RT-PCR and Western blot analysis, transfection of mouse ECs with RIG-I specific agonist, 5’-triphosphate double-stranded RNA (3pRNA, 0.5 mg/L) increased RIG-I mRNA level by 106% and protein level by 81% compared to those in control double-stranded RNA (dsRNA) transfected ECs. ELISA analyses showed that 3pRNA significantly increased release of type I IFN alpha by 31 folds and IL-1 beta (a prototype cytokine from inflammasome activation) by 8 folds in these ECs. Proatherogenic stimulation of mouse ECs with cholesterol crystals or 7-ketocholesterol also markedly increased protein expression of RIG-I, but had no effect on RIG-I mRNA levels. Measurements of active caspase-1, an inflammasome activation marker using FLICA fluorescent probe that specifically binds to cleaved caspase-1, demonstrated that 3pRNA doubled FLICA positive cells compared to that in control dsRNA transfected ECs. Interestingly, cholesterol crystals significantly increased FLICA positive cells by 3 folds. This activation of caspase-1 in ECs by cholesterol crystals was further confirmed by increase in cleaved caspase-1 (p10) using Western blot analysis and by enhanced IL-1 beta release as detected by ELISA. In the presence of 3pRNA, cholesterol crystal-induced inflammasome activation was not further augmented. These data indicate that increased expression and activity of RIG-I activate IL-1 beta producing inflammasomes in ECs, which may represent an early molecular mechanism mediating vascular inflammation or injury upon atherogenic stimulations.

Inhibition of LPS-Induced Retinal Microglia Activation by Naloxone Does Not Prevent Photoreceptor Death

Microglia-associated inflammation is closely related to the pathogenesis of retinal degenerative disorders. We have previously shown in vivo that naloxone protected photoreceptors from light-induced apoptosis possibly through inhibiting microglial activation. In this study, we attempted to explore the effect of lipopolysaccharide (LPS)-activated microglia on photoreceptor death and the influence of naloxone treatment using an in vitro retinal microglia and 661 W photoreceptor co-culture system. Immunofluorescent staining and ELISA measurements demonstrated that LPS activated microglia by changing the morphology and increasing the production of proinflammatory factors interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. Flow cytometry analysis of annexin V/propidium iodide staining showed that LPS-activated microglia promoted the apoptosis of co-cultured 661 W photoreceptor cells. Naloxone inhibited microglial activation and decreased the release of IL-1beta and TNF-alpha but could not prevent photoreceptors from undergoing apoptosis. Considering the dual role of microglia-associated inflammation in both neurotoxicity and neuroprotection, modulating the function, rather than simply inhibiting their activation, might be a new therapeutic method for preventing photoreceptor degeneration.

IL-1 signaling contributes to the chronic inflammatory response to the oral pathogen, P. gingivalis (169.17)

Abstract Objective. Interleukin-1 signaling is an important mediator of the pro-inflammatory response to pathogens. We have shown that the oral pathogen P. gingivalis (Pg) induces the release of IL-1alpha (IL-1α) and IL-1beta (IL-1β) in cultured cells in vitro and is associated with chronic inflammation including platelet activation, oral bone loss and atherosclerosis. We sought to define the specificity (IL-1α vs. IL-1β) of IL-1 receptor signaling on platelet function and oral bone loss in response to Pg. Methods. Wild type (WT), IL-1β knockout (IL-1β KO), and IL-1 receptor knockout (IL1R KO) mice were challenged orally with Pg or control (Con) 3 times/week for one week. Platelet activation (flow cytometry) was determined one day (acute, N=3/group) and 6 weeks (chronic, N=5/group) after the third Pg challenge. Oral bone loss (micro-CT analysis) was determined 6 weeks after the third Pg challenge (N=5/group). Results. Challenge with Pg resulted in significant platelet activation in acute and chronic samples in WT but not in IL-1β KO or IL1R KO mice. Challenge with Pg resulted in significant oral bone loss in WT mice. Inflammatory bone loss was prevented in IL-1R KO mice but was not prevented in IL-1β KO mice. Conclusions. Platelet activation following Pg challenge is likely mediated by Pg-induced IL-1β release and signaling. In contrast, oral bone loss does not appear to be mediated by IL-1β and may involve Pg-induced IL-1α release and signaling through IL1R.

Role of inflammasome in HSP-mediated innate immunity (114.5)

Abstract The immunogenic heat shock proteins gp96, hsp70 and calreticulin stimulate antigen presenting cells to provide co-stimulation in the form of cytokines and co-stimulatory molecules. The unique pattern of co-stimulation is dependent on the type of antigen presenting cell and the HSP that is used as the stimulant. The pattern of co-stimulation also determines the T helper response that is primed. A cytokine that is routinely released by HSP-stimulated APCs is IL-1beta. Here we investigate the role of (components of) the inflammasome pathway in HSP-mediated IL-1beta release. Our results show that in response to HSP stimulation and in a CD91-dependent manner, first, antigen presenting cells secrete robust amounts of IL-1β comparable to LPS stimulation, second, the HSP receptor CD91 is phosphorylated, and third, downstream p38 and NF-kB are activated. The source of the second signal which allows for the conversion of the pro-IL1beta to the mature form in this system is being investigated.

107 Immunomodulatory Effects of Manumycin-type Antibiotics on Human Macrophages

BackgroundPolyketide-derived antibiotics including macrolides are known to exert potent anti-inflammatory and immunomodulatory effects beyond their purely antibacterial action. The mechanisms of their biological activities are still being investigated but the effect on signalling pathways of transcription factors which regulate a number of pro-inflammatory and/or pro-fibrotic genes might be preferentially involved. The aim of our study was to assess the effect of manumycin and structurally related compounds asukamycin and collabomycin on a release of proinflammatory cytokines IL-1beta and IL-18 from THP-1 monocyte/macrophage cell line. Furthermore, the level of mRNA expression of multiple genes associated with immune regulation has been studied.MethodsThe THP-1 cells were cultured in RPMI1640 with 5% fetal calf serum and then stimulated with TNF alpha (20 ng/mL) under serum free conditions in the presence or absence of manumycin and asukamycin (both at 0.3 μg/mL). The concentrations of cytokines in culture supernatants were measured by ELISA (IL-18, MBL) or Luminex (IL-1 beta, R&D). Quantitative RT-PCR (SABiosciences) was used for the evaluation of 84 different gene expressions in TNF alpha and manumycin stimulated cultures.ResultsIL-1 beta was not detectable in culture supernatants of unstimulated THP-1 cells but appeared in response to TNF alpha (4.96 + 0.59 pg/mL). Both manumycin (0.34 + 0.48 pg/mL) and asukamycin (1.06 + 0.81) inhibited IL-1 beta release induced by TNF alpha. IL-18 was found to be constitutively produced (14.68 + 7.83 pg/mL) and the release was doubled by TNF alpha (30.98 + 2.21 pg/mL) and inhibited to basal values by both manumycin (18.04 + 10.21 pg/mL) and asukamycin (12.96 + 2.32 pg/mL). Manumycin inhibited mRNA expression of several genes associated with proinflammatory responses including IL-1 beta, IL-6, and TLR8. Among the genes upregulated in response to manumycin, HMOX1, gene for heme oxigenase 1, showed the highest mRNA induction.ConclusionsWe assume from our study that manumycin and asukamycin represent potent inhibitors of IL-1 beta and IL-18 release from human macrophages. Some of the potentially proinflammatory genes are regulated on the level of transcription.

Response to 'Plasma proteins present in osteoarthritic synovial fluid can stimulate cytokine production via Toll-like receptor 4'

We would like to add some comments with regard to our own experimental data concerning the immunomodulatory effect of plasma on calcium crystal-induced inflammation to findings outlined by Sohn and colleagues. Our results show that healthy human plasma in THP1 cells is able to enhance the release of IL-1beta and IL-8 in a dose dependent manner but the effect is higher when plasma is replaced by serum. We noted that fibrinogen is able to increase the inflammatory action of crystals with a more marked effect on IL-8. Showing a direct effect both on cells and on crystals, fibrinogen has an overall effect on the modulation of the inflammatory response to calcium crystals.

Nanotechnology-based drug delivery in mucosal immune diseases: hype or hope?

Nanotechnology-based drug delivery in mucosal immune diseases: hype or hope?

Glibenclamide reduces proinflammatory cytokines in an ex vivo model of human endotoxinaemia under hypoxaemic conditions

Aims In vivo application of the K ATP-channel blocker glibenclamide can reverse endotoxin-induced hypotension, vascular hyporeactivity and shock in experimental animals. The hypothesis of the present study is, that the drug effects might not only be based on direct inhibition of K ATP-channels of vascular smooth muscle cells, but might also reflect reduction of shock-induced excess proinflammatory cytokines and procoagulatory molecules produced in the blood monocytes. Main methods Human whole blood (normoxaemic or hypoxaemic) supplemented ex vivo with 100 ng/ml LPS was used to assess glibenclamide (3–100 μM) effects on IL-1beta, IL-6, TNF-alpha, tissue factor, and plasminogen-activator-inhibitor-2 (PAI-2). Co-incubations with monocytes and erythrocytes and cytosolic calcium measurements were performed to reveal their purinergic intercellular interaction. Key findings In heparinized blood, glibenclamide reduced LPS-induced release of IL-1beta and TNF-alpha, tissue factor and PAI-2 mRNA in a concentration-dependent manner. When samples were subjected to strong hypoxemia using 95% N 2/5% CO 2, these parameters became even more sensitive to the drug. No drug effect was observable in citrated blood or in isolated monocytes. IL-1beta mRNA inhibition by glibenclamide appeared to be dependent on P2X7-receptor activation of monocytes by ATP-releasing erythrocytes during hypoxia. Cytosolic calcium values as well as the duration of calcium transients elicited by P2X7-receptor stimulation in isolated monocytes were strongly increased during hypoxia, both of which could be abolished by glibenclamide. Significance We conclude that the anti-inflammatory effect of glibenclamide is mainly based on the reduction of calcium entry by drug-induced depolarization of hypoxic monocytes. Thus, glibenclamide possesses a potentially beneficial shock-specific anti-inflammatory action.

Size dependent induction of proinflammatory cytokines and cytotoxicity of particulate beta-tricalciumphosphate in vitro

Cellular responses to particulate calcium phosphate ceramics can lead to inflammatory reactions under certain conditions that depend on particle composition, size and morphology. In this context, the potential influence of varying sizes of particulate beta-tricalciumphosphate (beta-TCP) on the induction of inflammation and cytotoxicity remains to be determined. The present work investigates the effects of beta-TCP particles of five different sizes (1, 3, 13, 32 and 40 μm) on human peripheral blood mononuclear cells (PBMC) in vitro concerning the release of TNF-alpha, IL-1beta and IL-8 after six and 18 h of incubation (ELISA) as well as intracellular TNF-alpha, IFN-gamma, IL-1alpha, IL-1beta and IL-8 levels within distinct PBMC subpopulations after 12 h (FACS). Potential cytotoxic effects were determined by assaying lactate dehydrogenase (LDH) and morphological analyses (electron microscopy). Beta-TCP 1 μm did not induce any cytokine after 6 h but slightly increases TNF-alpha, IL-1beta and IL-8 release after 18 h. Larger particles (32 and 40 μm) consistently caused higher levels of cytokine release by increasing the fraction of cytokine producing monocytes. They also caused higher levels of LDH release as did smaller, phagocytosable particles. These data suggest a less inflammatory and cytotoxic profile of beta-TCP devices with a smaller primary particle size when compared to larger particles.

Modulation of lipopolysaccharide-induced pro-inflammatory mediators by an extract of Glycyrrhiza glabra and its phytoconstituents

To evaluate the inhibitory property of de-glycyrrhizinated extract of Glycyrrhiza glabra root and its phytoconstituents (glabridin, isoliquiritigenin and glycyrrhizin) on LPS-induced production of pro-inflammatory mediators. Inhibitory effect of G. glabra extract and its phytoconstituents were studied on lipopolysaccharide (LPS)-induced nitric oxide (NO), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) levels in J774A.1 murine macrophages. G. glabra and isoliquiritigenin significantly inhibited LPS stimulated NO, IL-1 beta and IL-6 production. Glabridin showed significant inhibition of NO and IL-1 beta release, but failed to attenuate IL-6 levels at the tested concentrations. In addition, glycyrrhizin did not exhibit inhibitory response towards any of the LPS-induced pro-inflammatory mediators at the tested concentrations. From the results we speculate that the inhibitory effect of G. glabra extract on LPS-induced pro-inflammatory mediators is influenced by glabridin and isoliquiritigenin and is not contributed by glycyrrhizin.

Realgar is active ingredient of Angong Niuhuang pill in protection against LPS-induced neuroinflammation

To determine the effects of Angong Niuhuang pill (AGNHW) on lipopolysaccharide (LPS)-induced neuroinflammation and further to investigate the role of realgar and cinnabar on AGNHW-mediated neuroprotection. Primary rat midbrain neuron-glia cultures were used as an in vitro model to examine the effects of AGNHW on LPS-induced dopamine (DA) neuronal damage. Cultures were divided randomly into five groups: control, LPS, LPS plus AGNHW, LPS plus realgar and LPS plus cinnabar. Dopaminergic neurotoxicity was measured by [3H] DA uptake assay. The production of intracellular reactive oxygen species (ROS) was quantified via the DCFH-DA probe. Real-time RT-PCR was applied to detect the mRNA expression of pro-inflammatory factors. Then the protein levels of these factors were determined by ELISA and western blot assay. Compared with the control group, LPS apparently decreased DA uptake capacity (P < 0.05); induced the production of intracellular ROS (P < 0.05); enhanced the mRNA expression of TNF-alpha, iNOS, IL-13 and COX-2 (P < 0.05) and the release of TNF-alpha, IL-1beta and PGE2 in the supernatant of cultures (P < 0.05); and also increased the level of iNOS protein (P < 0.05). Compared with the LPS group, AGNHW and realgar significantly inhibited LPS-induced reduction of DA uptake (P < 0.05); attenuated the production of intracellular ROS (P < 0.05) and the mRNA expression of TNF-alpha, iNOS, IL-1beta and COX-2 (P < 0.05) and the release of TNF-alpha, IL-1beta and PGE2 (P < 0.05) and the level of iNOS protein (P < 0.05). However, there was no significant difference between LPS group and LPS plus cinnabar group. AGNHW is effective in protecting against LPS-induced neuroinflammation, and realgar is one of active components for AGNHW to produce anti-inflammatory effects.

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X7-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X(7)-invalidated (KO) mice was tested. Low concentrations (1-100μM) of ATP transiently increased the intracellular concentration of calcium ([Ca(2+)](i)) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X(7) receptors. One millimolar ATP provoked a sustained increase in the [Ca(2+)](i) only in WT mice. The response to 10μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K(+)](i)) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X(4) receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K(+)](i) triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X(4) receptors. Higher concentrations of ATP elicited similar responses after binding to P2X(7) receptors. The expression of the P2X(7) receptors was also coupled to a better response to P2Y receptors.

Role of the NF-κB transcription pathway in the haemozoin- and 15-HETE-mediated activation of matrix metalloproteinase-9 in human adherent monocytes

Haemozoin (HZ, malarial pigment) is a crystalline ferriprotoporphyrin IX polymer derived from undigested host haemoglobin haem, present in late stages of Plasmodium falciparum-parasitized RBCs and in residual bodies shed after schizogony. It was shown previously that phagocytosed HZ or HZ-containing trophozoites increased monocyte matrix metalloproteinase-9 (MMP-9) activity and enhanced production of MMP-9-related cytokines TNF and IL-1beta. Here we show that in human monocytes the HZ/trophozoite phagocytosis effects and their recapitulation by 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid (15-HETE), a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem catalysis, were mediated via activation of NF-κB transcription pathway. After phagocytosis of HZ/trophozoites or treatment with 15-HETE, the NF-κB complex migrated to the nuclear fraction while the inhibitory cytosolic IκBalpha protein was phosphorylated and degraded. All HZ/trophozoite/15-HETE effects on MMP-9 activity and TNF/IL-1beta production were abrogated by quercetin, artemisinin and parthenolide, inhibitors of IκBalpha phosphorylation and subsequent degradation, NF-κB nuclear translocation, and NF-κB-p65 binding to DNA respectively. In conclusion, enhanced activation of MMP-9, and release of pro-inflammatory cytokines TNF and IL-1beta, a triad of effects involved in malaria pathogenesis, elicited in human monocytes by trophozoite and HZ phagocytosis and recapitulated by 15-HETE, appear to be causally connected to persisting activation of the NF-κB system.

Relevance of Epidermal Growth Factor to Improve Steatotic Liver Preservation in IGL-1 Solution

Aim Static preservation solution is critical for liver graft outcomes, especially when steatosis is present. Institut Georges Lopez (IGL)-1 solution protects fatty livers effectively against cold ischemia reperfusion injury. Its benefits are mediated by nitric oxide and prevention of oxidative stress. The supplementation of IGL-1 with epidermal growth factor (EGF) enhances steatotic graft preservation by increasing adenosine triphosphate content, thereby mitigating oxidative stress and mitochondrial damage. Methods After steatotic livers were preserved for 24 hours in IGL-1 solution with or without EGF supplements, they were perfused ex vivo for 2 hours at 37°C. The benefits of EGF were assessed by evidences of hepatic damage and function—transaminases, bile production, and flow rate—as well as by other factors presumably associated with the poor tolerance of fatty livers toward cold ischemia-reperfusion injury (IRI)—energy metabolism, mitochondrial damage, oxidative stress, eNOS activity and proinflammatory interleukin (IL) beta content. Results Steatotic livers preserved in IGL-1 solutions supplemented with EGF (10 μg/L) showed lower transaminase levels, greater bile production, and ameliorated flow rates when compared to IGL-1 alone. In addition, energy metabolism deterioration, mitochondrial damage, oxidative stress, and cytokine IL-1 beta release were prevented. Conclusion EGF addition to IGL-1 increased fatty liver graft preservation, thereby reducing steatotic liver damage against cold IRI.

Effect of Sulfated Modification on the Molecular Characteristics and Biological Activities of Polysaccharides fromHypsizigus marmoreus

The effect of sulfated modification on polysaccharides from Hypsizigus marmoreus was examined by determining their molecular structures and bioactivities. The sulfation, which was implemented by using an orthogonal array design, produced polysaccharides with varying degrees of substitution (DS) ranging from 0.11 to 1.06. The sulfated polysaccharides exhibited a lower average molecular weight (M(w)) and considerably higher radius of gyration (R(g)) than those of native polysaccharide, suggesting that the conformation of the sulfated polysaccharides had been changed towards a more extended type. The inhibitory activity toward cancer cell growth was enhanced by treating with the sulfated polysaccharides by up to 34%, as compared to the native polysaccharide. In addition, treating with the sulfated polysaccharides increased the nitric oxide (NO) and cytokine (IL-1beta and TNF-alpha) release to levels comparable to those detected in the positive control, lipopolysaccharide (LPS), suggesting that the sulfated polysaccharides might have strong immunomodulatory activity.

Regulation of interleukin‐1β by interferon‐γ is species specific, limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon-gamma (IFNgamma) on interleukin-1beta (IL-1beta) production are conflicting. We resolve this controversy by showing that IFNgamma potentiates IL-1beta release from human cells, but transiently inhibits the production of IL-1beta from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL-1beta and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNgamma and IFNbeta are anti-inflammatory. We observed that these cytokines suppress IL-1beta production in response to MTB, resulting in a reduced number of IL-17-producing cells. In human cells, IFNgamma increased IL-1beta production, and this might explain why IFNgamma is detrimental for multiple sclerosis. In mice, IFNgamma decreased IL-1beta and subsequently IL-17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.

Temporal Interleukin-1β Secretion from Primary Human Peripheral Blood Monocytes by P2X7-independent and P2X7-dependent Mechanisms

The processing and regulated secretion of IL-1β are critical points of control of the biological activity of this important pro-inflammatory cytokine. IL-1β is produced by both monocytes and macrophages, but the rate and mechanism of release differ according to the differentiation status and the origin of these cells. We aimed to study the control of processing and release in human blood monocytes and human monocyte-derived macrophages. Toll-like receptor (TLR)-induced IL-1β production and release were investigated for dependence upon caspase-1, P2X7 receptor activation, and loss of membrane asymmetry associated with microvesicle shedding. TLR agonists induced P2X7 receptor-dependent IL-1β release in both monocytes and macrophages; however, only monocytes also showed P2X7 receptor-independent release of mature IL-1β. Furthermore, in monocytes ATP-mediated PS exposure could be activated independently of IL-1β production. Release of IL-1β from monocytes showed selectivity for specific TLR agonists and was accelerated by P2X7 receptor activation. Human monocytes released more IL-1β/cell than macrophages. These data have important implications for inflammatory diseases that involve monocyte activation and IL-1 release.

Peroxisome-proliferator-activated receptors γ and peroxisome-proliferator-activated receptors β/δ and the regulation of interleukin 1 receptor antagonist expression by pioglitazone in ischaemic brain

The imbalance between the production and release of interleukin-1 (IL-1) ligands, IL-1alpha, IL-1beta and IL-1 receptor antagonist (IL-1ra) in ischaemic brain exaggerates inflammatory responses and contributes to neuronal death. Cerebral ischaemia also upregulates the peroxisome-proliferator-activated receptor (PPAR) gamma. We studied in rats the effects of the PPARgamma agonist, pioglitazone, on the regulation of IL-1beta, IL-1ra and IL-1 receptor I (IL-1RI) expression in ischaemic brain after occlusion of the middle cerebral artery for 90 min. Pioglitazone or vehicle was infused intracerebroventricularly over a 5-day period before, during and 24 or 48 h after middle cerebral artery occlusion. The expression of IL-1beta, IL-1ra and IL-1RI in the peri-infarct cortex was investigated by immunohistochemistry, Western blotting and immunofluorescence staining. The mechanisms of the IL-1ra regulation by pioglitazone and the neuroprotection under excitotoxic neuronal injury were studied in primary cortical neurones expressing PPARgamma and PPAR beta/delta. Cerebral ischaemia increased the expression of IL-1beta, IL-1RI and IL-1ra in the ischaemic cortex. Pioglitazone reduced IL-1beta, but upregulated IL-1ra and increased the number of IL-1ra immunoreactive cells. In primary cortical neurones, pioglitazone stimulated the IL-1ra production via activation of the PPARbeta/delta, but prevented excitotoxic neuronal injury and death by a PPARgamma-dependent mechanism. Our data demonstrate that activation of PPARgamma and PPAR beta/delta by proglitazone in neurones triggers diverse neuroprotective mechanisms. The restoration of the equilibrium between I1-1beta and IL-1ra in ischaemic brain tissue limits IL-1beta signalling, reduces inflammatory responses and is an important mechanism by which thiazolidinediones improve the recovery from ischaemic stroke.

Two adult siblings with atypical cryopyrin‐associated periodic syndrome due to a novel M299V mutation in NLRP3

The NALP3 inflammasome is a multiprotein complex that triggers caspase 1-mediated interleukin-1beta (IL-1beta) release. Mutations in the gene encoding NALP3 (NLRP3) underlie the cryopyrin-associated periodic syndrome (CAPS). The aim of this study was to report a novel NLRP3 mutation in 2 siblings of Swedish descent in whom symptoms first presented in adulthood. Mutation analysis of NLRP3 was performed on DNA from patients with CAPS and 100 control subjects. For assessment of caspase 1 and IL-1beta, blood was collected from patients and age- and sex-matched healthy control subjects. Genetic constructs containing mutant or wild-type NLRP3 were transduced into THP-1 cells, followed by assessment of IL-1beta levels in cell supernatant. Both siblings carried a novel M299V mutation in NLRP3, which was not present in the control population. The samples obtained from the patients displayed increased caspase 1 activity and elevated IL-1beta levels at basal conditions as compared with healthy control subjects. THP-1 cells expressing mutated M299V revealed almost 10-fold higher IL-1beta production compared with the wild-type construct. M299V is an activating mutation in NLRP3 resulting in elevated spontaneous caspase 1 activity and IL-1beta levels. The classic CAPS phenotype was lacking in these adult siblings. Whereas one sibling displayed a milder phenotype that has so far responded satisfactorily to oral nonsteroidal antiinflammatory drugs in combination with low-dose corticosteroids, the inflammatory symptoms in the sibling with the more severe case responded well to IL-1beta blockade. Understanding the pathogenic mechanism underlying such disorders can be helpful for the physician. Our study reinforces the importance of genetic testing and laboratory investigations in combination with careful phenotypic evaluation for the diagnosis of such patients.