ASC mRNA Expression Research Articles

Saikosaponin‑d alleviates carbon‑tetrachloride induced acute hepatocellular injury by inhibiting oxidative stress and NLRP3 inflammasome activation in the HL‑7702 cell line.

Saikosaponin‑d (SSd) the primary active component of triterpene saponin derived from Bupleurum falcatum L., possesses anti‑inflammatory and antioxidant properties. The present study aimed to examine the potential therapeutic effects of SSd on carbon tetrachloride (CCl4)‑induced acute hepatocellular injury in the HL‑7702 cell line and its underlying mechanisms. HL‑7702 cells were treated with SSd at different doses (0.5, 1 or 2µmol/l). Cell viability was determined using an MTT assay. Injury was assessed by the levels of serum alanine aminotransferase (ALT) and aspartate transaminase (AST). Oxidative stress was assessed using malondialdehyde (MDA) content and total‑superoxide dismutase (T‑SOD) activity. The expression of nucleotide‑binding domain, leucine‑rich‑containing family, pyrin domain‑containing‑3 (NLRP3), apoptosis‑associated speck‑like protein (ASC), caspase‑1 and high mobility group protein B1 (HMGB1) was assessed by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. Interleukin (IL)‑1β and IL‑18 were determined by RT‑qPCR and ELISA. SSd attenuated the inhibition of cell viability and the high AST and ALT levels induced by CCl4 in HL‑7702 cells. Oxidative stress was induced in HL‑7702 cells by CCl4, as demonstrated by the increase of MDA and the decrease of T‑SOD activity. These changes were reversed by SSd. SSd significantly downregulated the mRNA and protein expression of NLRP3, ASC, caspase‑1, IL‑1β, IL‑18 and HMGB1 induced by CCl4. In conclusion SSd alleviated CCl4‑induced acute hepatocellular injury, possibly by inhibiting oxidative stress and NLRP3 inflammasome activation in the HL‑7702 cell line.

Mesenchymal stromal cells inhibit NLRP3 inflammasome activation in a model of Coxsackievirus B3-induced inflammatory cardiomyopathy

Inflammation in myocarditis induces cardiac injury and triggers disease progression to heart failure. NLRP3 inflammasome activation is a newly identified amplifying step in the pathogenesis of myocarditis. We previously have demonstrated that mesenchymal stromal cells (MSC) are cardioprotective in Coxsackievirus B3 (CVB3)-induced myocarditis. In this study, MSC markedly inhibited left ventricular (LV) NOD2, NLRP3, ASC, caspase-1, IL-1β, and IL-18 mRNA expression in CVB3-infected mice. ASC protein expression, essential for NLRP3 inflammasome assembly, increased upon CVB3 infection and was abrogated in MSC-treated mice. Concomitantly, CVB3 infection in vitro induced NOD2 expression, NLRP3 inflammasome activation and IL-1β secretion in HL-1 cells, which was abolished after MSC supplementation. The inhibitory effect of MSC on NLRP3 inflammasome activity in HL-1 cells was partly mediated via secretion of the anti-oxidative protein stanniocalcin-1. Furthermore, MSC application in CVB3-infected mice reduced the percentage of NOD2-, ASC-, p10- and/or IL-1β-positive splenic macrophages, natural killer cells, and dendritic cells. The suppressive effect of MSC on inflammasome activation was associated with normalized expression of prominent regulators of myocardial contractility and fibrosis to levels comparable to control mice. In conclusion, MSC treatment in myocarditis could be a promising strategy limiting the adverse consequences of cardiac and systemic NLRP3 inflammasome activation.

Isoliquiritigenin Ameliorates Indomethacin-Induced Small Intestinal Damage by Inhibiting NOD-Like Receptor Family, Pyrin Domain-Containing 3 Inflammasome Activation

Activation of the NOD-Like Receptor Family, Pyrin Domain-Containing 3 (NLRP3) inflammasome, which consists of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1, triggers pro-caspase-1 cleavage promoting the processing of pro-interleukin (IL)-1β into mature IL-1β, which is critical for the development of non-steroidal anti-inflammatory drug (NSAID)-induced enteropathy. We investigated the effects of isoliquiritigenin, a flavonoid derived from the roots of Glycyrrhiza species, on NSAID-induced small intestinal damage and the inflammasome activation. To induce enteropathy, mice were administered indomethacin by gavage with or without isoliquiritigenin pretreatment. Some mice received an intraperitoneal injection of recombinant murine IL-1β in addition to isoliquiritigenin and indomethacin. Indomethacin induced small intestinal damage and increased protein levels of cleaved caspase-1 and mature IL-1β in the small intestine. Treatment with 7.5 and 75 mg/kg isoliquiritigenin inhibited indomethacin-induced small intestinal damage by 40 and 56%, respectively. Isoliquiritigenin also inhibited the indomethacin-induced increase in cleaved caspase-1 and mature IL-1β protein levels, whereas it did not affect the mRNA expression of NLRP3, ASC, caspase-1, and IL-1β. Protection against intestinal damage in isoliquiritigenin-treated mice was completely abolished with exogenous IL-1β. NLRP3^–/– and caspase-1^–/– mice exhibited resistance to intestinal damage, and isoliquiritigenin treatment failed to inhibit the damage in NLRP3^–/– and caspase-1^–/– mice. Isoliquiritigenin prevents NSAID-induced small intestinal damage by inhibiting NLRP3 inflammasome activation.

Anti-Inflammatory Action of Sitagliptin and Linagliptin in Doxorubicin Nephropathy

Background/Aims: Dipeptidyl peptidase-4 (DPP4) inhibitors are known to have a protective effect on diabetic kidney disease, possibly via reduction of oxidative stress and inflammation in the kidney. However, whether these potential mechanisms play a role in non-diabetic proteinuric kidney diseases is not clear. Methods: Two different animal experiments were carried out using sitagliptin and linagliptin for DPP4 inhibition. In each experiment, male Sprague-Dawley rats were uninephrectomized and randomly divided into vehicle-treated and doxorubicin-treated rats, with or without DPP4 inhibition. Administration of a DPP4 inhibitor was performed daily by oral gavage over six weeks. Results: A single intravenous injection of doxorubicin resulted in hypertension and remarkable proteinuria. Linagliptin, but not sitagliptin, lowered systolic blood pressure in rats with doxorubicin nephropathy. By contrast, sitagliptin ameliorated tubulointerstitial injury, inflammatory cell infiltration, and interstitial fibrosis in rat kidneys with doxorubicin nephropathy. Quantitative polymerase chain reaction analysis revealed that mRNA expression of NLRP3, caspase-1, ASC, and IL-1β was remarkably increased in rat kidneys with doxorubicin nephropathy, and that this upregulation of the major components of the NLRP3 inflammasome was effectively suppressed by treatment with either sitagliptin or linagliptin. Additionally, upregulation of IL-6 was reversed by linagliptin, but not by sitagliptin. On the other hand, sitagliptin, but not linagliptin, reversed the increase in mRNA expression of gp91^phox, p47^phox, and p67^phox in rat kidneys with doxorubicin nephropathy. Conclusion: NLRP3 inflammasome activation was shown in our rat model of doxorubicin nephropathy. DPP4 inhibitors can suppress the activity of NLRP3, with or without relieving NADPH oxidase 2-related oxidative stress.

The PYHIN Protein p205 Regulates the Inflammasome by Controlling Asc Expression.

Members of the IFN-inducible PYHIN protein family, such as absent in melanoma-2 and IFN-γ-inducible protein (IFI)16, bind dsDNA and form caspase-1-activating inflammasomes that are important in immunity to cytosolic bacteria, DNA viruses, or HIV. IFI16 has also been shown to regulate transcription of type I IFNs during HSV infection. The role of other members of the PYHIN protein family in the regulation of immune responses is much less clear. In this study, we identified an immune-regulatory function for a member of the murine PYHIN protein family, p205 (also called Ifi205). Examination of immune responses induced by dsDNA and other microbial ligands in bone marrow-derived macrophages lacking p205 revealed that inflammasome activation by dsDNA, as well as ligands that engage the NLRP3 inflammasome, was severely compromised in these cells. Further analysis revealed that p205-knockdown cells showed reduced expression of apoptosis-associated speck-like molecule containing CARD domain (Asc) at the protein and RNA levels. p205 knockdown resulted in reduced binding of actively transcribing RNA polymerase II to the endogenous Asc gene, resulting in decreased transcription and processing of Asc pre-mRNA. Deletion of p205 in B16 melanoma cells using CRISPR/Cas9 showed a similar loss of Asc expression. Ectopic expression of p205 induced expression of an Asc promoter-luciferase reporter gene. Together, these findings suggest that p205 controls expression of Asc mRNA to regulate inflammasome responses. These findings expand on our understanding of immune-regulatory roles for the PYHIN protein family.

Significance of the NLRP3 inflammasome expression in rats with acute lung injury induced by phosgene

Objective: To investigate changes of NLRP3 signal transduction pathway of acute lung injury induced by phosgene to analyze NLRP3-mediated IL-1β release inflammatory process in rats. Methods: Rats were randomly divided into two groups, 10 rats in the Air group that consists of the rats with air exposure, 10 rats in the Psg group that consists of the rats with phosgene exposure at 8.33 g/m(3) for 5 min. The specimens of serum, bronchoalveolar lavage fluid (BALF) and lung were collected after 6h. Morphological changes were observed by HE staining. The expression of NLRP3 in the lung of two groups was detected by immunohistochemistry. NLRP3、ASC and caspase-1 expression in the lung tissue was quantified by Western blot. Reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of NLRP3、ASC and caspase-1 mRNA in the lung tissue. The concentrations of IL-1β、IL-18 and IL-33 in the serum and BALF were measured by enzyme-linked immunosorbent assay. RT-PCR were used to detect the expression of IL-1β、IL-18 and IL-33 mRNA in the lung tissue. Results: We successfully replicated the model of phosgene-induced ALI in rats. Morphological of HE staining after phosgene exposure to 6 h observed inflammatory cell infiltration in lung tissue in Phosgene group. Immunohistochemical staining results showed that there were many NLRP3 positive cells in lung tissue in Phosgene group. The levels of NLRP3 and caspase-1 mRNA and protein expression in lung were significantly increased (P<0.05) in Phosgene group, but no significant change was observed in lung ASC mRNA and protein expression (P>0.05) . Compared with Air group, the serum, BALF and lung tissue of IL-1β、IL-18 and IL-33 mRNA and protein expression were significantly increased (P<0.01) in Phosgene group. Conclusion: NLRP3-mediated inflammatory response probably involved in the process of the phosgene, so it maybe one of the pathogenesis of acute lung injury.

Activation of the TXNIP/NLRP3 inflammasome pathway contributes to inflammation in diabetic retinopathy: a novel inhibitory effect of minocycline.

Chronic low-grade inflammation occurs in diabetic retinopathy (DR), but the underlying mechanism(s) remains (remain) unclear. NLRP3 inflammasome activation is involved in several other inflammatory diseases. Thus, we investigated the role of the NLRP3 inflammasome signaling pathway in the pathogenesis of DR. Diabetes was induced in rats by streptozotocin treatment for 8weeks. They were treated with NLRP3 shRNA or minocycline during the last 4weeks. High glucose-exposed human retinal microvascular endothelial cells (HRMECs) were co-incubated with antioxidants or subjected to TXNIP or NLRP3 shRNA interference. In high glucose-exposed HRMECs and retinas of diabetic rats, mRNA and protein expression of NLRP3, ASC, and proinflammatory cytokines were induced significantly by hyperglycemia. Upregulated interleukin (IL)-1β maturation, IL-18 secretion, and caspase-1 cleavage were also observed with increased cell apoptosis and retinal vascular permeability, compared with the control group. NLRP3 silencing blocked these effects in the rat model and HRMECs, confirming that inflammasome activation contributed to inflammation in DR. TXNIP expression was increased by reactive oxygen species (ROS) overproduction in animal and cell models, whereas antioxidant addition or TXNIP silencing blocked IL-1β and IL-18 secretion in high glucose-exposed HRMECs, indicating that the ROS-TXNIP pathway mediates NLRP3 inflammasome activation. Minocycline significantly downregulated ROS generation and reduced TXNIP expression, subsequently inhibited NLRP3 activation, and further decreased inflammatory factors, which were associated with a decrease in retinal vascular permeability and cell apoptosis. Together, our data suggest that the TXNIP/NLRP3 pathway is a potential therapeutic target for the treatment of DR, and the use of minocycline specifically for such therapy may be a new avenue of investigation in inflammatory disease.

Expression of dectin-1 and enhanced activation of NALP3 inflammasome are associated with resistance to paracoccidioidomycosis.

Dectin-1 is a pattern recognition receptor (PRR) that recognizes β-glucans and plays a major role in the immunity against fungal pathogens. Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis, has a sugar-rich cell wall mainly composed of mannans and glucans. To investigate the role of dectin-1 in the innate immunity of resistant (A/J) and susceptible (B10.A) mice to P. brasiliensis infection, we evaluated the role of curdlan (a dectin-1 agonist) and laminarin (a dectin-1 antagonist) in the activation of macrophages from both mouse strains. We verified that curdlan has a negligible role in the activation of B10.A macrophages but enhances the phagocytic and fungicidal abilities of A/J macrophages. Curdlan up-regulated the expression of costimulatory molecules and PRRs in A/J macrophages that express elevated levels of dectin-1, but not in B10.A cells. In addition, curdlan treatment inhibited arginase-1 and enhanced NO-synthase mRNA expression in infected A/J macrophages but had not effect in B10.A cells. In contrast, laminarin reinforced the respective M2/M1 profiles of infected A/J and B10.A macrophages. Following curdlan treatment, A/J macrophages showed significantly higher Syk kinase phosphorylation and expression of intracellular pro-IL-1β than B10.A cells. These findings led us to investigate if the NRLP3 inflammasome was differently activated in A/J and B10.A cells. Indeed, compared with B10.A cells A/J macrophages showed an increased expression of NALP3, ASC, and IL-1β mRNA. They also showed elevated caspase-1 activity and secreted high levels of mature IL-β and IL-18 after curdlan treatment and P. brasiliensis infection. Our data demonstrate that soluble and particulate β-glucans exert opposed modulatory activities on macrophages of diverse genetic patterns. Moreover, the synergistic action of dectin-1 and NALP3 inflammasome were for the first time associated with the innate response of resistant hosts to P. brasiliensis infection.

Abstract 428: DNA Methylation of ASC is Associated with Decreased ASC and IL-1β Expression in Heart Failure

Introduction: Heart failure (HF) is associated with formation and activation of inflammasome, a complex of intracellular interaction proteins that trigger maturation of inflammatory cytokines to initiate inflammatory response. ASC, a vital component of the inflammasome, is controlled through epigenetic modification via methylation of CpG islands surrounding exon 1. Methods: To assess the relationships between DNA methylation of ASC, ASC expression, and inflammatory cytokines IL-1β and IL-18 in HF, stored samples from 155 chronic HF patients (age 56.9±12.0 yr, 64% male, 47% black, and ejection fraction 29.9±14.9) were analyzed. DNA extracted from PMBCs were analyzed by pyrosequencing for percent methylation of seven CpG sites in the intron region preceding exon 1 of the ASC gene. ASC mRNA was quantified via real-time PCR and analyzed as the ratio ASC:GAPDH. Serum ASC, IL-1β, and IL-18 were measured by ELISA. Results: Higher ASC methylation was associated with lower ASC mRNA (r=0-.328, p&lt;0.001) and protein (r=-.464, p&lt;0.001) expression. Lower ASC mRNA expression was associated with lower ASC protein expression (r=0.494, p&lt;0.001). Decreased IL-1β expression was associated with higher ASC methylation (r=-.424, p=0.005) and lower ASC mRNA (r=.619, p&lt;0.001) and ASC protein (r=.433, p&lt;0.001). IL-18 expression was not significantly associated with ASC methylation or expression. Conclusions: Increased ASC methylation was associated with lower IL-1β, likely via decreased ASC gene expression. As ASC is required for inflammasome activation of IL-1β, this study implicates the inflammasome pathway as a driver of inflammation in HF, proving a potential target for novel interventions.

Thymoquinone ameliorates NLRP3-mediated inflammation in the pancreas of albino Wistar rats fed ethanol and high-fat diet.

Inflammasomes are protein complexes that mediate the process of inflammation and tissue injury by regulating the level of cytokine production. Pancreatitis is a major gastrointestinal disorder characterized by painful inflammation in the pancreas. The aim of this study was to evaluate whether thymoquinone (TQ) exerts anti-inflammatory activity by influencing the expression of the apoptosis-associated speck-like protein (ASC) complex of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasomes in rats subjected to experimental pancreatitis. Male albino Wistar rats were randomly separated into four groups. Rats in groups 1 and 2 were fed with a normal diet for 90 days, and rats in groups 3 and 4 were administered with ethanol (EtOH) 8-12 g/kg/day orally and fed with a high-fat diet (HFD) for 90 days. In addition, rats in groups 2 and 4 were administered with 100 mg/kg body weight of TQ from the 31st day. The serum lipase (L)/amylase (A) ratio; the oxidative stress markers; the GSH/GSSG ratio; the mRNA expression of ASC, caspase-1, IL-1β, IL-18, and TNF-α; and the protein expression of ASC and caspase-1 in the pancreas were assessed. We observed a significant increase in the serum L/A ratio and oxidative stress, a decrease in the GSH/GSSG ratio, and a GST activity in EtOH- and HFD-fed rats. The mRNA expression of IL-1β, IL-18, and TNF-α was significantly reduced in TQ-coadministered rats than that in EtOH- and HFD-fed rats. The upregulation of mRNA and the protein expression of ASC and caspase-1 were significantly reduced in TQ-coadministered rats. TQ exerts the anti-inflammatory activity probably by downregulating the ASC expression to minimize the maturation of proinflammatory cytokines.

Expression of Nucleotide-oligomerization Domain (NOD) and Related Genes in Mouse Tissues Infected with Mycobacterium leprae.

The nucleotide-oligomerization domain (NOD) is an important molecule involved in host defense against bacterial infection. To study the role of NODs in the host response to Mycobacterium leprae, we measured the mRNA levels of NODs and related genes in infected mouse tissues. The mRNA expression of NOD1, NOD2, caspase-1 and ASC was increased in mouse footpads. Whereas NOD2 expression in macrophages was increased at 2 and 24 h post-infection with M. leprae, there was no expression of NOD1 at these time points. An increase in caspase-1 expression was observed at 2 h and continued at 24 h. However, the expression of ASC was increased only at the early time point. The expression of caspase-1 is regulated by NOD2-dependent pathway in established HEK 293. Our results suggest NOD2, rather than NOD1, may be associated with the host response to M. leprae and that caspase-1 activation is essential for the host response.

NLRP3 inflammasome activation is essential for paraquat-induced acute lung injury.

The innate immune response is important in paraquat-induced acute lung injury, but the exact pathways involved are not elucidated. The objectives of this study were to determine the specific role of the NLRP3 inflammasome in the process. Acute lung injury was induced by administering paraquat (PQ) intraperitoneally. NLRP3 inflammasome including NLRP3, ASC, and caspase-1 mRNA and protein expression in lung tissue and IL-1β and IL-18 levels in BALF were detected at 4, 8, 24, and 72 h after PQ administration in rats. Moreover, rats were pretreated with 10, 30, and 50 mg/kg NLRP3 inflammasome blocker glybenclamide, respectively, 1 h before PQ exposure. At 72 h after PQ administration, lung histopathology changes, NLRP3, ASC, and caspase-1 protein expression, as well as secretion of cytokines including IL-1β and IL-18 in BALF were investigated. The NLRP3 inflammasome including NLRP3, ASC, caspase-1 expression, and cytokines IL-1β and IL-18 levels in PQ poisoning rats were significantly higher than that in the control group. NLRP3 inflammasome blocker glybenclamide pretreatment attenuated lung edema, inhibited the NLRP3, ASC, and caspase-1 activation, and reduced IL-1β and IL-18 levels in BALF. In the in vitro experiments, IL-1β and IL-18 secreted from RAW264.7 mouse macrophages treated with paraquat were attenuated by glybenclamide. In conclusion, paraquat can induce IL-1β/IL-18 secretion via NLRP3-ASC-caspase-1 pathway, and the NLRP3 inflammasome is essential for paraquat-induced acute lung injury.

PS-336 Influence Of N-acetylcysteine Amide (naca) On The Inflammasome Pathway. A Study On Neonatal Pigs

Background and aims Severe perinatal hypoxia contributes to approximately 6% of spastic cerebral palsy (CP). Studies have indicated an association between elevation of IL-1beta after perinatal asphyxia and the development of CP. The NLRP3 Inflammasome complex may lead to release of the cytokines IL-1beta and IL-18 and cell death. Reactive oxygen species (ROS) have been proposed to be an upstream inducer of this complex and the anti-oxidant N-Acetylcysteine amide (NACA) may provide organ protection after hypoxia. Objectives To map inflammasome activation in specific brain regions of the pig after neonatal hypoxia-reoxygenation and to investigate if the expression of different proteins in this pathway are modulated by NACA. Study design ([Table 1][1]). ELISA was used to measure IL-1b protein in cerebral cortex and Realtime PCR for mRNA expression of NLRP3, ASC, IL-1b and Il18 in cortex, cerebellum, hippocampus and striatum. ![Abstract PS-336 Table 1][2] Abstract PS-336 Table 1 Study design: Fifty-four newborn piglets, age 1236h, were included. Invasive blood pressure, EEG and ECG were measured continuously. One control group (n = 6) and 4 experimental groups (n = 12), exposed to global hypoxia, until BE was either -15 or -20 mmol/l (moderate/severe asphyxia with or without NACA) The pigs were observed for 9.5 h Results After severe hypoxia the protein expression of IL-1b in cerebral cortex was reduced for the NACA treated pigs vs. saline, p < 0.05. When comparing all the pigs treated with NACA vs. saline after hypoxia Fold Change of ASC in cortex was significantly reduced, p ([Table 2][3] ). ![Abstract PS-336 Table 2][2] Abstract PS-336 Table 2 Fold change for NLRP3, ASC, IL-18 and IL-1b measured in cerebral cortex. Significant difference in Fold Change of ASC (*), p < 0.05. All values are Median values In hippocampus, cortex and Striatum Fold Change of IL-1b was elevated in all the hypoxia groups compared with the control group, p Conclusion NACA reduces the protein expression of Il-1beta and mRNA-expression of ASC in cortex after hypoxia. This may indicate that NACA has some neuroprotective abilities after perinatal asphyxia. Upcoming analyses of histopathology and injury markers will elucidate possible neuroprotective effects of NACA treatment following birth asphyxia. [1]: #F1 [2]: pending:yes [3]: #F2

Role of NLRP3 and NLRP1 inflammasomes signaling pathways in pathogenesis of rheumatoid arthritis

ObjectivesTo investigate the role of NLRP3 and NLRP1 inflammasomes signaling pathways in rheumatoid arthritis (RA). MethodsA total of 36 patients with RA were selected, peripheral blood mononuclear cell (PBMC) and granulocyte were separated from venous blood. RT-qPCR method was used to detect the expression level and diversity of NLRP3 and NLRP1 in PBMC and granulocyte mRNA in patients with RA, and detect the mRNA expression of downstream factor IL-1α. The correlation between RA and the expression of NLRP3 and NLRP1 was analyzed. Normal 30 cases were set as control group. ResultsExpression levels of NLRP1, and caspase-1 mRNA in PBMC of RA group were significantly lower than those of control group (P<0.05), while there was no significant difference in expression levels of NLRP3, ASC, IL-1α mRNA between these two groups (P>0.05); NLRP3, caspase-1, and ASC mRNA expression in granulocyte of RA patients were significantly lower than those in control group (P<0.05). There was no correlation between rheumatoid factor and expression levels of NLRP3, ASC, caspase-1 mRNA in RA group (P>0.05); NLRP1, IL-1α mRNA expression level had a negative correlation with anti-rheumatoid factor antibody (P=0.033 2, 0.034 0). ConclusionsNLRP3 and NLRP1 inflammasomes signaling pathways are involved in RA inflammatory reaction process as protective factors, and play an important role in RA inflammatory mechanisms.

Nigella sativa Linn. seed extract modulates the activity of ASC complex of NLRP3 inflammasome in rats subjected to experimental pancreatitis

NLRP3 inflammasome, a multi-protein complex containing ASC as a linker protein influences the process of inflammation and tissue injury in pancreas. In this study, the effect of methanolic seed extract of Nigella sativa (MENS) on the expression of ASC protein of NLRP3 inflammasome was investigated in rats subjected to experimental pancreatitis. Male albino Wistar rats were divided into 4 groups. Group 1 and 2 rats were fed with normal diet; group 3 and 4 rats were administered with ethanol (EtOH) and fed high fat diet (HFD) for 90 days. In addition, group 2 and 4 rats were administered orally with 200mg/kg body weight of MENS for the last 60 days. We measured serum lipase and amylase activities, oxidative stress markers and inflammatory markers. The mRNA expression of caspase-1, ASC, pro-inflammatory cytokines, IL-1β, IL-18, TNF-α and protein expression of caspase-1 and ASC were determined in pancreas. Our study indicated that MENS co-administration significantly decreased the level of serum lipase and amylase activities, oxidative stress markers and inflammatory markers in EtOH and HFD fed rats. mRNA and protein level expression of caspase-1 and ASC were found to be downregulated in MENS co-administered rats. Spearman's rank correlation test showed that ASC mRNA expression has significant positive correlation with the serum levels of caspase-1 (rs=0.885, P<0.01), IL-1β (rs=0.828, P<0.05) and IL-18 (rs=0.943, P=0.01) in MENS co-administered rats. The present study showed that MENS exhibits anti-inflammatory activity probably by downregulating the expression of ASC protein of NLRP3 inflammasome in pancreas to minimize the activation of caspase-1.

The PYHIN family member IFI205 regulates immune signaling via transcriptional regulation of the inflammasome adapter ASC (P1280)

Abstract Members of the PYHIN protein family such as AIM2 and IFI16, function as DNA sensors in innate immunity. Here we investigated the role of Ifi205, a murine PYHIN family member in innate immunity by generating murine macrophages lacking Ifi205 and monitoring immune signaling in these cells. Macrophages lacking Ifi205 had abrogated caspase-1 dependent processing and production of the pro-inflammatory cytokine IL-1β in response to cytosolic double stranded DNA and Nigericin, that activate the AIM2 and NLRP3 inflammasomes, respectively. Surprisingly, expression of the inflammasome adapter ASC was completely abolished at both the protein and mRNA levels in all Ifi205 knockdown cells generated. Reconstitution of the knockdown cells with ASC rescued IL-1β production attributing the knockdown phenotype to the loss of ASC expression. We observed a similar defect in ASC expression in cell lines lacking Ifi205 by targeting the 3’-UTR of the ifi205 gene. Reconstitution of these cells with Ifi205 rescued ASC mRNA expression. We also found that ectopic expression of Ifi205 augmented the activation of an ASC promoter-luciferase reporter gene in a dose-dependent manner in HEK 293T cells. The data suggest that ASC expression might be controlled at the transcriptional level. Thus, Ifi205 regulates inflammasome activation by controlling transcriptional regulation of ASC.

Apoptosis-associated speck-like protein containing a CARD (ASC) expression profiles in familial Mediterranean fever (FMF) patients with different MEFV mutation patterns

Objectives: The inflammasome complex and the inflammatory pathway have been implicated in the pathogenesis of the most common autoinflammatory disorder, familial Mediterranean fever (FMF). Pyrin, the protein product of the FMF gene MEFV, interacts with the inflammasome complex adaptor protein ASC/PYCARD (apoptosis-associated speck-like protein with a CARD). Pyrin and ASC can both function as either inducers or suppressors of the cellular inflammatory response. We aimed to characterize ASC-induced gene expression profiles in FMF patients with different MEFV mutation patterns.Methods: A total of 165 Caucasian patients with clinical and molecular FMF diagnoses were enrolled in the study. ASC gene expression was quantified by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).Results: ASC mRNA expression was increased in the MEFV mutation-positive group compared to the mutation-negative group (p = 0.001). The fold changes of ASC expression in the M694V homozygous (p = 0.02), M694V heterozygous (p = 0.012), compound heterozygous (p = 0.002), and R202Q/P369S/R408Q (p = 0.00) groups relative to the MEFV mutation-negative group were +2.4, +2.7, +3, and +3.4, respectively. qRT-PCR did not reveal a significant difference in ASC mRNA expression levels among the MEFV mutation-positive groups (p > 0.05).Conclusion: ASC mRNA expression was up-regulated in patients carrying MEFV mutations independent of mutation type. There was no significant relationship between specific MEFV genotypes and the level of ASC expression in the patient group analysed. Thus, the findings of this work may suggest a crucial relationship between mutant MEFV/pyrin and remarkable ASC up-regulation in FMF inflammation.

The NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation

BackgroundPrion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal disease-associated prion protein, PrPSc. In prion-infected brains, activated microglia are often present in the vicinity of PrPSc aggregates, and microglial activation is thought to play a key role in the pathogenesis of prion diseases. Although interleukin (IL)-1β release by prion-induced microglia has been widely reported, the mechanism by which primed microglia become activated and secrete IL-1β in prion diseases has not yet been elucidated. In this study, we investigated the role of the NACHT, LRR and PYD domains-containing protein (NALP)3 inflammasome in IL-1β release from lipopolysaccharide (LPS)-primed microglia after exposure to a synthetic neurotoxic prion fragment (PrP106-126).MethodsThe inflammasome components NALP3 and apoptosis-associated speck-like protein (ASC) were knocked down by gene silencing. IL-1β production was assessed using ELISA. The mRNA expression of NALP3, ASC, and pro-inflammatory factors was measured by quantitative PCR. Western blot analysis was used to detect the protein level of NALP3, ASC, caspase-1 and nuclear factor-κB.ResultsWe found that that PrP106-126-induced IL-1β release depends on NALP3 inflammasome activation, that inflammasome activation is required for the synthesis of pro-inflammatory and chemotactic factors by PrP106-126-activated microglia, that inhibition of NF-κB activation abrogated PrP106-126-induced NALP3 upregulation, and that potassium efflux and production of reactive oxygen species were implicated in PrP106-126-induced NALP3 inflammasome activation in microglia.ConclusionsWe conclude that the NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation. To our knowledge, this is the first time that strong evidence for the involvement of NALP3 inflammasome in prion-associated inflammation has been found.

Activation of Inflammasomes as a Triggering Mechanism of Glomerular Injury in Mice on the High Fat Diet

Inflammasome, an intracellular inflammatory machinery, is a multiprotein complex consisting of PYCARD, NALP, ASC and caspase 5 or caspase-1, which functions to switch on inflammatory process. The present study hypothesizedthat the formation and activation of inflammasomes are an important initiating mechanismresulting in obesity-associated glomerular injury. To test this hypothesis, C57BL/6J WT mice were fed control or high fat diet (HFD) for 0, 1, 3 and 5 weeks. HFD significantly increased the body weight, unfasted blood glucose levels and plasma visfatin concentration at week 3 and 5. RT-PCR showed that HFD increased the mRNA expression of NALP3, ASC and caspase-1 compared to control mice. Confocal fluorescent microscopy showed the presence of ASC, NALP3 and caspase-1 molecules in the glomeruli, but they were not colocalized in control mice. In the HFD mice, however, colocalization of NALP3, ASC and Caspase-1 was largely increased at week 3 and 5, accompanied by enhanced caspase-1 activity (by 1.3 fold), indicating the assembling and activation of NALP3inflammasomes or the inflammatory machinery. HFD treatment for 5 weeks further enhanced activation of inflammasomes and led to significant increases in the urinary total protein excretion, glomerular damage index, desmin expression and renal IL-1β production compared to control mice. In in vitro studies of podocytes, visfatin treatment increased the colocalization of NALP3 and ASC or Caspase 1 and it significantly enhanced the caspase activity, desmin expression, and IL-1β production. These effects of visfatin were substantially attenuated by prior treatment of ASC siRNA or WEHD, a caspase-1 inhibitor. We conclude thatthe formation and activation of inflammasomes in podocytes by adipokines such as visfatin may be importantly implicated in the development of obesity-associated glomerular injury (supported by NIH grants HL-091464, HL-75316 and DK54927).

Airborne Asian sand dust enhances murine lung eosinophilia

There is no experimental study demonstrating the effects of airborne Asian sand dust (AASD) on allergic lung eosinophilia. The organic substances adsorbed onto AASD collected from the atmosphere of Iki-island in Japan were excluded by heat treatment at 360°C for 30 min. The effects of AASD or heated-AASD (H-AASD) towards allergic lung inflammation were compared in murine lungs to investigate the role of organic substances. ICR mice were administrated with the two kinds of AASD and/or ovalbumin (OVA) intratracheally four times at 2-week intervals. AASD and H-AASD enhanced eosinophil recruitment induced by OVA in the alveoli and in the submucosa of the airway, which has a goblet cell proliferation in the bronchial epithelium. AASD and H-AASD synergistically increased Th2 cytokines—interleukin-13 (IL-13), eosinophil-relevant cytokine and chemokine, such as IL-5, and monocyte chemotactic protein-3 (MCP-3) induced by OVA in whole lung lavage fluid. The enhancing effects were much greater in AASD than in H-AASD. AASD induced adjuvant effects on OVA-specific immunoglobulin E (IgE) and IgG1 production. In an in vitro study using RAW264.7 cells, AASD increased the expression of Toll-like receptors 2 (TLR2) mRNA, but not TLR4 mRNA. AASD increased mRNA expression of NALP3, ASC, and IL-1β compared with the control. H-AASD caused no expression of either mRNA. These results suggest that the aggravated lung eosinophilia in AASD is due to activation of a Th2-associated immune response and that the activation of TLR2 and NALP3 inflammasome by microbial materials could be participating in this phenomenon.