Inhibiting Inflammasome Activation Research Articles

Dichotomy between Receptor-Interacting Protein 1– and Receptor-Interacting Protein 3–Mediated Necroptosis in Experimental Pancreatitis

Pancreatic acinar cell necrosis and inflammatory responses are two key pathologic processes in acute pancreatitis (AP), which determines the severity and outcome of the disease. Recent studies suggest that necroptosis, a programed form of necrosis, is involved in the pathogenesis of AP, but the underlying mechanisms remain unknown. We investigated the expression of necrosome components, including receptor-interacting protein (RIP) 1, RIP3, and mixed lineage kinase domain-like (MLKL), and the molecular mechanisms in pancreatitis-associated necroptosis. We found that RIP3 and phosphorylated MLKL expression was positively related to the degree of necrosis, whereas RIP1 expression was negatively related to the degree of necrosis. Pharmacologic inhibition of RIP1 kinase activity exerted no protection against caerulein/cholecystokinin-8-induced AP, but knockdown of RIP1 with siRNA increased acinar cell necrosis and inhibition of NF-κB activation. RIP1 inhibition led to enhanced RIP3 expression. RIP3 and MLKL inhibition decreased acinar cell necrosis, in which the inhibition of RIP3 reduced the phosphorylation level of MLKL. RIP3 inhibition had no effect on trypsinogen activation but partly inhibited inflammasome activation. Our study strongly suggests that the imbalance between RIP1and RIP3 shifts the cell death to necrosis, which unravels a new molecular pathogenesis of mechanism of AP and may provide insight into the development of novel therapeutic agent for other necrosis-related diseases.

Interleukin-1beta may act on hepatocytes to boost plasma homocysteine – The increased cardiovascular risk associated with elevated homocysteine may be mediated by this cytokine

The results of multi-center trials of B vitamin supplementation reveal that, whereas moderately elevated homocysteine predicts increased risk for coronary disease, it does not play a mediating role in this regard. This essay proposes that interleukin-1beta can act on hepatocytes to suppress expression of the hepatocyte-specific forms of methionine adenosyltransferase; this in turn can be expected to decrease hepatic activity of cystathionine-β-synthase, leading to an increase in plasma homocysteine. It is further proposed that interleukin-1beta (IL-1β) is a true mediating risk factor for cardiovascular disease, and that elevated homocysteine predicts coronary disease because it can serve as a marker for increased IL-1β activity. Potent statin therapy may decrease IL-1β production by suppressing inflammasome activation – thereby accounting for the marked protection from cardiovascular events observed in the classic JUPITER study, in which the enrolled subjects had low-normal Low Density Lipoprotein cholesterol but elevated C-reactive protein.

Inflammasome activation and innate immunity in Alzheimer's disease.

Activation of innate immunity and the assembly of microglial cells at sites of Alzheimer disease pathology has long been regarded as bystander phenomenon, which does not actively contribute to disease pathogenesis and progression. Recent data emerging from genetics, clinical imaging and animal experimentation point to an intimate and mutual interaction of innate immune mechanisms and neurodegenerative processes. NOD-like receptor (NLR) family, pyrin domain containing 3 and 1 inflammasomes, present in myeloid cells and neurons, respectively, represent key components of the innate immune reaction observed in Alzheimer patient brains. Inhibition of inflammasome activation just begins to prove beneficial and protective from cognitive deficits and neuronal death in cell culture and animal models of Alzheimer's disease, thereby opening a new avenue for therapeutic intervention.

Quercetin Inhibits Inflammasome Activation by Interfering with ASC Oligomerization and Prevents Interleukin-1 Mediated Mouse Vasculitis

Interleukin-1β (IL-1β) is a highly inflammatory cytokine that significantly contributes to both acute and chronic inflammatory diseases. The secretion of IL-1β requires a unique protease, caspase-1, which is activated by various protein platforms called inflammasomes. Data suggests a key role for mitochondrial reactive oxygen species for inflammasome activation. Flavonoids constitute a group of naturally occurring polyphenolic molecules with many biological activities, including antioxidant effects. In this study, we investigated the effect of three flavonoids, quercetin (QUC), naringenin, and silymarim on inflammasome activation. We found that QUC inhibits IL-1β secretion by both the NLRP3 and AIM2 inflammasome in a dose dependent manner, but not the NLRC4 inflammasome. QUC inhibition of the inflammasome was still observed in Atg16l1 knockout macrophages, indicating that QUC’s effect was autophagy independent. Since QUC inhibited both NLRP3 and AIM2 inflammasomes but not NLRC4, we assessed ASC speck formation. QUC reduced ASC speck formation and ASC oligomerization compared with controls. Additionally, QUC inhibited IL-1β in Cryopyrin-Associated Periodic Syndromes (CAPS) macrophages, where NLRP3 inflammasome is constitutively activated. In conclusion, QUC inhibits both the NLRP3 and AIM2 inflammasome by preventing ASC oligomerization and may be a potential therapeutic candidate for Kawasaki disease vasculitis and other IL-1 mediated inflammatory diseases.

Off-Target Anti-Inflammatory Activity of the P2X7 Receptor Antagonist AZ11645373

We have found that a well-characterized P2X7 receptor antagonist AZ11645373 blocked production of pro-inflammatory chemokine IL-8 in endothelial cells treated with OxPAPC. The effect was not due to toxicity of AZ11645373 as documented by cellular metabolic activity assay. The mechanism of inhibition by AZ11645373 was apparently independent of the P2X7 receptor because this receptor was not involved in induction of IL-8 under our experimental conditions. In support of this notion, two P2X7 agonists ATP and BzATP did not upregulate IL-8. On the other hand, a chemically different P2X7 receptor antagonist A740003 did not inhibit OxPAPC-induced production of IL-8. The inhibitory action of AZ11645373 was observed at the level of IL-8 protein and messenger RNA (mRNA) induction. Furthermore, AZ11645373 inhibited induction of mRNA encoding for COX-2 (PTGS2) suggesting that its anti-inflammatory potential is not limited to suppression of IL-8 production. In addition to inhibiting stimulation by OxPAPC, AZ11645373 suppressed induction of IL-8 by TNFα and LPS. To summarize, AZ11645373 inhibits in a P2X7-independent manner action of chemically different inflammatory agonists such as OxPLs, LPS, and TNFα. Thus, AZ11645373 may be especially effective for treatment of inflammatory disorders due to a beneficial combination of P2X7 receptor-dependent effects (inhibition of inflammasome activation, antinociceptive effects) with P2X7-independent general anti-inflammatory action described in this paper.

NLRP3 Inflammasome Activation-Mediated Pyroptosis Aggravates Myocardial Ischemia/Reperfusion Injury in Diabetic Rats.

The reactive oxygen species- (ROS-) induced nod-like receptor protein-3 (NLRP3) inflammasome triggers sterile inflammatory responses and pyroptosis, which is a proinflammatory form of programmed cell death initiated by the activation of inflammatory caspases. NLRP3 inflammasome activation plays an important role in myocardial ischemia/reperfusion (MI/R) injury. Our present study investigated whether diabetes aggravated MI/R injury through NLRP3 inflammasome-mediated pyroptosis. Type 1 diabetic rat model was established by intraperitoneal injection of streptozotocin (60 mg/kg). MI/R was induced by ligating the left anterior descending artery (LAD) for 30 minutes followed by 2 h reperfusion. H9C2 cardiomyocytes were exposed to high glucose (HG, 30 mM) conditions and hypoxia/reoxygenation (H/R) stimulation. The myocardial infarct size, CK-MB, and LDH release in the diabetic rats subjected to MI/R were significantly higher than those in the nondiabetic rats, accompanied with increased NLRP3 inflammasome activation and increased pyroptosis. Inhibition of inflammasome activation with BAY11-7082 significantly decreased the MI/R injury. In vitro studies showed similar effects, as BAY11-7082 or the ROS scavenger N-acetylcysteine, attenuated HG and H/R-induced H9C2 cell injury. In conclusion, hyperglycaemia-induced NLRP3 inflammasome activation may be a ROS-dependent process in pyroptotic cell death, and NLRP3 inflammasome-induced pyroptosis aggravates MI/R injury in diabetic rats.

Pathogen-Mediated Inhibition of Anorexia Promotes Host Survival and Transmission

Sickness-induced anorexia is a conserved behavior induced during infections. Here, we report that an intestinal pathogen, Salmonella Typhimurium, inhibits anorexia by manipulating the gut-brain axis. Inhibition of inflammasome activation by the S. Typhimurium effector, SlrP, prevented anorexia caused by IL-1β-mediated signaling to the hypothalamus via the vagus nerve. Rather than compromising host defenses, pathogen-mediated inhibition of anorexia increased host survival. SlrP-mediated inhibition of anorexia prevented invasion and systemic infection by wild-type S. Typhimurium, reducing virulence while increasing transmission to new hosts, suggesting that there are trade-offs between transmission and virulence. These results clarify the complex and contextual role of anorexia in host-pathogen interactions and suggest that microbes have evolved mechanisms to modulate sickness-induced behaviors to promote health of their host and their transmission at the expense of virulence.

Inhibition of Rac1 Signaling Downregulates Inflammasome Activation and Attenuates Lung Injury in Neonatal Rats Exposed to Hyperoxia

Background: Inflammatory injury, particularly the production of active interleukin (IL)-1β plays a major role in the pathogenesis of bronchopulmonary dysplasia (BPD) in preterm infants. The release of active IL-1β is controlled by posttranscriptional modifications of its proform (pro-IL-1β) through the inflammasome. Rac1 is a member of the Rho family of GTPases that regulate the inflammatory process. Objective: This study tested the hypothesis that Rac1 signaling increases inflammasome activation that results in damaging inflammation, and that the inhibition of Rac1 signaling prevents lung injury, by inhibiting inflammasome activation in a newborn rat model of BPD induced by hyperoxia. Methods: Newborn rat pups were exposed to room air or hyperoxia (85% O₂) and received daily intraperitoneal injections of placebo (normal saline) or NSC23766, a specific Rac1 inhibitor, for 10 days. The effects on lung inflammation, alveolarization, vascular development, vascular remodeling, right ventricular systolic pressure, and right ventricular hypertrophy (RVH) were then assessed. Results: Hyperoxia exposure upregulated Rac1 and increased the production of active IL-1β, which was accompanied by increasing expression of the inflammasome. In addition, hyperoxia induced the pathological hallmarks of BPD. However, treatment with NSC23766 significantly decreased inflammasome activation and macrophage infiltration, improved alveolar and vascular development, and reduced pulmonary vascular remodeling and RVH. Conclusion: These results indicate that Rac1 signaling regulates the expression of the inflammasome and plays a pivotal role in the pathogenesis of hyperoxia-induced neonatal lung injury. Therefore, targeting Rac1 signaling may provide a novel strategy to prevent and treat BPD in preterm infants.

Inhibiting the NLRP3 inflammasome with MCC950 promotes non-phlogistic clearance of amyloid-β and cognitive function in APP/PS1 mice

Activation of the inflammasome is implicated in the pathogenesis of an increasing number of inflammatory diseases, including Alzheimer’s disease (AD). Research reporting inflammatory changes in post mortem brain tissue of individuals with AD and GWAS data have convincingly demonstrated that neuroinflammation is likely to be a key driver of the disease. This, together with the evidence that genetic variants in the NLRP3 gene impact on the risk of developing late-onset AD, indicates that targetting inflammation offers a therapeutic opportunity. Here, we examined the effect of the small molecule inhibitor of the NLRP3 inflammasome, MCC950, on microglia in vitro and in vivo. The findings indicate that MCC950 inhibited LPS+Aβ-induced caspase 1 activation in microglia and this was accompanied by IL-1β release, without inducing pyroptosis. We demonstrate that MCC950 also inhibited inflammasome activation and microglial activation in the APP/PS1 mouse model of AD. Furthermore, MCC950 stimulated Aβ phagocytosis in vitro, and it reduced Aβ accumulation in APP/PS1 mice, which was associated with improved cognitive function. These data suggest that activation of the inflammasome contributes to amyloid accumulation and to the deterioration of neuronal function in APP/PS1 mice and demonstrate that blocking assembly of the inflammasome may prove to be a valuable strategy for attenuating changes that negatively impact on neuronal function.

Liver X receptors agonists suppress NLRP3 inflammasome activation

Inflammasomes are multiprotein complexes that control the production of IL-1β and IL-18. NLRP3 inflammasome, the most characterized inflammasome, plays prominent roles in defense against infection, however aberrant activation is deleterious and leads to diseases. Therefore, its tight control offers therapeutic promise. Liver X receptors (LXRs) have significant anti-inflammatory properties. Whether LXRs regulate inflammasome remains unresolved. We thus tested the hypothesis that LXR’s anti-inflammatory properties may result from its ability to suppress inflammasome activation. In this study, LXRs agonists inhibited the induction of IL-1β production, caspase-1 cleavage and ASC oligomerization by NLRP3 inflammasome. The agonists also inhibited inflammasome-associated mtROS production. Importantly, the agonists inhibited the priming of inflammasome activation. In vivo data also showed that LXRs agonist prevented NLRP3-dependent peritonitis. In conclusion, LXRs agonists are identified to potently suppress NLRP3 inflammasome and the regulation of LXRs signaling is a potential therapeutic for inflammasome-driven diseases.

Dandelion extract suppresses reactive oxidative species and inflammasome in intestinal epithelial cells

Dandelion (Taraxacum spp.) has received increasing attention in western countries due to its health beneficial properties and pharmaceutical functions. In the current study, we evaluated compounds extracted from different parts of dandelion. Dandelion leaf had the highest, while root had the lowest level of total phenolic and flavonoid contents. Using HPLC, eleven phenolic acids and flavonoids were identified, among which chicoric acid was the main component in all parts of dandelion. Consistent with the total content of phenolics and flavonoids, leaf extract had the highest total antioxidant and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. Dandelion leaf extract at 400μg/ml suppressed reactive oxygen species (ROS) in HT-29 cells induced by hydrogen peroxide, equivalent to the ROS scavenging activity of 20μg/ml of pure chicoric acid. Importantly, both leaf extract and chicoric acid mitigated inflammatory signaling NF-κB p65 and cyclooxygenase-2 activity induced by lipopolysaccharide, demonstrated by suppressed LRR and PYD domains-containing protein 3 (NLRP3) inflammasome activation and caspase-1 activity, as well as NLRP3 inflammasome–mediated interleukin (IL)-1β and IL-18 secretion. In conclusion, dandelion extract inhibited inflammasome activation through ROS scavenging and anti-inflammatory activity; and chicoric acid, a major component of dandelion, is mainly responsible for the beneficial effects.

Isorhamnetin and hyperoside derived from water dropwort inhibits inflammasome activation

BackgroundWater dropwort (Oenanthe javanica), an umbelliferous plant, has been reported as hypolipidemic, antiplatelet, antitumor, or immune-stimulating agents and it has been suggested to cure cardiovascular disease and cancer. PurposePresent study aimed to evaluate the effect of the extracts of water dropwort (EWD) and its pharmacological molecules, hyperoside and isorhamnetin, on inflammatory response, especially inflammasome activation. Study design/MethodsThe anti-inflammasome properties of EWD, isorhamnetin, and hyperoside were elucidated by human and mouse macrophages. ResultsEWD attenuated secretion of interleukin (IL)-1β and formation of Asc pyroptosome resulting from NLRP3, NLRC4, and AIM2 inflammasome activation without interruption of cytokine transcription. Isorhamnetin selectively inhibited NLRP3 and AIM2 inflammasome activation and down-regulated expression of pro-inflammatory cytokines. Hyperoside selectively interrupted NLRC4 and AIM2 inflammasome activation but did not alter cytokine expression. In addition, EWD, isorhamnetin, and hyperoside inhibited caspase-1. ConclusionIsorhamnetin and hyperoside, a key molecule of water dropwort, have been suggested as candidates to attenuate inflammasome inhibition.

MPS 01-07 ENDOTHELIAL CELLS REGULATE INFLAMMASOME ACTIVATION THROUGH NITRIC OXIDE IN HYPERTENSIVE KIDNEY DISEASE

Objective: It is well known that chronic inflammation is an important common pathway of various progressive kidney disease. It has also been reported that inflammasome activation, which is part of the innate immune system, plays an important role in such chronic inflammation. However, the mechanism involved in the prolongation of inflammasome activation is unknown. We focused on nitric oxide (NO) as a regulatory mechanism of inflammasome. To test the hypothesis that “Endothelial cells-induced nitric oxide inhibits inflammasome activation and regulates chronic inflammation,” we used wild-type (WT) and eNOS knockout (eNOSKO) mice. Design and Method: [1] After unilateral nephrectomy (Nx), aldosterone (Ald) was continuously administered (0.25 mg/kg/day) using an infusion pump with 1% NaCl drinking water. The mice were sacrificed after 4 weeks of Ald administration, and the kidney tissue was examined. [2] We used bone-marrow-derived macrophages (BMDMs) to examine the molecular mechanism. After LPS priming (200 ng/mL 3 h), ATP (5 mM 60 min) was added to activate NLRP3 inflammasome. At the same time, we also performed priming with the NO donor, S-nitrosoglutathione (GSNO) to confirm the role of NO. Results: [1] In the WT mice, administration of Ald-Nx-Nacl caused an increase in blood pressure, kidney tissue fibrosis and inflammasome activation. The eNOSKO mice showed a similar tendency, but the inflammation in kidney was even worse. Since blood pressure was higher in the eNOSKO mice, we administered hydralazine to both groups to lower the blood pressure to the same extent before the examination. Even when the blood pressure was lowered to the same extent, renal inflammasome activation was significantly enhanced in the eNOSKO mice. [2] BMDM stimulation with LPS-ATP caused NLRP3 inflammasome-dependent cell death and IL1beta secretion. GSNO inhibited this activation. Conclusions: Endothelial cells-induced NO inhibits inflammasome activation to regulate chronic intrarenal inflammation. Endothelial dysfunction might promote chronic inflammation in CKD.

High-density lipoprotein reduces inflammation from cholesterol crystals by inhibiting inflammasome activation.

Interleukin-1β (IL-1β), a potent pro-inflammatory cytokine, has been implicated in many diseases, including atherosclerosis. Activation of IL-1β is controlled by a multi-protein complex, the inflammasome. The exact initiating event in atherosclerosis is unknown, but recent work has demonstrated that cholesterol crystals (CC) may promote atherosclerosis development by activation of the inflammasome. High-density lipoprotein (HDL) has consistently been shown to be anti-atherogenic and to have anti-inflammatory effects, but its mechanism of action is unclear. We demonstrate here that HDL is able to suppress IL-1β secretion in response to cholesterol crystals in THP-1 cells and in human-monocyte-derived macrophages. HDL is able to blunt inflammatory monocyte cell recruitment in vivo following intraperitoneal CC injection in mice. HDL appears to modulate inflammasome activation in several ways. It reduces the loss of lysosomal membrane integrity following the phagocytosis of CC, but the major mechanism for the suppression of inflammasome activation by HDL is decreased expression of pro-IL-1β and NLRP3, and reducing caspase-1 activation. In summary, we have described a novel anti-inflammatory effect of HDL, namely its ability to suppress inflammasome activation by CC by modulating the expression of several key components of the inflammasome.

Benzyl isothiocyanate inhibits inflammasome activation in E. coli LPS-stimulated BV2 cells

Inflammasomes are multi-protein complexes that play a crucial role in innate immune responses. Benzyl isothiocyanate(BITC) is a naturally occurring compound found in cruciferous vegetables, and BITC exhibits potential as a chemopreventive agent. However, whether BITC exerts inflammasome-mediated regulatory effects on neuroinflammation is unknown. In this study, we examined the effects of BITC on inflammasome-mediated interleukin-1β(IL-1β) production in E.coli lipopolysaccharide (LPS)-stimulated BV2 microglial cells. IL-1β production is tightly regulated at the post-translational level through the inflammasoume. We measured the levels of IL-1β produced from the LPS-exposed BV2 microglial cells using enzyme-linked immunosorbent assays(ELISAs). The BITC regulatory mechanisms in inflammasome-mediated cellular signaling pathways were examined by RT-PCR, western blot analysis and electrophoretic mobility shift assays. BITC inhibited the secretion of IL-1β induced by LPS in the BV2 microglial cells. BITC inhibited inflammasome activation and NLRfamily, pyrin domain containing3(NLRP3)-mediated caspase-1 activation, and decreased the levels of inflammasome activation pro-inflammatory mediators, including mitochondrial reactive oxygen species(ROS) and adenosine triphosphate(ATP) secretion in the LPS-stimulated BV2 microglial cells. Furthermore, we demonstrated that nuclear factor-κB(NF-κB) activation induced by LPS was inhibited by BITC, which may contribute to the attenuated secretion of IL-1β. These BITC-mediated inhibitory effects on IL-1β expression may thus regulate neuroinflammation through the inflammasome-mediated signaling pathway.

Inflammasome priming increases retinal pigment epithelial cell susceptibility to lipofuscin phototoxicity by changing the cell death mechanism from apoptosis to pyroptosis

Progressive death of retinal pigment epithelium (RPE) cells is a hallmark of age-related macular degeneration (AMD), the leading cause of blindness in all developed countries. Photooxidative damage and activation of the NLRP3 inflammasome have been suggested as contributing factors to this process. We investigated the effects of inflammasome activation on oxidative damage-induced RPE cell death. In primary human RPE cells and ARPE-19 cells, lipofuscin accumulated following incubation with oxidatively modified photoreceptor outer segments. Oxidative stress was induced by blue light irradiation (dominant wavelength: 448nm, irradiance: 0.8mW/cm2, duration: 3 to 6h) of lipofuscin-loaded cells and resulted in cell death by apoptosis. Prior inflammasome priming by IL-1α or complement activation product C5a altered the cell death mechanism to pyroptosis and resulted in a significant increase of the phototoxic effect. Following IL-1α priming, viability 24h after irradiation was reduced in primary RPE cells and ARPE-19 cells from 65.3% and 56.7% to 22.6% (p=0.003) and 5.1% (p=0.0002), respectively. Inflammasome-mediated IL-1β release occurred only in association with pyroptotic cell lysis. Inflammasome priming by conditioned media of pyroptotic cells likewise increased cell death. Suppression of inflammasome activation by inhibition of caspase-1 or cathepsins B and L significantly reduced cell death in primed cells. In summary, inflammasome priming by IL-1α, C5a, or conditioned media of pyroptotic cells increases RPE cell susceptibility to photooxidative damage-mediated cell death and changes the mechanism of induced cell death from apoptosis to pyroptosis. This process may contribute to RPE degeneration in AMD and provide new targets for intervention.

Inflammasome-associated inflammation inhibits cell-mediated immune responses to Listeria monocytogenes

Abstract Listeria monocytogenes is a genetically tractable Gram-positive intracellular bacterium that activates a robust cell-mediated immune response capable of breaking self-tolerance. These properties contribute to its success as a cancer immunotherapeutic platform; however, despite these attributes, the specific mechanisms by which L. monocytogenes stimulates a robust adaptive immune response remain largely unknown. We hypothesized that cytosolic innate immune activation, specifically inflammasome activation, by L. monocytogenes would be critical for triggering a robust CD8+ T-cell response. Surprisingly, we found that inflammasome activation impairs the induction of antigen specific T-cells and ultimately the generation of protective immunity. To determine if this phenomenon is unique to inflammasome-induced pyroptosis or a general outcome of pathogen induced host cell death, we designed strains of L. monocytogenes to uniquely activate pyroptosis, necrosis, or apoptosis. Contrary to previous studies of immunity in the context of sterile cell death, pathogen induced pyroptosis, necrosis and apoptosis each impaired the generation of cell-mediated immunity. Importantly, the mechanism of impairment was unique for each form of cell death, although in each case it was independent of dendritic cell depletion or deficits in antigen presentation. Apoptosis and necrosis impaired dendritic cell co-stimulatory molecule expression, while the pyroptotic inflammatory milieu independently impaired the generation of cell-mediated immunity. These results suggest that inhibition of inflammasome activation, or specifically certain inflammatory components, is critical for the success of L. monocytogenes immunotherapy.

Chitohexaose protects against acetaminophen-induced hepatotoxicity in mice.

Acetaminophen (N-acetyl-para-aminophenol (APAP)) toxicity causes acute liver failure by inducing centrilobular hepatic damage as a consequence of mitochondrial oxidative stress. Sterile inflammation, triggered by hepatic damage, facilitates gut bacterial translocation leading to systemic inflammation; TLR4-mediated activation by LPS has been shown to have a critical role in APAP-mediated hepatotoxicity. In this study, we demonstrate significant protection mediated by chitohexaose (Chtx) in mice challenged with a lethal dose of APAP (400 mg/kg b.w.). Decreased mortality by Chtx was associated with reduced hepatic damage, increased peritoneal migration of neutrophils, decreased mRNA expression of IL-1β as well as inhibition of inflammasome activation in liver. Further, an alternate mouse model of co-administration of a sublethal doses of APAP (200 mg/kg b.w.) and LPS (5 mg/kg b.w.) operating synergistically and mediating complete mortality was developed. Overwhelming inflammation, characterized by increased inflammatory cytokines (TNF-α, IL-1β and so on) in liver as well as in circulation and mortality was demonstrable in this model. Also, Chtx administration mediated significant reversal of mortality in APAP+LPS co-administered mice, which was associated with reduced IL-1β in liver and plasma cytokines in this model. In conclusion, Chtx being a small molecular weight linear carbohydrate offers promise for clinical management of liver failure associated with APAP overdose.

A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.

Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci.

The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation.

SummaryThe integrated stress response (ISR) is a homeostatic mechanism by which eukaryotic cells sense and respond to stress-inducing signals, such as amino acid starvation. General controlled nonrepressed (GCN2) kinase is a key orchestrator of the ISR, and modulates cellular metabolism in response to amino acid starvation. Here we demonstrate that GCN2 controls intestinal inflammation by suppressing inflammasome activation. Enhanced activation of ISR was observed in intestinal antigen presenting cells (APCs) and epithelial cells during amino acid starvation, or intestinal inflammation. Genetic deletion of GCN2 in CD11c+ APCs or intestinal epithelial cells resulted in enhanced intestinal inflammation and Th17 responses, due to enhanced inflammasome activation and IL-1β production. This was caused by reduced autophagy in GCN2−/− intestinal APCs and epithelial cells, leading to increased reactive oxygen species (ROS), a potent activator of inflammasomes1. Thus, conditional ablation of Atg5 and Atg7 in intestinal APCs resulted in enhanced ROS and Th17 responses. Furthermore, in vivo blockade of ROS and IL-1β resulted in inhibition of Th17 responses and reduced inflammation in GCN2−/− mice. Importantly, acute amino acid starvation suppressed intestinal inflammation via a mechanism dependent on GCN2. These results reveal a mechanism that couples amino acid sensing with control of intestinal inflammation via GCN2.