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NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.

ABSTRACT: Inflammasome activation plays a central role in the development of drug-induced and obesity-associated liver disease. However, the sources and mechanisms of inflammasome-mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell-specific conditional mutant Nlrp3 knock-in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3), resulting in a hyperactive NLRP3. To study the presence and significance of NLRP3-initiated pyroptotic cell death, we separated hepatocytes from nonparenchymal cells and developed a novel flow-cytometry-based (fluorescence-activated cell sorting; FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase 1 (Casp1)- and propidium iodide (PI)-positive cells. Liver inflammation was quantified histologically by FACS and gene expression analysis. Liver fibrosis was assessed by Sirius Red staining and quantitative polymerase chain reaction for markers of hepatic stellate cell (HSC) activation. NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation; characterized by neutrophilic infiltration and HSC activation with collagen deposition in the liver. These changes were partially attenuated by treatment with anakinra, an interleukin-1 receptor antagonist. Notably, hepatocytes from global Nlrp3-mutant mice showed marked hepatocyte pyroptotic cell death, with more than a 5-fold increase in active Casp1/PI double-positive cells. Myeloid cell-restricted mutant NLRP3 activation resulted in a less-severe liver phenotype in the absence of detectable pyroptotic hepatocyte cell death.Our data demonstrate that global and, to a lesser extent, myeloid-specific NLRP3 inflammasome activation results in severe liver inflammation and fibrosis while identifying hepatocyte pyroptotic cell death as a novel mechanism of NLRP3-mediated liver damage.

SUBMITTER: Wree A

PROVIDER: S-EPMC4008151 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.

Wree Alexander A Eguchi Akiko A McGeough Matthew D MD Pena Carla A CA Johnson Casey D CD Canbay Ali A Hoffman Hal M HM Feldstein Ariel E AE

Hepatology (Baltimore, Md.) 20140130 3

<h4>Unlabelled</h4>Inflammasome activation plays a central role in the development of drug-induced and obesity-associated liver disease. However, the sources and mechanisms of inflammasome-mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell-specific conditional mutant Nlrp3 knock-in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP ...[more]

PMID: 23813842

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Project description:Background and Aims: The activation of stimulator of interferon genes (STING) and NOD-like receptors protein 3 (NLRP3) inflammasomes-mediated pyroptosis signaling pathways represent two distinct central mechanisms in liver disease. However, the interconnection between these two pathways and the epigenetic regulation of the STING-NLRP3 axis in hepatocyte pyroptosis during liver fibrosis remain unknown and is the focus of this study. Approach and Results: Liver fibrosis was induced in Sting knockout, Gasdermin D (Gsdmd) knockout mice, and in mice with hepatocyte-specific Nlrp3 deletion. RNA-sequencing, metabolomics, epigenetic compound screening system, and chromatin immunoprecipitation were utilized. STING and NLRP3 inflammasome signaling pathways were activated in cirrhotic livers but were suppressed by Sting knockout. Sting knockout also ameliorated hepatic pyroptosis, inflammation, and fibrosis in the murine cirrhotic model. In vitro, STING induced pyroptosis in primary murine hepatocytes via activating the NLRP3 inflammasome. H3K4-specific histone methyltransferase WD repeat-containing protein 5 (WDR5) and DOT1-like histone H3K79 methyltransferase (DOT1L) were identified to regulate NLRP3 expression in STING-overexpressed AML12 hepatocytes. WDR5/DOT1L-mediated histone methylation enhanced interferon regulatory transcription factor 3 (IRF3) binding to the Nlrp3 promoter and promoted STING-induced Nlrp3 transcription in hepatocytes. The RNA-sequencing and metabolomics analysis in murine livers and primary hepatocytes showed that metabolic reprogramming might participate in NLRP3-mediated hepatocyte pyroptosis and liver fibrosis. Moreover, hepatocyte-specific Nlrp3 deletion and downstream Gsdmd knockout attenuated hepatic pyroptosis, inflammation, and fibrosis in murine cirrhotic models. Conclusions: This study describes a novel epigenetic mechanism by which the STING-WDR5/DOT1L/IRF3-NLRP3 signaling pathway enhances hepatocyte pyroptosis and hepatic inflammation in liver fibrosis.

Project description:Background & aimsIncreased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown.MethodsWe used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia).ResultsWe demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1β secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B.ConclusionsThese results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development.Lay summaryOur findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.

Project description:BackgroundAlthough diabetes mellitus has been reported to aggravate liver ischaemia and reperfusion (IR) injury, the basic mechanism remains largely unknown. The object of the present study was to determine the role of oxidative stress and hepatocellular pyroptosis in liver IR injury in diabetic mice.MethodsDb/db and C57BL/6 mice at 8 weeks of age were subjected to liver IR injury. Liver injury and hepatocyte cell death were analyzed. A NOD-like receptor family pyrin domain-containing 3 protein (NLRP3) inflammasome antagonist (CY09) and a reactive oxygen species (ROS) antagonist (N-Acetyl-L-cysteine, NAC) were used to determine the role of ROS-mediated hepatocellular pyroptosis in diabetic mice post-IR.ResultsAggravated liver IR injury was found in db/db mice compared to C57BL/6 control mice, as demonstrated by increased serum alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) levels, liver architecture damage and Suzuki scores. Interestingly, IR induces the pyroptosis of hepatocytes in db/db mice, as evidenced by enhanced NLRP3 inflammasome activation, increased numbers of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive hepatocytes and increased gene expression of interleukin-1β (IL-1β) and IL-18 in livers post-IR. The inhibitory effect of CY09, an NLRP3 antagonist, efficiently abrogated the exacerbation effects of diabetes on liver IR injury in db/db mice. Furthermore, increased ROS expression was detected in db/db mice compared to control mice after IR. ROS scavenging by NAC pretreatment markedly inhibited hepatocellular NLRP3 inflammasome activation and pyroptosis in the db/db mice post-IR, indicating that ROS play an essential role in mediating hepatocyte pyroptosis in the setting of diabetes mellitus.ConclusionsOur results demonstrate that diabetes induces hepatocyte pyroptosis by promoting oxidative stress-mediated NLRP3 inflammasome activation during liver IR injury. Strategies targeting ROS and NLRP3 inflammasome activation would be beneficial for preventing liver IR injury in diabetic patients.

Project description:BACKGROUND & AIMS:NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. METHODS:We fed foz/foz and wild-type mice an atherogenic diet for 16weeks, gavaged MCC950 or vehicle until 24weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6weeks and determined the effects on liver fibrosis. RESULTS:In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1?, active caspase-1 and IL-1? increased at 24weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1?, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1? expression, plasma IL-1?, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1?; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1?, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. CONCLUSION:MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. LAY SUMMARY:Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis.

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NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.

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NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.

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