Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 16 de 16
Filter
Add more filters










Publication year range
2.
Cell Mol Gastroenterol Hepatol ; 8(4): 609-623, 2019.
Article in English | MEDLINE | ID: mdl-31401214

ABSTRACT

BACKGROUND & AIM: Ammonia is central in the pathogenesis of brain edema in acute liver failure (ALF) with infection and systemic inflammation expediting development of intracranial hypertension (ICH). Patients with acetaminophen-induced ALF have increased neutrophil TLR9 expression which can be induced by ammonia. We determined whether ammonia-induced brain edema and immune dysfunction are mediated by TLR9 and if this could be prevented in a TLR9-deficient mouse model. METHODS: Ammonium acetate (NH4-Ac; 4mmol/kg) was injected intraperitoneally in wild type (WT), Tlr9-/- and Lysm-Cre Tlr9fl/fl mice (TLR9 absent in neutrophils and macrophages including Kupffer cells) and compared to controls. Six hours after NH4-Ac injection, intracellular cytokine production was determined in splenic macrophages, CD4+ and CD8+ T cells. Brain water (BW) and total plasma DNA (tDNA) were also measured. The impact of the TLR9 antagonist ODN2088 (50µg/mouse) was evaluated. RESULTS: Following NH4-Ac injection, BW, macrophage and T cell cytokine production increased (P < .0001) in WT but not Tlr9-/- mice (P < .001). ODN2088 inhibited macrophage and T cell cytokine production (P < .05) and prevented an increase in BW (P < .0001). Following NH4-Ac injection, macrophage cytokine production and BW were ameliorated in Lysm-Cre Tlr9fl/fl mice compared to WT mice (P < .05) but there was no difference compared to Tlr9-/- mice. Following NH4-Ac injection, plasma tDNA levels increased in WT and Tlr9-/- mice (P < .05) suggesting that TLR9 may be activated by DNA released from ammonia-stimulated cells. CONCLUSION: Ammonia-induced brain edema requires macrophage and T cell expression of TLR9. Amelioration of brain edema and lymphocyte cytokine production by ODN2088 supports exploration of TLR9 antagonism in early ALF to prevent progression to ICH.


Subject(s)
Ammonia/toxicity , Brain Edema/metabolism , Macrophages/metabolism , T-Lymphocytes/metabolism , Toll-Like Receptor 9/metabolism , Acetaminophen/pharmacology , Animals , Brain Edema/chemically induced , Brain Edema/drug therapy , Brain Edema/immunology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cytokines/metabolism , Disease Models, Animal , Liver Failure, Acute/metabolism , Macrophages/drug effects , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/metabolism , Oligodeoxyribonucleotides/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Toll-Like Receptor 9/antagonists & inhibitors , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/immunology
4.
J Hepatol ; 69(3): 687-696, 2018 09.
Article in English | MEDLINE | ID: mdl-29705237

ABSTRACT

BACKGROUND & AIMS: Sterile inflammation resulting in alcoholic hepatitis (AH) occurs unpredictably after many years of excess alcohol intake. The factors responsible for the development of AH are not known but mitochondrial damage with loss of mitochondrial function are common features. Hcar2 is a G-protein coupled receptor which is activated by ß-hydroxybutyrate (BHB). We aimed to determine the relevance of the BHB-Hcar2 pathway in alcoholic liver disease. METHODS: We tested if loss of BHB production can result in increased liver inflammation. We further tested if BHB supplementation is protective in AH through interaction with Hcar2, and analyzed the immune and cellular basis for protection. RESULTS: Humans with AH have reduced hepatic BHB, and inhibition of BHB production in mice aggravated ethanol-induced AH, with higher plasma alanine aminotransferase levels, increased steatosis and greater neutrophil influx. Conversely supplementation of BHB had the opposite effects with reduced alanine aminotransferase levels, reduced steatosis and neutrophil influx. This therapeutic effect of BHB is dependent on the receptor Hcar2. BHB treatment increased liver Il10 transcripts, and promoted the M2 phenotype of intrahepatic macrophages. BHB also increased the transcriptional level of M2 related genes in vitro bone marrow derived macrophages. This skewing towards M2 related genes is dependent on lower mitochondrial membrane potential (Δψ) induced by BHB. CONCLUSIONS: Collectively, our data shows that BHB production during excess alcohol consumption has an anti-inflammatory and hepatoprotective role through an Hcar2 dependent pathway. This introduces the concept of metabolite-based therapy for AH. LAY SUMMARY: Alcoholic hepatitis is a life-threatening condition with no approved therapy that occurs unexpectedly in people who consume excess alcohol. The liver makes many metabolites, and we demonstrate that loss of one such metabolite ß-hydroxybutyrate occurs in patients with alcoholic hepatitis. This loss can increase alcohol-induced liver injury, and ß-hydroxybutyrate can protect from alcohol-induced liver injury via a receptor on liver macrophages. This opens the possibility of metabolite-based therapy for alcoholic hepatitis.


Subject(s)
3-Hydroxybutyric Acid/metabolism , Cyclic AMP/metabolism , Liver Diseases, Alcoholic , Liver , Mitochondria, Liver , Receptors, G-Protein-Coupled/metabolism , Animals , Central Nervous System Depressants/adverse effects , Central Nervous System Depressants/metabolism , Ethanol/adverse effects , Ethanol/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Inflammation/metabolism , Liver/metabolism , Liver/pathology , Liver Diseases, Alcoholic/metabolism , Liver Diseases, Alcoholic/pathology , Liver Diseases, Alcoholic/prevention & control , Liver Function Tests , Macrophages/drug effects , Macrophages/metabolism , Mice , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Protective Agents/metabolism
5.
Cell Metab ; 27(2): 339-350.e3, 2018 02 06.
Article in English | MEDLINE | ID: mdl-29414684

ABSTRACT

Sterile inflammation after tissue damage is a ubiquitous response, yet it has the highest amplitude in the liver. This has major clinical consequences, for alcoholic and non-alcoholic steatohepatitis (ASH and NASH) account for the majority of liver disease in industrialized countries and both lack therapy. Requirements for sustained sterile inflammation include increased oxidative stress and activation of the HIF-1α signaling pathway. We demonstrate the ability of digoxin, a cardiac glycoside, to protect from liver inflammation and damage in ASH and NASH. Digoxin was effective in maintaining cellular redox homeostasis and suppressing HIF-1α pathway activation. A proteomic screen revealed that digoxin binds pyruvate kinase M2 (PKM2), and independently of PKM2 kinase activity results in chromatin remodeling and downregulation of HIF-1α transactivation. These data identify PKM2 as a mediator and therapeutic target for regulating liver sterile inflammation, and demonstrate a novel role for digoxin that can effectively protect the liver from ASH and NASH.


Subject(s)
Digoxin/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Non-alcoholic Fatty Liver Disease/genetics , Pyruvate Kinase/metabolism , Transcriptional Activation/drug effects , Amino Acid Sequence , Animals , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Chromatin/metabolism , Disease Models, Animal , Endotoxins , Histones/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Inflammation/genetics , Inflammation/pathology , Liver/drug effects , Liver/metabolism , Liver/pathology , Oxidation-Reduction , Protein Binding/drug effects , Pyruvate Kinase/chemistry , THP-1 Cells , Transcription, Genetic/drug effects
6.
J Clin Invest ; 126(3): 859-64, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26808498

ABSTRACT

Nonalcoholic steatohepatitis (NASH) is the most common liver disease in industrialized countries. NASH is a progressive disease that can lead to cirrhosis, cancer, and death, and there are currently no approved therapies. The development of NASH in animal models requires intact TLR9, but how the TLR9 pathway is activated in NASH is not clear. Our objectives in this study were to identify NASH-associated ligands for TLR9, establish the cellular requirement for TLR9, and evaluate the role of obesity-induced changes in TLR9 pathway activation. We demonstrated that plasma from mice and patients with NASH contains high levels of mitochondrial DNA (mtDNA) and intact mitochondria and has the ability to activate TLR9. Most of the plasma mtDNA was contained in microparticles (MPs) of hepatocyte origin, and removal of these MPs from plasma resulted in a substantial decrease in TLR9 activation capacity. In mice, NASH development in response to a high-fat diet required TLR9 on lysozyme-expressing cells, and a clinically applicable TLR9 antagonist blocked the development of NASH when given prophylactically and therapeutically. These data demonstrate that activation of the TLR9 pathway provides a link between the key metabolic and inflammatory phenotypes in NASH.


Subject(s)
DNA, Mitochondrial/physiology , Non-alcoholic Fatty Liver Disease/immunology , Toll-Like Receptor 9/metabolism , Adolescent , Cells, Cultured , Child , Diet, High-Fat/adverse effects , Female , Gene Expression , Hepatocytes/metabolism , Humans , Kupffer Cells/immunology , Male , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/metabolism , Toll-Like Receptor 9/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
7.
Am J Physiol Gastrointest Liver Physiol ; 307(7): G732-40, 2014 Oct 01.
Article in English | MEDLINE | ID: mdl-25104498

ABSTRACT

Activation of the cytosolic inflammasome machinery is responsible for acute and chronic liver inflammation, but little is known about its regulation. The N-methyl-d-aspartate (NMDA) receptor families are heterotetrameric ligand-gated ion channels that are activated by a range of metabolites, including aspartate, glutamate, and polyunsaturated fatty acids. In the brain NMDA receptors are present on neuronal and nonneuronal cells and regulate a diverse range of functions. We tested the role of the NMDA receptor and aspartate in inflammasome regulation in vitro and in models of acute hepatitis and pancreatitis. We demonstrate that the NMDA receptor is present on Kupffer cells, and their activation on primary mouse and human cells limits inflammasome activation by downregulating NOD-like receptor family, pyrin domain containing 3 and procaspase-1. The NMDA receptor pathway is active in vivo, limits injury in acute hepatitis, and can be therapeutically further activated by aspartate providing protection in acute inflammatory liver injury. Downregulation of inflammasome activation by NMDA occurs via a ß-arrestin-2 NF-kß and JNK pathway and not via Ca(2+) mobilization. We have identified the NMDA receptor as a regulator of inflammasome activity in vitro and in vivo. This has identified a new area of immune regulation associated by metabolites that may be relevant in a diverse range of conditions, including nonalcoholic steatohepatitis and total parenteral nutrition-induced immune suppression.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Arrestins/metabolism , Aspartic Acid/pharmacology , Chemical and Drug Induced Liver Injury/prevention & control , Excitatory Amino Acid Agonists/pharmacology , Inflammasomes/drug effects , Liver/drug effects , Macrophages/drug effects , Receptors, N-Methyl-D-Aspartate/agonists , Animals , Arrestins/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspase 1/genetics , Caspase 1/metabolism , Cell Line , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/immunology , Chemical and Drug Induced Liver Injury/metabolism , Disease Models, Animal , Humans , Inflammasomes/immunology , Inflammasomes/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Liver/immunology , Liver/metabolism , Macrophages/immunology , Macrophages/metabolism , Male , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Pancreatitis/immunology , Pancreatitis/metabolism , Pancreatitis/prevention & control , Protein Precursors/genetics , Protein Precursors/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction/drug effects , Time Factors , beta-Arrestin 2 , beta-Arrestins
8.
Dig Dis ; 32(5): 507-15, 2014.
Article in English | MEDLINE | ID: mdl-25034283

ABSTRACT

The liver possesses a strong inflammatory response, as seen experimentally and clinically with liver inflammation due to toxic and metabolic stress, sepsis and ischemia. Initiation of this inflammatory response requires the interaction of two types of extracellular signals which collectively upregulate and activate a cytosolic molecular complex termed the inflammasome. Signal 1 is via activation of pattern recognition receptors, and signal 2 is delivered by diverse stimuli including particulates and adenosine triphosphate. The common end result of inflammasome activation is the activation of the protease caspase-1 with release of active interleukin-1ß. The inflammasome is important in a wide range of conditions including alcoholic and non-alcoholic steatohepatitis. Kupffer cells are known to be important, but the consequences of inflammasome activation in other hepatic immune cells have not been well characterized. The inflammasome pathway is also known to be required for a full fibrotic response, as demonstrated by reduced lung, skin and liver fibrosis in inflammasome-deficient mice. Identification of the inflammasome machinery has opened up novel therapeutic avenues by the use of antagonists for Toll-like receptors as well as the adenosine triphosphate receptor P2X7, and the interleukin-1 receptor. There is now great interest in how inflammasome pathways are regulated. The initial challenge is to understand how an acute inflammatory response is sustained. This is a significant issue as the known stimuli result in an acute response that is self-limited to under 24 h. This suggests that there are significant regulators which allow sustained inflammasome activation in conditions such as non-alcoholic steatohepatitis and alcoholic hepatitis.


Subject(s)
Inflammasomes/metabolism , Liver/injuries , Liver/pathology , Non-alcoholic Fatty Liver Disease/pathology , Animals , Humans , Inflammation/pathology , Liver Cirrhosis/pathology , Receptors, Pattern Recognition/metabolism
9.
Gastroenterology ; 146(7): 1763-74, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24657625

ABSTRACT

BACKGROUND & AIMS: The NACHT, LRR, and pyrin domain-containing protein 3 (NLRP3) inflammasome induces inflammation in response to organ injury, but little is known about its regulation. Toll-like receptors (TLRs) provide the first signal required for activation of the inflammasome and stimulate aerobic glycolysis to generate lactate. We examined whether lactate and the lactate receptor, Gi-protein-coupled receptor 81 (GPR81), regulate TLR induction of signal 1 and limit inflammasome activation and organ injury. METHODS: Primary mouse macrophages and human monocytes were incubated with TLR4 agonists and lactate and assayed for levels of pro-interleukin (IL)1ß, NLRP3, and caspase-1 (CASP1); release of IL1ß; and activation of nuclear factor-κB (NF-κB) and caspase-1. Small interfering RNAs were used to reduce levels of GPR81 and arrestin ß-2 (ARRB2), and an NF-κB luciferase reporter transgene was transfected in RAW 264.7 cells. Cell lysates were analyzed by immunoprecipitation with an antibody against GPR81. Acute hepatitis was induced in C56BL/6N mice by administration of lipopolysaccharide and D-galactosamine. Acute pancreatitis was induced by administration of lipopolysaccharide and cerulein. Some mice were given intraperitoneal injections of sodium lactate or small interfering RNA against Gpr81. Activation of NF-κB in tissue macrophages was assessed in mice that expressed a reporter transgene. RESULTS: In macrophages and monocytes, increasing concentrations of lactate reduced TLR4-mediated induction of Il1B, Nlrp3, and Casp1; activation of NF-κB; release of IL1ß; and cleavage of CASP1. GPR81 and ARRB2 physically interacted and were required for these effects. The administration of lactate reduced inflammation and organ injury in mice with immune hepatitis; this reduction required Gpr81 dependence in vivo. Lactate also prevented activation of NF-κB in macrophages of mice, and, when given after injury, reduced the severity of acute pancreatitis and acute liver injury. CONCLUSIONS: Lactate negatively regulates TLR induction of the NLRP3 inflammasome and production of IL1ß, via ARRB2 and GPR81. Lactate could be a promising immunomodulatory therapy for patients with acute organ injury.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Chemical and Drug Induced Liver Injury/prevention & control , Immunity, Innate/drug effects , Inflammasomes/drug effects , Liver/drug effects , Pancreas/drug effects , Pancreatitis/prevention & control , Receptors, G-Protein-Coupled/metabolism , Sodium Lactate/pharmacology , Toll-Like Receptors/drug effects , Animals , Anti-Inflammatory Agents/administration & dosage , Arrestins/metabolism , Carrier Proteins/metabolism , Cell Line , Ceruletide , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/immunology , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Cytoprotection , Disease Models, Animal , Dose-Response Relationship, Drug , Down-Regulation , Galactosamine , Humans , Inflammasomes/immunology , Inflammasomes/metabolism , Injections, Intraperitoneal , Interleukin-1beta/metabolism , Lipopolysaccharides , Liver/immunology , Liver/metabolism , Liver/pathology , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , NF-kappa B/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , Pancreas/immunology , Pancreas/metabolism , Pancreas/pathology , Pancreatitis/chemically induced , Pancreatitis/genetics , Pancreatitis/immunology , Pancreatitis/metabolism , Pancreatitis/pathology , RNA Interference , RNA, Small Interfering/metabolism , Receptors, G-Protein-Coupled/genetics , Signal Transduction/drug effects , Sodium Lactate/administration & dosage , Toll-Like Receptor 4/drug effects
10.
Nat Commun ; 4: 2909, 2013.
Article in English | MEDLINE | ID: mdl-24352507

ABSTRACT

Inflammasome pathways are important in chronic diseases; however, it is not known how the signalling is sustained after initiation. Inflammasome activation is dependent on stimuli such as lipopolysaccharide (LPS) and ATP that provide two distinct signals resulting in rapid production of interleukin (IL)-1ß, with the lack of response to repeat stimulation. Here we report that adenosine is a key regulator of inflammasome activity, increasing the duration of the inflammatory response via the A(2A) receptor. Adenosine does not replace signals provided by stimuli such as LPS or ATP but sustains inflammasome activity via a cAMP/PKA/CREB/HIF-1α pathway. In the setting of the lack of IL-1ß responses after previous exposure to LPS, adenosine can supersede this tolerogenic state and drive IL-1ß production. These data reveal that inflammasome activity is sustained, after initial activation, by A(2A) receptor-mediated signalling.


Subject(s)
Adenosine/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Inflammasomes/metabolism , Receptor, Adenosine A2A/metabolism , Adenosine/pharmacology , Adenosine Triphosphate/metabolism , Animals , Carrier Proteins/metabolism , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Inflammasomes/drug effects , Interleukin-1beta/metabolism , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , Liver/drug effects , Liver/immunology , Liver/metabolism , Liver/pathology , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Receptor, Adenosine A2A/genetics , Signal Transduction/immunology
11.
Dig Dis Sci ; 57(9): 2371-8, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22573344

ABSTRACT

BACKGROUND AND AIMS: Adenine is a uric acid pathway metabolite of no known function, and has recently been identified as a ligand for a rat G protein-coupled receptor. Due to the known role of other uric acid pathway metabolites in HSC biology, we tested the ability of adenine to induce HSC differentiation. METHODS: RT-PCR was performed for adenine receptor expression in T-6 and primary rat HSC. T-6 and primary rats HSC were cultured with and without adenine, and stellation examined. Next, we examined inhibition of calcium signaling using caged IP(3). To test if adenine inhibits HSC chemotaxis T-6 cells and rat HSCs were cultured with or without adenine for 24 h in a transwell assay with PDGF as the chemoattractant. cDNA was prepared from T-6 and primary HSC for quantification of collagen 1 mRNA using real-time PCR. RESULTS: We found that mRNA for the adenine receptor is expressed in T-6 cells and primary rat HSC. Also, adenine induces HSC stellation and adenine inhibits IP(3) mediated increase in cytosolic [Ca(2+)](i) and inhibits chemotaxis in T-6 cells and primary rat HSC. Adenine was also shown to up-regulate α-SMA and collagen 1, and this effect is lost by using specific si-RNA for the adenine receptor. Finally, adenine inhibits endothelin-1-induced gel contraction. CONCLUSIONS: The adenine receptor is present in T-6 cells and primary rats HSC. Adenine, via the adenine receptor, induces morphological change, and cytosolic calcium signaling, inhibits chemotaxis, and up-regulates collagen 1 mRNA in HSCs.


Subject(s)
Adenine/pharmacology , Hepatic Stellate Cells/drug effects , Actins/genetics , Actins/metabolism , Adenine/metabolism , Animals , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cell Differentiation , Chemotaxis , Collagen Type I/genetics , Collagen Type I/metabolism , DNA, Complementary/genetics , DNA, Complementary/metabolism , Endothelin-1/genetics , Endothelin-1/metabolism , Gene Expression Regulation , Hepatic Stellate Cells/cytology , Inositol 1,4,5-Trisphosphate/genetics , Inositol 1,4,5-Trisphosphate/metabolism , Metalloporphyrins , Platelet-Derived Growth Factor , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, Purinergic/drug effects , Receptors, Purinergic/genetics , Receptors, Purinergic/metabolism , Reverse Transcriptase Polymerase Chain Reaction
12.
Am J Physiol Gastrointest Liver Physiol ; 296(6): G1248-57, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19359429

ABSTRACT

The inflammasome is a cytoplasmic multiprotein complex that has recently been identified in immune cells as an important sensor of signals released by cellular injury and death. Analogous to immune cells, hepatic stellate cells (HSC) also respond to cellular injury and death. Our aim was to establish whether inflammasome components were present in HSC and could regulate HSC functionality. Monosodium urate (MSU) crystals (100 microg/ml) were used to experimentally induce inflammasome activation in LX-2 and primary mouse HSC. Twenty-four hours later primary mouse HSC were stained with alpha-smooth muscle actin and visualized by confocal microscopy, and TGF-beta and collagen1 mRNA expression was quantified. LX-2 cells were further cultured with or without MSU crystals for 24 h in a transwell chemotaxis assay with PDGF as the chemoattractant. We also examined inhibition of calcium (Ca(2+)) signaling in LX-2 cells treated with or without MSU crystals using caged inositol 1,4,5-triphosphate (IP(3)). Finally, we confirmed an important role of the inflammasome in experimental liver fibrosis by the injection of carbon tetrachloride (CCl(4)) or thioacetamide (TAA) in wild-type mice and mice lacking components of the inflammasome. Components of the inflammasome are expressed in LX-2 cells and primary HSC. MSU crystals induced upregulation of TGF-beta and collagen1 mRNA and actin reorganization in HSCs from wild-type mice but not mice lacking inflammasome components. MSU crystals inhibited the release of Ca(2+) via IP(3) in LX-2 cells and also inhibited PDGF-induced chemotaxis. Mice lacking the inflammasome-sensing and adaptor molecules, NLRP3 and apoptosis-associated speck-like protein containing CARD, had reduced CCl(4) and TAA-induced liver fibrosis. We concluded that inflammasome components are present in HSC, can regulate a variety of HSC functions, and are required for the development of liver fibrosis.


Subject(s)
Hepatic Stellate Cells/physiology , Inflammation/physiopathology , Actins/metabolism , Animals , Apoptosis Regulatory Proteins , CARD Signaling Adaptor Proteins , Calcium Signaling/drug effects , Carbon Tetrachloride/pharmacology , Carrier Proteins/genetics , Cell Line, Transformed , Chemotaxis/drug effects , Chemotaxis/genetics , Collagen Type I/genetics , Cytoskeletal Proteins/genetics , Gene Expression/drug effects , Gene Expression/genetics , Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/drug effects , Humans , Inflammation/chemically induced , Inflammation/metabolism , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/pharmacology , Liver/metabolism , Liver/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/etiology , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein , Platelet-Derived Growth Factor/pharmacology , Thioacetamide/pharmacology , Transforming Growth Factor beta/genetics , Uric Acid/pharmacology
13.
Hepatology ; 46(5): 1509-18, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17705260

ABSTRACT

UNLABELLED: Apoptosis of hepatocytes results in the development of liver fibrosis, but the molecular signals mediating this are poorly understood. Degradation and modification of nuclear DNA is a central feature of apoptosis, and DNA from apoptotic mammalian cells is known to activate immune cells via Toll-like receptor 9 (TLR9). We tested if DNA from apoptotic hepatocytes can induce hepatic stellate cell (HSC) differentiation. Our data show that apoptotic hepatocyte DNA and cytidine-phosphate-guanosine oligonucleotides induced up-regulation of transforming growth factor beta1 and collagen 1 messenger RNA both in the human HSC line LX-2 and in primary mouse HSCs. These effects were opposed by TLR9 antagonists. We have recently shown that adenosine inhibits HSC chemotaxis, and we now show that apoptotic hepatocyte DNA also inhibits platelet-derived growth factor (PDGF)-mediated HSC chemotaxis. Inhibition of HSC chemotaxis by PDGF was blocked by TLR9 antagonists, and was absent in primary HSCs from mice deficient in TLR9 or the TLR adaptor molecule MyD88. Stimulation of TLR9 on HSCs blocked signaling by the PDGF signaling molecule inositol 1,4,5-triphosphate and reduced PDGF-mediated increase in cytosolic Ca(2+). CONCLUSION: DNA from apoptotic hepatocytes acts as an important mediator of HSC differentiation by (1) providing a stop signal to mobile HSCs when they have reached an area of apoptosing hepatocytes and (2) inducing a stationary phenotype-associated up-regulation of collagen production.


Subject(s)
Cell Differentiation/physiology , Cell Migration Inhibition/physiology , DNA/metabolism , Liver/cytology , Toll-Like Receptor 9/metabolism , Actins/metabolism , Animals , Apoptosis/physiology , Calcium Signaling/physiology , Cell Line , Collagen Type I/metabolism , Gene Expression Regulation , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Liver/metabolism , Liver/physiology , Liver Cirrhosis/metabolism , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/metabolism , Oligodeoxyribonucleotides/metabolism , Platelet-Derived Growth Factor/metabolism , RNA, Messenger/metabolism , Toll-Like Receptor 9/antagonists & inhibitors , Transforming Growth Factor beta1/metabolism
14.
Dig Dis Sci ; 51(10): 1697-705, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16957995

ABSTRACT

Hepatic fibrogenesis is reduced in the absence of leptin. We hypothesized that leptin protects hepatic stellate cells (HSCs) from apoptosis and tested this in in vitro and in vivo systems. (i) Fas ligand (fas-L)-mediated apoptosis was induced in vitro in activated HSCs in the absence and presence of leptin. (ii) HSC apoptosis was also induced by UV irradiation in the absence and presence of leptin. (iii) Fas-L-mediated apoptosis was induced in vitro in HSCs from db/db mice in the absence and presence of leptin. (iv) Liver fibrosis was induced in wt and db/db mice. (v) Liver fibrosis was induced in wild-type mice with TAA, and mice received additional leptin or a control solution. HSC apoptosis was assessed by TUNEL staining. Western blot for alpha-SMA was used to determine differences in HSC activation. Results were as follows. (i) Fas-L induced significant apoptosis of HSC, and preincubation with leptin reduced this approximately threefold. (ii) Leptin provided no protection from UV-induced apoptosis. (iii) HSCs from db/db mice were not protected by leptin against fas-L-induced apoptosis. (iv) TAA-induced fibrosis was significantly less in db/db mice compared to wild type. (v) Wild-type mice receiving leptin had less apoptosis and more alpha-SMA than controls. We conclude that leptin protects HSC from in vitro and in vivo apoptosis. The antiapoptotic effect of leptin requires the long form of the leptin receptor and interacts with the apoptotic pathway proximal to mitochondrial activation.


Subject(s)
Apoptosis/drug effects , Hepatocytes/drug effects , Hepatocytes/physiology , Leptin/pharmacology , Liver/drug effects , Actins/metabolism , Animals , Cell Culture Techniques , Fas Ligand Protein , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Receptors, Cell Surface/metabolism , Receptors, Leptin , Thioacetamide
15.
Cell Immunol ; 238(1): 31-7, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16445896

ABSTRACT

BACKGROUND: Activated CD8(+) T cells are retained by the healthy liver where the majority undergo apoptosis. The intrahepatic apoptosis of activated CD8(+) T cells is enhanced by the presence of SIINFEKL peptide. It is of great interest to identify strategies for maintaining intrahepatic T cell number and function in the presence of SIINFEKL peptides. AIM: Our aim was to test if low affinity peptides can block SIINFEKL peptide induced T cell deletion. METHODS: We used an in vivo model of intrahepatic CD8(+) T cell deletion with peptides of different affinities. RESULTS AND DISCUSSION: We show that the intrahepatic deletion of CD8(+) T cells by SIINFEKL peptide results in loss of in vivo cytotoxic T lymphocyte function. In contrast we show that a low affinity peptide (G4) does not result in intrahepatic deletion of CD8(+) T cells. High concentrations G4 peptide can however block intrahepatic deletion of activated CD8(+) T cells, and prevent loss of in vivo cytotoxicity due to SIINFEKL peptide. This is the first demonstration of blocking of SIINFEKL peptide induced CD8(+) T cell deletion in the liver, with enhancement of in vivo cytotoxicity.


Subject(s)
Apoptosis/drug effects , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Egg Proteins/pharmacology , Liver/immunology , Ovalbumin/pharmacology , Adoptive Transfer , Animals , Apoptosis/immunology , CD8-Positive T-Lymphocytes/cytology , Flow Cytometry , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/immunology , Ligands , Liver/cytology , Liver/drug effects , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments , Specific Pathogen-Free Organisms , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology
16.
Hepatology ; 39(4): 1017-27, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15057906

ABSTRACT

The immune response to foreign antigens in the liver is often suboptimal and this is clinically relevant in chronic persistence of hepatotropic viruses. In chronic infection with the hepatitis C virus, activated CD8+ T cells specific for viral epitopes are present in the peripheral blood and the liver, yet viral clearance is unusual. To define the fate of activated CD8+ entering the liver, we developed a mouse model of portal vein injection of activated CD8+ T cells in vivo. Activated CD8+ T cells are retained very efficiently by the liver and undergo an approximately 8-fold expansion in the first 48 hours. This expansion is followed by apoptosis and a decline in numbers of the retained cells over the next 4 days. The presence of high affinity (HA) antigen does not affect the initial retention by the liver but greatly limits the expansion in the first 48 hours by increasing apoptosis of the retained cells. In the absence of Kupffer cells, the initial retention and expansion are unchanged, but HA antigen does not limit the expansion of the liver CD8+ T cell pool. In conclusion, these data identify a previously unknown phase of CD8+ T cell expansion after entering the liver, demonstrate that HA antigen limits the hepatic CD8+ T cell pool by inducing apoptosis, and that this effect requires Kupffer cells. Interfering with antigen presentation by Kupffer cells may be a strategy to limit HA antigen-induced deletion of activated CD8+ T cells entering the liver.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Kupffer Cells/immunology , Liver/immunology , Animals , Apoptosis/immunology , CD8-Positive T-Lymphocytes/cytology , Cell Division/immunology , Cells, Cultured , Histocompatibility Antigens/immunology , Kupffer Cells/cytology , Liver/cytology , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunology
SELECTION OF CITATIONS
SEARCH DETAIL
...