RESUMEN
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.
Asunto(s)
Ácido 3-Hidroxibutírico/metabolismo , AMP Cíclico/metabolismo , Hepatopatías Alcohólicas , Hígado , Mitocondrias Hepáticas , Receptores Acoplados a Proteínas G/metabolismo , Animales , Depresores del Sistema Nervioso Central/efectos adversos , Depresores del Sistema Nervioso Central/metabolismo , Etanol/efectos adversos , Etanol/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Inflamación/metabolismo , Hígado/metabolismo , Hígado/patología , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/patología , Hepatopatías Alcohólicas/prevención & control , Pruebas de Función Hepática , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Sustancias Protectoras/metabolismoRESUMEN
PURPOSE OF REVIEW: Acute pancreatitis is a major cause of gastrointestinal morbidity for which specific therapy is greatly needed to prevent progression to and induce resolution of severe disease. RECENT FINDINGS: Innate immune components and metabolite signaling are recently identified as strong determinants of disease severity and resolution in acute pancreatitis and this work will be discussed herein. SUMMARY: Targeting innate immune cell populations and metabolite signaling pathways in acute pancreatitis may result in broader and ultimately more efficacious re-direction of the inflammatory programme toward disease resolution and improved clinical outcomes.
Asunto(s)
Inmunidad Innata/fisiología , Pancreatitis/inmunología , Enfermedad Aguda , Animales , Humanos , Mediadores de Inflamación/metabolismo , Metabolismo de los Lípidos/inmunología , Terapia Molecular Dirigida , Pancreatitis/metabolismo , Pancreatitis/terapia , Transducción de Señal/inmunologíaRESUMEN
In this review we summarize the role of inflammasomes in pancreatic physiology and disease with a focus on acute pancreatitis where much recent progress has been made. New findings have identified inducers of and cell specificity of inflammasome component expression in the pancreas, the contribution of inflammasome-regulated effectors to pancreatitis, and metabolic regulation of inflammasome activation, which are strong determinants of injury in pancreatitis. New areas of pancreatic biology will be highlighted in the context of our evolving understanding of gut microbiome- and injury-induced inflammasome priming, pyroptosis, and innate immune-mediated regulation of cell metabolism.
Asunto(s)
Inflamasomas/inmunología , Páncreas/inmunología , Enfermedades Pancreáticas/inmunología , Animales , Humanos , Inmunidad Innata , Inflamasomas/metabolismo , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Páncreas/metabolismo , Páncreas/patología , Enfermedades Pancreáticas/metabolismo , Enfermedades Pancreáticas/patología , Transducción de SeñalRESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Inmunidad Innata/efectos de los fármacos , Inflamasomas/efectos de los fármacos , Hígado/efectos de los fármacos , Páncreas/efectos de los fármacos , Pancreatitis/prevención & control , Receptores Acoplados a Proteínas G/metabolismo , Lactato de Sodio/farmacología , Receptores Toll-Like/efectos de los fármacos , Animales , Antiinflamatorios/administración & dosificación , Arrestinas/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Ceruletida , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Citoprotección , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Galactosamina , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Inyecciones Intraperitoneales , Interleucina-1beta/metabolismo , Lipopolisacáridos , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Páncreas/inmunología , Páncreas/metabolismo , Páncreas/patología , Pancreatitis/inducido químicamente , Pancreatitis/genética , Pancreatitis/inmunología , Pancreatitis/metabolismo , Pancreatitis/patología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacos , Lactato de Sodio/administración & dosificación , Receptor Toll-Like 4/efectos de los fármacosRESUMEN
TLR9 is a key determinant of the innate immune responses in both infectious and sterile injury. Specific antagonism of TLR9 is of great clinical interest to reduce tissue damage in a wide range of pathologies, and has been approached by modification of nucleic acids, the recognized ligand for TLR9. Such oligonucleotide-derived pharmacotherapeutics have limitations in specificity for nucleic acid receptors, significant potential for immunologic recognition with generation of innate and adaptive immune responses, and limited bioavailability. We have identified enantiomeric analogues of traditional (-)-morphinans as having TLR9 antagonist properties on reporter cell lines. One of these analogues (COV08-0064) is demonstrated to be a novel small-molecule antagonist of TLR9 with greater specificity for TLR9 than oligo-based antagonists. COV08-0064 has wide bioavailability, including the s.c. and oral routes. It specifically inhibits the action of TLR9 antagonists on reporter cells lines and the production of cytokines by TLR9 agonists from primary cells. It also has efficacy in limiting TLR9-mediated sterile inflammation in in vivo models of acute liver injury and acute pancreatitis. The identification of a morphinan-based novel small-molecule structure with TLR9 antagonism is a significant step in expanding therapeutic strategies in the field of sterile inflammatory injury.
Asunto(s)
Mediadores de Inflamación/uso terapéutico , Morfinanos/química , Morfinanos/uso terapéutico , Receptor Toll-Like 9/antagonistas & inhibidores , Acetaminofén/uso terapéutico , Animales , Ensayos Clínicos Fase I como Asunto/métodos , Células HEK293 , Humanos , Ligandos , Fallo Hepático Agudo/inmunología , Fallo Hepático Agudo/patología , Fallo Hepático Agudo/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Pancreatitis/inmunología , Pancreatitis/patología , Pancreatitis/prevención & control , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/metabolismo , Estereoisomerismo , Receptor Toll-Like 9/fisiologíaRESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Arrestinas/metabolismo , Ácido Aspártico/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Agonistas de Aminoácidos Excitadores/farmacología , Inflamasomas/efectos de los fármacos , Hígado/efectos de los fármacos , Macrófagos/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/agonistas , Animales , Arrestinas/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caspasa 1/genética , Caspasa 1/metabolismo , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Hígado/inmunología , Hígado/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Pancreatitis/inmunología , Pancreatitis/metabolismo , Pancreatitis/prevención & control , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Arrestina beta 2 , beta-ArrestinasRESUMEN
Implantation of biomaterials and devices into soft tissues leads to the development of the foreign body response (FBR), which can interfere with implant function and eventually lead to failure. The FBR consists of overlapping acute and persistent inflammatory phases coupled with collagenous encapsulation and currently there are no therapeutic options. Initiation of the FBR involves macrophage activation, proceeding to giant cell formation, fibroblast activation, and collagen matrix deposition. Despite the recognition of this sequence of events, the molecular pathways required for the FBR have not been elucidated. We have identified that the acute inflammatory response to biomaterials requires nucleotide-binding domain and leucine-rich repeat-containing 3 (Nlrp3), apoptosis-associated speck-like protein containing CARD (Asc), and caspase-1, as well as plasma membrane cholesterol, and Syk signaling. Full development of the FBR is dependent on Asc and caspase-1, but not Nlrp3. The common antiinflammatory drug aspirin can reduce inflammasome activation and significantly reduce the FBR. Taken together, these findings expand the role of the inflammasome from one of sensing damage associated molecular patterns (DAMPs) to sensing all particulate matter irrespective of size. In addition, implication of the inflammasome in biomaterial recognition identifies key pathways, which can be targeted to limit the FBR.
Asunto(s)
Materiales Biocompatibles/efectos adversos , Caspasa 1/metabolismo , Proteínas del Citoesqueleto/metabolismo , Reacción a Cuerpo Extraño/patología , Inflamasomas/metabolismo , Inflamación/patología , Administración Oral , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Aspirina/administración & dosificación , Aspirina/efectos adversos , Proteínas Adaptadoras de Señalización CARD , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Análisis por Conglomerados , Reacción a Cuerpo Extraño/complicaciones , Reacción a Cuerpo Extraño/enzimología , Reacción a Cuerpo Extraño/inmunología , Células Gigantes/efectos de los fármacos , Células Gigantes/inmunología , Células Gigantes/patología , Inflamación/complicaciones , Inflamación/enzimología , Inflamación/inmunología , Interleucina-1beta/biosíntesis , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microesferas , Proteína con Dominio Pirina 3 de la Familia NLR , Polimetil Metacrilato/efectos adversosRESUMEN
Tissue stress and cell death result in inflammation even in the absence of pathogens. Such sterile inflammation is dependent on a cytosolic complex of proteins inside immune cells termed the inflammasome. This complex converts two groups of extracellular signals into an inflammatory response via activation of caspase-1 and secretion of IL-1ß and IL-18. Group 1 signals are typically TOLL like receptor agonists and result in transcriptional upregulation of inflammasome components and pro-cytokines. Group 2 signals are diverse, ranging from uric acid to ATP, and lead to assembly and activation of the inflammasome complex. Inflammasome components are required for a wide range of acute and chronic pathologies, including experimental alcoholic and non-alcoholic steatohepatitis, and drug-induced liver injury. Collectively, group 1 and 2 signals, inflammasome components, and cytokine receptors provide a rich source of therapeutic targets. Many of the advances in the field have come from standard reductionist experiments. Progress in the understanding of complex human systems will, however, be dependent on novel strategies such as systems analysis, which analyze large data sets to provide new insights.
Asunto(s)
Inflamasomas/fisiología , Hepatopatías/fisiopatología , Hepatopatías/terapia , Transducción de Señal/fisiología , Animales , Caspasa 1/fisiología , Citocinas/fisiología , Modelos Animales de Enfermedad , Humanos , Inflamación/fisiopatologíaRESUMEN
The ability of tissue injury to result in inflammation is a well-recognized phenomenon and is central to a number of common liver and pancreatic diseases including alcoholic steatohepatitis and pancreatitis, as well as drug-induced liver injury, non-alcoholic steatohepatitis, and pancreatitis from other causes. The requirements of extracellular damage-associated molecules and a cytosolic machinery labeled the inflammasome have been established in in vitro culture systems and in vivo disease models. This has provided a generic insight into the pathways involved, and the challenge now is to understand the specifics of these mechanisms in relation to the particular insults and organs involved. One reason for the excitement in this field is that a number of therapeutic candidates such a toll-like receptor antagonists and interleukin-1R antagonists are either approved or in clinical trials for other indications.
Asunto(s)
Inflamasomas , Inflamación/complicaciones , Hepatopatías/tratamiento farmacológico , Hepatopatías/etiología , Terapia Molecular Dirigida , Enfermedades Pancreáticas/tratamiento farmacológico , Enfermedades Pancreáticas/etiología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas , Citosol , Descubrimiento de Drogas , Hígado Graso , Hígado Graso Alcohólico , Humanos , Inflamación/genética , Hepatopatías/genética , Enfermedad del Hígado Graso no Alcohólico , Enfermedades Pancreáticas/genética , Pancreatitis , Receptores de Interleucina-1/antagonistas & inhibidores , Receptores Toll-Like/antagonistas & inhibidoresRESUMEN
Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7-/-), and in wild-type mice. P2X7-/- mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre- or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7-/- mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39-/-) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39-/- mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1ß release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP-induced hepatotoxicity.
Asunto(s)
Acetaminofén/efectos adversos , Antipiréticos/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Receptores Purinérgicos P2X7/fisiología , Acetaminofén/antagonistas & inhibidores , Animales , Antígenos CD/metabolismo , Apirasa/metabolismo , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hemorragia/inducido químicamente , Hemorragia/tratamiento farmacológico , Hemorragia/patología , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , NAD/análogos & derivados , NAD/metabolismo , Necrosis/metabolismo , Piridinas/farmacología , Receptores Purinérgicos P2X7/genética , Transducción de Señal/efectos de los fármacos , Tetrazoles/farmacologíaRESUMEN
BACKGROUND & AIMS: Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) in the biliary epithelium reduces bile flow and alkalinization in patients with cystic fibrosis (CF). Liver damage is believed to result from ductal cholestasis, but only 30% of patients with CF develop liver defects, indicating that another factor is involved. We studied the effects of CFTR deficiency on Toll-like receptor 4 (TLR4)-mediated responses of the biliary epithelium to endotoxins. METHODS: Dextran sodium sulfate (DSS) was used to induce colitis in C57BL/6J-Cftrtm1Unc (Cftr-KO) mice and their wild-type littermates. Ductular reaction and portal inflammation were quantified by keratin-19 and CD45 immunolabeling. Cholangiocytes isolated from wild-type and Cftr-KO mice were challenged with lipopolysaccharide (LPS); cytokine secretion was quantified. Activation of nuclear factor κB (NF-κB), phosphorylation of TLR4, and activity of Src were determined. HEK-293 that expressed the secreted alkaline phosphatase reporter and human TLR4 were transfected with CFTR complementary DNAs. RESULTS: DSS-induced colitis caused biliary damage and portal inflammation only in Cftr-KO mice. Biliary damage and inflammation were not attenuated by restoring biliary secretion with 24-nor-ursodeoxycholic acid but were significantly reduced by oral neomycin and polymyxin B, indicating a pathogenetic role of gut-derived bacterial products. Cftr-KO cholangiocytes incubated with LPS secreted significantly higher levels of cytokines regulated by TLR4 and NF-κB. LPS-mediated activation of NF-κB was blocked by the TLR4 inhibitor TAK-242. TLR4 phosphorylation by Src was significantly increased in Cftr-KO cholangiocytes. Expression of wild-type CFTR in the HEK293 cells stimulated with LPS reduced activation of NF-κB. CONCLUSIONS: CFTR deficiency alters the innate immunity of the biliary epithelium and reduces its tolerance to endotoxin, resulting in an Src-dependent inflammatory response mediated by TLR4 and NF-κB. These findings might be used to develop therapies for CF-associated cholangiopathy.
Asunto(s)
Conductos Biliares/inmunología , Colangitis/inmunología , Colitis/inmunología , Células Epiteliales/inmunología , Inmunidad Innata , Mediadores de Inflamación/metabolismo , FN-kappa B/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Antibacterianos/farmacología , Conductos Biliares/efectos de los fármacos , Conductos Biliares/metabolismo , Conductos Biliares/microbiología , Colagogos y Coleréticos/farmacología , Colangitis/inducido químicamente , Colangitis/genética , Colangitis/metabolismo , Colangitis/microbiología , Colangitis/prevención & control , Colitis/inducido químicamente , Colitis/genética , Colitis/metabolismo , Colitis/microbiología , Citocinas/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células HEK293 , Humanos , Inmunidad Innata/efectos de los fármacos , Queratina-19/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CFTR , Ratones Noqueados , Neomicina/farmacología , Fosforilación , Polimixina B/farmacología , Factores de Tiempo , Receptor Toll-Like 4/genética , Transfección , Ácido Ursodesoxicólico/análogos & derivados , Ácido Ursodesoxicólico/farmacología , Familia-src QuinasasRESUMEN
BACKGROUND & AIMS: Acute pancreatitis is characterized by early activation of intracellular proteases followed by acinar cell death and inflammation. Activation of damage-associated molecular pattern (DAMP) receptors and a cytosolic complex termed the inflammasome initiate forms of inflammation. In this study, we examined whether DAMP-receptors and the inflammasome provide the link between cell death and the initiation of inflammation in pancreatitis. METHODS: Acute pancreatitis was induced by caerulein stimulation in wild-type mice and mice deficient in components of the inflammasome (apoptosis-associated speck-like protein containing a caspase recruitment domain [ASC], NLRP3, caspase-1), Toll-like receptor 9 (TLR9), or the purinergic receptor P2X(7). Resident and infiltrating immune cell populations and pro-interleukin-1ß expression were characterized in control and caerulein-treated adult murine pancreas. TLR9 expression was quantified in pancreatic cell populations. Additionally, wild-type mice were pretreated with a TLR9 antagonist before induction of acute pancreatitis by caerulein or retrograde bile duct infusion of taurolithocholic acid 3-sulfate. RESULTS: Caspase-1, ASC, and NLRP3 were required for inflammation in acute pancreatitis. Genetic deletion of Tlr9 reduced pancreatic edema, inflammation, and pro-IL-1ß expression in pancreatitis. TLR9 was expressed in resident immune cells of the pancreas, which are predominantly macrophages. Pretreatment with the TLR9 antagonist IRS954 reduced pancreatic edema, inflammatory infiltrate, and apoptosis. Pretreatment with IRS954 reduced pancreatic necrosis and lung inflammation in taurolithocholic acid 3-sulfate-induced acute pancreatitis. CONCLUSIONS: Components of the inflammasome, ASC, caspase-1, and NLRP3, are required for the development of inflammation in acute pancreatitis. TLR9 and P2X(7) are important DAMP receptors upstream of inflammasome activation, and their antagonism could provide a new therapeutic strategy for treating acute pancreatitis.
Asunto(s)
Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Páncreas/inmunología , Pancreatitis/inmunología , Transducción de Señal , Receptor Toll-Like 9/metabolismo , Enfermedad Aguda , Animales , Antiinflamatorios/farmacología , Apoptosis , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Caspasa 1/genética , Caspasa 1/metabolismo , Ceruletida , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , ADN/farmacología , Modelos Animales de Enfermedad , Inflamasomas/deficiencia , Inflamasomas/genética , Interleucina-1/metabolismo , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Necrosis , Infiltración Neutrófila , Páncreas/efectos de los fármacos , Páncreas/patología , Pancreatitis/inducido químicamente , Pancreatitis/genética , Pancreatitis/patología , Pancreatitis/prevención & control , Neumonía/inmunología , Neumonía/prevención & control , Precursores de Proteínas/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , ARN Mensajero/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Ácido Taurolitocólico/análogos & derivados , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/deficiencia , Receptor Toll-Like 9/genéticaRESUMEN
BACKGROUND & AIMS: Inflammation plays an important role in the pathogenesis of cholestatic liver injury, but it is unclear whether the inflammasome is involved and is the objective of this study. METHODS: Gene expression was analyzed in the livers of patients with primary biliary cholangitis (n = 15) and primary sclerosing cholangitis (n = 15). Bile duct ligation (BDL) or sham operation was performed in wild-type (WT) and Caspase-1-/- (Casp1-/-) mice for 7 days. Mouse hepatocytes and macrophages were treated with bile acids. RESULTS: Caspase-1, NLRP1, NLRP3 and IL-1ß were significantly increased in the livers of cholestatic patients when compared to healthy control subjects (n = 9). Significantly higher levels of plasma IL-1ß (826 vs 345 pg/ml), ALT (674 vs 482 U/L) and ALP (900 vs 622 U/L) were seen in WT BDL mice compared to Casp1-/- BDL mice. Caspase-1 cleavage was found only in WT BDL livers. Assessment of liver histology indicated more fibrosis in Casp1-/- BDL mice than in WT BDL mice, confirmed by analyses of liver hydroxyproline content and the expression of fibrotic genes. Profiling of immune cells revealed that there were more macrophages in Casp1-/- BDL livers than in WT BDL livers. Further macrophage phenotype characterization indicated that Casp1-/- BDL livers had more M2 anti-inflammatory macrophages evidenced by more CD206 positive cells and higher expression of IL-4, CD163, Fizz1 and IL-33. When mouse hepatocytes and peritoneal macrophages were exposed to cholestatic levels of major endogenous bile acids (300µM TCA), neither IL-1ß induction nor procaspase-1 cleavage were detected. CONCLUSIONS: The inflammasome exacerbates cholestatic liver injury, but bile acids do not directly activate the inflammasome.
Asunto(s)
Colangitis/complicaciones , Colestasis/inmunología , Inflamasomas/inmunología , Fallo Hepático Agudo/inmunología , Hígado/patología , Animales , Conductos Biliares/cirugía , Caspasa 1/genética , Células Cultivadas , Colangitis/inmunología , Colangitis/patología , Colangitis Esclerosante , Colestasis/patología , Modelos Animales de Enfermedad , Hepatocitos , Humanos , Inflamasomas/genética , Ligadura , Hígado/inmunología , Fallo Hepático Agudo/patología , Macrófagos , Ratones , Ratones Noqueados , Cultivo Primario de CélulasRESUMEN
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.
Asunto(s)
Amoníaco/toxicidad , Edema Encefálico/metabolismo , Macrófagos/metabolismo , Linfocitos T/metabolismo , Receptor Toll-Like 9/metabolismo , Acetaminofén/farmacología , Animales , Edema Encefálico/inducido químicamente , Edema Encefálico/tratamiento farmacológico , Edema Encefálico/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Fallo Hepático Agudo/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Oligodesoxirribonucleótidos/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunologíaRESUMEN
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.
Asunto(s)
Digoxina/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Piruvato Quinasa/metabolismo , Activación Transcripcional/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Modelos Animales de Enfermedad , Endotoxinas , Histonas/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/genética , Inflamación/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Oxidación-Reducción , Unión Proteica/efectos de los fármacos , Piruvato Quinasa/química , Células THP-1 , Transcripción Genética/efectos de los fármacosRESUMEN
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.
Asunto(s)
ADN Mitocondrial/fisiología , Enfermedad del Hígado Graso no Alcohólico/inmunología , Receptor Toll-Like 9/metabolismo , Adolescente , Células Cultivadas , Niño , Dieta Alta en Grasa/efectos adversos , Femenino , Expresión Génica , Hepatocitos/metabolismo , Humanos , Macrófagos del Hígado/inmunología , Masculino , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Receptor Toll-Like 9/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The initial injury in acute pancreatitis is characteristically sterile and results in acinar cells necrosis. Intracellular contents released from damaged cells into the extracellular space serve as damage-associated molecular patterns (DAMPs) that trigger inflammation. There is increasing evidence that this sterile inflammatory response mediated through DAMPs released from necrotic acinar cells is a key determinant of further pancreatic injury, remote organ injury, and disease resolution in experimental models. A number of DAMPS, including high-mobility group box protein 1, DNA, adenosine triphosphate and heat shock protein 70, have been shown to have a role in experimental pancreatitis. Many of these DAMPs are also detectable in the human pancreatitis. Genetic deletion and pharmacologic antagonism demonstrate that specific DAMP receptors, including Toll-like receptor (TLR) 4, TLR9, and P2X7, are also required for inflammation in experimental acute pancreatitis. Downstream DAMP-sensing components include nod-like receptor protein 3, caspase 1, interleukin-1ß (IL-1), IL-18, and IL-1 receptor, and also are required for full experimental pancreatitis. These DAMP-mediated pathways provide novel therapeutic targets using antagonists of TLRs and other receptors.