RESUMO
Sepsis represents a major health problem worldwide because of high mortality rates and cost-intensive therapy. Immunomodulatory strategies as a means of controlling overshooting inflammatory responses during sepsis have thus far not been effective, and there is a general paucity of new therapies. Regulatory immune cells have been shown to play important roles in limiting systemic inflammation. However, the signals inducing a regulatory phenotype in myeloid cells during infection are unknown. Here, we report that myeloid cell-intrinsic glycoprotein 130 (gp130) signals constitute a critical element for immune homeostasis during polymicrobial sepsis. We identify an essential role for gp130 signaling in myeloid cells during M2 macrophage polarization in vitro and in vivo. Myeloid cell-specific deletion of gp130 signaling leads to a defective M2 macrophage polarization followed by exacerbated inflammatory responses and increased mortality during sepsis. These data provide new insights into the molecular basis of M1 and M2 phenotypic dichotomy and identify gp130 as a key regulator of immune homeostasis during sepsis. Our study highlights the Janus-faced role of IL-6 family cytokines during inflammation, which may explain the failure of IL-6-targeted anti-inflammatory approaches in the treatment of sepsis.-Sackett, S. D., Otto, T., Mohs, A., Sander, L. E., Strauch, S., Streetz, K. L., Kroy, D. C., Trautwein, C. Myeloid cells require gp130 signaling for protective anti-inflammatory functions during sepsis.
Assuntos
Receptor gp130 de Citocina/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Células Mieloides/metabolismo , Sepse/metabolismo , Animais , Citocinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Homeostase , Humanos , Sistema Imunitário , Interleucina-10/metabolismo , Ativação de Macrófagos , Camundongos , Camundongos Knockout , Fenótipo , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
Tumor necrosis factor (TNF) is a well-known mediator of sepsis. In many cases, sepsis results in multiple organ injury including the lung with acute respiratory distress syndrome (ARDS). More than 20-year-old studies have suggested that TNF may be directly responsible for organ injury during sepsis. However, these old studies are inconclusive, because they relied on human rather than conspecific TNF, which was contaminated with endotoxin in most studies. In this study, we characterized the direct effects of intravenous murine endotoxin-free TNF on cardiovascular functions and organ injury in mice with a particular focus on the lungs. Because of the relevance of the acid sphingomyelinase in sepsis, ARDS, and caspase-independent cell death, we also included acid sphingomyelinase-deficient (ASM-/-) mice. ASM-/- and wild-type (WT) mice received 50 µg endotoxin-free murine TNF intravenously alone or in combination with the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD) and were ventilated at low tidal volume while lung mechanics were followed. Blood pressure was stabilized by intra-arterial fluid support, and body temperature was kept at 37°C to delay lethal shock and to allow investigation of blood gases, lung histopathology, proinflammatory mediators, and microvascular permeability 6 hours after TNF application. Besides the lungs, also the kidneys and liver were examined. TNF elicited the release of inflammatory mediators and a high mortality rate, but failed to injure the lungs, kidneys, or liver of healthy mice significantly within 6 hours. Mortality in WT mice was most likely due to sepsis-like shock, as indicated by metabolic acidosis, high procalcitonin levels, and cardiovascular failure. ASM-/- mice were protected from TNF-induced hypotension and reflex tachycardia and also from mortality. In WT mice, intravenous exogenous TNF does not cause organ injury but induces a systemic inflammatory response with cardiovascular failure, in which the ASM plays a role.
Assuntos
Lesão Pulmonar/metabolismo , Choque/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Permeabilidade Capilar , Endotoxinas/metabolismo , Feminino , Inflamação , Mediadores da Inflamação/metabolismo , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação , Neutrófilos/metabolismo , Oligopeptídeos/farmacologia , Permeabilidade , Respiração Artificial , SepseRESUMO
BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease in Western countries. It is unclear how infiltrating leukocytes affect NASH-development. Our study aims to investigate the role of the homing/receptor, pair mucosal addressin cell adhesion molecule-1 (MAdCAM-1)/ß7-Integrin, on immune cell recruitment and disease progression in a steatohepatitis model. METHODS: Constitutive ß7-Integrin deficient (ß7-/-) and MAdCAM-1 deficient (MAdCAM-1-/-) mice were fed a high fat diet (HFD) for 26weeks or methionine-choline-deficient-diet (MCD) for 4weeks. RESULTS: ß7-/- mice displayed earlier and more progressive steatohepatitis during HFD- and MCD-treatment, while MAdCAM-1-/- mice showed less histomorphological changes. The anti-oxidative stress response was significantly weaker in ß7-/- mice as reflected by a significant downregulation of the transcription factors nuclear-factor(erythroid-derived 2)-like 2 (Nrf2) and heme-oxigenase-1 (HO-1). Additionally, stronger dihydroethidium-staining revealed an increased oxidative stress response in ß7-/- animals. In contrast, MAdCAM-1-/- mice showed an upregulation of the anti-oxidative stress response. ß7-/- animals exhibited stronger hepatic infiltration of inflammatory cells, especially neutrophils, reflecting earlier steatohepatitis initiation. Expression of regulatory T cell (TReg) markers as well as numbers of anti-inflammatory macrophages was significantly enhanced in MAdCAM-1-/- mice. Those changes finally resulted in earlier and stronger collagen accumulation in ß7-/- mice, whereas MAdCAM-1-/- mice were protected from fibrosis initiation. CONCLUSIONS: Adhesion molecule mediated effector cell migration contributes to the outcome of steatohepatitis in the HFD- and the MCD model. While MAdCAM-1 promotes steatohepatitis, ß7-Integrin unexpectedly exerts protective effects. ß7-/- mice show earlier steatohepatitis initiation and significantly stronger fibrosis progression. Accordingly, the interaction of ß7-Integrins and their receptor MAdCAM-1 provide novel targets for therapeutic interventions in steatohepatitis. LAY SUMMARY: The mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is expressed in livers upon diet-induced non-alcoholic steatohepatitis (NASH). Loss of MAdCAM-1 has beneficial effects regarding the development of NASH - manifested by reduced hepatic oxidative stress and decreased inflammation. In contrast, ß7-Integrin-deficiency results in increased steatohepatitis.
Assuntos
Moléculas de Adesão Celular/metabolismo , Cadeias beta de Integrinas/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Animais , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Deficiência de Colina/complicações , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Progressão da Doença , Cadeias beta de Integrinas/genética , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/patologia , Masculino , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucoproteínas , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse OxidativoRESUMO
BACKGROUND & AIMS: Non-alcoholic-fatty-liver disease (NAFLD) is part of the metabolic syndrome. The spectrum of NAFLD includes NASH (non-alcoholic steatohepatitis), which is characterised by progressive inflammation associated with oxidative stress and apoptosis, finally triggering liver cirrhosis and hepatocellular carcinoma. HGF (hepatocyte growth factor)/mesenchymal-epithelial transition factor (c-Met) receptor signalling is known to activate distinct intracellular pathways mediating among others anti-apoptotic properties to hepatocytes. Therefore, the aim was to characterise the role of c-Met during NASH development. METHODS: Hepatocyte specific c-Met knockout mice (c-MetΔ(hepa)) using the cre-loxP system and wild type controls (c-Met(loxP/loxP)) were fed a methionine-choline deficient (MCD) diet. RESULTS: MCD feeding triggered massive steatosis, decreased survival and higher transaminases in c-MetΔ(hepa) livers compared to c-Met(loxP/loxP). Gene array analysis demonstrated that genes involved in fatty acid metabolism were strongly upregulated in c-MetΔ(hepa) livers correlating with higher amounts of hepatic free fatty acids. Consequently, c-MetΔ(hepa) mice showed significantly more TUNEL positive cells and more superoxide anion production than c-Met(loxPloxP) animals. Additionally, c-MetΔ(hepa) livers showed significantly larger fractions of infiltrating neutrophils, macrophages, and cytotoxic T cells. These changes correlated with an enhanced progression of liver fibrosis as evidenced by higher collagen deposition in c-MetΔ(hepa) livers. As increased apoptosis was a prominent feature in c-MetΔ(hepa) livers, we generated c-Met/Casp8Δ(hepa) double knockout mice. In these animals compared to c-MetΔ(hepa) animals the increase in apoptosis could be reverted. CONCLUSIONS: c-Met deletion in hepatocytes triggers NASH progression. A prominent mechanism is higher fatty acid accumulation and increased apoptosis, which in part can be reverted by blocking caspase 8.
Assuntos
Apoptose , Deficiência de Colina , Dieta , Fator de Crescimento de Hepatócito/metabolismo , Inflamação/metabolismo , Cirrose Hepática , Metionina , Hepatopatia Gordurosa não Alcoólica , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Caspase 8/metabolismo , Deficiência de Colina/metabolismo , Dieta/efeitos adversos , Dieta/métodos , Hepatócitos/metabolismo , Lipotrópicos/metabolismo , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/prevenção & controle , Metionina/deficiência , Metionina/metabolismo , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
UNLABELLED: Aberrant expression of the chemokine CXC chemokine ligand (CXCL)10 has been linked to the severity of hepatitis C virus (HCV)-induced liver injury, but the underlying molecular mechanisms remain unclear. In this study, we describe a yet-unknown proapoptotic effect of CXCL10 in hepatocytes, which is not mediated through its cognate chemokine receptor, but the lipopolysaccharide receptor Toll-like receptor 4 (TLR4). To this end, we investigated the link of CXCL10 expression with apoptosis in HCV-infected patients and in murine liver injury models. Mice were treated with CXCL10 or neutralizing antibody to systematically analyze effects on hepatocellular apoptosis in vivo. Direct proapoptotic functions of CXCL10 on different liver cell types were evaluated in detail in vitro. The results showed that CXCL10 expression was positively correlated with liver cell apoptosis in humans and mice. Neutralization of CXCL10 ameliorated concanavalin A-induced tissue injury in vivo, which was strongly associated with reduced liver cell apoptosis. In vitro, CXCL10 mediated the apoptosis of hepatocytes involving TLR4, but not CXC chemokine receptor 3 signaling. Specifically, CXCL10 induced long-term protein kinase B and Jun N-terminal kinase activation, leading to hepatocyte apoptosis by caspase-8, caspase-3, and p21-activated kinase 2 cleavage. Accordingly, systemic application of CXCL10 led to TLR4-induced liver cell apoptosis in vivo. CONCLUSION: The results identify CXCL10 and its noncognate receptor, TLR4, as a proapoptotic signaling cascade during liver injury. Antagonism of the CXCL10/TLR4 pathway might be a therapeutic option in liver diseases associated with increased apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Quimiocina CXCL10/farmacologia , Hepatócitos/patologia , Receptor 4 Toll-Like/fisiologia , Animais , Intoxicação por Tetracloreto de Carbono/patologia , Caspases/metabolismo , Doença Hepática Induzida por Substâncias e Drogas , Quimiocina CXCL10/antagonistas & inibidores , Quimiocina CXCL10/biossíntese , Concanavalina A , Hepatite C/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Humanos , Fígado/patologia , Camundongos , Receptores CXCR3/fisiologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND & AIMS: Receptor mediated cell death through the activation of caspases has been identified as an important mechanism to control life and death in various tissues and is thus crucial for the maintenance of liver tissue homeostasis. Here we investigated how caspase 8 (Casp8) differentially regulates immune-mediated liver injury and regeneration in distinct liver cell types during chronic liver injury. METHODS: Conditional knockout mice with hepatocellular (Casp8(Δhepa)) and ubiquitous deletion of Casp8 (Casp8(ΔMx)) were used in models of cholestatic hepatitis [(DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) treatment, bile duct ligation (BDL) and choline deficient diet with ethionine supplementation (CDE)]. RESULTS: Mice with a hepatocellular deletion of Casp8 (Casp8(Δhepa)) were protected after DDC-treatment. Animals with a ubiquitous conditional Casp8 knockout (Casp8(ΔMx)) displayed a significantly enhanced liver injury in various models of cholestatic liver injury. This was associated with higher transaminases, bilirubin levels and finally more liver fibrosis. However, caspase 3 (Casp3) activity was reduced in both knockout strains, suggesting additionally mechanisms contributing to the phenotype. Casp8(ΔMx) mice displayed a stronger infiltration of mononuclear immune cells and more proliferation of liver-parenchymal cells in periportal areas. Further analysis confirmed that these infiltrating immune cells are resistant against extrinsic apoptosis. Bone-marrow-transplantation (BMT) experiments demonstrated that Casp8-deficient bone marrow derived cells are responsible for increased liver injury in DDC fed mice. CONCLUSIONS: Our results demonstrate that cell-type specific differences in apoptosis resistance mediated by Casp8 deletion are of significant relevance for the outcome of chronic liver injury.
Assuntos
Caspase 8/fisiologia , Colestase/patologia , Hepatócitos/patologia , Animais , Apoptose , Caspase 8/genética , Colangite Esclerosante/complicações , Doença Crônica , Citoproteção , Camundongos , Camundongos Knockout , Piridinas/toxicidadeRESUMO
BACKGROUND: At present hepatocyte transplantation is a promising option for cellular therapy of end-stage liver diseases. However, the underlying molecular mechanisms need to be better defined in order to translate this technique into clinical use. This study investigated the cursiv relevance of hepatocyte growth factor (HGF)/c-Met signalling for hepatocyte repopulation after transplantion. METHODS: Wild-type mice (c-Met(loxP/loxP)) and hepatocyte-specific conditional c-Met (HGF receptor) knockout (c-Met(Δhepa)) mice were used as donors and recipients for hepatocyte transplantation. RESULTS: Transplantation experiments revealed two major findings. First it was demonstrated that c-Met is indispensable in donor cells, as c-Met(Δhepa) cells did not repopulate recipient livers after transplantation. Second, genetic deletion of c-Met in recipient hepatocytes resulted in enhanced expansion of unmodified donor cells in host livers (up to 250-fold after 12 weeks). The relevant mechanisms for this observation in c-Met(Δhepa) host hepatocytes could be defined. c-Met(Δhepa) hepatocytes showed enhanced apoptosis, reduced cellular proliferation and a lack of AKT-kinase and STAT3 activation. In addition, tissue remodelling was changed in c-Met(Δhepa) recipient livers. Therefore, the lack of pro-proliferative transcription factors, increased apoptosis and changes in matrix-remodelling inhibit host cell proliferation in c-Met(Δhepa) recipient livers and thus favour repopulation of transplanted hepatocytes. Therapeutically liver repopulation could be increased through adenoviral expression of NK-4--an inhibitor of HGF signalling--in host hepatocytes. CONCLUSION: HGF/c-Met plays a crucial role in host and donor cells of the liver for the cursiv selection of transplanted hepatocytes. Modulating HGF-dependent signalling seems a promising therapeutic option to favour expansion of transplanted hepatocytes.
Assuntos
Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Hepatócitos/transplante , Regeneração Hepática , Transplante de Fígado/métodos , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Apoptose , Western Blotting , Comunicação Celular , Proliferação de Células , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Fator de Crescimento de Hepatócito/biossíntese , Hepatócitos/citologia , Marcação In Situ das Extremidades Cortadas , Falência Hepática/genética , Falência Hepática/metabolismo , Falência Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Receptores Proteína Tirosina Quinases/biossíntese , Transdução de SinaisRESUMO
The prognosis of liver failure is often determined by infectious and cholestatic complications. As HGF/c-Met and interleukin (IL)-6/gp130 control hepatic cytoprotective pathways, we here investigated their cooperative role during the onset of cholestatic liver injury. Conditional hepatocyte-specific ((Δhepa)) c-Met, gp130 and c-Met/gp130 knockout mice (Cre-loxP system) were subjected to bile duct ligation (BDL) and lipopolysaccharide (LPS) stimulation. gp130(Δhepa) and c-Met/gp130(Δhepa) mice displayed increased lethality associated with severe bacteraemia early after BDL, whereas c-Met(Δhepa) and wild-type mice showed normal survival. Analysis of the innate immune response and the regulation of hepatic antibacterial pathways showed that the LPS-triggered hepatocellular response via the Toll-like receptor-4 pathway was regulated differentially by HGF/c-Met and IL-6/gp130. Activation of p38MAPK, c-Jun N-terminal kinase and signalling transducer and activator of transcription-3 was impaired in gp130(Δ) and c-Met(Δhepa) livers. In addition, the acute-phase response (APR) was reduced in c-Met(Δhepa) livers, whereas gp130(Δhepa) displayed a completely abolished APR. In contrast, TNF-α-dependent NF-κB activation was enhanced in gp130(Δhepa) and c-Met(Δhepa) mice and it was associated with a higher rate of apoptosis and inflammation. Moreover, expression of the neutrophil produced and secreted cathelin-related antimicrobial peptide and of genes related to the inflammasome complex correlated with the strength of the bacterial infection and with TNF-α expression. In conclusion, Gp130 and c-Met are involved in the hepatic antibacterial and innate immune response, control the APR and thus prevent sepsis and liver injury during cholestatic conditions.
Assuntos
Bacteriemia/metabolismo , Ductos Biliares/metabolismo , Ductos Biliares/cirurgia , Receptor gp130 de Citocina/deficiência , Fígado/metabolismo , Proteínas Proto-Oncogênicas c-met/deficiência , Reação de Fase Aguda/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos , Apoptose/fisiologia , Bacteriemia/microbiologia , Carga Bacteriana , Ductos Biliares/microbiologia , Catelicidinas/genética , Catelicidinas/metabolismo , Proliferação de Células , Colestase/metabolismo , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Imunidade Inata/fisiologia , Estimativa de Kaplan-Meier , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Ligadura , Lipopolissacarídeos/farmacologia , Fígado/lesões , Fígado/microbiologia , Fígado/patologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Although acute liver failure is a rare disease, its presence is associated with high morbidity and mortality in affected patients. While a contribution of the immune system to the outcome of toxic liver failure is anticipated, functionally relevant immune cell receptors for liver cell damage need to be better defined. We here investigate the relevance of the chemokine receptor CXCR3, which is important for hepatic immune cell infiltration, in a model of experimental acute liver failure. Liver injury was induced by a single intraperitoneal injection of carbon tetrachloride (CCl(4)) in CXCR3(-/-), CCR1(-/-), CCR5(-/-) and wild-type mice. In this model, CXCR3(-/-) mice displayed augmented liver damage compared with all other mouse strains as assessed by liver histology and serum transaminases 24 and 72 h after injury. Phenotypically, CXCR3(-/-) mice had significantly reduced intrahepatic NK and NKT cells after injury at all investigated time points (all P<0.05), but strongly elevated expression levels of IL1-ß, TNF-α and IFN-γ. In line with a functional role of innate immune cells, wild-type mice depleted for NK cells with an anti-ASIALO GM1 antibody before liver injury also displayed increased liver injury after CCl(4) challenge. CXCR3(-/-) and NK cell-depleted mice show reduced apoptotic liver cells (TUNEL assay), but more necrotic hepatocytes. Functionally, the augmented liver cell necrosis in CXCR3(-/-) and NK cell-depleted mice was associated with increased expression of high mobility group 1 (HMGB1) protein and a consecutive enhanced infiltration of neutrophils into the liver. In conclusion, the results demonstrate a primarily unexpected beneficial role of CXCR3 in acute toxic liver injury. These findings should be taken into account when planning trials with CXCR3 antagonists.
Assuntos
Proteína HMGB1/metabolismo , Falência Hepática Aguda/imunologia , Falência Hepática Aguda/metabolismo , Receptores CXCR3/metabolismo , Animais , Tetracloreto de Carbono/farmacologia , Proteína HMGB1/imunologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Falência Hepática Aguda/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Receptores CCR1/imunologia , Receptores CCR1/metabolismo , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Transaminases/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND & AIMS: Disruption of the nuclear factor-κB (NF-κB) essential modulator (NEMO) in hepatocytes of mice (NEMO(Δhepa) mice) results in spontaneous liver apoptosis and chronic liver disease involving inflammation, steatosis, fibrosis, and development of hepatocellular carcinoma. Activation of caspase-8 (Casp8) initiates death receptor-mediated apoptosis. We investigated the pathogenic role of this protease in NEMO(Δhepa) mice or after induction of acute liver injury. METHODS: We created mice with conditional deletion of Casp8 in hepatocytes (Casp8(Δhepa)) and Casp8(Δhepa)NEMO(Δhepa) double knockout mice. Acute liver injury was induced by Fas-activating antibodies, lipopolysaccharides, or concanavalin A. Spontaneous hepatocarcinogenesis was monitored by magnetic resonance imaging. RESULTS: Hepatocyte-specific deletion of Casp8 protected mice from induction of apoptosis and liver injury by Fas or lipopolysaccharides but increased necrotic damage and reduced survival times of mice given concanavalin A. Casp8(Δhepa)NEMO(Δhepa) mice were protected against steatosis and hepatocarcinogenesis but had a separate, spontaneous phenotype that included massive liver necrosis, cholestasis, and biliary lesions. The common mechanism by which inactivation of Casp8 induces liver necrosis in both injury models involves the formation of protein complexes that included the adaptor protein Fas-associated protein with death domain and the kinases receptor-interacting protein (RIP) 1 and RIP3-these have been shown to be required for programmed necrosis. We demonstrated that hepatic RIP1 was proteolytically cleaved by Casp8, whereas Casp8 inhibition resulted in accumulation of RIP complexes and subsequent liver necrosis. CONCLUSIONS: Inhibition of Casp8 protects mice from hepatocarcinogenesis following chronic liver injury mediated by apoptosis of hepatocytes but can activate RIP-mediated necrosis in an inflammatory environment.
Assuntos
Carcinoma Hepatocelular/enzimologia , Caspase 8/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Neoplasias Hepáticas Experimentais/enzimologia , Animais , Apoptose , Inibidores de Caspase , Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatite Animal/enzimologia , Inflamação/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Knockout , Necrose/enzimologiaRESUMO
RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.
Assuntos
MicroRNAs/metabolismo , MicroRNAs/toxicidade , Conformação de Ácido Nucleico , RNA/metabolismo , RNA/toxicidade , Animais , Vírus da Hepatite B/fisiologia , Humanos , Carioferinas/metabolismo , Fígado/efeitos dos fármacos , Fígado/lesões , Fígado/metabolismo , Fígado/patologia , Regeneração Hepática , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , RNA/química , RNA/genética , Interferência de RNA , Análise de SobrevidaRESUMO
The 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) model leads to chronic cholestatic liver injury and therefore resembles human diseases such as sclerosing cholangitis and forms of metabolic liver diseases. The role of the interleukin-6/glycoprotein 130 (gp130) system in this context is still undefined. Therefore, conditional gp130 knockout and knockin mice were used to achieve hepatocyte-specific deletions of gp130 (gp130(Deltahepa)), gp130-dependent ras (gp130(DeltahepaRas)), and signal transducer and activator of transcription (STAT) (gp130(DeltahepaSTAT)) activation. These mice were treated with a DDC-containing diet and analyzed over time. Mice deficient in hepatic gp130 and STAT signaling showed increased and earlier mortality than wild-type and gp130(DeltahepaRas) animals. Over time, significantly more apoptosis and cholestasis became evident in gp130(Deltahepa) and gp130(DeltahepaSTAT) mice. These mice also displayed increased tumor necrosis factor-alpha expression, a diminished acute-phase response (lack of STAT3 and serum amyloid A activation), and enhanced immune cell infiltration in the liver. These were associated with stronger periportal oval cell activation. In addition, DDC treatment in gp130(Deltahepa) and gp130(DeltahepaSTAT) mice resulted in significantly stronger hepatic stellate cell activation. Long-term analysis revealed the development of severe liver fibrosis in gp130(Deltahepa) and gp130(DeltahepaSTAT) animals, as evidenced by increased collagen accumulation. Here we demonstrate that gp130/STAT signaling in hepatocytes provides protection in a cholestatic hepatitis mouse model. STAT3-dependent signaling pathways in hepatocytes protect from apoptosis and tissue injury, which subsequently reduce oval cell activation and prevent fibrosis progression.
Assuntos
Colangite Esclerosante/patologia , Receptor gp130 de Citocina/metabolismo , Fibrose/metabolismo , Hepatócitos/citologia , Fígado/lesões , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Alelos , Animais , Apoptose , Colestase/patologia , Progressão da Doença , Fígado/patologia , Masculino , Camundongos , Camundongos TransgênicosRESUMO
UNLABELLED: A deregulated cytokine balance is involved in triggering the sequence from steatosis to nonalcoholic steatohepatitis, ultimately leading to liver fibrosis and cancer. To better define the role of proinflammatory interleukin-6 (IL-6)-type cytokines in hepatocytes we investigated the role of IL-6 and its shared receptor, glycoprotein 130 (gp130), in a mouse model of steatohepatitis. IL-6(-/-) mice were fed a choline-deficient, ethionine-supplemented (CDE) diet. Conditional gp130 knockout and knockin mice were used to achieve hepatocyte-specific deletion of gp130 (gp130(Deltahepa)), gp130-dependent rat sarcoma (Ras)-(gp130(DeltahepaRas)), and signal transducers and activators of transcription (STAT)-(gp130(DeltahepaSTAT)) activation. CDE-treated IL-6(-/-) mice showed a significant hepatic steatosis at 2 weeks after feeding. The mice rapidly developed elevated fasting blood glucose, insulin serum levels, and transaminases. To better define IL-6-dependent intracellular pathways, specifically in hepatocytes, we next treated gp130(Deltahepa) mice with a CDE diet. These animals also developed a marked steatosis with hyperglycemia and displayed elevated insulin serum levels. Additionally, gp130(Deltahepa) animals showed an imbalanced inflammatory response with increased hepatic tumor necrosis factor-alpha and decreased adiponectin messenger RNA levels. Dissecting the hepatocyte-specific gp130-dependent pathways revealed a similar disease phenotype in gp130(DeltahepaSTAT) mice, whereas gp130(DeltahepaRas) animals were protected. In CDE-treated mice lack of gp130-STAT3 signaling was associated with immune-cell-infiltration, jun kinase-activation, a blunted acute-phase-response, and elevated transaminases. Furthermore, gp130(Deltahepa) and gp130(DeltahepaSTAT) mice showed beginning signs of liver fibrosis compared to gp130(DeltahepaRas) mice and controls. CONCLUSION: During CDE treatment mice lacking IL-6 and gp130-STAT signaling in hepatocytes are prone to hepatic metabolic changes and inflammation. This ultimately leads to progressive steatohepatitis with signs of liver remodeling. Thus, the presented model allows one to further dissect the role of IL-6/gp130-type signaling in hepatocytes during fatty liver degeneration to define new therapeutic targets in metabolic liver diseases.
Assuntos
Fator 3 Ativador da Transcrição/fisiologia , Receptor gp130 de Citocina/fisiologia , Fígado Gorduroso/etiologia , Hepatite/etiologia , Hepatócitos , Transdução de Sinais/fisiologia , Animais , Causalidade , CamundongosRESUMO
UNLABELLED: Liver fibrosis is a major cause of morbidity and mortality worldwide. Platelets are involved in liver damage, but the underlying molecular mechanisms remain elusive. Here, we investigate the platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) as a molecular mediator of fibrotic liver damage. Serum concentrations and intrahepatic messenger RNA of CXCL4 were measured in patients with chronic liver diseases and mice after toxic liver injury. Platelet aggregation in early fibrosis was determined by electron microscopy in patients and by immunohistochemistry in mice. Cxcl4(-/-) and wild-type mice were subjected to two models of chronic liver injury (CCl(4) and thioacetamide). The fibrotic phenotype was analyzed by histological, biochemical, and molecular analyses. Intrahepatic infiltration of immune cells was investigated by fluorescence-activated cell sorting, and stellate cells were stimulated with recombinant Cxcl4 in vitro. The results showed that patients with advanced hepatitis C virus-induced fibrosis or nonalcoholic steatohepatitis had increased serum levels and intrahepatic CXCL4 messenger RNA concentrations. Platelets were found directly adjacent to collagen fibrils. The CCl(4) and thioacetamide treatment led to an increase of hepatic Cxcl4 levels, platelet activation, and aggregation in early fibrosis in mice. Accordingly, genetic deletion of Cxcl4 in mice significantly reduced histological and biochemical liver damage in vivo, which was accompanied by changes in the expression of fibrosis-related genes (Timp-1 [tissue inhibitor of matrix metalloproteinase 1], Mmp9 [matrix metalloproteinase 9], Tgf-beta [transforming growth factor beta], IL10 [interleukin 10]). Functionally, Cxcl4(-/-) mice showed a strongly decreased infiltration of neutrophils (Ly6G) and CD8(+) T cells into the liver. In vitro, recombinant murine Cxcl4 stimulated the proliferation, chemotaxis, and chemokine expression of hepatic stellate cells. CONCLUSION: The results underscore an important role of platelets in chronic liver damage and imply a new target for antifibrotic therapies.
Assuntos
Plaquetas/fisiologia , Cirrose Hepática Experimental/etiologia , Fator Plaquetário 4/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Quimiotaxia , Células Estreladas do Fígado/fisiologia , Hepatite C/complicações , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Ativação Plaquetária , Fator Plaquetário 4/sangue , Fator Plaquetário 4/genética , RNA Mensageiro/análiseRESUMO
BACKGROUND & AIMS: The hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-Met) system is an essential inducer of hepatocyte growth and proliferation. Although a fundamental role for the HGF receptor c-Met has been shown in acute liver regeneration, its cell-specific role in hepatocytes during chronic liver injury and fibrosis progression has not been determined. METHODS: Hepatocyte-specific c-Met knockout mice (c-Met(Delta hepa)) using the Cre-loxP system were studied in a bile duct ligation (BDL) model. Microarray analyses were performed to define HGF/c-Met-dependent gene expression. RESULTS: Two strategies for c-Met deletion in hepatocytes to generate hepatocyte-specific c-Met knockout mice were tested. Early deletion during embryonic development was lethal, whereas post-natal Cre expression was successful, leading to the generation of viable c-Met(Delta hepa) mice. BDL in these mice resulted in extensive necrosis and lower proliferation rates of hepatocytes. Gene array analysis of c-Met(Delta hepa) mice revealed a significant reduction of anti-apoptotic genes in c-Met-deleted hepatocytes. These findings could be tested functionally because c-Met(Delta hepa) mice showed a stronger apoptotic response after BDL and Jo-2 stimulation. The phenotype was associated with increased expression of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and an enhanced recruitment of neutrophils. Activation of these mechanisms triggered a stronger profibrogenic response as evidenced by increased transforming growth factor-beta(1), alpha-smooth muscle actin, collagen-1alpha messenger RNA expression, and enhanced collagen-fiber staining in c-Met(Delta hepa) mice. CONCLUSIONS: Our results show that deletion of c-Met in hepatocytes leads to more liver cell damage and fibrosis in a chronic cholestatic liver injury model because c-Met triggers survival signals important for hepatocyte recovery.
Assuntos
Apoptose , Colestase Extra-Hepática/complicações , Cirrose Hepática/prevenção & controle , Fígado/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Apoptose/genética , Proliferação de Células , Colestase Extra-Hepática/genética , Colestase Extra-Hepática/metabolismo , Colestase Extra-Hepática/patologia , Doença Crônica , Ducto Colédoco/cirurgia , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica/métodos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatite/metabolismo , Hepatite/patologia , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Mediadores da Inflamação/metabolismo , Ligadura , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Knockout , Necrose , Infiltração de Neutrófilos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-met/deficiência , Proteínas Proto-Oncogênicas c-met/genética , Fatores de TempoRESUMO
BACKGROUND & AIMS: Fibrosis is the hallmark of chronic liver diseases, yet many aspects of its mechanism remain to be defined. Chemokines are ubiquitous chemotactic molecules that mediate many acute and chronic inflammatory conditions, and CXC chemokine genes colocalize with a locus previously shown to include fibrogenic genes. We investigated the roles of the chemokine CXCL9 and its receptor CXCR3 in liver fibrosis. METHODS: The effects of CXCL variants on fibrogenesis were analyzed using samples from patients with hepatitis C virus infection and by induction of fibrosis in CXCR3(-/-) and wild-type mice. In mice, intrahepatic immune cell subsets were investigated and interferon gamma messenger RNA levels were measured at baseline and after injury. Human serum CXCL9 levels were measured and correlated with CXCL9 variant and fibrosis severity. The effects of stimulation with CXCL9 were investigated on human hepatic stellate cells (LX-2). RESULTS: Specific CXCL9 variants were associated with liver fibrosis in mice and humans; CXCL9 serum concentrations correlated with genotypes and levels of fibrosis in patients. In contrast to other chemokines, CXCL9 exerted antifibrotic effects in vitro, suppressing collagen production in LX-2 cells. CXCR3(-/-) mice had increased liver fibrosis; progression was associated with decreased numbers of intrahepatic interferon gamma-positive T cells and reduced interferon gamma messenger RNA, indicating that CXCL9-CXCR3 regulates Th1-associated immune pathways. CONCLUSIONS: This is the first description of a chemokine-based antifibrotic pathway in the liver; antifibrotic therapies might be developed to modulate CXC chemokine levels.
Assuntos
Quimiocina CXCL9/metabolismo , Cirrose Hepática/prevenção & controle , Fígado/imunologia , Receptores CXCR3/metabolismo , Animais , Tetracloreto de Carbono , Células Cultivadas , Quimiocina CXCL9/sangue , Quimiocina CXCL9/genética , Estudos de Coortes , Colágeno/metabolismo , Modelos Animais de Doenças , Genótipo , Células Estreladas do Fígado/imunologia , Hepatite C Crônica/complicações , Hepatite C Crônica/imunologia , Humanos , Interferon gama/metabolismo , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , Receptores CXCR3/deficiência , Receptores CXCR3/genética , Índice de Gravidade de Doença , Células Th1/imunologiaRESUMO
Hepatocyte transplantation (HT) is still restricted by the limited amount of transplantable cells. Therefore, a better understanding of the mechanisms involved in cellular engraftment, proliferation, and in vivo selection is important. Here we aimed to evaluate the role of the interleukin 6 (IL-6)/glycoprotein 130 (gp130) system for liver repopulation. Mice carrying a conditional hepatocyte-specific deletion of the common IL-6 signal transducer gp130 (gp130(Deltahepa)) were used for HT. First, we compared bone marrow transplantation (BMT), partial hepatectomy (PH), and retrorsine treatment of recipient mice to optimize the in vivo selection of transplanted hepatocytes. BMT combined with PH was sufficient to induce a 30-fold increase in the number of transplanted donor hepatocytes, whereas additional retrorsine pretreatment led to an up to 40-fold increase. Next, the influence of gp130 signaling in hepatocytes on cell selection was evaluated. Wild-type (WT) hepatocytes repopulated WT recipients at the same rate as gp130(Deltahepa) cells. In contrast, liver repopulation by transplanted cells was enhanced in gp130(Deltahepa) recipient mice. This was associated with higher proliferation of donor hepatocytes and enhanced apoptosis in gp130(Deltahepa) recipient livers. Additionally, the acute phase response was strongly induced after HT in WT recipients but blunted in gp130(Deltahepa) recipients. As a result, significantly more liver remodeling, evidenced by stronger hepatic stellate cell activation and collagen accumulation, was found in gp130(Deltahepa) mice after HT. In conclusion, the HT model established here can be efficiently applied to investigate cell-specific mechanisms in liver repopulation. Moreover, we have shown that gp130-dependent pathways in host hepatocytes are important for controlling liver repopulation.
Assuntos
Receptor gp130 de Citocina/metabolismo , Hepatócitos/transplante , Fígado/metabolismo , Reação de Fase Aguda/metabolismo , Animais , Colágeno/metabolismo , Células Estreladas do Fígado/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Transdução de SinaisRESUMO
IL-6 is known to play a crucial role in the pathogenesis of chronic intestinal inflammation by modulating T cell functions. In this study, we investigated the role of gp130, the common signal transducer for all IL-6 cytokines, in a murine model of acute T cell independent colitis to better characterize the impact of gp130 on innate immune cells and the early stages of inflammation. Experimental colitis was induced by dextran sulfate sodium treatment of mice with inducible systemic deletion of gp130 (MxCre/gp130(-/-)), macrophage/neutrophil-specific gp130-deficiency (LysCre/gp130(-/-)), or bone marrow chimeric mice and compared with wild-type controls (gp130(f/f)). Systemic deletion of gp130 (MxCre/gp130(-/-)) protected mice from severe colitis and wasting and attenuated the mucosal inflammatory infiltrate as well as local cytokine, chemokine, and adhesion molecule expression. Experiments in newly generated macrophage/neutrophil-specific gp130-deleted animals (LysCre/gp130(-/-)) and gp130 bone marrow chimeric mice, revealed a dual mechanism of proinflammatory effects mediated by gp130. Leukocyte recruitment was impaired in gp130-deleted animals and gp130-deleted recipients of wild-type bone marrow, demonstrating a central role of gp130-dependent signals in nonmyeloid cells for directing leukocytes to sites of inflammation, which was further confirmed in a model of sterile peritonitis. In contrast, macrophage/neutrophil-specific gp130 deficiency delayed and attenuated the disease but only marginally affected the inflammatory infiltrate, indicating a defective activation of mucosal leukocytes. We provide evidence that IL-6 cytokines acting via gp130 are required in the acute stages of intestinal inflammation by modulating the dynamics of innate immune cell recruitment and activation.
Assuntos
Quimiotaxia de Leucócito , Colite/etiologia , Receptor gp130 de Citocina/fisiologia , Ativação de Macrófagos , Ativação de Neutrófilo , Doença Aguda , Animais , Receptor gp130 de Citocina/deficiência , Modelos Animais de Doenças , Imunidade Inata , Inflamação , Interleucina-6/fisiologia , Camundongos , Camundongos Knockout , Transdução de Sinais/imunologiaRESUMO
UNLABELLED: The lack of adequate donor organs is a major limitation to the successful widespread use of liver transplantation for numerous human hepatic diseases. A desirable alternative therapeutic option is hepatocyte transplantation (HT), but this approach is similarly restricted by a shortage of donor cells and by immunological barriers. Therefore, in vivo expansion of tolerized transplanted cells is emerging as a novel and clinically relevant potential alternative cellular therapy. Toward this aim, in the present study we established a new mouse model that combines HT with prior bone marrow transplantation (BMT). Donor hepatocytes were derived from human alpha(1)-antitrypsin (hAAT) transgenic mice of the FVB strain. Serial serum enzyme-linked immunosorbent assays for hAAT protein were used to monitor hepatocyte engraftment and expansion. In control recipient mice lacking BMT, we observed long-term yet modest hepatocyte engraftment. In contrast, animals undergoing additional syngeneic BMT prior to HT showed a 3- to 5-fold increase in serum hAAT levels after 24 weeks. Moreover, complete liver repopulation was observed in hepatocyte-transplanted Balb/C mice that had been transplanted with allogeneic FVB-derived bone marrow. These findings were validated by a comparison of hAAT levels between donor and recipient mice and by hAAT-specific immunostaining. Taken together, these findings suggest a synergistic effect of BMT on transplanted hepatocytes for expansion and tolerance induction. Livers of repopulated animals displayed substantial mononuclear infiltrates, consisting predominantly of CD4(+) cells. Blocking the latter prior to HT abrogated proliferation of transplanted hepatocytes, and this implied an essential role played by CD4(+) cells for in vivo hepatocyte selection following allogeneic BMT. CONCLUSION: The present mouse model provides a versatile platform for investigation of the mechanisms governing HT with direct relevance to the development of clinical strategies for the treatment of human hepatic failure.
Assuntos
Transplante de Medula Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Hepatócitos/transplante , Animais , Citometria de Fluxo , Humanos , Camundongos , Camundongos Transgênicos , Doadores de Tecidos , Imunologia de Transplantes , Tolerância ao Transplante/imunologia , Transplante Homólogo , alfa 1-Antitripsina/genéticaRESUMO
The Nrf2 pathway protects against oxidative stress and induces regeneration of various tissues. Here, we investigated whether Nrf2 protects from sclerosing cholangitis and biliary fibrosis and simultaneously induces liver regeneration. Diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was fed to Nrf2-KO mice (Nrf2-/-), mice with liver-specific hyperactivated Nrf2 (HKeap1-/-) and wild-type (WT) littermates to induce cholangitis, liver fibrosis, and oval cell expansion. HKeap1-/--mice were protected from almost all DDC-induced injury compared with WT and Nrf2-/-. Liver injury in Nrf2-/- and WT mice was mostly similar, albeit Nrf2-/- suffered more from DDC diet as seen for several parameters. Nrf2 activity was especially important for the expression of the hepatic efflux transporters Abcg2 and Abcc2-4, which are involved in hepatic toxin elimination. Surprisingly, cell proliferation was more enhanced in Nrf2-/-- and HKeap1-/--mice compared with WT. Interestingly, Nrf2-/--mice failed to sufficiently activate oval cell expansion after DDC treatment and showed almost no resident oval cell population under control conditions. The resident oval cell population of untreated HKeap1-/--mice was increased and DDC treatment resulted in a stronger oval cell expansion compared with WT. We provide evidence that Nrf2 activation protects from DDC-induced sclerosing cholangitis and biliary fibrosis. Moreover, our data establish a possible role of Nrf2 in oval cell expansion.