RESUMEN
Acute-phase proteins (APPs) are an evolutionarily conserved family of proteins produced mainly in the liver in response to infection and inflammation. Despite vast pro- and antiinflammatory properties ascribed to individual APPs, their collective function during infections remains poorly defined. Using a mouse model of polymicrobial sepsis, we show that abrogation of APP production by hepatocyte-specific gp130 deletion, the signaling receptor shared by IL-6 family cytokines, strongly increased mortality despite normal bacterial clearance. Hepatic gp130 signaling through STAT3 was required to control systemic inflammation. Notably, hepatic gp130-STAT3 activation was also essential for mobilization and tissue accumulation of myeloid-derived suppressor cells (MDSCs), a cell population mainly known for antiinflammatory properties in cancer. MDSCs were critical to regulate innate inflammation, and their adoptive transfer efficiently protected gp130-deficient mice from sepsis-associated mortality. The hepatic APPs serum amyloid A and Cxcl1/KC cooperatively promoted MDSC mobilization, accumulation, and survival, and reversed dysregulated inflammation and restored survival of gp130-deficient mice. Thus, gp130-dependent communication between the liver and MDSCs through APPs controls inflammatory responses during infection.
Asunto(s)
Proteínas de Fase Aguda/inmunología , Inmunidad Innata/inmunología , Hígado/inmunología , Hígado/microbiología , Células Mieloides/inmunología , Sepsis/inmunología , Sepsis/microbiología , Animales , Apoptosis/genética , Apoptosis/inmunología , Bacterias/inmunología , Antígeno CD11b/metabolismo , Movimiento Celular/genética , Movimiento Celular/inmunología , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Receptor gp130 de Citocinas/genética , Receptor gp130 de Citocinas/metabolismo , Perfilación de la Expresión Génica , Hepatocitos/inmunología , Hepatocitos/metabolismo , Hepatocitos/microbiología , Hepatocitos/patología , Inflamación/complicaciones , Inflamación/genética , Inflamación/prevención & control , Hígado/patología , Masculino , Ratones , Células Mieloides/citología , Células Mieloides/metabolismo , Factor de Transcripción STAT3/metabolismo , Sepsis/complicaciones , Sepsis/genética , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Bazo/inmunología , Bazo/microbiología , Bazo/patologíaRESUMEN
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.
Asunto(s)
Colangitis Esclerosante/patología , Receptor gp130 de Citocinas/metabolismo , Fibrosis/metabolismo , Hepatocitos/citología , Hígado/lesiones , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Alelos , Animales , Apoptosis , Colestasis/patología , Progresión de la Enfermedad , Hígado/patología , Masculino , Ratones , Ratones TransgénicosRESUMEN
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.
Asunto(s)
Quimiotaxis de Leucocito , Colitis/etiología , Receptor gp130 de Citocinas/fisiología , Activación de Macrófagos , Activación Neutrófila , Enfermedad Aguda , Animales , Receptor gp130 de Citocinas/deficiencia , Modelos Animales de Enfermedad , Inmunidad Innata , Inflamación , Interleucina-6/fisiología , Ratones , Ratones Noqueados , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND AND OBJECTIVES: This study provides a one-step transcription/real-time (TaqMan probe) PCR assay (TM-PCR) with new consensus primer and probe sequences for generic detection of human pathogenic enteroviruses including difficult to detect ones like for instance Echovirus 30. The amplicon included parts of domain IV and V of the highly conserved internal ribosomal entry site. Generic detection was confirmed by testing a panel of 41 prototypes representing all five human enterovirus/poliovirus species. STUDY DESIGN AND RESULTS: The 95% detection limit was found to be 100 copies per run using in vitro transcribed coxsackievirus B3 RNA. TM-PCR was compared to an in house nested-PCR assay implemented in detecting enterovirus RNA from CSF samples of patients suffering from meningitis and encephalitis. Concordant results were obtained in all samples (11 positive, 101 negative). Specificity was confirmed with laboratory strains of other neurotropic viruses, and by testing 76 CSF samples of patients with encephalomyelitis disseminata, which all gave negative results. CONCLUSIONS: The new TM-PCR is a convincing alternative to conventional PCR protocols for the diagnosis of enterovirus meningitis. The one-step strategy limits hands on time and cross contamination risk combined with accelerated assay procedure of only 100 min.
Asunto(s)
Líquido Cefalorraquídeo/virología , Encefalitis Viral/diagnóstico , Infecciones por Enterovirus/diagnóstico , Enterovirus/aislamiento & purificación , Meningitis Viral/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Secuencia de Bases , Cartilla de ADN/genética , Encefalitis Viral/virología , Enterovirus/clasificación , Enterovirus/genética , Infecciones por Enterovirus/virología , Humanos , Meningitis Viral/virología , Datos de Secuencia Molecular , Sensibilidad y Especificidad , Alineación de SecuenciaRESUMEN
Interleukin-6 (IL-6) via its signal transducer gp130 is an important mediator of liver regeneration involved in protecting from lipopolysaccharide (LPS)-induced liver injury after partial hepatectomy (PH). Here we generated mice either defective (Delta) in hepatocyte-specific gp130-dependent Ras or STAT activation to define their role during liver regeneration. Deletion of gp130-dependent signaling had major impact on acute phase gene (APG) regulation after PH. APG expression was blocked in gp130-DeltaSTAT animals, whereas gp130-DeltaRas mice showed an enhanced APG response and stronger SOCS3 regulation correlating with delayed hepatocyte proliferation. To define the role of SOCS3 during hepatocyte proliferation, primary hepatocytes were co-stimulated with IL-6 and hepatocyte growth factor. Higher SOCS3 expression in gp130-DeltaRas hepatocytes correlated with delayed hepatocyte proliferation. Next, we tested the impact of LPS, mimicking bacterial infection, on liver regeneration. LPS and PH induced SOCS3 and APG in all animal strains and delayed cell cycle progression. Additionally, IL-6/gp130-dependent STAT3 activation in hepatocytes was essential in mediating protection and thus required for maximal proliferation. Unexpectedly, oncostatin M was most strongly induced in gp130-DeltaSTAT animals after PH/LPS-induced stress and was associated with hepatocyte proliferation in this strain. In summary, gp130-dependent STAT3 activation and concomitant SOCS3 during liver regeneration is involved in timing of DNA synthesis and protects hepatocyte proliferation during stress conditions.
Asunto(s)
Receptor gp130 de Citocinas/metabolismo , Hepatocitos/metabolismo , Interleucina-6/metabolismo , Regeneración Hepática/fisiología , Hígado/metabolismo , Reacción de Fase Aguda/genética , Reacción de Fase Aguda/metabolismo , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/metabolismo , Proliferación Celular/efectos de los fármacos , Receptor gp130 de Citocinas/genética , ADN/biosíntesis , Femenino , Hepatectomía , Interleucina-6/genética , Lipopolisacáridos/toxicidad , Hígado/lesiones , Regeneración Hepática/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
BACKGROUND: Sepsis and septic shock caused by gram-positive bacteria have become increasingly frequent clinical problems. These conditions are accompanied by an overwhelming inflammation in which the liver plays a central role as a source and target of inflammatory mediators. Sepsis is still associated with high mortality rates, and new intervention strategies directed at ameliorating the extent of the inflammatory reaction are strongly needed. Here, we investigated whether blockage of the transducer gp130, a receptor involved in the regulation of the inflammatory response, might be useful in the treatment of experimental gram-positive sepsis. METHODS: An experimental model of gram-positive sepsis was used in which liver-specific gp130-deficient mice (FVB/n alfpCre+ gp130(LoxP/LoxP)) and wild-type mice (FVB/n gp130(LoxP/LoxP)) were intravenously infected with Streptococcus pyogenes. The following parameters were monitored: mortality, bacterial loads in systemic organs, serum inflammatory cytokine levels, and organ damage. RESULTS: We show that infected gp130-deficient mice survived significantly longer, had lower bacterial loads, and developed organ damage more slowly than infected wild-type mice. Furthermore, levels of interferon- gamma , interleukin-6, and the chemokine cytokine-induced neutrophil chemoattractant were significantly lower in gp130-deficient mice than in wild-type mice. Histopathological examination of livers showed lower amounts of neutrophil infiltration, apoptosis, and tissue damage in infected gp130-deficient mice than in wild-type mice. CONCLUSION: Our results demonstrate that the gp130 receptor is involved in the regulation of inflammation during gram-positive sepsis and that blockage of gp130 signaling in hepatocytes could constitute a novel target for adjunctive therapy in patients with sepsis.
Asunto(s)
Receptor gp130 de Citocinas/metabolismo , Hepatocitos/metabolismo , Inflamación/metabolismo , Interleucina-6/metabolismo , Infecciones Estreptocócicas/metabolismo , Streptococcus pyogenes/fisiología , Animales , Receptor gp130 de Citocinas/genética , Eliminación de Gen , Regulación de la Expresión Génica , Ratones , Sepsis/metabolismo , Sepsis/microbiología , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: Hepcidin is a peptide hormone that is central to the regulation of iron homeostasis. In response to interleukin 6 (IL-6), hepatocytes produce hepcidin that decreases iron release/transfer from enterocytes and macrophages and causes hypoferremia. To clarify the molecular pathways involved in hepcidin activation by IL-6, we used different mice strains in which the main IL-6/gp130 signaling pathways have been genetically disrupted. METHODS: We generated mice with hepatocyte-specific deletion of the IL-6 signal-transducing gp130 receptor (alfpgp130 (LoxP/LoxP)), with a gp130 receptor lacking the essential region for STAT1 and -3 activation (alfpCre gp130(DeltaSTAT/LoxP)) or mice expressing a gp130 allele lacking the essential tyrosine for RAS-MAPK activation (alfpCregp130(Y757F/LoxP)). We studied gp130-dependent pathways and hepcidin mRNA expression by Western blot, reverse-transcription polymerase chain reaction, and Northern blot in vivo and ex vivo. RESULTS: IL-6 stimulated phospho STAT3, serum amyloid A (SAA), and suppressor of cytokine signaling 3 (SOCS3) expression in livers of wild-type and alfpCregp130(Y757F/LoxP) mice, whereas this response was blocked in alfpCre gp130(LoxP/LoxP) and alfpCre gp130(DeltaSTAT/LoxP) mice. In wild-type and alfpCregp130(Y757F/LoxP) animals, significantly higher hepcidin mRNA expression was found 3 to 6 hours after IL-6 stimulation. In contrast, no IL-6-dependent regulation of hepcidin mRNA expression was found in alfpgp130 (DeltaSTAT/LoxP) and AlfpCre gp130 (LoxP/LoxP) animals. In primary hepatocytes, higher hepcidin mRNA expression after IL-6 stimulation was only observed when gp130-STAT3-dependent signaling was intact. CONCLUSIONS: We have demonstrated that both in vivo and in vitro STAT3 is the key transcription factor responsible for IL-6 activation of hepcidin gene expression in the liver.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Receptor gp130 de Citocinas/metabolismo , Hepatocitos/fisiología , Factor de Transcripción STAT3/metabolismo , Animales , Receptor gp130 de Citocinas/genética , Hepcidinas , Interleucina-6/metabolismo , Interleucina-6/farmacología , Hierro/metabolismo , Ratones , Ratones Mutantes , Fosforilación , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Cyclin E1 controls G1/S phase transition of the eukaryotic cell cycle. We report the impact of alternative spliced cyclin E1 isoforms on cell cycle regulation in hepatocytes. We show that expression of new cyclin E1 mRNA variants IN3, Delta4, and Delta5 is associated with retarded proliferation in murine hepatocellular carcinoma. Additionally, we demonstrate that a new cyclin E1 isoform Delta3/8 lacking the central part of wild-type mRNA is expressed predominantly in nonproliferating murine hepatocytes. Following partial hepatectomy, Delta3/8 is downregulated when hepatocytes enter the cell cycle from quiescence. The Delta3/8 protein does not exhibit any cyclin box motif but binds cyclin-dependent kinase 2 without stimulating kinase activity. We demonstrate that Delta3/8 lacks any nuclear localization signal and is exclusively located in the cytoplasm. Overexpression of Delta3/8 in cultured cells leads to a delayed G0-G1 transition, indicating that this splice variant helps to maintain a quiescent state of hepatocytes. In conclusion, we identified an isoform of cyclin E1 involved in G0 maintenance and suggest an additional mechanism for cell cycle control.