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BACKGROUND: Macrophages play an important role in maintaining liver homeostasis and regeneration. However, it is not clear to what extent the different macrophage populations of the liver differ in terms of their activation state and which other liver cell populations may play a role in regulating the same. METHODS: Reverse transcription PCR, flow cytometry, transcriptome, proteome, secretome, single cell analysis, and immunohistochemical methods were used to study changes in gene expression as well as the activation state of macrophages in vitro and in vivo under homeostatic conditions and after partial hepatectomy. RESULTS: We show that F4/80+/CD11bhi/CD14hi macrophages of the liver are recruited in a C-C motif chemokine receptor (CCR2)-dependent manner and exhibit an activation state that differs substantially from that of the other liver macrophage populations, which can be distinguished on the basis of CD11b and CD14 expressions. Thereby, primary hepatocytes are capable of creating an environment in vitro that elicits the same specific activation state in bone marrow-derived macrophages as observed in F4/80+/CD11bhi/CD14hi liver macrophages in vivo. Subsequent analyses, including studies in mice with a myeloid cell-specific deletion of the TGF-ß type II receptor, suggest that the availability of activated TGF-ß and its downregulation by a hepatocyte-conditioned milieu are critical. Reduction of TGF-ßRII-mediated signal transduction in myeloid cells leads to upregulation of IL-6, IL-10, and SIGLEC1 expression, a hallmark of the activation state of F4/80+/CD11bhi/CD14hi macrophages, and enhances liver regeneration. CONCLUSIONS: The availability of activated TGF-ß determines the activation state of specific macrophage populations in the liver, and the observed rapid transient activation of TGF-ß may represent an important regulatory mechanism in the early phase of liver regeneration in this context.
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Regeneración Hepática , Factor de Crecimiento Transformador beta , Animales , Ratones , Expresión Génica , Hepatocitos/metabolismo , Macrófagos/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The expression of acute-phase proteins (APP's) maintains homeostasis and tissue repair, but also represents a central component of the organism's defense strategy, especially in the context of innate immunity. Accordingly, an inflammatory response is accompanied by significant changes in the serum protein composition, an aspect that is also used diagnostically. As the main site of APP synthesis the liver is constantly exposed to antigens or pathogens via blood flow, but also to systemic inflammatory signals originating either from the splanchnic area or from the circulation. Under both homeostatic and acute-phase response (APR) conditions the composition of APP's is determined by the pattern of regulatory mediators derived from the systemic circulation or from local cell populations, especially liver macrophages. The key regulators mentioned here most frequently are IL-1ß, IL-6 and TNF-α. In addition to a variety of molecular mediators described mainly on the basis of in vitro studies, recent data emphasize the in vivo relevance of cellular key effectors as well as molecular key mediators and protein modifications for the regulation and function of APP's. These are aspects, on which the present review is primarily focused.
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Proteínas de Fase Aguda , Animales , Humanos , Hígado , Factor de Necrosis Tumoral alfaRESUMEN
The induction of an interferon-mediated response is the first line of defense against pathogens such as viruses. Yet, the dynamics and extent of interferon alpha (IFNα)-induced antiviral genes vary remarkably and comprise three expression clusters: early, intermediate and late. By mathematical modeling based on time-resolved quantitative data, we identified mRNA stability as well as a negative regulatory loop as key mechanisms endogenously controlling the expression dynamics of IFNα-induced antiviral genes in hepatocytes. Guided by the mathematical model, we uncovered that this regulatory loop is mediated by the transcription factor IRF2 and showed that knock-down of IRF2 results in enhanced expression of early, intermediate and late IFNα-induced antiviral genes. Co-stimulation experiments with different pro-inflammatory cytokines revealed that this amplified expression dynamics of the early, intermediate and late IFNα-induced antiviral genes can also be achieved by co-application of IFNα and interleukin1 beta (IL1ß). Consistently, we found that IL1ß enhances IFNα-mediated repression of viral replication. Conversely, we observed that in IL1ß receptor knock-out mice replication of viruses sensitive to IFNα is increased. Thus, IL1ß is capable to potentiate IFNα-induced antiviral responses and could be exploited to improve antiviral therapies.
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Regulación Viral de la Expresión Génica/efectos de los fármacos , Factor 2 Regulador del Interferón/metabolismo , Interferón-alfa/farmacología , Coriomeningitis Linfocítica/tratamiento farmacológico , Virus de la Coriomeningitis Linfocítica/efectos de los fármacos , Receptores Tipo I de Interleucina-1/fisiología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Hepatocitos/virología , Humanos , Factor 2 Regulador del Interferón/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/patología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/aislamiento & purificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estabilidad del ARNRESUMEN
The p38MAPK downstream targets MAPKAP kinases (MK) 2 and 3 are critical for the regulation of the macrophage response to LPS. The extents to which these two kinases act cooperatively and distinctly in regulating LPS-induced inflammatory cytokine expression are still unclear. To address this uncertainty, whole transcriptome analyses were performed using bone marrow-derived macrophages (BMDM) generated from MK2-/- or MK2/3-/- animals and their wild-type littermates. The results suggest that in BMDM, MK2 and MK3 not only cooperatively regulate the transcript expression of signaling intermediates, including IL-10, IL-19, CXCL2 and the IL-4 receptor (IL-4R)α subunit, they also exert distinct regulatory effects on the expression of specific transcripts. Based on the differential regulation of gene expression by MK2 and MK3, at least six regulatory patterns were identified. Importantly, we confirmed our previous finding, which showed that in the absence of MK2, MK3 negatively regulates IFN-ß. Moreover, this genome-wide analysis identified the regulation of Cr1A, NOD1 and Serpina3f as similar to that of IFN-ß. In the absence of MK2, MK3 also delayed the nuclear translocation of NFκB by delaying the ubiquitination and subsequent degradation of IκBß, reflecting the substantial plasticity of the response of BMDM to LPS.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transcriptoma , Animales , Células Cultivadas , Quimiocina CXCL2 , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND & AIMS: To affect immune response and inflammation, the hepatitis C virus (HCV) substantially influences intercellular communication pathways that are decisive for immune cell recruitment. The present study investigates mechanisms by which HCV modulates chemokine-mediated intercellular communication from infected cells. METHODS: Chemokine expression was studied in HCVcc-infected cell lines or cell lines harbouring a subgenomic replicon, as well as in serum samples from patients. Expression or activity of mediators and signalling intermediates was manipulated using knockdown approaches or specific inhibitors. RESULTS: HCV enhances expression of CXCR2 ligands in its host cell via the induction of epidermal growth factor (EGF) production. Knockdown of EGF or of the p65 subunit of the NF-κB complex results in a substantial downregulation of HCV-induced CXCR2 ligand expression, supporting the involvement of an EGF-dependent mechanism as well as activation of NF-κB. Furthermore, HCV upregulates expression of CXCR2 ligands in response to EGF stimulation via downregulation of the T-cell protein tyrosine phosphatase (TC-PTP [PTPN2]), activation of NF-κB, and enhancement of EGF-inducible signal transduction via MEK1 (MAP2K1). This results in the production of a cytokine/chemokine pattern by the HCV-infected cell that can recruit neutrophils but not monocytes. CONCLUSIONS: These data reveal a novel EGF-dependent mechanism by which HCV influences chemokine-mediated intercellular communication. We propose that this mechanism contributes to modulation of the HCV-induced inflammation and the antiviral immune response. LAY SUMMARY: In most cases hepatitis C virus (HCV) results in chronic infection and persistent viral replication, taking decades until development of overt disease. To achieve such a course, the respective virus must have developed mechanisms to circumvent antiviral response, to modulate the inflammatory response and to utilise the infrastructure of its host with moderate effect on its viability. The present study provides novel data indicating that HCV induces epidermal growth factor production in its host cell, enhancing epidermal growth factor-inducible expression of chemokines that bind to the CXCR2 receptor and recruit neutrophile granulocytes. Importantly, chemokines are critical mediators determining the pattern of immune cells recruited to the site of injury and thereby the local inflammatory and immunological milieu. These data strongly suggest that HCV triggers mechanisms that enable the virus to influence the inflammatory and immunological processes of its host.
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Comunicación Celular/inmunología , Factor de Crecimiento Epidérmico , Hepacivirus/fisiología , Hepatitis C Crónica , Inflamación , Receptores de Interleucina-8B/inmunología , Transducción de Señal/inmunología , Línea Celular , Factor de Crecimiento Epidérmico/inmunología , Factor de Crecimiento Epidérmico/metabolismo , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunidad Celular , Inflamación/inmunología , Inflamación/virología , Regulación hacia Arriba , Replicación Viral/fisiologíaRESUMEN
Cholestasis is caused by autoimmune reactions, drug-induced hepatotoxicity, viral infections of the liver and the obstruction of bile ducts by tumours or gallstones. Cholestatic conditions are associated with impaired innate and adaptive immunity, including alterations of the cellular functions of monocytes, macrophages, NK cells and T-cells. Bile acids act as signalling molecules, affecting lipopolysaccharide (LPS)-induced cytokine expression in primary human macrophages. The present manuscript investigates the impact of bile acids, such as taurolithocholic acid (TLC), on the transcriptome of human macrophages in the presence or absence of LPS. While TLC itself has almost no effect on gene expression under control conditions, this compound modulates the expression of 202 out of 865 transcripts in the presence of LPS. Interestingly, pathway analysis revealed that TLC specifically supressed the expression of genes involved in mediating pro-inflammatory effects, phagocytosis, interactions with pathogens and autophagy as well as the recruitment of immune cells, such as NK cells, neutrophils and T cells. These data indicate a broad influence of bile acids on inflammatory responses and immune functions in macrophages. These findings may contribute to the clinical observation that patients with cholestasis present a lack of response to bacterial or viral infections.
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Ácidos y Sales Biliares/metabolismo , Reprogramación Celular , Macrófagos/metabolismo , Fenotipo , Ácidos y Sales Biliares/farmacología , Biomarcadores , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Quimiotaxis , Citocinas/genética , Citocinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Lipopolisacáridos/inmunología , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , TranscriptomaRESUMEN
IL-6 is a central mediator of the immediate induction of hepatic acute phase proteins (APP) in the liver during infection and after injury, but increased IL-6 activity has been associated with multiple pathological conditions. In hepatocytes, IL-6 activates JAK1-STAT3 signaling that induces the negative feedback regulator SOCS3 and expression of APPs. While different inhibitors of IL-6-induced JAK1-STAT3-signaling have been developed, understanding their precise impact on signaling dynamics requires a systems biology approach. Here we present a mathematical model of IL-6-induced JAK1-STAT3 signaling that quantitatively links physiological IL-6 concentrations to the dynamics of IL-6-induced signal transduction and expression of target genes in hepatocytes. The mathematical model consists of coupled ordinary differential equations (ODE) and the model parameters were estimated by a maximum likelihood approach, whereas identifiability of the dynamic model parameters was ensured by the Profile Likelihood. Using model simulations coupled with experimental validation we could optimize the long-term impact of the JAK-inhibitor Ruxolitinib, a therapeutic compound that is quickly metabolized. Model-predicted doses and timing of treatments helps to improve the reduction of inflammatory APP gene expression in primary mouse hepatocytes close to levels observed during regenerative conditions. The concept of improved efficacy of the inhibitor through multiple treatments at optimized time intervals was confirmed in primary human hepatocytes. Thus, combining quantitative data generation with mathematical modeling suggests that repetitive treatment with Ruxolitinib is required to effectively target excessive inflammatory responses without exceeding doses recommended by the clinical guidelines.
RESUMEN
OBJECTIVE: Bone morphogenetic protein (BMP)-9, a member of the transforming growth factor-ß family of cytokines, is constitutively produced in the liver. Systemic levels act on many organs and tissues including bone and endothelium, but little is known about its hepatic functions in health and disease. DESIGN: Levels of BMP-9 and its receptors were analysed in primary liver cells. Direct effects of BMP-9 on hepatic stellate cells (HSCs) and hepatocytes were studied in vitro, and the role of BMP-9 was examined in acute and chronic liver injury models in mice. RESULTS: Quiescent and activated HSCs were identified as major BMP-9 producing liver cell type. BMP-9 stimulation of cultured hepatocytes inhibited proliferation, epithelial to mesenchymal transition and preserved expression of important metabolic enzymes such as cytochrome P450. Acute liver injury caused by partial hepatectomy or single injections of carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) into mice resulted in transient downregulation of hepatic BMP-9 mRNA expression. Correspondingly, LPS stimulation led to downregulation of BMP-9 expression in cultured HSCs. Application of BMP-9 after partial hepatectomy significantly enhanced liver damage and disturbed the proliferative response. Chronic liver damage in BMP-9-deficient mice or in mice adenovirally overexpressing the selective BMP-9 antagonist activin-like kinase 1-Fc resulted in reduced deposition of collagen and subsequent fibrosis. CONCLUSIONS: Constitutive expression of low levels of BMP-9 stabilises hepatocyte function in the healthy liver. Upon HSC activation, endogenous BMP-9 levels increase in vitro and in vivo and high levels of BMP-9 cause enhanced damage upon acute or chronic injury.
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Lesión Pulmonar Aguda/fisiopatología , Factor 2 de Diferenciación de Crecimiento/metabolismo , Factor 2 de Diferenciación de Crecimiento/farmacología , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/fisiología , Cirrosis Hepática/metabolismo , Regeneración Hepática/efectos de los fármacos , Lesión Pulmonar Aguda/genética , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Factor 2 de Diferenciación de Crecimiento/antagonistas & inhibidores , Factor 2 de Diferenciación de Crecimiento/genética , Hepatectomía , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Lipopolisacáridos/farmacología , Cirrosis Hepática/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The IL-1ß induced activation of the p38MAPK/MAPK-activated protein kinase 2 (MK2) pathway in hepatocytes is important for control of the acute phase response and regulation of liver regeneration. Many aspects of the regulatory relevance of this pathway have been investigated in immune cells in the context of inflammation. However, very little is known about concentration-dependent activation kinetics and signal propagation in hepatocytes and the role of MK2. We established a mathematical model for IL-1ß-induced activation of the p38MAPK/MK2 pathway in hepatocytes that was calibrated to quantitative data on time- and IL-1ß concentration-dependent phosphorylation of p38MAPK and MK2 in primary mouse hepatocytes. This analysis showed that, in hepatocytes, signal transduction from IL-1ß via p38MAPK to MK2 is characterized by strong signal amplification. Quantification of p38MAPK and MK2 revealed that, in hepatocytes, at maximum, 11.3% of p38MAPK molecules and 36.5% of MK2 molecules are activated in response to IL-1ß. The mathematical model was experimentally validated by employing phosphatase inhibitors and the p38MAPK inhibitor SB203580. Model simulations predicted an IC50 of 1-1.2 µm for SB203580 in hepatocytes. In silico analyses and experimental validation demonstrated that the kinase activity of p38MAPK determines signal amplitude, whereas phosphatase activity affects both signal amplitude and duration. p38MAPK and MK2 concentrations and responsiveness toward IL-1ß were quantitatively compared between hepatocytes and macrophages. In macrophages, the absolute p38MAPK and MK2 concentration was significantly higher. Finally, in line with experimental observations, the mathematical model predicted a significantly higher half-maximal effective concentration for IL-1ß-induced pathway activation in macrophages compared with hepatocytes, underscoring the importance of cell type-specific differences in pathway regulation.
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Hepatocitos/metabolismo , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Células Cultivadas , Hepatocitos/citología , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
BACKGROUND/AIMS: Viral infections represent a global health problem with the need for new viral therapies and better understanding of the immune response during infection. The most immediate and potent anti-viral defense mechanism is the production of type I interferon (IFN-I) which are activated rapidly following recognition of viral infection by host pathogen recognition receptors (PRR). The mechanisms of innate cellular signaling downstream of PRR activation remain to be fully understood. In the present study, we demonstrate that CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD/RAIDD) is a critical component in type I IFN production. METHODS: The role of RAIDD during IFN-I production was investigated using western blot, shRNA mediated lentiviral knockdown, immunoprecipitation and IFN-I driven dual luciferase assay. RESULTS: Immunoprecipitation analysis revealed the molecular interaction of RAIDD with interferon regulatory factor 7 (IRF7) and its phosphorylating kinase IKKε. Using an IFN-4α driven dual luciferase analysis in RAIDD deficient cells, type I IFN activation by IKKε and IRF7 was dramatically reduced. Furthermore, deletion of either the caspase recruitment domain (CARD) or death domain (DD) of RAIDD inhibited IKKε and IRF7 mediated interferon-4α activation. CONCLUSION: We have identified that the adaptor molecule RAIDD coordinates IKKε and IRF7 interaction to ensure efficient expression of type I interferon.
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Proteína Adaptadora de Señalización CRADD/genética , Quinasa I-kappa B/genética , Factor 7 Regulador del Interferón/genética , Receptor Toll-Like 3/genética , Animales , Proteína Adaptadora de Señalización CRADD/inmunología , Dominio de Reclutamiento y Activación de Caspasas , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Quinasa I-kappa B/inmunología , Factor 7 Regulador del Interferón/inmunología , Interferón-alfa/genética , Interferón-alfa/inmunología , Interferón beta/genética , Interferón beta/inmunología , Lentivirus/genética , Lentivirus/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Plásmidos/química , Plásmidos/metabolismo , Poli I-C/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transducción de Señal , Receptor Toll-Like 3/inmunologíaRESUMEN
Macrophages are cells with remarkable plasticity. They integrate signals from their microenvironment leading to context-dependent polarization into classically (M1) or alternatively (M2) activated macrophages, representing two extremes of a broad spectrum of divergent phenotypes. Thereby, macrophages deliver protective and pro-regenerative signals towards injured tissue but, depending on the eliciting damage, may also be responsible for the generation and aggravation of tissue injury. Although incompletely understood, there is emerging evidence that macrophage polarization is critical for these antagonistic roles. To identify activation-specific expression patterns of chemokines and cytokines that may confer these distinct effects a systems biology approach was applied. A comprehensive literature-based Boolean model was developed to describe the M1 (LPS-activated) and M2 (IL-4/13-activated) polarization types. The model was validated using high-throughput transcript expression data from murine bone marrow derived macrophages. By dynamic modeling of gene expression, the chronology of pathway activation and autocrine signaling was estimated. Our results provide a deepened understanding of the physiological balance leading to M1/M2 activation, indicating the relevance of co-regulatory signals at the level of Akt1 or Akt2 that may be important for directing macrophage polarization.
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Citocinas/genética , Expresión Génica/genética , Inflamación/genética , Activación de Macrófagos/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Animales , Células Cultivadas , Biología Computacional , Citocinas/inmunología , Citocinas/metabolismo , Expresión Génica/inmunología , Perfilación de la Expresión Génica , Inflamación/metabolismo , Activación de Macrófagos/inmunología , Ratones , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND & AIMS: The kinase p38(MAPK) and its downstream target MAPKAP kinase (MK) 2 are critical regulators of inflammatory responses towards pathogens. To date, the relevance of MK2 for regulating IL-10 expression and other cytokine responses towards cytomegalovirus (CMV) infection and the impact of this pathway on viral replication in vitro and in vivo is unknown and the subject of this study. METHODS: The effect of MK2, interferon-α receptor (IFNAR)1, tristetraprolin (TTP) and IL-10 on mouse (M)CMV virus titres, cytokine expression, signal transduction, transcript stability, liver enzymes release, immune cell recruitment and aggregation in response to MCMV infection were studied ex vivo in hepatocytes and macrophages, as well as in vivo. RESULTS: MK2 is critical for MCMV-induced production of IL-10, IFN-α2 and 4, IFN-ß, IL-6, and TNF-α but not for IFN-γ. The MCMV-induced IL-10 production requires activation of IFNAR1 and is further regulated by MK2 and TTP-dependent stabilization of IL-10 transcripts. MK2(-/-) mice are able to control acute MCMV replication, despite deregulated cytokine production. This may be related to the observation that MCMV-infected MK2(-/-) mice show enhanced formation of focal intrahepatic lymphocyte infiltrates resembling intrahepatic myeloid cell aggregates of T cell expansion (iMATEs), which were also observed in MCMV-infected IL-10(-/-) mice but are almost absent in MCMV-infected wild-type controls. CONCLUSIONS: The data suggest that MK2 is critical for regulating cytokine responses towards acute MCMV infection, including that of IL-10 via IFNARI-mediated circuits. MCMV stimulates expression of MK2-dependent cytokines, in particular IL-10 and thereby prevents enhanced formation of intrahepatic iMATE-like cellular aggregates.
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Infecciones por Citomegalovirus , Interleucina-10/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado , Células Mieloides/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Agregación Celular/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/patología , Interferón-alfa/metabolismo , Hígado/metabolismo , Hígado/patología , Ratones , Receptor de Interferón alfa y beta/metabolismo , Tristetraprolina/metabolismoRESUMEN
The induction of suppressor of cytokine signalling (SOCS)3 expression context dependently involves regulation of SOCS3 transcript stability as previously demonstrated for MAPK activated protein kinase (MK)2-dependent regulation of SOCS3 expression by TNFα (Ehlting et al., 2007). In how far the IL-6-type cytokine OSM, which in contrast to IL-6 is a strong activator of p38(MAPK)/MK2 signalling, also involves regulation of transcript stability and activation of MK2 to induce SOCS3 expression is unclear. In contrast to IL-6, OSM induces SOCS3 expression in murine fibroblasts and in primary human and murine hepatocytes, but not in macrophages because the latter lack the OSM receptor (OSMR)ß subunit. Evidence is provided that regulation of OSM-induced expression of SOCS3 involves MEK1- and Erk1/2-mediated stabilization of the SOCS3 transcript. Consistently, OSM-induced stabilization of the SOCS3 transcript is impaired in the presence of inhibitors that specifically block activation of MEK1/2 (U0126) and ERK1/2 (FR180204) or upon knock-down of ERK1/2 expression using specific siRNA. As a potential target site that integrates the stability regulating effect of OSM and OSM-induced activation of MEK1/2 and ERK1/2 a region containing three copies of a pentameric AUUUA motif located within position 2422 and 2541 in closed proximity to the 3' UTR of the SOCS3 transcript has been identified. Unexpectedly, activation of the p38(MAPK)/MK2 pathway, which apart from STAT3 and ERK1/2, is also strongly activated by OSM in human and murine hepatocytes and murine fibroblasts is dispensable for stabilization of the SOCS3 transcript as suggested from inhibitor studies using the p38(MAPK) inhibitor SB203580 or from the analysis of MK2-deficient hepatocytes. However, analysis of MK2-deficient macrophages and hepatocytes revealed that, although MK2 is dispensable for regulation of OSM-induced SOCS3 expression, MK2 is essential for LPS-induced OSM production in macrophages and limits the overall availability of the OSMRß subunit in hepatocytes. Thus MK2 plays a role for the induction and sensing of OSM-mediated intercellular signalling between macrophages and hepatocytes during LPS-induced inflammation.
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Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Oncostatina M/farmacología , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Antineoplásicos/farmacología , Secuencia de Bases , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Interleucina-6/farmacología , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidad beta del Receptor de Oncostatina M/genética , Subunidad beta del Receptor de Oncostatina M/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
That cholestatic conditions are accompanied by an enhanced susceptibility to bacterial infection in human and animal models is a known phenomenon. This correlates with the observation that bile acids have suppressive effects on cells of innate and adaptive immunity. The present study provides evidence that in human macrophages, bile acids inhibit the LPS-induced expression of proinflammatory cytokines without affecting the expression of the anti-inflammatory cytokine IL-10. This results in a macrophage phenotype that is characterized by an increased IL-10/IL-12 ratio. Correspondingly, bile acids suppress basal phagocytic activity of human macrophages. These effects of bile acids can be mimicked by cAMP, which is presumably induced TGR5-dependently. The data provided further suggest that in primary human macrophages, modulation of the macrophage response toward LPS by bile acids involves activation of CREB, disturbed nuclear translocation of NF-κB, and PKA-dependent enhancement of LPS-induced cFos expression. The increase in cFos expression is paralleled by an enhanced formation of a protein complex comprising cFos and the p65 subunit of NF-κB. In summary, the data provided suggest that in human macrophages, bile acids induce an anti-inflammatory phenotype characterized by an increased IL-10/IL-12 ratio via activation of PKA and thereby, prevent their activation as classically activated macrophages. This bile acid-induced modulation of macrophage function may also be responsible for the experimentally and clinically observed anti-inflammatory and immunosuppressive effects of bile acids.
Asunto(s)
Ácidos y Sales Biliares/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , Interleucina-10/inmunología , Interleucina-12/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Ácidos y Sales Biliares/metabolismo , AMP Cíclico/genética , AMP Cíclico/inmunología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Activación Enzimática/inmunología , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/citología , Macrófagos/enzimología , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-fyn/inmunología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunologíaRESUMEN
In macrophages detection of gram-negative bacteria particularly involves binding of the outer-wall component lipopolysaccharide (LPS) to its cognate receptor complex, comprising Toll like receptor 4 (TLR4), CD14 and MD2. LPS-induced formation of the LPS receptor complex elicits a signaling network, including intra-cellular signal-transduction directly activated by the TLR4 receptor complex as well as successional induction of indirect autocrine and paracrine signaling events. All these different pathways are integrated into the macrophage response towards an inflammatory stimulus by a highly complex cross-talk of the pathways engaged. This also includes a tight control by several intra- and inter-cellular feedback loops warranting an inflammatory response sufficient to battle invading pathogens and to avoid non-essential tissue damage caused by an overwhelming inflammatory response. Several evidences indicate that the reciprocal cross-talk between the p38(MAPK)-pathway and signal transducer and activator of transcription (STAT)3-mediated signal-transduction forms a critical axis successively activated by LPS. The balanced activation of this axis is essential for both induction and propagation of the inflammatory macrophage response as well as for the control of the resolution phase, which is largely driven by IL-10 and sustained STAT3 activation. In this context regulation of suppressor of cytokine signaling (SOCS)3 expression and the recently described divergent regulatory roles of the two p38(MAPK)-activated protein kinases MK2 and MK3 for the regulation of LPS-induced NF-κB- and IRF3-mediated signal-transduction and gene expression, which includes the regulation of IFNß, IL-10 and DUSP1, appears to play an important role.
Asunto(s)
Lipopolisacáridos/inmunología , Macrófagos/inmunología , Factor de Transcripción STAT3/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Animales , Humanos , Transducción de SeñalRESUMEN
UNLABELLED: Glucocorticoids are known to be potent regulators of inflammation and have been used pharmacologically against inflammatory, immune, and lymphoproliferative diseases for more than 50 years. Due to their possible and well-documented side effects, it is crucial to understand the molecular mechanisms and targets of glucocorticoid action in detail. Several modes of action have been discussed; nevertheless, none of them fully explain all the functions of glucocorticoids. Therefore, we analyzed the cross-talk between glucocorticoids and interleukin-6 (IL-6) in the liver. IL-6 exerts pro-inflammatory as well as anti-inflammatory properties and is a main inducer of the acute-phase response. The balance between the proinflammatory and anti-inflammatory activities of IL-6 is tightly regulated by suppressor of cytokine signaling 3 (SOCS3), a well-known feedback inhibitor of IL-6 signaling. Here, it is demonstrated that glucocorticoids enhance IL-6-dependent γ-fibrinogen expression. Studying of the underlying mechanism revealed prolonged activation of signal transducer and activator of transcription 3 (STAT3) caused by down-regulation of SOCS3 protein expression. Consequently, in SOCS3-deficient cells glucocorticoids do not affect IL-6-induced signal transduction. Moreover, in hepatocytes lacking the SOCS3 recruiting motif within gp130, IL-6-dependent γ-fibrinogen expression is not influenced by glucocorticoid treatment. CONCLUSION: Glucocorticoids interfere with IL-6-induced expression of the feedback inhibitor SOCS3, thereby leading to enhanced expression of acute-phase genes in hepatocytes. This mechanism contributes to the explanation of how glucocorticoids affect inflammation and acute-phase gene induction.
Asunto(s)
Glucocorticoides/inmunología , Hepatocitos/metabolismo , Interleucina-6/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/genética , Reacción de Fase Aguda/inmunología , Animales , Células Cultivadas , Dexametasona/inmunología , Dexametasona/metabolismo , Dexametasona/farmacología , Retroalimentación Fisiológica/efectos de los fármacos , Glucocorticoides/metabolismo , Glucocorticoides/farmacología , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Humanos , Interleucina-6/inmunología , Ratones , Ratones Mutantes , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/inmunologíaRESUMEN
In LPS-treated macrophages, activation of STAT3 is considered to be crucial for terminating the production of inflammatory cytokines. By analyzing the role of MAPK-activated protein kinase (MK) 2 and MK3 for LPS-induced STAT3 activation in macrophages, the present study provides evidence that MK2 is crucial for STAT3 activation in response to LPS because it prevents MK3 from impeding IFNß gene expression. Accordingly, LPS-induced IFNß gene expression is down-regulated in MK2-deficient macrophages and can be reconstituted by additional ablation of the MK3 gene in MK2/3(-/-) macrophages. This is in contrast to LPS-induced IL-10 expression, which essentially requires the presence of MK2. Further analysis of downstream signaling events involved in the transcriptional regulation of IFNß gene expression suggests that, in the absence of MK2, MK3 impairs interferon regulatory factor 3 protein expression and activation and inhibits nuclear translocation of p65. This inhibition of p65 nuclear translocation coincides with enhanced expression and delayed degradation of IκBß, whereas expression of IκBα mRNA and protein is impaired in the absence of MK2. The observation that siRNA directed against IκBß is able to reconstitute IκBα expression in MK2(-/-) macrophages suggests that enhanced expression and delayed degradation of IκBß and impaired NFκB-dependent IκBα expression are functionally linked. In summary, evidence is provided that MK2 regulates LPS-induced IFNß expression and downstream STAT3 activation as it restrains MK3 from mediating negative regulatory effects on NFκB- and interferon regulatory factor 3-dependent LPS signaling.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/biosíntesis , Interferón beta/genética , Interleucina-10/biosíntesis , Interleucina-10/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/citología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
The potential of some proinflammatory mediators to inhibit gp130-dependent STAT3 activation by enhancing suppressor of cytokine signaling (SOCS) 3 expression represents an important molecular mechanism admitting the modulation of the cellular response toward gp130-mediated signals. Thus, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by proinflammatory mediators. In this study, we investigate SOCS3 expression initiated by the proinflammatory cytokine TNF-alpha. In contrast to IL-6, TNF-alpha increases SOCS3 expression by stabilizing SOCS3 mRNA. Activation of the MAPK kinase 6 (MKK6)/p38(MAPK)-cascade is required for TNF-alpha-mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38(MAPK) cascade, basal SOCS3-expression is strongly reduced and TNF-alpha-induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38(MAPK)-dependent signals. As a target for SOCS3 mRNA stability-regulating signals, a region containing three copies of a pentameric AUUUA motif in close proximity to a U-rich region located between positions 2422 and 2541 of the 3' untranslated region of SOCS3 is identified. One factor that could target this region is the zinc finger protein tristetraprolin (TTP), which is shown to be capable of destabilizing SOCS3 mRNA via this region. However, data from TTP-deficient cells suggest that TTP does not play an irreplaceable role in the regulation of SOCS3 mRNA stability by TNF-alpha. In summary, these data indicate that TNF-alpha regulates SOCS3 expression on the level of mRNA stability via activation of the MKK6/p38(MAPK) cascade and that the activation of MK2, a downstream target of p38(MAPK), is important for the regulation of SOCS3 expression.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Estabilidad del ARN/fisiología , ARN Mensajero/biosíntesis , Proteínas Supresoras de la Señalización de Citocinas/biosíntesis , Factor de Necrosis Tumoral alfa/inmunología , Células 3T3 , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular , MAP Quinasa Quinasa 6/inmunología , MAP Quinasa Quinasa 6/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/inmunología , Ratones , Proteínas Quinasas/inmunología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/inmunología , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Tristetraprolina/inmunología , Tristetraprolina/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Cytokine-induced expression of SOCS (suppressor of cytokine signalling) molecules is important for the negative regulatory control of STAT (signal transduction and activators of transcription)-dependent cytokine signalling, e.g. for the signal transduction of IL-6 (interleukin-6)-type cytokines through the JAK (Janus kinase)/STAT cascade. STAT activation itself represents an important step in the transcriptional activation of SOCS3 gene expression. However, downstream of the STAT-responsive element, the SOCS3 gene contains a GC-rich element in its 5'-upstream region. The aim of the present study was to investigate the implications of this GC-rich element in the transcriptional control of SOCS3 gene expression. In the present study, we show that mutation of this GC-rich element abolishes IL-6-dependent transcriptional activation of the SOCS3 promoter and that Sp3 (specificity protein 3), a ubiquitously expressed transcription factor, but not Sp1 binds to this GC-rich motif, suggesting that Sp3 is involved in the regulation of SOCS3 expression. The results suggest that Sp3 is important for IL-6-induced transcriptional activation of the SOCS3 (gene) promoter and acts as an enhancer of basal as well as induced transcriptional activity, resulting in enhanced SOCS3 mRNA and protein expression. Mutation of Lys-483, a potential target for Sp3 acetylation, inhibited Sp3-mediated enhancement of SOCS3 mRNA expression and SOCS3 promoter activation, indicating that the acetylation of this lysine residue of Sp3 is important for the enhancing effect of Sp3 on SOCS3 expression.