RESUMO
Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies.
Assuntos
Proteína ADAM17/antagonistas & inibidores , Células Endoteliais/metabolismo , Necroptose , Neoplasias/etiologia , Neoplasias/patologia , Animais , Antineoplásicos/farmacologia , Biomarcadores , Biomarcadores Tumorais , Comunicação Celular , Morte Celular , Suscetibilidade a Doenças/imunologia , Humanos , Necroptose/genética , Invasividade Neoplásica , Metástase Neoplásica , Inoculação de Neoplasia , Neoplasias/metabolismo , Neoplasias/terapia , Proteólise , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is an idiopathic, chronic cholestatic liver disorder characterized by biliary inflammation and fibrosis. Increased numbers of intrahepatic interferon-γ- (IFNγ) producing lymphocytes have been documented in patients with PSC, yet their functional role remains to be determined. METHODS: Liver tissue samples were collected from patients with PSC. The contribution of lymphocytes to liver pathology was assessed in Mdr2-/- x Rag1-/- mice, which lack T and B cells, and following depletion of CD90.2+ or natural killer (NK)p46+ cells in Mdr2-/- mice. Liver pathology was also determined in Mdr2-/- x Ifng-/- mice and following anti-IFNγ antibody treatment of Mdr2-/- mice. Immune cell composition was analysed by multi-colour flow cytometry. Liver injury and fibrosis were determined by standard assays. RESULTS: Patients with PSC showed increased IFNγ serum levels and elevated numbers of hepatic CD56bright NK cells. In Mdr2-/- mice, hepatic CD8+ T cells and NK cells were the primary source of IFNγ. Depletion of CD90.2+ cells reduced hepatic Ifng expression, NK cell cytotoxicity and liver injury similar to Mdr2-/- x Rag1-/- mice. Depletion of NK cells resulted in reduced CD8+ T cell cytotoxicity and liver fibrosis. The complete absence of IFNγ in Mdr2-/-x Ifng-/- mice reduced NK cell and CD8+ T cell frequencies expressing the cytotoxic effector molecules granzyme B and TRAIL and prevented liver fibrosis. The antifibrotic effect of IFNγ was also observed upon antibody-dependent neutralisation in Mdr2-/- mice. CONCLUSION: IFNγ changed the phenotype of hepatic CD8+ T cells and NK cells towards increased cytotoxicity and its absence attenuated liver fibrosis in chronic sclerosing cholangitis. Therefore, unravelling the immunopathogenesis of PSC with a particular focus on IFNγ might help to develop novel treatment options. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by biliary inflammation and fibrosis, whose current medical treatment is hardly effective. We observed an increased interferon (IFN)-γ response in patients with PSC and in a mouse model of sclerosing cholangitis. IFNγ changed the phenotype of hepatic CD8+ T lymphocytes and NK cells towards increased cytotoxicity, and its absence decreased liver cell death, reduced frequencies of inflammatory macrophages in the liver and attenuated liver fibrosis. Therefore, IFNγ-dependent immune responses may disclose checkpoints for future therapeutic intervention strategies in sclerosing cholangitis.
Assuntos
Colangite Esclerosante/imunologia , Interferon gama , Células Matadoras Naturais , Cirrose Hepática , Fígado , Linfócitos T Citotóxicos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imunidade Celular/imunologia , Fatores Imunológicos/imunologia , Fatores Imunológicos/farmacologia , Interferon gama/imunologia , Interferon gama/farmacologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Cirrose Hepática/terapia , Camundongos , Camundongos Knockout , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Tumour necrosis factor α receptor 1 (TNFR1) activation is known to induce cell death, inflammation, and fibrosis but also hepatocyte survival and regeneration. The multidrug resistance protein 2 knockout (Mdr2-/) mice are a model for chronic hepatitis and inflammation-associated hepatocellular carcinoma (HCC) development. This study analysed how the absence of TNFR1 mediated signalling shapes cytokine and chemokine production, immune cell recruitment and ultimately influences liver injury and fibrotic tissue remodelling in the Mdr2-/- mouse model. We show that Tnfr1-/-/Mdr2-/- mice displayed increased plasma levels of ALT, ALP, and bilirubin as well as a significantly higher collagen content, and markers of fibrosis than Mdr2-/- mice. The expression profile of inflammatory cytokines (Il1b, Il23, Tgfb1, Il17a), chemokines (Ccl2, Cxcl1, Cx3cl1) and chemokine receptors (Ccr6, Cxcr6, Cx3cr1) in livers of Tnfr1-/-/Mdr2-/- mice indicated TH17 cell infiltration. Flow cytometric analysis confirmed that the aggravated tissue injury in Tnfr1-/-/Mdr2-/- mice strongly correlated with increased hepatic recruitment of TH17 cells and enhanced IL-17 production in the injured liver. Moreover, we observed increased hepatic activation of RIPK3 in Tnfr1-/-/Mdr2-/- mice, which was not related to necroptotic cell death. Rather, frequencies of infiltrating CX3CR1+ monocytes increased over time in livers of Tnfr1-/-/Mdr2-/- mice, which expressed significantly higher levels of Ripk3 than those of Mdr2-/- mice. Overall, we conclude that the absence of TNFR1-mediated signalling did not improve the pathological phenotype of Mdr2-/- mice. It instead caused enhanced infiltration of TH17 cells and CX3CR1+ monocytes into the injured tissue, which was accompanied by increased RIPK3 activation and IL-17 production.
Assuntos
Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/imunologia , Fígado/imunologia , Proteínas de Neoplasias , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Células Th17/imunologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Doença Crônica , Deleção de Genes , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Th17/patologiaRESUMO
Multi drug resistance protein 2 knockout mice (Mdr2-/-) are a mouse model of chronic liver inflammation and inflammation-induced tumour development. Here we investigated the kinetics of early heme oxygenase 1 (HO-1) induction on inflammation, tumour development, and DNA damage in Mdr2-/- mice. HO-1 was induced by intraperitoneal injection of cobalt protoporphyrin IX (CoPP) twice weekly for 9 consecutive weeks. Immediately after HO-1 induction, liver function improved and infiltration of CD4+ and CD8+ T cells was reduced. Furthermore, we observed increased p38 activation with concomitant reduction of Cyclin D1 expression in aged Mdr2-/- mice. Long-term effects of HO-1 induction included increased CD8+ T cell infiltration as well as delayed and reduced tumour growth in one-year-old animals. Unexpectedly, DNA double-strand breaks were detected predominantly in macrophages of 65-week-old Mdr2-/- mice, while DNA damage was reduced in response to early HO-1 induction in vivo and in vitro. Overall, early induction of HO-1 in Mdr2-/- mice had a beneficial short-term effect on liver function and reduced hepatic T cell accumulation. Long-term effects of early HO-1 induction were increased CD8+ T cell numbers, decreased proliferation as wells as reduced DNA damage in liver macrophages of aged animals, accompanied by delayed and reduced tumour growth.
Assuntos
Reparo do DNA/efeitos dos fármacos , Ativadores de Enzimas/administração & dosagem , Heme Oxigenase-1/metabolismo , Hepatite/tratamento farmacológico , Neoplasias Hepáticas/prevenção & controle , Proteínas de Membrana/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Dano ao DNA , Modelos Animais de Doenças , Feminino , Hepatite/genética , Hepatite/imunologia , Hepatite/patologia , Humanos , Injeções Intraperitoneais , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Protoporfirinas/administração & dosagem , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
UNLABELLED: A Disintegrin And Metalloprotease (ADAM) 10 exerts essential roles during organ development and tissue integrity in different organs, mainly through activation of the Notch pathway. However, only little is known about its implication in liver tissue physiology. Here we show that in contrast to its role in other tissues, ADAM10 is dispensable for the Notch2-dependent biliary tree formation. However, we demonstrate that expression of bile acid transporters is dependent on ADAM10. Consequently, mice deficient for Adam10 in hepatocytes, cholangiocytes and liver progenitor cells develop spontaneous hepatocyte necrosis and concomitant liver fibrosis. We furthermore observed a strongly augmented ductular reaction in 15-week old ADAM10(Δhep/Δch) mice and demonstrate that c-Met dependent liver progenitor cell activation is enhanced. Additionally, liver progenitor cells are primed to hepatocyte differentiation in the absence of ADAM10. These findings show that ADAM10 is a novel central node controlling liver tissue homeostasis. HIGHLIGHTS: Loss of ADAM10 in murine liver results in hepatocyte necrosis and concomitant liver fibrosis. ADAM10 directly regulates expression of bile acid transporters but is dispensable for Notch2-dependent formation of the biliary system. Activation of liver progenitor cells is enhanced through increased c-Met signalling, in the absence of ADAM10. Differentiation of liver progenitor cells to hepatocytes is augmented in the absence of ADAM10.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Proteína ADAM10/deficiência , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/genética , Animais , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Regulação para Baixo , Hepatócitos/metabolismo , Hepatócitos/patologia , Homeostase , Fígado/citologia , Fígado/patologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Necrose , Receptor Notch2/metabolismo , Transdução de SinaisRESUMO
Acetaminophen (APAP; ie, Paracetamol or Tylenol) is generally self-medicated to treat fever or pain and recommended to pregnant women by their physicians. Recent epidemiological studies reveal an association between prenatal APAP use and an increased risk for asthma. Our aim was to identify the effects of APAP in pregnancy using a mouse model. Allogeneically mated C57Bl/6J females were injected i.p. with 50 or 250 mg/kg APAP or phosphate-buffered saline on gestation day 12.5; nonpregnant females served as controls. Tissue samples were obtained 1 or 4 days after injection. APAP-induced liver toxicity was mirrored by significantly increased plasma alanine aminotransferase levels. In uterus-draining lymph nodes of pregnant dams, the frequencies of mature dendritic cells and regulatory T cells significantly increased on 250 mg/kg APAP. Plasma progesterone levels significantly decreased in dams injected with APAP, accompanied by a morphologically altered placenta. Although overall litter sizes and number of fetal loss remained unaltered, a reduced fetal weight and a lower frequency of hematopoietic stem cells in the fetal liver were observed on APAP treatment. Our data provide strong evidence that prenatal APAP interferes with maternal immune and endocrine adaptation to pregnancy, affects placental function, and impairs fetal maturation and immune development. The latter may have long-lasting consequences on children's immunity and account for the increased risk for asthma observed in humans.
Assuntos
Acetaminofen/farmacologia , Adaptação Fisiológica , Feto/efeitos dos fármacos , Placenta/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Feto/embriologia , Feto/metabolismo , Camundongos Endogâmicos C57BL , Placenta/efeitos dos fármacos , Placenta/imunologia , GravidezRESUMO
Intrauterine growth restriction (IUGR) affects up to 10% of pregnancies in Western societies. IUGR is a strong predictor of reduced short-term neonatal survival and impairs long-term health in children. Placental insufficiency is often associated with IUGR; however, the molecular mechanisms involved in the pathogenesis of placental insufficiency and IUGR are largely unknown. Here, we developed a mouse model of fetal-growth restriction and placental insufficiency that is induced by a midgestational stress challenge. Compared with control animals, pregnant dams subjected to gestational stress exhibited reduced progesterone levels and placental heme oxygenase 1 (Hmox1) expression and increased methylation at distinct regions of the placental Hmox1 promoter. These stress-triggered changes were accompanied by an altered CD8+ T cell response, as evidenced by a reduction of tolerogenic CD8+CD122+ T cells and an increase of cytotoxic CD8+ T cells. Using progesterone receptor- or Hmox1-deficient mice, we identified progesterone as an upstream modulator of placental Hmox1 expression. Supplementation of progesterone or depletion of CD8+ T cells revealed that progesterone suppresses CD8+ T cell cytotoxicity, whereas the generation of CD8+CD122+ T cells is supported by Hmox1 and ameliorates fetal-growth restriction in Hmox1 deficiency. These observations in mice could promote the identification of pregnancies at risk for IUGR and the generation of clinical interventional strategies.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Desenvolvimento Fetal/fisiologia , Retardo do Crescimento Fetal/prevenção & controle , Heme Oxigenase-1/fisiologia , Proteínas de Membrana/fisiologia , Placenta/imunologia , Insuficiência Placentária/imunologia , Complicações na Gravidez/imunologia , Progesterona/fisiologia , Estresse Psicológico/imunologia , Animais , Metilação de DNA , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Retardo do Crescimento Fetal/imunologia , Feto/imunologia , Feto/patologia , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Ruído/efeitos adversos , Placenta/metabolismo , Circulação Placentária , Insuficiência Placentária/etiologia , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/psicologia , Progesterona/biossíntese , Progesterona/uso terapêutico , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Estresse Psicológico/genéticaRESUMO
BACKGROUND & AIMS: During pregnancy, acetaminophen is one of the very few medications recommended by physicians to treat fever or pain. Recent insights from epidemiological studies suggest an association between prenatal acetaminophen medication and an increased risk for development of asthma in children later in life. The underlying pathogenesis of such association is still unknown. METHODS: We aimed to develop a mouse model to provide insights into the effect of prenatal acetaminophen on maternal, fetal and adult offspring's health. The toxic effect of acetaminophen was studied in mice on 1) maternal liver; mirrored by biomarkers of liver injury, centrilobular necrosis, and infiltration of granulocytes; 2) fetal liver; reflected by the frequency of hematopoietic stem cells, and 3) postnatal health; evaluated by the severity of allergic airway inflammation among offspring. RESULTS: We observed an increased susceptibility towards acetaminophen-induced liver damage in pregnant mice compared to virgins. Moreover, hematopoietic stem cell frequency in fetal liver declined in response to acetaminophen. Furthermore, a greater severity of airway inflammation was observed in offspring of dams upon prenatal acetaminophen treatment, identified lung infiltration by leukocytes and eosinophil infiltration into the airways. CONCLUSION: Our newly developed mouse model on prenatal use of acetaminophen reflects findings from epidemiological studies in humans. The availability of this model will allow improvement in our understanding of how acetaminophen-related hepatotoxicity is operational in pregnant individuals and how an increased risk for allergic diseases in response to prenatal acetaminophen is mediated. Such insights, once available, may change the recommendations for prenatal acetaminophen use.
Assuntos
Acetaminofen , Asma , Doença Hepática Induzida por Substâncias e Drogas , Células-Tronco Fetais , Efeitos Tardios da Exposição Pré-Natal , Acetaminofen/administração & dosagem , Acetaminofen/efeitos adversos , Adulto , Filhos Adultos , Analgésicos não Narcóticos/administração & dosagem , Analgésicos não Narcóticos/efeitos adversos , Animais , Asma/etiologia , Asma/fisiopatologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Modelos Animais de Doenças , Feminino , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/patologia , Humanos , Inflamação/etiologia , Inflamação/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Índice de Gravidade de DoençaRESUMO
UNLABELLED: Induction or overexpression of the heme-degrading enzyme, heme oxygenase 1 (HO-1), has been shown to protect mice from liver damage induced by acute inflammation. We have investigated the effects of HO-1 induction in a mouse model of chronic liver inflammation and fibrogenesis with progression to hepatocellular carcinoma (HCC) (Mdr2ko; FVB.129P2-Abcb4(tm1Bor)). HO-1 was induced in vivo by treatment with cobalt protoporphyrin IX, starting at week 5 or 12 of mice lifespan, and continued for 7 weeks. Our results showed that HO-1 induction reduced liver damage and chronic inflammation by regulating immune cell infiltration or proliferation as well as tumor necrosis factor receptor signaling. Fibrosis progression was significantly reduced by HO-1 induction in mice with mild, as well as established, portal and lobular fibrosis. HO-1 induction significantly suppressed hepatic stellate cell activation. During established fibrosis, HO-1 induction was able to revert portal inflammation and fibrosis below levels observed at the start of treatment. Moreover, hepatocellular proliferation and signs of dysplasia were decreased after HO-1 induction. CONCLUSION: Induction of HO-1 interferes with chronic inflammation and fibrogenesis and, in consequence, might delay progression to HCC.
Assuntos
Heme Oxigenase-1/metabolismo , Cirrose Hepática/enzimologia , Proteínas de Membrana/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Proliferação de Células , Progressão da Doença , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Cirrose Hepática/imunologia , Camundongos , Camundongos Knockout , Fosforilação , Protoporfirinas , Receptores do Fator de Necrose Tumoral/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Protein ubiquitylation is a key post-translational control mechanism contributing to different physiological processes, such as signal transduction and ageing. The size and linkage of a ubiquitin chain, which determines whether a substrate is efficiently targeted for proteasomal degradation, is determined by the interplay between ubiquitylation and deubiquitylation. A conserved factor that orchestrates distinct substrate-processing co-regulators in diverse species is the ubiquitin-selective chaperone CDC-48 (also known as p97). Several deubiquitylation enzymes (DUBs) have been shown to interact with CDC-48/p97, but the mechanistic and physiological relevance of these interactions remained elusive. Here we report a synergistic cooperation between CDC-48 and ATX-3 (the Caenorhabditis elegans orthologue of ataxin-3) in ubiquitin-mediated proteolysis and ageing regulation. Surprisingly, worms deficient for both cdc-48.1 and atx-3 demonstrated extended lifespan by up to 50%, mediated through the insulin-insulin-like growth factor 1 (IGF-1) signalling pathway. As lifespan extension specifically depends on the deubiquitylation activity of ATX-3, our findings identify a mechanistic link between protein degradation and longevity through editing of the ubiquitylation status of substrates involved in insulin-IGF-1 signalling.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica , Doença de Machado-Joseph/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Ataxina-3 , Caenorhabditis elegans , Proteínas de Ciclo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Insulina/metabolismo , Longevidade , Modelos Biológicos , Mutação , Transdução de Sinais , Fatores de Tempo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Proteína com ValosinaRESUMO
Protein degradation in eukaryotes often requires the ubiquitin-selective chaperone p97 for substrate recruitment and ubiquitin-chain assembly. However, the physiological relevance of p97, and its role in developmental processes, remain unclear. Here, we discover an unanticipated function for CDC-48/p97 in myosin assembly and myofibril organization, both in Caenorhabditis elegans and humans. The developmentally regulated assembly of a CDC-48-UFD-2-CHN-1 complex links turnover of the myosin-directed chaperone UNC-45 to functional muscle formation. Our data suggest a similarly conserved pathway regulating myosin assembly in humans. Remarkably, mutations in human p97, known to cause hereditary inclusion-body myopathy, abrogate UNC-45 degradation and result in severely disorganized myofibrils, detrimental towards sarcomeric function. These results identify a key role for CDC-48/p97 in the process of myofibre differentiation and maintenance, which is abolished during pathological conditions leading to protein aggregation and inclusion-body formation in human skeletal muscle.