RESUMEN
ABSTRACT: Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse. SIGNIFICANCE: CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171.
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Neoplasias de la Mama , Células Supresoras de Origen Mieloide , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/radioterapia , Femenino , Haptoglobinas , Humanos , Células Supresoras de Origen Mieloide/metabolismo , Recurrencia Local de Neoplasia/genética , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismoRESUMEN
KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers.
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Neoplasias Pulmonares/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/genética , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Mutación , Oncogenes/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Interferencia de ARN , Tratamiento con ARN de Interferencia/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND & AIMS: Liver regeneration after partial hepatectomy (PH) increases the protein folding burden at the endoplasmic reticulum of remnant hepatocytes, resulting in induction of the unfolded protein response. We investigated the role of the core unfolded protein response transcription factor X-box binding protein 1 (XBP1) in liver regeneration using genome-wide chromatin immunoprecipitation analysis. METHODS: We performed studies with C57Bl6-J (control) and interleukin 6-knockout mice. Mice underwent PH or sham surgeries. In some mice, hepatic expression of XBP1 was knocked down by injection of adenoviral vectors encoding small hairpin RNAs against Xbp1 messenger RNA. Liver tissues were collected before surgery and at 6 and 48 hours after surgery and analyzed by chromatin immunoprecipitation followed by sequencing. We also performed functional analyses of HepG2 cells. RESULTS: Expression of XBP1 by hepatocytes increased immediately after PH (priming phase of liver regeneration) in control mice, but this effect was delayed in interleukin 6-deficient mice. In mice with knockdown of XBP1, we observed of liver tissue persistent endoplasmic reticulum stress, defects in acute-phase response, and increased hepatocellular damage, compared with control mice. Chromatin immunoprecipitation analyses of liver tissue showed that at 6 hours after PH, liver XBP1 became bound to a large set of genes implicated in proteostasis, the acute-phase response, metabolism, and the DNA damage response (DDR). At this time point, XBP1 bound the promoter of the signal transducer and activator of transcription 3 gene (Stat3). Livers of XBP1-knockdown mice showed reduced expression of STAT3 and had lower levels of STAT3 phosphorylation at Ser727, a modification that promotes cell proliferation and the DDR. Regenerating livers from XBP1-knockdown mice expressed high levels of a marker of DNA double-strand breaks, phosphorylated histone 2A, member X (H2AX), compared with control mice. The inhibition of XBP1 expression caused a reduced up-regulation of DDR messenger RNAs in regenerating hepatocytes. CONCLUSION: In livers of mice, we found that PH induces expression of XBP1, and that this activity requires interleukin 6. XBP1 expression regulates the unfolded protein response, acute-phase response, and DDR in hepatocytes. In regenerating livers, XBP1 deficiency leads to endoplasmic reticulum stress and DNA damage.
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Reacción de Fase Aguda/genética , Daño del ADN/genética , Estrés del Retículo Endoplásmico/genética , Regeneración Hepática/genética , Hígado/metabolismo , Respuesta de Proteína Desplegada/genética , Proteína 1 de Unión a la X-Box/genética , Animales , Células Hep G2 , Hepatectomía , Humanos , Interleucina-6/genética , Ratones , Ratones Noqueados , Fosforilación , Factor de Transcripción STAT3/metabolismoRESUMEN
UNLABELLED: In chronic hepatitis B (CHB), failure to control hepatitis B virus (HBV) is associated with T cell dysfunction. HBV transgenic mice mirror many features of the human disease, including T cell unresponsiveness, and thus represent an appropriate model in which to test novel therapeutic strategies. To date, the tolerant state of CD8(+) T cells in these animals could be altered only by strong immunogens or by immunization with HBV antigen-pulsed dendritic cells; however, the effectors induced were unable to suppress viral gene expression or replication. Because of the known stimulatory properties of alpha interferon (IFN-α) and interleukin-15 (IL-15), this study explored the therapeutic potential of liver-directed gene transfer of these cytokines in a murine model of CHB using adeno-associated virus (AAV) delivery. This combination not only resulted in a reduction in the viral load in the liver and the induction of an antibody response but also gave rise to functional and specific CD8(+) immunity. Furthermore, when splenic and intrahepatic lymphocytes from IFN-α- and IL-15-treated animals were transferred to new HBV carriers, partial antiviral immunity was achieved. In contrast to previous observations made using either cytokine alone, markedly attenuated PD-L1 induction in hepatic tissue was observed upon coadministration. An initial study with CHB patient samples also gave promising results. Hence, we demonstrated synergy between two stimulating cytokines, IL-15 and IFN-α, which, given together, constitute a potent approach to significantly enhance the CD8(+) T cell response in a state of immune hyporesponsiveness. Such an approach may be useful for treating chronic viral infections and neoplastic conditions. IMPORTANCE: With 350 million people affected worldwide and 600,000 annual deaths due to HBV-induced liver cirrhosis and/or hepatocellular carcinoma, chronic hepatitis B (CHB) is a major health problem. However, current treatment options are costly and not very effective and/or need to be administered for life. The unprecedented efficacy of the strategy described in our paper may offer an alternative and is relevant for a broad spectrum of readers because of its clear translational importance to other chronic viral infections in which a hyporesponsive antigen-specific T cell repertoire prevents clearance of the pathogen.
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Linfocitos T CD8-positivos/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Interferón-alfa/administración & dosificación , Interleucina-15/administración & dosificación , Adenoviridae/genética , Animales , Modelos Animales de Enfermedad , Portadores de Fármacos , Terapia Genética , Anticuerpos contra la Hepatitis B/sangre , Interferón-alfa/genética , Interleucina-15/genética , Hígado/virología , Ratones Transgénicos , Resultado del Tratamiento , Carga ViralRESUMEN
BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in the ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs and zinc salts, which may cause side effects and do not restore normal copper metabolism. In this work we assessed the efficacy of gene therapy to treat this condition. METHODS: We transduced the liver of the Atp7b(-/-) WD mouse model with an adeno-associated vector serotype 8 (AAV8) encoding the human ATP7B cDNA placed under the control of the liver-specific α1-antitrypsin promoter (AAV8-AAT-ATP7B). After vector administration we carried out periodic evaluation of parameters associated with copper metabolism and disease progression. The animals were sacrificed 6months after treatment to analyze copper storage and hepatic histology. RESULTS: We observed a dose-dependent therapeutic effect of AAV8-AAT-ATP7B manifested by the reduction of serum transaminases and urinary copper excretion, normalization of serum holoceruloplasmin, and restoration of physiological biliary copper excretion in response to copper overload. The liver of treated animals showed normalization of copper content and absence of histological alterations. CONCLUSIONS: Our data demonstrate that AAV8-AAT-ATP7B-mediated gene therapy provides long-term correction of copper metabolism in a clinically relevant animal model of WD providing support for future translational studies.
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Cobre/metabolismo , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Degeneración Hepatolenticular , Hígado , Adenosina Trifosfatasas/genética , Animales , Proteínas de Transporte de Catión/genética , ATPasas Transportadoras de Cobre , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Técnicas de Transferencia de Gen , Degeneración Hepatolenticular/genética , Degeneración Hepatolenticular/metabolismo , Degeneración Hepatolenticular/terapia , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Fragmentos de Péptidos/genética , Resultado del Tratamiento , alfa 1-Antitripsina/genéticaRESUMEN
BACKGROUND & AIMS: Oncostatin M (OSM) is an inflammatory cytokine which interacts with a heterodimeric receptor formed by gp130 and either OSMRß or LIFR. Here we have analysed OSM and its receptors in livers with chronic hepatitis C (CHC) and studied the factors that regulate this system. METHODS: OSM, OSM receptors and OSM-target molecules were studied by immunohistochemistry and/or qPCR analysis in livers from CHC patients and controls. We determined the production of OSM by CD40L-stimulated antigen presenting cells (APC) and its biological effects on HuH7 cells containing HCV replicon (HuH7 Core-3'). RESULTS: OSM was upregulated in livers with CHC and its production was mapped to CD11c+ cells. OSM levels correlated directly with inflammatory activity and CD40L expression. In vitro studies showed that OSM is released by APC upon interaction with activated CD4+ T cells in a CD40L-dependent manner. Culture of HuH7 Core-3' cells with supernatant from CD40L-stimulated APC repressed HCV replication and induced IL-7 and IL-15Rα. These effects were dampened by antibodies blocking OSM or gp130 and by silencing OSMRß. In CHC livers OSMRß and LIFR were significantly downregulated and their values correlated with those of OSM-induced molecules. Experiments in HuH7 cells showed that impaired STAT3 signaling and exposure to TGFß1, two findings in CHC, are factors involved in repressing OSMRß and LIFR, respectively. CONCLUSIONS: OSM is a cytokine possessing vigorous antiviral and immunostimulatory properties which is released by APC upon interaction with CD40L present on activated CD4+ T cells. In livers with CHC, OSM is overexpressed but its biological activity appears to be hampered because of downregulation of its receptor subunits.
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Ligando de CD40/fisiología , Hepatitis C Crónica/inmunología , Subunidad beta del Receptor de Oncostatina M/fisiología , Oncostatina M/fisiología , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Humanos , Monocitos/inmunología , Factor de Transcripción STAT3/fisiologíaRESUMEN
BACKGROUND: IL-7 and IL-15 are produced by hepatocytes and are critical for the expansion and function of CD8 T cells. IL-15 needs to be presented by IL-15Rα for efficient stimulation of CD8 T cells. METHODS: We analysed the hepatic levels of IL-7, IL-15, IL-15Rα and interferon regulatory factors (IRF) in patients with chronic hepatitis C (CHC) (78% genotype 1) and the role of IRF1 and IRF2 on IL-7 and IL-15Rα expression in Huh7 cells with or without hepatitis C virus (HCV) replicon. RESULTS: Hepatic expression of both IL-7 and IL-15Rα, but not of IL-15, was reduced in CHC. These patients exhibited decreased hepatic IRF2 messenger RNA levels and diminished IRF2 staining in hepatocyte nuclei. We found that IRF2 controls basal expression of both IL-7 and IL-15Rα in Huh7 cells. IRF2, but not IRF1, is downregulated in cells with HCV genotype 1b replicon and this was accompanied by decreased expression of IL-7 and IL-15Rα, a defect reversed by overexpressing IRF2. Treating Huh7 cells with IFNα plus oncostatin M increased IL-7 and IL-15Rα mRNA more intensely than either cytokine alone. This effect was mediated by strong upregulation of IRF1 triggered by the combined treatment. Induction of IRF1, IL-7 and IL-15Rα by IFNα plus oncostatin M was dampened in replicon cells but the combination was more effective than either cytokine alone. CONCLUSIONS: HCV genotype 1 infection downregulates IRF2 in hepatocytes attenuating hepatocellular expression of IL-7 and IL-15Rα. Our data reveal a new mechanism by which HCV abrogates specific T-cell responses and point to a novel therapeutic approach to stimulate anti-HCV immunity.
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Hepacivirus/fisiología , Hepatitis C Crónica/fisiopatología , Hepatocitos/fisiología , Factores Reguladores del Interferón/fisiología , Western Blotting , Linfocitos T CD8-positivos/fisiología , Regulación Viral de la Expresión Génica/genética , Regulación Viral de la Expresión Génica/fisiología , Genotipo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatitis C Crónica/metabolismo , Hepatitis C Crónica/virología , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Factor 1 Regulador del Interferón/biosíntesis , Factor 1 Regulador del Interferón/fisiología , Factor 2 Regulador del Interferón/biosíntesis , Factor 2 Regulador del Interferón/fisiología , Interleucina-15/biosíntesis , Interleucina-15/fisiología , Subunidad alfa del Receptor de Interleucina-15/biosíntesis , Subunidad alfa del Receptor de Interleucina-15/fisiología , Interleucina-7/biosíntesis , Interleucina-7/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Replicación Viral/fisiologíaRESUMEN
Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies (AU)
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Animales , Ratas , Células Madre Pluripotentes , Hepatocitos/trasplante , Hepatopatías/terapia , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Modelos Animales de EnfermedadRESUMEN
It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/-) γc(-/-) mice using an adenovirus encoding herpes virus thymidine kinase (AdTk) and two consecutive doses of ganciclovir (GCV). We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.
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Adenoviridae/metabolismo , Hepatocitos/trasplante , Hígado/patología , Timidina Quinasa/metabolismo , Trasplante Heterólogo , Albúminas/metabolismo , Animales , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Ganciclovir/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Hepatitis B/patología , Hepatitis C/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante HomólogoRESUMEN
Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.
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Supervivencia de Injerto/inmunología , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/trasplante , Albúminas/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Tratamiento Basado en Trasplante de Células y Tejidos , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/farmacología , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/inmunología , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Hígado/inmunología , Hígado/lesiones , Ratones , Ratones Transgénicos , Bazo , Teratoma/inmunología , Teratoma/metabolismo , Factores de Tiempo , Urea/metabolismoRESUMEN
Recombinant adeno-associated virus 5 (rAAV5) represents a candidate vector with unique advantages for the treatment of hepatic disorders because of its narrow hepatic tropism. Noninvasive in vivo imaging of transgene expression provides an important tool with which to quantify the transduction efficiency, and duration and location, of transgene expression. In this study, we used positron emission tomography (PET) and positron emission tomography-computed tomography (PET-CT) imaging to monitor liver transduction efficacy in rodents and nonhuman primates that received rAAV5 vector encoding herpes simplex virus thymidine kinase (HSV-TK). HSV-TK expression in liver was also measured by immunohistochemistry. Notable differences in liver transduction efficiency were found, dependent on the animal species and sex. Male rodents were better transduced than females, as previously described. Moreover, male nonhuman primates also displayed increased hepatic expression of the rAAV5-delivered transgene, indicating that differences in rAAV-mediated liver transduction can be anticipated in humans. Our results demonstrate the high sensitivity and reproducibility of PET, using HSV-TK and [(18)F]FHBG, to detect gene expression after rAAV vector administration into living animals, confirming the utility of this technology in the quantification of transgene expression, even at low expression levels. However, we also describe how an immune response against HSV-TK hampered analysis of long-term expression in nonhuman primates.
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Dependovirus/genética , Hígado/metabolismo , Animales , Femenino , Radioisótopos de Flúor , Genes Reporteros , Vectores Genéticos , Guanina/análogos & derivados , Hígado/diagnóstico por imagen , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Tomografía de Emisión de Positrones , Radiofármacos , Ratas , Ratas Sprague-Dawley , Factores Sexuales , Simplexvirus/enzimología , Simplexvirus/inmunología , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Transducción Genética , TransgenesRESUMEN
BACKGROUND & AIMS: The mechanisms by which Foxp3+ T regulatory cells (Treg) accumulate in HCV infected livers are not known. Here, we studied the role of chemokines CCL17 and CCL22 in this process. METHODS: Chemokine mRNA levels were determined by qPCR in liver biopsies from 26 HCV chronically infected patients (CHC), 11 patients with treatment-induced sustained virological response (SVR), 16 patients with other liver diseases unrelated to HCV, and 24 normal livers. Double-immunofluorescence Foxp3/CD3 or CD11c/CCL22 was performed in liver sections. Chemokine production by monocyte-derived dendritic cells (MDDC) co-cultured with uninfected or HCV-JFH1 infected Huh7 cells was measured by qPCR and ELISA. Chemotactic activity of culture supernatants was also tested. RESULTS: Foxp3+ Treg were increased in CHC livers as compared to controls. Patients with CHC showed elevated intrahepatic levels of CCL17 mRNA compared to normal livers or livers from subjects with SVR or other forms of liver disease. Intrahepatic CCL22 expression was also higher in CHC than in healthy subjects or SVR patients but similar to that observed in other liver diseases. Dendritic cells producing CCL22 could be found inside the hepatic lobule in CHC patients. Contact between MDDC and HCV-JFH1-infected Huh7 cells induced the expression of CCL17 and CCL22 in a process partially dependent on ICAM-1. Transwell experiments showed that upregulation of these chemokines enhanced Treg migration. CONCLUSIONS: Contact of HCV-infected cells with dendritic cells induces the production of Treg-attracting chemokines, an effect which may favour liver accumulation of Treg in CHC. Our findings contribute to explain the mechanism by which HCV escapes the immune response and thus reveals novel therapeutic targets.
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Quimiocina CCL17/biosíntesis , Quimiocina CCL17/genética , Quimiocina CCL22/biosíntesis , Quimiocina CCL22/genética , Hepacivirus/inmunología , Hepacivirus/patogenicidad , Hepatitis C Crónica/inmunología , Linfocitos T Reguladores/inmunología , Secuencia de Bases , Estudios de Casos y Controles , Adhesión Celular/inmunología , Técnicas de Cocultivo , Estudios de Cohortes , Cartilla de ADN/genética , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/metabolismo , Hepatitis C Crónica/patología , Hepatitis C Crónica/virología , Humanos , Hígado/inmunología , Hígado/patología , Hígado/virología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Regulación hacia ArribaRESUMEN
Immunosuppressive activity of regulatory T cells (Treg) may contribute to the progression of cancer or infectious diseases by preventing the induction of specific immune responses. Using a phage-displayed random peptide library, we identified a 15-mer synthetic peptide, P60, able to bind to forkhead/winged helix transcription factor 3 (FOXP3), a factor required for development and function of Treg. P60 enters the cells, inhibits FOXP3 nuclear translocation, and reduces its ability to suppress the transcription factors NF-κB and NFAT. In vitro, P60 inhibited murine and human-derived Treg and improved effector T cell stimulation. P60 administration to newborn mice induced a lymphoproliferative autoimmune syndrome resembling the reported pathology in scurfy mice lacking functional Foxp3. However, P60 did not cause toxic effects in adult mice and, when given to BALB/c mice immunized with the cytotoxic T cell epitope AH1 from CT26 tumor cells, it induced protection against tumor implantation. Similarly, P60 improved the antiviral efficacy of a recombinant adenovirus expressing NS3 protein from hepatitis C virus. Functional inhibition of Treg by the FOXP3-inhibitory peptide P60 constitutes a strategy to enhance antitumor and antiviral immunotherapies.
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Factores de Transcripción Forkhead/antagonistas & inhibidores , Péptidos/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Vacunas/inmunología , Animales , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Microscopía Confocal , Biblioteca de Péptidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/metabolismo , TransfecciónRESUMEN
UNLABELLED: We investigated whether gene transfer of insulin-like growth factor I (IGF-I) to the hepatic tissue was able to improve liver histology and function in established liver cirrhosis. Rats with liver cirrhosis induced by carbon tetrachloride (CCl(4)) given orally for 8 weeks were injected through the hepatic artery with saline or with Simian virus 40 vectors encoding IGF-I (SVIGF-I), or luciferase (SVLuc). Animals were sacrificed 8 weeks after vector injection. In cirrhotic rats we observed that, whereas IGF-I was synthesized by hepatocytes, IGF-I receptor was predominantly expressed by nonparenchymal cells, mainly in fibrous septa surrounding hepatic nodules. Rats treated with SVIGF-I showed increased hepatic levels of IGF-I, improved liver function tests, and reduced fibrosis in association with diminished alpha-smooth muscle actin expression, up-regulation of matrix metalloproteases (MMPs) and decreased expression of the tissue inhibitors of MMPs TIM-1 and TIM-2. SVIGF-I therapy induced down-regulation of the profibrogenic molecules transforming growth factor beta (TGFbeta), amphiregulin, platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), and vascular endothelium growth factor (VEGF) and induction of the antifibrogenic and cytoprotective hepatocyte growth factor (HGF). Furthermore, SVIGF-I-treated animals showed decreased expression of Wilms tumor-1 (WT-1; a nuclear factor involved in hepatocyte dedifferentiation) and up-regulation of hepatocyte nuclear factor 4 alpha (HNF4alpha) (which stimulates hepatocellular differentiation). The therapeutic potential of SVIGF-I was also tested in rats with thioacetamide-induced liver cirrhosis. Also in this model, SVIGF-I improved liver function and reduced liver fibrosis in association with up-regulation of HGF and MMPs and down-regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1). CONCLUSION: IGF-I gene transfer to cirrhotic livers induces MMPs and hepatoprotective factors leading to reversion of fibrosis and improvement of liver function. IGF-I gene therapy may be a useful alternative therapy for patients with advanced cirrhosis without timely access to liver transplantation.
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Técnicas de Transferencia de Gen , Terapia Genética/métodos , Factor I del Crecimiento Similar a la Insulina/genética , Cirrosis Hepática/terapia , Animales , Células Cultivadas , Cirrosis Hepática/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Prion diseases are believed to develop from the conformational change of normal cellular prion protein (PrPc) to a pathogenic isoform (PrPsc). PrPc is present in both the central nervous system and many peripheral tissues, although protein concentration is significantly lower in non-neuronal tissues. PrPc expression is essential for internalization and replication of the infectious agent. Several works have pointed to the gastrointestinal (GI) tract as the principal site of entry of PrPsc, but how passage through the GI mucosa occurs is not yet known. Here we studied PrPc expression using Western blot, RT-PCR, and immunohistochemistry in rat GI tract. PrPc mRNA and protein were detected in corpus, antrum, duodenum, and colon. Immunoreactivity was found in scattered cells of the GI epithelium. With double immunofluorescence, these cells have been identified as neuroendocrine cells. PrPc immunostaining was found in subsets of histamine, somatostatin (Som), ghrelin, gastrin (G), and serotonin (5HT) cells in stomach. In small and large bowel, PrPc cells co-localized with subpopulations of 5HT-, Som-, G-, and peptide YY-immunolabeled cells. Our results provide evidence for a possible and important role of endocrine cells in the internalization of PrPsc from gut lumen.
Asunto(s)
Células Enteroendocrinas/metabolismo , Tracto Gastrointestinal/metabolismo , Sistemas Neurosecretores/metabolismo , Priones/biosíntesis , Animales , Técnica del Anticuerpo Fluorescente , Tracto Gastrointestinal/citología , Inmunohistoquímica , Sistemas Neurosecretores/citología , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In the present review we will summarise the current knowledge about the cells comprising the Diffuse Endocrine System (DES) in mammalian organs. We will describe the morphological, histochemical and functional traits of these cells in three major systems gastrointestinal, respiratory and prostatic. We will also focus on some aspects of their ontogeny and differentiation, as well as to their relevance in carcinogenesis, especially in neuroendocrine tumors. The first chapter describes the characteristics of DES cells and some of their specific biological and biochemical traits. The second chapter deals with DES in the gastrointestinal organs, with special reference to the new data on the differentiation mechanisms that leads to the appearance of endocrine cells from an undifferentiated stem cell. The third chapter is devoted to DES of the respiratory system and some aspects of its biological role, both, during development and adulthood. Neuroendocrine hyperplasia and neuroendocrine lung tumors are also addressed. Finally, the last chapter deals with the prostatic DES, discussing its probable functional role and its relevance in hormone-resistant prostatic carcinomas.