RESUMEN
ËCCL24 is a pro-fibrotic, pro-inflammatory chemokine expressed in several chronic fibrotic diseases. In the liver, CCL24 plays a role in fibrosis and inflammation, and blocking CCL24 led to reduced liver injury in experimental models. We studied the role of CCL24 in primary sclerosing cholangitis (PSC) and evaluated the potential therapeutic effect of blocking CCL24 in this disease. Multidrug resistance gene 2-knockout (Mdr2-/-) mice demonstrated CCL24 expression in liver macrophages and were used as a relevant experimental PSC model. CCL24-neutralizing monoclonal antibody, CM-101, significantly improved inflammation, fibrosis, and cholestasis-related markers in the biliary area. Moreover, using spatial transcriptomics, we observed reduced proliferation and senescence of cholangiocytes following CCL24 neutralization. Next, we demonstrated that CCL24 expression was elevated under pro-fibrotic conditions in primary human cholangiocytes and macrophages, and it induced proliferation of primary human hepatic stellate cells and cholangiocytes, which was attenuated following CCL24 inhibition. Correspondingly, CCL24 was found to be highly expressed in liver biopsies of patients with PSC. CCL24 serum levels correlated with Enhanced Liver Fibrosis score, most notably in patients with high alkaline phosphatase levels. These results suggest that blocking CCL24 may have a therapeutic effect in patients with PSC by reducing liver inflammation, fibrosis, and cholestasis.
Asunto(s)
Quimiocina CCL24 , Colangitis Esclerosante , Colestasis , Animales , Humanos , Ratones , Colangitis Esclerosante/complicaciones , Fibrosis , Inflamación , HígadoRESUMEN
BACKGROUND & AIMS: C-C motif chemokine ligand 24 (CCL24) is a chemokine that regulates inflammatory and fibrotic activities through its receptor, C-C motif chemokine receptor (CCR3). The aim of the study was to evaluate the involvement of the CCL24-CCR3 axis in liver fibrosis and inflammation and to assess the potential of its blockade, by a monoclonal anti-CCL24 antibody, as a therapeutic strategy for non-alcoholic steatohepatitis (NASH) and liver fibrosis. METHODS: Expression of CCL24 and CCR3 was evaluated in liver biopsies and blood samples. CCL24 involvement in NAFLD/NASH pathogenesis was assessed in Ccl24 knockout mouse using the methionine-choline deficient (MCD) diet experimental model. Antifibrotic and anti-inflammatory effects of CM-101 were tested in the MCD and STAM mouse models and in the thioacetamide (TAA) model in rats. Liver enzymes, liver morphology, histology and collagen deposition, as well as fibrosis- and inflammation-related protein expression were assessed. Activation of hepatic stellate cells (HSCs) was evaluated in the human LX2 cell line. RESULTS: Patients with NASH and advanced NAFLD exhibited significant expression of both CCL24 and CCR3 in liver and blood samples. In the experimental MCD-diet model, Ccl24 knockout mice showed an attenuated liver damage response compared to wild-type mice, exhibiting reduced histological NAFLD activity scores and fibrosis, as well as lower levels of liver enzymes. Blocking CCL24 using CM-101 robustly reduced liver damage in 3 experimental animal models (MCD, STAM and TAA), as demonstrated by attenuation of liver fibrosis and NAFLD activity score. Furthermore, blocking CCL24 by CM-101 significantly inhibited CCL24-induced HSC motility, α-SMA expression and pro-collagen I secretion. CONCLUSION: Our results reveal that blocking CCL24 significantly attenuates liver fibrosis and inflammation and may have a potential therapeutic effect in patients with NASH and/or liver fibrosis. LAY SUMMARY: CCL24 is a chemokine that regulates inflammation and fibrosis. It was found to be significantly expressed in patients with non-alcoholic steatohepatitis, in whom it regulates profibrotic processes in the liver. Herein, we show that blockade of CCL24 using a monoclonal antibody robustly attenuated liver fibrosis and inflammation in animal models, thus suggesting a potential therapeutic role for an anti-CCL24 agent.
RESUMEN
OBJECTIVES: We aimed to assess the expression of the CCL24 chemokine in systemic sclerosis (SSc) and to evaluate the possible pathogenic implications of the CCL24/CCR3 axis using both in vitro and in vivo models. We further investigated the efficacy of an anti-CCL24 monoclonal antibody (mAb), CM-101, in inhibiting cell activation as well as dermal and pulmonary inflammation and fibrosis in experimental animal models. METHODS: We used ELISA and fluorescence immunohistochemistry to determine CCL24 levels in serum and CCL24/CCR3 expression in skin biopsies of SSc patients. Skin fibroblasts and endothelial cells treated with CCL24 or SSc serum with or without CM-101 were used to follow cell activation and differentiation. Prevention and treatment in vivo bleomycin (BLM)-induced models were used to evaluate experimental dermal and pulmonary fibrosis progression following treatment with the CM-101 mAb. RESULTS: CCL24 circulating levels were significantly elevated in SSc patients. CCL24/CCR3 expression was strongly increased in SSc skin. Blockade of CCL24 with CM-101 significantly reduced the activation of dermal fibroblasts and their transition to myofibroblasts induced by SSc serum. CM-101 was also able to significantly inhibit endothelial cell activation induced by CCL24. In BLM-induced experimental animal models, CM-101 profoundly inhibited both dermal and pulmonary fibrosis and inflammation. CONCLUSIONS: CCL24 plays an important role in pathological processes of skin and lung inflammation and fibrosis. Inhibition of CCL24 by CM-101 mAb can be potentially beneficial for therapeutic use in SSc patients.
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Anticuerpos Monoclonales/farmacología , Quimiocina CCL24/antagonistas & inhibidores , Fibrosis Pulmonar/tratamiento farmacológico , Piel/patología , Animales , Diferenciación Celular , Células Cultivadas , Quimiocina CCL24/biosíntesis , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Fibrosis/tratamiento farmacológico , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Polisacáridos Bacterianos/inmunología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
CXCR4 expression in neuroblastoma tumors correlates with disease severity. In this study, we describe mechanisms by which CXCR4 signaling controls neuroblastoma tumor growth and response to therapy. We found that overexpression of CXCR4 or stimulation with CXCL12 supports neuroblastoma tumorigenesis. Moreover, CXCR4 inhibition with the high-affinity CXCR4 antagonist BL-8040 prevented tumor growth and reduced survival of tumor cells. These effects were mediated by the upregulation of miR-15a/16-1, which resulted in downregulation of their target genes BCL-2 and cyclin D1, as well as inhibition of ERK. Overexpression of miR-15a/16-1 in cells increased cell death, whereas antagomirs to miR-15a/16-1 abolished the proapoptotic effects of BL-8040. CXCR4 overexpression also increased miR-15a/16-1, shifting their oncogenic dependency from the BCL-2 to the ERK signaling pathway. Overall, our results demonstrate the therapeutic potential of CXCR4 inhibition in neuroblastoma treatment and provide a rationale to test combination therapies employing CXCR4 and BCL-2 inhibitors to increase the efficacy of these agents.Significance: These results provide a mechanistic rationale for combination therapy of CXCR4 and BCL-2 inhibitors to treat a common and commonly aggressive pediatric cancer.Cancer Res; 78(6); 1471-83. ©2017 AACR.
Asunto(s)
Neoplasias Encefálicas/patología , MicroARNs/metabolismo , Neuroblastoma/patología , Receptores CXCR4/metabolismo , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Ratones , MicroARNs/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time.
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Hipocampo/metabolismo , MicroARNs/genética , Microglía/fisiología , Neuronas/fisiología , Ribonucleasa III/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Reparación del ADN , Femenino , Hipocampo/embriología , Hipocampo/crecimiento & desarrollo , Humanos , Imagenología Tridimensional , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/metabolismo , Actividad Motora , Plasticidad Neuronal , Ribonucleasa III/genéticaRESUMEN
UNLABELLED: Human hepatocellular carcinoma (HCC) is an inflammation-induced cancer, which is the third-leading cause of cancer mortality worldwide. We investigated the role of the chemokine receptors, CCR5 and CCR1, in regulating inflammation and tumorigenesis in an inflammation-induced HCC model in mice. Multidrug resistance 2 gene (Mdr2)-knockout (Mdr2-KO) mice spontaneously develop chronic cholestatic hepatitis and fibrosis that is eventually followed by HCC. We generated two new strains from the Mdr2-KO mouse, the Mdr2:CCR5 and the Mdr2:CCR1 double knockouts (DKOs), and set out to compare inflammation and tumorigenesis among these strains. We found that in Mdr2-KO mice lacking the chemokine receptor, CCR5 (Mdr2:CCR5 DKO mice), but not CCR1 (Mdr2:CCR1 DKO), macrophage recruitment and trafficking to the liver was significantly reduced. Furthermore, in the absence of CCR5, reduced inflammation was also associated with reduced periductal accumulation of CD24(+) oval cells and abrogation of fibrosis. DKO mice for Mdr2 and CCR5 exhibited a significant decrease in tumor incidence and size. CONCLUSIONS: Our results indicate that CCR5 has a critical role in both the development and progression of liver cancer. Therefore, we propose that a CCR5 antagonist can serve for HCC cancer prevention and treatment.
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Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/fisiopatología , Hepatitis Crónica/complicaciones , Cirrosis Hepática/complicaciones , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/fisiopatología , Receptores CCR5/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Animales , Carcinoma Hepatocelular/epidemiología , Quimiocina CCL5/fisiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hepatitis Crónica/genética , Incidencia , Hígado/patología , Hígado/fisiopatología , Cirrosis Hepática/genética , Neoplasias Hepáticas/epidemiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR1/deficiencia , Receptores CCR1/genética , Receptores CCR1/fisiología , Receptores CCR5/deficiencia , Receptores CCR5/genética , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
UNLABELLED: Chronic inflammation is strongly associated with an increased risk for hepatocellular carcinoma (HCC) development. The multidrug resistance 2 (Mdr2)-knockout (KO) mouse (adenosine triphosphate-binding cassette b4(-/-) ), a model of inflammation-mediated HCC, develops chronic cholestatic hepatitis at an early age and HCC at an adult age. To delineate factors contributing to hepatocarcinogenesis, we compared the severity of early chronic hepatitis and late HCC development in two Mdr2-KO strains: Friend virus B-type/N (FVB) and C57 black 6 (B6). We demonstrated that hepatocarcinogenesis was significantly less efficient in the Mdr2-KO/B6 mice versus the Mdr2-KO/FVB mice; this difference was more prominent in males. Chronic hepatitis in the Mdr2-KO/B6 males was more severe at 1 month of age but was less severe at 3 months of age in comparison with age-matched Mdr2-KO/FVB males. A comparative genome-scale gene expression analysis of male livers of both strains at 3 months of age revealed both common and strain-specific aberrantly expressed genes, including genes associated with the regulation of inflammation, the response to oxidative stress, and lipid metabolism. One of these regulators, galectin-1 (Gal-1), possesses both anti-inflammatory and protumorigenic activities. To study its regulatory role in the liver, we transferred the Gal-1-KO mutation (lectin galactoside-binding soluble 1(-/-) ) from the B6 strain to the FVB strain, and we demonstrated that endogenous Gal-1 protected the liver against concanavalin A-induced hepatitis with the B6 genetic background but not the FVB genetic background. CONCLUSION: Decreased chronic hepatitis in Mdr2-KO/B6 mice at the age of 3 months correlated with a significant retardation of liver tumor development in this strain versus the Mdr2-KO/FVB strain. We found candidate factors that may determine strain-specific differences in the course of chronic hepatitis and HCC development in the Mdr2-KO model, including inefficient anti-inflammatory activity of the endogenous lectin Gal-1 in the FVB strain.
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Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica/patología , Galectina 1/fisiología , Hepatitis Crónica/patología , Neoplasias Hepáticas/patología , Ratones Endogámicos/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Carcinoma Hepatocelular/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Concanavalina A , Hepatitis Crónica/complicaciones , Hepatitis Crónica/etiología , Hígado/metabolismo , Neoplasias Hepáticas/etiología , Masculino , Metionina Adenosiltransferasa/biosíntesis , Ratones , Ratones Noqueados , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Chemokines were shown to govern the trafficking of immune cells and may also play important roles in the survival and activation of these cells. We report here that under physiological conditions, the bone marrow (BM), spleen, blood and liver of Ccr5, but not of Ccr1-deficient mice, contain reduced numbers of NK cells. NK cells in the BM of Ccr5-deficient mice proliferate to a lesser extent compared to WT mice. Furthermore, spleen NK cells derived from Ccr5-deficient mice that were transplanted into irradiated recipients failed to proliferate in the host. Ccr5, but not Ccr1-deficient NK cells, failed to migrate in vitro in response to RANTES and MIP-1ß but not MIP-1ß or SDF-1 and had reduced activation, lower expression levels of NK cell markers and a slightly reduced capacity to adhere to target cells and stimulate their killing. Using the polyI:C mouse model for NK trafficking, we found that in the absence of Ccr5, but not Ccr1, NK cells failed to accumulate in the liver. In contrast, using the influenza viral infection as a model to evaluate NK cell proliferation, we found that Ccr5-deficient NK cells in the BM had a higher proliferation rate than WT NK cells. These results suggest a role for Ccr5 in NK cell proliferation and circulation under physiological conditions and a complex role for Ccr5 in determining the fate of NK cells under pathological conditions.