RESUMO
OBJECTIVE: We evaluated the influence of sex on the pathophysiology of non-alcoholic fatty liver disease (NAFLD). We investigated diet-induced phenotypic responses to define sex-specific regulation between healthy liver and NAFLD to identify influential pathways in different preclinical murine models and their relevance in humans. DESIGN: Different models of diet-induced NAFLD (high-fat diet, choline-deficient high-fat diet, Western diet or Western diet supplemented with fructose and glucose in drinking water) were compared with a control diet in male and female mice. We performed metabolic phenotyping, including plasma biochemistry and liver histology, untargeted large-scale approaches (liver metabolome, lipidome and transcriptome), gene expression profiling and network analysis to identify sex-specific pathways in the mouse liver. RESULTS: The different diets induced sex-specific responses that illustrated an increased susceptibility to NAFLD in male mice. The most severe lipid accumulation and inflammation/fibrosis occurred in males receiving the high-fat diet and Western diet, respectively. Sex-biased hepatic gene signatures were identified for these different dietary challenges. The peroxisome proliferator-activated receptor α (PPARα) co-expression network was identified as sexually dimorphic, and in vivo experiments in mice demonstrated that hepatocyte PPARα determines a sex-specific response to fasting and treatment with pemafibrate, a selective PPARα agonist. Liver molecular signatures in humans also provided evidence of sexually dimorphic gene expression profiles and the sex-specific co-expression network for PPARα. CONCLUSIONS: These findings underscore the sex specificity of NAFLD pathophysiology in preclinical studies and identify PPARα as a pivotal, sexually dimorphic, pharmacological target. TRIAL REGISTRATION NUMBER: NCT02390232.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Humanos , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , PPAR alfa/metabolismoRESUMO
Bone deficits are frequent in HIV-1-infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1-induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef.
Assuntos
Reabsorção Óssea/etiologia , Infecções por HIV/complicações , HIV-1/fisiologia , Osteoclastos/virologia , Actinas/metabolismo , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Osso e Ossos/metabolismo , Adesão Celular , Feminino , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Immune response against pathogens is a tightly regulated process that must ensure microbial control while preserving integrity of the infected organs. Tuberculosis (TB) is a paramount example of a chronic infection in which antimicrobial immunity is protective in the vast majority of infected individuals but can become detrimental if not finely tuned. Here, we report that C-type lectin dendritic cell (DC) immunoreceptor (DCIR), a key component in DC homeostasis, is required to modulate lung inflammation and bacterial burden in TB. DCIR is abundantly expressed in pulmonary lesions in Mycobacterium tuberculosis-infected nonhuman primates during both latent and active disease. In mice, we found that DCIR deficiency impairs STAT1-mediated type I IFN signaling in DCs, leading to increased production of IL-12 and increased differentiation of T lymphocytes toward Th1 during infection. As a consequence, DCIR-deficient mice control M. tuberculosis better than WT animals but also develop more inflammation characterized by an increased production of TNF and inducible NOS (iNOS) in the lungs. Altogether, our results reveal a pathway by which a C-type lectin modulates the equilibrium between infection-driven inflammation and pathogen's control through sustaining type I IFN signaling in DCs.
Assuntos
Células Dendríticas/imunologia , Interferon Tipo I/imunologia , Lectinas Tipo C/imunologia , Tuberculose/imunologia , Animais , Feminino , Lectinas Tipo C/genética , Macaca mulatta , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Fator de Transcrição STAT1/imunologia , Transdução de SinaisRESUMO
Breast cancer (BC) remains the primary cause of death from cancer among women worldwide. Cholesterol-5,6-epoxide (5,6-EC) metabolism is deregulated in BC but the molecular origin of this is unknown. Here, we have identified an oncometabolism downstream of 5,6-EC that promotes BC progression independently of estrogen receptor α expression. We show that cholesterol epoxide hydrolase (ChEH) metabolizes 5,6-EC into cholestane-3ß,5α,6ß-triol, which is transformed into the oncometabolite 6-oxo-cholestan-3ß,5α-diol (OCDO) by 11ß-hydroxysteroid-dehydrogenase-type-2 (11ßHSD2). 11ßHSD2 is known to regulate glucocorticoid metabolism by converting active cortisol into inactive cortisone. ChEH inhibition and 11ßHSD2 silencing inhibited OCDO production and tumor growth. Patient BC samples showed significant increased OCDO levels and greater ChEH and 11ßHSD2 protein expression compared with normal tissues. The analysis of several human BC mRNA databases indicated that 11ßHSD2 and ChEH overexpression correlated with a higher risk of patient death, highlighting that the biosynthetic pathway producing OCDO is of major importance to BC pathology. OCDO stimulates BC cell growth by binding to the glucocorticoid receptor (GR), the nuclear receptor of endogenous cortisol. Interestingly, high GR expression or activation correlates with poor therapeutic response or prognosis in many solid tumors, including BC. Targeting the enzymes involved in cholesterol epoxide and glucocorticoid metabolism or GR may be novel strategies to prevent and treat BC.
Assuntos
Neoplasias da Mama/metabolismo , Carcinógenos/metabolismo , Colesterol/metabolismo , Receptores de Glucocorticoides/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Colesterol/análogos & derivados , Epóxido Hidrolases/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Células HEK293 , Humanos , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , RNA Mensageiro/metabolismoRESUMO
Fumonisin B1 (FB1), a congener of fumonisins produced by Fusarium species, is the most abundant and most toxicologically active fumonisin. FB1 causes severe mycotoxicosis in animals, including nephrotoxicity, hepatotoxicity, and disruption of the intestinal barrier. However, mechanisms associated with FB1 toxicity are still unclear. Preliminary studies have highlighted the role of liver X receptors (LXRs) during FB1 exposure. LXRs belong to the nuclear receptor family and control the expression of genes involved in cholesterol and lipid homeostasis. In this context, the toxicity of FB1 was compared in female wild-type (LXR+/+) and LXRα,ß double knockout (LXR-/-) mice in the absence or presence of FB1 (10 mg/kg body weight/day) for 28 days. Exposure to FB1 supplemented in the mice's drinking water resulted in more pronounced hepatotoxicity in LXR-/- mice compared to LXR+/+ mice, as indicated by hepatic transaminase levels (ALT, AST) and hepatic inflammatory and fibrotic lesions. Next, the effect of FB1 exposure on the liver transcriptome was investigated. FB1 exposure led to a specific transcriptional response in LXR-/- mice that included altered cholesterol and bile acid homeostasis. ELISA showed that these effects were associated with an elevated FB1 concentration in the plasma of LXR-/- mice, suggesting that LXRs participate in intestinal absorption and/or clearance of the toxin. In summary, this study demonstrates an important role of LXRs in protecting the liver against FB1-induced toxicity, suggesting an alternative mechanism not related to the inhibition of sphingolipid synthesis for mycotoxin toxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fumonisinas/toxicidade , Receptores X do Fígado/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Feminino , Fumonisinas/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/fisiologia , Receptores X do Fígado/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esfingolipídeos/metabolismoRESUMO
RATIONALE: In addition to their well-known function as antibody-producing cells, B lymphocytes can markedly influence the course of infectious or noninfectious diseases via antibody-independent mechanisms. In tuberculosis (TB), B cells accumulate in lungs, yet their functional contribution to the host response remains poorly understood. OBJECTIVES: To document the role of B cells in TB in an unbiased manner. METHODS: We generated the transcriptome of B cells isolated from Mycobacterium tuberculosis (Mtb)-infected mice and validated the identified key pathways using in vitro and in vivo assays. The obtained data were substantiated using B cells from pleural effusion of patients with TB. MEASUREMENTS AND MAIN RESULTS: B cells isolated from Mtb-infected mice displayed a STAT1 (signal transducer and activator of transcription 1)-centered signature, suggesting a role for IFNs in B-cell response to infection. B cells stimulated in vitro with Mtb produced type I IFN, via a mechanism involving the innate sensor STING (stimulator of interferon genes), and antagonized by MyD88 (myeloid differentiation primary response 88) signaling. In vivo, B cells expressed type I IFN in the lungs of Mtb-infected mice and, of clinical relevance, in pleural fluid from patients with TB. Type I IFN expression by B cells induced an altered polarization of macrophages toward a regulatory/antiinflammatory profile in vitro. In vivo, increased provision of type I IFN by B cells in a murine model of B cell-restricted Myd88 deficiency correlated with an enhanced accumulation of regulatory/antiinflammatory macrophages in Mtb-infected lungs. CONCLUSIONS: Type I IFN produced by Mtb-stimulated B cells favors macrophage polarization toward a regulatory/antiinflammatory phenotype during Mtb infection.
Assuntos
Linfócitos B/metabolismo , Interferon Tipo I/metabolismo , Macrófagos/metabolismo , Tuberculose/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Transdução de Sinais , Baço/metabolismo , Baço/microbiologiaRESUMO
OBJECTIVE: Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor expressed in tissues with high oxidative activity that plays a central role in metabolism. In this work, we investigated the effect of hepatocyte PPARα on non-alcoholic fatty liver disease (NAFLD). DESIGN: We constructed a novel hepatocyte-specific PPARα knockout (Pparα(hep-/-)) mouse model. Using this novel model, we performed transcriptomic analysis following fenofibrate treatment. Next, we investigated which physiological challenges impact on PPARα. Moreover, we measured the contribution of hepatocytic PPARα activity to whole-body metabolism and fibroblast growth factor 21 production during fasting. Finally, we determined the influence of hepatocyte-specific PPARα deficiency in different models of steatosis and during ageing. RESULTS: Hepatocyte PPARα deletion impaired fatty acid catabolism, resulting in hepatic lipid accumulation during fasting and in two preclinical models of steatosis. Fasting mice showed acute PPARα-dependent hepatocyte activity during early night, with correspondingly increased circulating free fatty acids, which could be further stimulated by adipocyte lipolysis. Fasting led to mild hypoglycaemia and hypothermia in Pparα(hep-/-) mice when compared with Pparα(-/-) mice implying a role of PPARα activity in non-hepatic tissues. In agreement with this observation, Pparα(-/-) mice became overweight during ageing while Pparα(hep-/-) remained lean. However, like Pparα(-/-) mice, Pparα(hep-/-) fed a standard diet developed hepatic steatosis in ageing. CONCLUSIONS: Altogether, these findings underscore the potential of hepatocyte PPARα as a drug target for NAFLD.
Assuntos
Envelhecimento , Ácidos Graxos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Hepatócitos , Hepatopatia Gordurosa não Alcoólica/genética , PPAR alfa/genética , Adipócitos , Envelhecimento/fisiologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Família 4 do Citocromo P450/genética , Modelos Animais de Doenças , Jejum , Fenofibrato/farmacologia , Fatores de Crescimento de Fibroblastos/biossíntese , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Homeostase/genética , Hipoglicemia/genética , Hipolipemiantes/farmacologia , Hipotermia/genética , Metabolismo dos Lipídeos/genética , Lipólise/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , Sobrepeso/genética , PPAR alfa/metabolismo , RNA Mensageiro/metabolismo , Triglicerídeos/metabolismoRESUMO
Models of microbe-elicited peritonitis have been invaluable to identify mechanisms underlying inflammation resolution, but whether resolution mechanisms differ from an inflammatory agent to another has not been determined. Thus, we analyzed the cellular and molecular components of the resolution phase of non-microbe-induced inflammation. In thioglycollate (TG)-induced peritonitis, resolution started at 12 h (Tmax) and displayed a 22 h resolution interval (Ri). During resolution, lipoxin A4, resolvin (Rv) D1 and RvD2, protectin D1 (PD1), and maresin 1 (MaR1) were transiently produced while RvD5 was continually generated. In addition, docosahexaenoic acid (DHA)-derived mediators were produced to a higher extent than in microbial peritonitis. We also investigated leukocyte infiltration and clearance in peritoneal tissues surrounding the inflammatory site. In the omentum, resolution parameters, neutrophil apoptosis, and efferocytosis were similar to those of the peritoneal cavity. However, we noticed long-term persistence of M2-polarized macrophages and B-lymphocytes in the omentum after TG administration, whereas zymosan injection caused M1/M2-macrophage and T-lymphocyte persistence regardless of the magnitude of the inflammatory response. Our study indicates that some aspects of resolution are shaped in a stimulus-specific manner, and it ultimately argues that the tissues surrounding the inflammatory site must also be considered to address the inflammatory response globally.
Assuntos
Linfócitos B/imunologia , Inflamação/imunologia , Leucócitos/imunologia , Macrófagos/imunologia , Peritonite/imunologia , Peritonite/metabolismo , Tioglicolatos/toxicidade , Animais , Apoptose/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Western Blotting , Células Cultivadas , Ácidos Docosa-Hexaenoicos/genética , Ácidos Docosa-Hexaenoicos/metabolismo , Feminino , Citometria de Fluxo , Técnicas Imunoenzimáticas , Inflamação/metabolismo , Inflamação/patologia , Leucócitos/metabolismo , Leucócitos/patologia , Lipídeos/análise , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Omento/imunologia , Omento/metabolismo , Omento/patologia , Peritonite/induzido quimicamente , Fagocitose/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Zimosan/toxicidadeRESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a mortality that is almost identical to incidence. Because early detected PDAC is potentially curable, blood-based biomarkers that could detect currently developing neoplasia would improve patient survival and management. PDAC develops from pancreatic intraepithelial neoplasia (PanIN) lesions, graded from low grade (PanIN1) to high grade (PanIN3). We made the hypothesis that specific proteomic signatures from each precancerous stage exist and are detectable in plasma. METHODS: We explored the peptide profiles of microdissected PanIN cells and of plasma samples corresponding to the different PanIN grade from genetically engineered mouse models of PDAC using capillary electrophoresis coupled to mass spectrometry (CE-MS) and Chip-MS/MS. RESULTS: We successfully characterised differential peptides profiles from PanIN microdissected cells. We found that plasma from tumor-bearing mice and age-matched controls exhibit discriminative peptide signatures. We also determined plasma peptide signatures corresponding to low- and high-grade precancerous step present in the mice pancreas using the two mass spectrometry technologies. Importantly, we identified biomarkers specific of PanIN3. CONCLUSIONS: We demonstrate that benign and advanced PanIN lesions display distinct plasma peptide patterns. This strongly supports the perspectives of developing a non-invasive screening test for prediction and early detection of PDAC.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma in Situ/sangue , Carcinoma Ductal Pancreático/sangue , Proteínas de Neoplasias/sangue , Neoplasias Pancreáticas/sangue , Peptídeos/sangue , Lesões Pré-Cancerosas/sangue , Animais , Biomarcadores Tumorais/análise , Carcinoma in Situ/química , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/química , Modelos Animais de Doenças , Camundongos , Proteínas de Neoplasias/análise , Neoplasias Pancreáticas/química , Peptídeos/análise , Lesões Pré-Cancerosas/química , Lesões Pré-Cancerosas/patologia , Análise Serial de Proteínas , Proteoma/análiseRESUMO
Tumor protein p53-induced nuclear protein 1 (TP53INP1) is involved in cell stress response. Its expression is lost at the pancreatic intraepithelial neoplasia 1b (PanIN1b)/PanIN2 stage of pancreatic carcinogenesis. Our objective was to determine whether TP53INP1 loss of expression contributes to pancreatic cancer formation in a conditional KrasG12D mouse model. We generated Kras-INP1KO mice using LSL-Kras(G12D/+);Pdx1-Cre(+/-) mice (Kras mice) and TP53INP1(-/-) mice. Analysis of pancreases during ageing shows that in the presence of activated Kras, TP53INP1 loss of expression accelerated PanIN formation and increased pancreatic injury and the number of high-grade lesions as compared with what occurs in Kras mice. Moreover, cystic lesions resembling intraductal papillary mucinous neoplasm (IPMN) were observed as early as 2 months of age. Remarkably, TP53INP1 is down-regulated in human IPMN. Activation of the small GTPase Rac1 shows that more oxidative stress is generated in Kras-INP1KO than in Kras mice pancreas despite elevated levels of the Nrf2 antioxidant regulator. We firmly establish the link between Kras-INP1KO pancreatic phenotype and oxidative stress with rescue of the phenotype by the antioxidant action of N-acetylcysteine. Our data provide in vivo functional demonstration that TP53INP1 deficiency accelerates progression of pancreatic cancer, underlining its role in the occurrence of IPMN and highlighting the importance of TP53INP1 in the control of oxidative status during development of pancreatic cancer.
Assuntos
Proteínas Nucleares/fisiologia , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Progressão da Doença , Regulação para Baixo/fisiologia , Humanos , Metaplasia/genética , Metaplasia/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismoRESUMO
In osteoclasts, Src controls podosome organization and bone degradation, which leads to an osteopetrotic phenotype in src(-/-) mice. Since this phenotype was even more severe in src(-/-)hck(-/-) mice, we examined the individual contribution of Hck in bone homeostasis. Compared to wt mice, hck(-/-) mice exhibited an osteopetrotic phenotype characterized by an increased density of trabecular bone and decreased bone degradation, although osteoclastogenesis was not impaired. Podosome organization and matrix degradation were found to be defective in hck(-/-) osteoclast precursors (preosteoclast) but were normal in mature hck(-/-) osteoclasts, probably through compensation by Src, which was specifically overexpressed in mature osteoclasts. As a consequence of podosome defects, the 3-dimensional migration of hck(-/-) preosteoclasts was strongly affected in vitro. In vivo, this translated by altered bone homing of preosteoclasts in hck(-/-) mice: in metatarsals of 1-wk-old mice, when bone formation strongly depends on the recruitment of these cells, reduced numbers of osteoclasts and abnormal developing trabecular bone were observed. This phenotype was still detectable in adults. In summmary, Hck is one of the very few effectors of preosteoclast recruitment described to date and thereby plays a critical role in bone remodeling.
Assuntos
Osso e Ossos/citologia , Osso e Ossos/metabolismo , Movimento Celular/fisiologia , Osteoclastos/citologia , Osteopetrose/metabolismo , Proteínas Proto-Oncogênicas c-hck/metabolismo , Animais , Movimento Celular/genética , Células Cultivadas , Feminino , Homeostase/genética , Homeostase/fisiologia , Masculino , Camundongos , Camundongos Knockout , Osteoclastos/metabolismo , Osteopetrose/genética , Proteínas Proto-Oncogênicas c-hck/genética , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
BACKGROUND & AIMS: Nutrients influence non-alcoholic fatty liver disease. Essential fatty acids deficiency promotes various syndromes, including hepatic steatosis, through increased de novo lipogenesis. The mechanisms underlying such increased lipogenic response remain unidentified. METHODS: We used wild type mice and mice lacking Liver X Receptors to perform a nutrigenomic study that aimed at examining the role of these transcription factors. RESULTS: We showed that, in the absence of Liver X Receptors, essential fatty acids deficiency does not promote steatosis. Consistent with this, Liver X Receptors are required for the elevated expression of genes involved in lipogenesis in response to essential fatty acids deficiency. CONCLUSIONS: This work identifies, for the first time, the central role of Liver X Receptors in steatosis induced by essential fatty acids deficiency.
Assuntos
Ácidos Graxos Essenciais/deficiência , Fígado Gorduroso/fisiopatologia , Expressão Gênica/fisiologia , Lipogênese/genética , Lipogênese/fisiologia , Receptores Nucleares Órfãos/fisiologia , Animais , Colesterol/metabolismo , Deficiências Nutricionais/fisiopatologia , Gorduras na Dieta/farmacologia , Modelos Animais de Doenças , Feminino , Expressão Gênica/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Fígado/metabolismo , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores Nucleares Órfãos/deficiência , Receptores Nucleares Órfãos/genética , Fatores de Transcrição/fisiologia , Triglicerídeos/metabolismo , Regulação para Cima/fisiologiaRESUMO
CD8+ CTLs are thought to play a role in the control of follicular lymphoma (FL). Yet, the link between CTL tissue distribution, activation status, ability to kill FL cells in vivo, and disease progression is still elusive. Pretreatment lymph nodes from FL patients were analyzed by IHC (n = 80) or by 3-color confocal microscopy (n = 10). IHC revealed a rich infiltrate of CD8+ granzyme B+ (GrzB) cells in FL interfollicular spaces. Accordingly, confocal microscopy showed an increased number of CD3+CD8+GrzB+ CTLs and a brighter GrzB staining in individual CTL in FL samples compared with reactive lymph nodes. CTLs did not penetrate tumor nodules. In 3-dimensional (3-D) image reconstructions, CTLs were detected at the FL follicle border where they formed lytic synapse-like structures with FL B cells and with apoptotic cells, suggesting an in situ cytotoxic function. Finally, although GrzB expression in CTLs did not correlate with risk factors, high GrzB content correlated with prolonged progression free-survival (PFS) after rituximab-combined chemotherapy. Our results show the recruitment of armed CTLs with a tumor-controlling potential into FL lymph nodes and suggest that CTL-associated GrzB expression could influence PFS in FL patients having received rituximab-combined chemotherapy.
Assuntos
Imageamento Tridimensional , Linfonodos/patologia , Linfoma Folicular/epidemiologia , Linfoma Folicular/patologia , Linfócitos T Citotóxicos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Biomarcadores Tumorais/metabolismo , Complexo CD3/metabolismo , Antígenos CD8/metabolismo , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Granzimas/metabolismo , Humanos , Linfoma Folicular/tratamento farmacológico , Masculino , Microscopia Confocal/métodos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prednisona/administração & dosagem , Prognóstico , Fatores de Risco , Rituximab , Linfócitos T Citotóxicos/metabolismo , Vincristina/administração & dosagemRESUMO
Tumor-associated macrophages are known to amplify the malignant potential of tumors by secreting a variety of cytokines and proteases involved in tumor cell invasion and metastasis, but how these macrophages infiltrate tumors and whether the macrophage migration process facilitates tumor cell invasion remain poorly documented. To address these questions, we used cell spheroids of breast carcinoma SUM159PT cells as an in vitro model of solid tumors. We found that macrophages used both the mesenchymal mode requiring matrix metalloproteinases (MMPs) and the amoeboid migration mode to infiltrate tumor cell spheroids. Whereas individual SUM159PT cells invaded Matrigel using an MMP-dependent mesenchymal mode, when they were grown as spheroids, tumor cells were unable to invade the Matrigel surrounding spheroids. When spheroids were infiltrated or in contact with macrophages, tumor cell invasiveness was restored. It was dependent on the capacity of macrophages to remodel the matrix and migrate in an MMP-independent mesenchymal mode. This effect of macrophages was much reduced when spheroids were infiltrated by Matrigel migration-defective Hck(-/-) macrophages. In the presence of macrophages, SUM159PT migrated into Matrigel in the proximity of macrophages and switched from an MMP-dependent mesenchymal migration to an amoeboid mode resistant to protease inhibitors.Thus, in addition to the well-described paracrine loop between macrophages and tumor cells, macrophages can also contribute to the invasiveness of tumor cells by remodeling the extracellular matrix and by opening the way to exit the tumor and colonize the surrounding tissues in an MMP-dispensable manner.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Macrófagos/metabolismo , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica/imunologia , Esferoides Celulares/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Macrófagos/imunologia , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Invasividade Neoplásica/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/ultraestruturaRESUMO
NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) and TPM3-ALK (nonmuscular tropomyosin 3-anaplastic lymphoma kinase) are oncogenic tyrosine kinases implicated in the pathogenesis of human ALK-positive lymphoma. We report here the development of novel conditional mouse models for ALK-induced lymphomagenesis, with the use of the tetracycline regulatory system under the control of the EmuSRalpha enhancer/promoter. The expression of either oncogene resulted in the arrest of the differentiation of early B cells and lymphomagenesis. We also observed the development of skin keratoacanthoma lesions, probably because of aberrant ALK expression in keratinocytes. The inactivation of the ALK oncogene on doxycycline treatment was sufficient to induce sustained regression of both hematopoietic tumors and skin disease. Importantly, treatment with the specific ALK inhibitor (PF-2341066) also reversed the pathologic states, showing the value of these mouse models for the validation of ALK tyrosine kinase inhibitors. Thus, our results show (1) that NPM-ALK and TPM3-ALK oncogenes are sufficient for lymphoma/leukemia development and required for tumor maintenance, hence validating ALK as potentially effective therapeutic target; and (2) for the first time, in vivo, the equal tumorigenic potential of the NPM-ALK and TPM3-ALK oncogenic tyrosine kinases. Our models offer a new tool to investigate in vivo the molecular mechanisms associated with ALK-induced lymphoproliferative disorders.
Assuntos
Leucemia de Células B/patologia , Linfoma de Células B/patologia , Proteínas Tirosina Quinases/genética , Tropomiosina/genética , Quinase do Linfoma Anaplásico , Animais , Apoptose , Western Blotting , Diferenciação Celular , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Integrases/metabolismo , Leucemia de Células B/genética , Leucemia de Células B/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tropomiosina/metabolismoRESUMO
Tissue infiltration of phagocytes exacerbates several human pathologies including chronic inflammations or cancers. However, the mechanisms involved in macrophage migration through interstitial tissues are poorly understood. We investigated the role of Hck, a Src-family kinase involved in the organization of matrix adhesion and degradation structures called podosomes. In Hck(-/-) mice submitted to peritonitis, we found that macrophages accumulated in interstitial tissues and barely reached the peritoneal cavity. In vitro, 3-dimensional (3D) migration and matrix degradation abilities, 2 protease-dependent properties of bone marrow-derived macrophages (BMDMs), were affected in Hck(-/-) BMDMs. These macrophages formed few and undersized podosome rosettes and, consequently, had reduced matrix proteolysis operating underneath despite normal expression and activity of matrix metalloproteases. Finally, in fibroblasts unable to infiltrate matrix, ectopic expression of Hck provided the gain-of-3D migration function, which correlated positively with formation of podosome rosettes. In conclusion, spatial organization of podosomes as large rosettes, proteolytic degradation of extracellular matrix, and 3D migration appeared to be functionally linked and regulated by Hck in macrophages. Hck, as the first protein combining a phagocyte-limited expression with a role in 3D migration, could be a target for new anti-inflammatory and antitumor molecules.
Assuntos
Movimento Celular/fisiologia , Matriz Extracelular/metabolismo , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/patologia , Peritonite/metabolismo , Proteínas Proto-Oncogênicas c-hck/metabolismo , Células 3T3 , Animais , Células da Medula Óssea/citologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Cavidade Peritoneal/patologia , Peritonite/patologia , Fagócitos/metabolismo , Fagócitos/patologia , Inibidores de Proteases/farmacologia , Proteínas Proto-Oncogênicas c-hck/genéticaRESUMO
OBJECTIVE: Sparse data are available on renal consequences of hemorrhagic shock in mice. This study aimed to extend the current knowledge on functional and morphologic renal impact of hemorrhagic shock in mice and to determine its ability to stand as an accurate model of acute kidney injury. DESIGN: In vivo study. SETTING: University research unit. SUBJECTS: C57/Bl6 mice. INTERVENTIONS: A model of controlled hemorrhagic shock was adapted to determine the renal impact of hemorrhagic shock in mice. MEASUREMENTS AND MAIN RESULTS: Renal functions and kidney morphology were followed up from 3 hrs to 21 days after hemorrhagic shock. When prolonged up to 2 hrs, hypotension (35 mm Hg mean arterial blood pressure) induced by temporary blood removal was responsible for an early and lasting increase in hypoxia-inducible factor-1α and kidney-inducible molecule-1 gene expression that paralleled acute tubular necrosis and renal failure. Two-hr hypotension induced an important but reversible decrease in glomerular filtration rate up to 6 days after hemorrhagic shock. Other renal dysfunctions included a renal loss of sodium, assessed by the increase in sodium excretion, and a decrease in urine concentration that persists up to day 21. Tissular damages prevailed in the outer medulla 2 days after hemorrhagic shock, being maximal at day 6. At day 21, renal healing was associated with epithelial recovery and a significant interstitial fibrosis. CONCLUSIONS: Our data indicate that apparent recovery of renal function after acute kidney injury can mask persisting dysfunctions and tissular damages that could predispose to chronic kidney disease. Prolonged hemorrhagic shock in mice closely mimics renal effects induced by similar situation in humans, thus providing a useful tool to investigate pathophysiological mechanisms and protection strategies against acute kidney injury in situations such as hemorrhagic shock.
Assuntos
Injúria Renal Aguda/etiologia , Choque Hemorrágico/complicações , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Animais , Modelos Animais de Doenças , Taxa de Filtração Glomerular , Receptor Celular 1 do Vírus da Hepatite A , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/patologia , Falência Renal Crônica/etiologia , Falência Renal Crônica/patologia , Falência Renal Crônica/fisiopatologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Choque Hemorrágico/patologia , Choque Hemorrágico/fisiopatologia , Fatores de TempoRESUMO
Impaired regulation of hepcidin expression in response to iron loading appears to be the pathogenic mechanism for hereditary hemochromatosis. Iron normally induces expression of the BMP6 ligand, which, in turn, activates the BMP/Smad signaling cascade directing hepcidin expression. The molecular function of the HFE protein, involved in the most common form of hereditary hemochromatosis, is still unknown. We have used Hfe-deficient mice of different genetic backgrounds to test whether HFE has a role in the signaling cascade induced by BMP6. At 7 weeks of age, these mice have accumulated iron in their liver and have increased Bmp6 mRNA and protein. However, in contrast to mice with secondary iron overload, levels of phosphorylated Smads 1/5/8 and of Id1 mRNA, both indicators of BMP signaling, are not significantly higher in the liver of these mice than in wild-type livers. As a consequence, hepcidin mRNA levels in Hfe-deficient mice are similar or marginally reduced, compared with 7-week-old wild-type mice. The inappropriately low levels of Id1 and hepcidin mRNA observed at weaning further suggest that Hfe deficiency triggers iron overload by impairing hepatic Bmp/Smad signaling. HFE therefore appears to facilitate signal transduction induced by the BMP6 ligand.
Assuntos
Proteína Morfogenética Óssea 6/metabolismo , Antígenos de Histocompatibilidade Classe I/fisiologia , Proteínas de Membrana/fisiologia , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Western Blotting , Proteína Morfogenética Óssea 6/genética , Proteína da Hemocromatose , Hepcidinas , Técnicas Imunoenzimáticas , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Ferro/administração & dosagem , Sobrecarga de Ferro , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína Smad1/genética , Proteína Smad5/genética , Proteína Smad8/genéticaRESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has the poorest overall prognosis among gastrointestinal cancers; however, curative resection in early-stage PDAC greatly improves survival rates, indicating the importance of early detection. Because abnormal microRNA production is commonly detected in cancer, we investigated noninvasive precursor pancreatic intraepithelial neoplasia (PanIN) lesions for microRNA production as a potential early biomarker of PDAC. METHODS: Pathologists identified and classified ductal lesions. We extracted total RNA from laser-capture microdissected PanIN tissue samples from a conditional KRAS(G12D) mouse model (n = 29) or of human origin (n = 38) (KRAS is v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). MicroRNA production was quantified by quantitative real-time PCR. Internal controls included 5S and U6 RNAs. RESULTS: Production of microRNAs miR-21, miR-205, and miR-200 paralleled PanIN progression in the KRAS(G12D) mouse model, compared with microRNA production in samples of nonpathologic ducts. miR-21 demonstrated the highest relative concentrations in the precursor lesions. Interestingly, miR-205 and miR-21 up-regulation preceded phenotypic changes in the ducts. The production of microRNAs miR-21, miR-221, miR-222, and let-7a increased with human PanIN grade, with peak production occurring in hyperplastic PanIN-2/3 lesions. In situ hybridization analysis indicated miR-21 production to be concentrated in pathologic ductal cells. miR-21 production was regulated by KRAS(G12D) and epidermal growth factor receptor in PDAC-derived cell lines. CONCLUSIONS: Aberrant microRNA production is an early event in the development of PanIN. Our findings indicate that miR-21 warrants further investigation as a marker for early detection of PDAC.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Animais , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/cirurgia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Knockout , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
Cell surface expression of MHC class I molecules by tumor cells is determinant in the interplay between tumor cells and the immune system. Nevertheless, the mechanisms which regulate MHCI expression on tumor cells are not clear. We previously showed that immune innate cells from the spleen can regulate MHCI expression on MHCI(low) tumor cells. Here, using the murine model of B16 melanoma, we demonstrate that the MHCI status of tumor cells in vivo is regulated by the microenvironment. In subcutaneous grafts, induction of MHCI molecules on tumor cells is concomitant to the recruitment of lymphocytes and relies on an IFNgamma-mediated mechanism. gammadelta T and NK cells are essential to this regulation. A small proportion of tumor-infiltrating NK cells and gammadelta T cells were found to produce IFNgamma, suggesting a possible direct participation to the MHCI increase on the tumor cells upon tumor cell recognition. Depletion of gammadelta T cells increases the tumor growth rate, confirming their anti-tumoral role in our model. Taken together, our results demonstrate that in vivo, NK and gammadelta T cells play a dual role during the early growth of MHCI(low) tumor cells. In addition to controlling the growth of tumor cells, they contribute to modifying the immunogenic profile of residual tumor cells.