RESUMO
BACKGROUND AND AIMS: Inflammatory bowel diseases (IBD) are characterized by mucosal inflammation and sequential fibrosis formation, but the exact role of the hyperactive NLRP3 inflammasome in these processes is unclear. Thus, we studied the expression and function of the NLRP3 inflammasome in the context of inflammation and fibrosis in IBD. METHODS: We analysed intestinal NLRP3 expression in mucosal immune cells and fibroblasts from IBD patients and NLRP3-associated gene expression via single-cell RNA sequencing and microarray analyses. Furthermore, cytokine secretion of NLRP3 inhibitor treated blood and mucosal cells, as well as proliferation, collagen production, and cell death of NLRP3 inhibitor treated intestinal fibroblasts from IBD patients were studied. RESULTS: We found increased NLRP3 expression in the inflamed mucosa of IBD patients and NLRP3 inhibition led to reduced IL-1ß and IL-18 production in blood cells and diminished the bioactive form of mucosal IL-1ß. Single cell analysis identified overlapping expression patterns of NLRP3 and IL-1ß in classically activated intestinal macrophages and we also detected NLRP3 expression in CD163+ macrophages. In addition, NLRP3 expression was also found in intestinal fibroblasts from IBD patients. Inhibition of NLRP3 led to reduced proliferation of intestinal fibroblasts, which was associated with a marked decrease in production of collagen type I and type VI in IBD patients. Moreover, NLRP3 inhibition in intestinal fibroblasts induced autophagy, a cellular process involved in collagen degradation. CONCLUSIONS: In the presented study, we demonstrate that inhibiting NLRP3 might pave the way for novel therapeutic approaches in IBD, especially to prevent the severe complication of intestinal fibrosis formation.
Assuntos
Doenças Inflamatórias Intestinais , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Mucosa/metabolismo , Interleucina-1beta/metabolismo , Inflamação , Fibroblastos/metabolismo , Colágeno , FibroseRESUMO
T cell proliferation and cytokine production are bioenergetically and biosynthetically costly. The inability to meet these metabolic demands results in altered differentiation, accompanied by impaired effector function, and attrition of the immune response. Interleukin-17-producing CD4 T cells (TH17s) are mediators of host defense, autoimmunity, and antitumor immunity in the setting of adoptive T cell therapy. TH17s are long-lived cells that require mitochondrial oxidative phosphorylation (OXPHOS) for effector function in vivo. Considering that TH17s polarized under standardized culture conditions are predominately glycolytic, little is known about how OXPHOS regulates TH17 processes, such as their ability to persist and thus contribute to protracted immune responses. Here, we modified standardized culture medium and identified a culture system that reliably induces OXPHOS dependence in TH17s. We found that TH17s cultured under OXPHOS conditions metabolically resembled their in vivo counterparts, whereas glycolytic cultures were dissimilar. OXPHOS TH17s exhibited increased mitochondrial fitness, glutamine anaplerosis, and an antiapoptotic phenotype marked by high BCL-XL and low BIM. Limited mitophagy, mediated by mitochondrial fusion regulator OPA-1, was critical to apoptotic resistance in OXPHOS TH17s. By contrast, glycolytic TH17s exhibited more mitophagy and an imbalance in BCL-XL to BIM, thereby priming them for apoptosis. In addition, through adoptive transfer experiments, we demonstrated that OXPHOS protected TH17s from apoptosis while enhancing their persistence in the periphery and tumor microenvironment in a murine model of melanoma. Together, our work demonstrates how metabolism regulates TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.
Assuntos
Fosforilação Oxidativa , Microambiente Tumoral , Camundongos , Animais , Mitocôndrias/metabolismo , Glicólise/genética , Diferenciação CelularRESUMO
Naive T cells are thought to be functionally quiescent. In this study, we studied and compared the phenotype, cytokine profile, and potential function of human naive CD4+ T cells in umbilical cord and peripheral blood. We found that naive CD4+ T cells, but not memory T cells, expressed high levels of chemokine CXCL8. CXCL8+ naive T cells were preferentially enriched CD31+ T cells and did not express T cell activation markers or typical Th effector cytokines, including IFN-γ, IL-4, IL-17, and IL-22. In addition, upon activation, naive T cells retained high levels of CXCL8 expression. Furthermore, we showed that naive T cell-derived CXCL8 mediated neutrophil migration in the in vitro migration assay, supported tumor sphere formation, and promoted tumor growth in an in vivo human xenograft model. Thus, human naive T cells are phenotypically and functionally heterogeneous and can carry out active functions in immune responses.
Assuntos
Células Sanguíneas/fisiologia , Interleucina-8/metabolismo , Neoplasias Experimentais/imunologia , Neutrófilos/fisiologia , Linfócitos T/fisiologia , Cordão Umbilical/patologia , Animais , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Interleucina-8/genética , Ativação Linfocitária , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma is a cancer of neural crest stem cell (NCSC) lineage. Signaling pathways that regulate NCSC differentiation have been implicated in neuroblastoma tumorigenesis. This is exemplified by MYCN oncogene targets that balance proliferation, differentiation, and cell death similarly in normal NCSC and in high-risk neuroblastoma. Our previous work discovered a survival mechanism by which MYCN-amplified neuroblastoma circumvents cell death by upregulating components of the error-prone non-canonical alternative nonhomologous end-joining (alt-NHEJ) DNA repair pathway. Similar to proliferating stem cells, high-risk neuroblastoma cells have enhanced DNA repair capacity, overcoming DNA damage with higher repair efficiency than somatic cells. Adequate DNA maintenance is required for lineage protection as stem cells proliferate and during tumor progression to overcome oncogene-induced replication stress. On this basis, we hypothesized that alt-NHEJ overexpression in neuroblastoma is a cancer cell survival mechanism that originates from DNA repair systems of NCSC, the presumed progenitor cell of origin. A human NCSC model was generated in which inducible MYCN triggered an immortalized phenotype capable of forming metastatic neuroectodermal tumors in mice, resembling human neuroblastoma. Critical alt-NHEJ components (DNA Ligase III, DNA Ligase I, and Poly [ADP-ribose polymerase 1]) were highly expressed in normal early NCSC, and decreased as cells became terminally differentiated. Constitutive MYCN expression maintained high alt-NHEJ protein expression, preserving the expression pattern of the immature neural phenotype. siRNA knockdown of alt-NHEJ components reversed MYCN effects on NCSC proliferation, invasion, and migration. DNA Ligase III, Ligase I, and PARP1 silencing significantly decreased neuroblastoma markers expression (TH, Phox2b, and TRKB). These results utilized the first human NCSC model of neuroblastoma to uncover an important link between MYCN and alt-NHEJ expression in developmental tumor initiation, setting precedence to investigate alt-NHEJ repair mechanics in neuroblastoma DNA maintenance.
Assuntos
Transformação Celular Neoplásica/genética , Reparo do DNA por Junção de Extremidades , Regulação Neoplásica da Expressão Gênica , Proteína Proto-Oncogênica N-Myc/genética , Células-Tronco Neurais/metabolismo , Neuroblastoma/genética , Animais , Diferenciação Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , DNA Ligase Dependente de ATP/antagonistas & inibidores , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteína Proto-Oncogênica N-Myc/metabolismo , Crista Neural/metabolismo , Crista Neural/patologia , Células-Tronco Neurais/patologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransgenesRESUMO
Integration of signaling and metabolic pathways enables and sustains lymphocyte function. Whereas metabolic changes occurring during T cell activation are well characterized, the metabolic demands of differentiated T lymphocytes are largely unexplored. In this study, we defined the bioenergetics of Th17 effector cells generated in vivo. These cells depend on oxidative phosphorylation (OXPHOS) for energy and cytokine production. Mechanistically, the essential role of OXPHOS in Th17 cells results from their limited capacity to increase glycolysis in response to metabolic stresses. This metabolic program is observed in mouse and human Th17 cells, including those isolated from Crohn disease patients, and it is linked to disease, as inhibiting OXPHOS reduces the severity of murine colitis and psoriasis. These studies highlight the importance of analyzing metabolism in effector lymphocytes within in vivo inflammatory contexts and suggest a therapeutic role for manipulating OXPHOS in Th17-driven diseases.
Assuntos
Diferenciação Celular/imunologia , Colite/imunologia , Ativação Linfocitária/imunologia , Fosforilação Oxidativa , Células Th17/imunologia , Animais , Separação Celular , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , TranscriptomaRESUMO
OBJECTIVES: To identify whether mast cell densities in vulvar biopsies from the vestibule are associated with vulvodynia. METHODS: We enrolled 100 women aged 19 to 59 years with confirmed vulvodynia cases, 100 racially matched controls, and 100 black control women. All had vulvar biopsies performed at the 7 o'clock position of the vestibule, which were then immunostained to detect c-KIT protein. The numbers of c-KIT positive mast cells per ×400 magnification field were manually counted, and t tests and logistic regression were used to assess the association with case-control status. RESULTS: Of the biopsies, 235 were adequate samples for c-KIT testing for mast cells. The mast cell density was substantially lower in black control women (13.9 ± 10.9) in comparison to white control women (22.5 ± 13.2 p < 0.001): hence the analysis was confined to white cases and racially matched control women. Compared with racially matched controls, cases were younger, more likely to be married, and reported a higher household income. The average number of mast cells per ×400 magnification field overall was 19.1 ± 13.2 (range, 0-62). There was no difference in the mast cell count between racially matched cases (22.4 ± 13.9 per ×400 field) and controls (22.5 ± 13.2) in either the univariate or multivariable analyses. Within the group of cases, there was no difference in mast cell density based on the presence or absence of a variety of urogenital symptoms. CONCLUSIONS: No difference in mast cell density in biopsies of the vestibule was found between white cases and racially matched controls. Black control women have a lower mast cell density compared with white control women.
Assuntos
Mastócitos/imunologia , Vulva/patologia , Vulvodinia/patologia , Adulto , Biópsia , Estudos de Casos e Controles , Feminino , Histocitoquímica , Humanos , Imuno-Histoquímica , Contagem de Leucócitos , Microscopia , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-kit/análise , Adulto JovemRESUMO
BACKGROUND: Resveratrol inhibits the growth of ovarian carcinoma cells in vitro through the inhibition of glucose metabolism and the induction of both autophagy and apoptosis. In the current study, we investigated the metabolic and therapeutic effects of resveratrol in vivo. METHODS: A fluorescent xenograft mouse model of ovarian cancer was used. Mice were treated with cisplatin, resveratrol, or vehicle alone. Tumor burden was assessed using whole-body imaging. The effect of resveratrol on glucose uptake in vivo was determined using micro-positron emission tomography scanning. To determine whether resveratrol could inhibit tumor regrowth, tumor-bearing mice were treated with cisplatin followed by either daily resveratrol or vehicle. Autophagic response in resected tumors taken from mice treated with resveratrol was examined by transmission electron microscopy. Glycolysis and mitochondrial respiration in ovarian tumor cells after treatment with resveratrol was assessed. RESULTS: Mice treated with resveratrol and cisplatin were found to have a significantly reduced tumor burden compared with control animals (P<.001). Resveratrol-treated mice demonstrated a marked decrease in tumor uptake of glucose compared with controls. After treatment with cisplatin, "maintenance" resveratrol resulted in the suppression of tumor regrowth compared with mice receiving vehicle alone (P<.01). Tumors resected from mice treated with resveratrol exhibited autophagosomes consistent with the induction of autophagy. Treatment with resveratrol inhibited glycolytic response in ovarian tumor cells with high baseline glycolytic rates. CONCLUSIONS: Treatment with resveratrol inhibits glucose uptake and has a significant antineoplastic effect in a preclinical mouse model of ovarian cancer. Resveratrol treatment suppresses tumor regrowth after therapy with cisplatin, suggesting that this agent has the potential to prolong disease-free survival. Cancer 2016;122:722-729. © 2015 American Cancer Society.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Epiteliais e Glandulares/diagnóstico por imagem , Neoplasias Ovarianas/diagnóstico por imagem , Estilbenos/farmacologia , Animais , Antineoplásicos/farmacologia , Carcinoma Epitelial do Ovário , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Feminino , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Camundongos , Transplante de Neoplasias , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Tomografia por Emissão de Pósitrons , Resveratrol , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The coinhibitory receptor programmed death-1 (PD-1) maintains immune homeostasis by negatively regulating T cell function and survival. Blockade of PD-1 increases the severity of graft-versus-host disease (GVHD), but the interplay between PD-1 inhibition and T cell metabolism is not well studied. We found that both murine and human alloreactive T cells concomitantly upregulated PD-1 expression and increased levels of reactive oxygen species (ROS) following allogeneic bone marrow transplantation. This PD-1(Hi)ROS(Hi) phenotype was specific to alloreactive T cells and was not observed in syngeneic T cells during homeostatic proliferation. Blockade of PD-1 signaling decreased both mitochondrial H2O2 and total cellular ROS levels, and PD-1-driven increases in ROS were dependent upon the oxidation of fatty acids, because treatment with etomoxir nullified changes in ROS levels following PD-1 blockade. Downstream of PD-1, elevated ROS levels impaired T cell survival in a process reversed by antioxidants. Furthermore, PD-1-driven changes in ROS were fundamental to establishing a cell's susceptibility to subsequent metabolic inhibition, because blockade of PD-1 decreased the efficacy of later F1F0-ATP synthase modulation. These data indicate that PD-1 facilitates apoptosis in alloreactive T cells by increasing ROS in a process dependent upon the oxidation of fat. In addition, blockade of PD-1 undermines the potential for subsequent metabolic inhibition, an important consideration given the increasing use of anti-PD-1 therapies in the clinic.
Assuntos
Sobrevivência Celular , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos/imunologia , Apoptose/genética , Apoptose/imunologia , Transplante de Medula Óssea/efeitos adversos , Sobrevivência Celular/genética , Ácidos Graxos/metabolismo , Feminino , Expressão Gênica , Doença Enxerto-Hospedeiro/etiologia , Xenoenxertos , Humanos , Camundongos , Camundongos Transgênicos , Oxirredução , Fenótipo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mitochondria are multifunctional organelles that play a central role in cellular homeostasis. Severe mitochondrial dysfunction leads to life-threatening diseases in humans and accelerates the aging process. Surprisingly, moderate reduction of mitochondrial function in different species has anti-aging effects. High-throughput screenings in the nematode Caenorhabditis elegans lead to the identification of several pro-longevity genetic and pharmacological interventions. Large-scale screens, however, are manual, subjective, time consuming and costly. These limitations could be reduced by the identification of automatically quantifiable biomarkers of healthy aging. In this study we exploit the distinct and reproducible phenotypes described in C. elegans upon different levels of mitochondrial alteration to develop an automated high-content strategy to identify new potential pro-longevity interventions. Utilizing the microscopy platform Cellomics ArrayScan Reader, we optimize a workflow to automatically and reliably quantify the discrete phenotypic readouts associated with different degrees of silencing of mitochondrial respiratory chain regulatory proteins, and validate the approach with mitochondrial-targeting drugs known to extend lifespan in C. elegans. Finally, we report that a new mitochondrial ATPase modulator matches our screening phenotypic criteria and extends nematode's lifespan thus providing the proof of principle that our strategy could be exploited to identify novel mitochondrial-targeted drugs with pro-longevity activity. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.
Assuntos
Caenorhabditis elegans/fisiologia , Longevidade , Mitocôndrias/fisiologia , Animais , Microscopia , Mitocôndrias/efeitos dos fármacos , FenótipoRESUMO
The 2006 National Institutes of Health (NIH) Consensus paper presented recommendations by the Ancillary Therapy and Supportive Care Working Group to support clinical research trials in chronic graft-versus-host disease (GVHD). Topics covered in that inaugural effort included the prevention and management of infections and common complications of chronic GVHD, as well as recommendations for patient education and appropriate follow-up. Given the new literature that has emerged during the past 8 years, we made further organ-specific refinements to these guidelines. Minimum frequencies are suggested for monitoring key parameters relevant to chronic GVHD during systemic immunosuppressive therapy and, thereafter, referral to existing late effects consensus guidelines is advised. Using the framework of the prior consensus, the 2014 NIH recommendations are organized by organ or other relevant systems and graded according to the strength and quality of supporting evidence.
Assuntos
Antineoplásicos/uso terapêutico , Doença Enxerto-Hospedeiro/terapia , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Imunossupressores/uso terapêutico , Transplante de Medula Óssea , Doença Crônica , Consenso , Contraindicações , Gerenciamento Clínico , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Humanos , Fármacos Fotossensibilizantes , Guias de Prática Clínica como Assunto , Ensaios Clínicos Controlados Aleatórios como Assunto , Transplante HomólogoRESUMO
UNLABELLED: In neuroblastoma, MYCN genomic amplification and segmental chromosomal alterations including 1p or 11q loss of heterozygocity and/or 17q gain are associated with progression and poor clinical outcome. Segmental alterations are the strongest predictor of relapse and result from unbalanced translocations attributable to erroneous repair of chromosomal breaks. Although sequence analysis of affected genomic regions suggests that these errors arise by nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB), abnormalities in NHEJ have not been implicated in neuroblastoma pathogenesis. On this basis, the hypothesis that an error-prone mechanism of NHEJ is critical for neuroblastoma cell survival was tested. Plasmid-based DSB repair assays demonstrated efficient NHEJ activity in human neuroblastoma cells with repair products that were error-prone relative to nontransformed cells. Neuroblastoma cells derived from tumorigenic neuroblastic phenotypes had differential DNA repair protein expression patterns compared with nontumorigenic cells. Tumorigenic neuroblastoma cells were deficient in DNA ligase IV (Lig4) and Artemis (DCLRE1C), mediators of canonical NHEJ. Conversely, enzymes required for an error-prone alternative NHEJ pathway (alt-NHEJ), DNA Ligase IIIα (Lig3), DNA Ligase I (Lig1), and PARP1 protein were upregulated. Inhibition of Lig3 and Lig1 led to DSB accumulation and cell death, linking alt-NHEJ to cell survival in neuroblastoma. Neuroblastoma cells demonstrated sensitivity to PARP1 inhibition (PARPi) that paralleled PARP1 expression. In a dataset of human neuroblastoma patient tumors, overexpression of genes encoding alt-NHEJ proteins associated with poor survival. IMPLICATIONS: These findings provide an insight into DNA repair fidelity in neuroblastoma and identify components of the alt-NHEJ pathway as promising therapeutic targets.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Neuroblastoma/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , PrognósticoRESUMO
T-cell activation requires increased ATP and biosynthesis to support proliferation and effector function. Most models of T-cell activation are based on in vitro culture systems and posit that aerobic glycolysis is employed to meet increased energetic and biosynthetic demands. By contrast, T cells activated in vivo by alloantigens in graft-versus-host disease (GVHD) increase mitochondrial oxygen consumption, fatty acid uptake, and oxidation, with small increases of glucose uptake and aerobic glycolysis. Here we show that these differences are not a consequence of alloactivation, because T cells activated in vitro either in a mixed lymphocyte reaction to the same alloantigens used in vivo or with agonistic anti-CD3/anti-CD28 antibodies increased aerobic glycolysis. Using targeted metabolic (13)C tracer fate associations, we elucidated the metabolic pathway(s) employed by alloreactive T cells in vivo that support this phenotype. We find that glutamine (Gln)-dependent tricarboxylic acid cycle anaplerosis is increased in alloreactive T cells and that Gln carbon contributes to ribose biosynthesis. Pharmacological modulation of oxidative phosphorylation rapidly reduces anaplerosis in alloreactive T cells and improves GVHD. On the basis of these data, we propose a model of T-cell metabolism that is relevant to activated lymphocytes in vivo, with implications for the discovery of new drugs for immune disorders.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Isoantígenos/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD28/imunologia , Complexo CD3/imunologia , Ciclo do Ácido Cítrico/imunologia , Feminino , Glutamina/metabolismo , Glicólise/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Camundongos , Fosforilação Oxidativa , Ribose/biossínteseRESUMO
Activated T cells require increased energy to proliferate and mediate effector functions, but the metabolic changes that occur in T cells following stimulation in vivo are poorly understood, particularly in the context of inflammation. We have previously shown that T cells activated during graft-versus-host disease (GVHD) primarily rely on oxidative phosphorylation to synthesize adenosine 5'-triphosphate. Here, we demonstrate that alloreactive effector T cells (Teff) use fatty acids (FAs) as a fuel source to support their in vivo activation. Alloreactive T cells increased FA transport, elevated levels of FA oxidation enzymes, up-regulated transcriptional coactivators to drive oxidative metabolism, and increased their rates of FA oxidation. Importantly, increases in FA transport and up-regulation of FA oxidation machinery occurred specifically in T cells during GVHD and were not seen in Teff following acute activation. Pharmacological blockade of FA oxidation decreased the survival of alloreactive T cells but did not influence the survival of T cells during normal immune reconstitution. These studies suggest that pathways controlling FA metabolism might serve as therapeutic targets to treat GVHD and other T-cell-mediated immune diseases.
Assuntos
Ácidos Graxos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Western Blotting , Transplante de Medula Óssea/métodos , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/imunologia , Carnitina O-Palmitoiltransferase/metabolismo , Inibidores Enzimáticos/farmacologia , Compostos de Epóxi/farmacologia , Ácidos Graxos/metabolismo , Feminino , Citometria de Fluxo , Doença Enxerto-Hospedeiro/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Transplante HomólogoRESUMO
Ku70 was originally described as an autoantigen, but it also functions as a DNA repair protein in the nucleus and as an antiapoptotic protein by binding to Bax in the cytoplasm, blocking Bax-mediated cell death. In neuroblastoma (NB) cells, Ku70's binding with Bax is regulated by Ku70 acetylation such that increasing Ku70 acetylation results in Bax release, triggering cell death. Although regulating cytoplasmic Ku70 acetylation is important for cell survival, the role of nuclear Ku70 acetylation in DNA repair is unclear. Here, we showed that Ku70 acetylation in the nucleus is regulated by the CREB-binding protein (CBP), and that Ku70 acetylation plays an important role in DNA repair in NB cells. We treated NB cells with ionization radiation and measured DNA repair activity as well as Ku70 acetylation status. Cytoplasmic and nuclear Ku70 were acetylated after ionization radiation in NB cells. Interestingly, cytoplasmic Ku70 was redistributed to the nucleus following irradiation. Depleting CBP in NB cells results in reducing Ku70 acetylation and enhancing DNA repair activity in NB cells, suggesting nuclear Ku70 acetylation may have an inhibitory role in DNA repair. These results provide support for the hypothesis that enhancing Ku70 acetylation, through deacetylase inhibition, may potentiate the effect of ionization radiation in NB cells.
Assuntos
Antígenos Nucleares/metabolismo , Proteína de Ligação a CREB/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neuroblastoma/metabolismo , Acetilação/efeitos da radiação , Antígenos Nucleares/genética , Apoptose/efeitos da radiação , Proteína de Ligação a CREB/genética , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Citoplasma/metabolismo , Citoplasma/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , DNA de Neoplasias/genética , DNA de Neoplasias/efeitos da radiação , Proteínas de Ligação a DNA/genética , Fibroblastos/efeitos da radiação , Humanos , Autoantígeno Ku , Neuroblastoma/genética , Radiação IonizanteRESUMO
For several decades, it has been known that T-cell activation in vitro leads to increased glycolytic metabolism that fuels proliferation and effector function. Recently, this simple model has been complicated by the observation that different T-cell subsets differentially regulate fundamental metabolic pathways under the control of distinct molecular regulators. Although the majority of these data have been generated in vitro, several recent studies have documented the metabolism of T cells activated in vivo. Here, we review the recent data surrounding the differential regulation of metabolism by distinct T-cell subsets in vitro and in vivo and discuss how differential metabolic regulation might facilitate T-cell function vis-à-vis proliferation, survival, and energy production. We further discuss the important therapeutic implications of differential metabolism across T-cell subsets and review recent successes in exploiting lymphocyte metabolism to treat immune-mediated diseases.
Assuntos
Imunomodulação , Ativação Linfocitária , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Metabolismo Energético , Glicólise , Humanos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ku70 was first characterized as a nuclear factor that binds DNA double-strand breaks in nonhomolog end-joining DNA repair. However, recent studies have shown that Ku70 is also found in the cytoplasm and binds Bax, preventing Bax-induced cell death. We have shown that, in neuroblastoma cells, the binding between Ku70 and Bax depends on the acetylation status of Ku70, such that, when Ku70 is acetylated, Bax is released from Ku70, triggering cell death. Thus, to survive, in neuroblastoma cells, cytoplasmic Ku70 acetylation status is carefully regulated such that Ku70 is maintained in a deacetylated state, keeping Bax complexed with Ku70. We have shown that overexpression of CREB-binding protein (CBP), a known acetyltransferase that acetylates Ku70, releases Bax from Ku70, triggering apoptosis. Although we have shown that blocking deacetylase activity using non-type-specific inhibitors also triggers Ku70 acetylation and Bax-dependent cell death, the targets of these deacetylase inhibitors in neuroblastoma cells remain unknown. Here, we demonstrate that, in neuroblastoma cells, histone deacetylase 6 (HDAC6) binds Ku70 and Bax in the cytoplasm and that knocking down HDAC6 or using an HDAC6-specific inhibitor triggers Bax-dependent cell death. Our results show that HDAC6 regulates the interaction between Ku70 and Bax in neuroblastoma cells and may be a therapeutic target in this pediatric solid tumor.
Assuntos
Antígenos Nucleares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/metabolismo , Neuroblastoma/metabolismo , Proteína X Associada a bcl-2/metabolismo , Acetilação/efeitos dos fármacos , Antígenos Nucleares/genética , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Autoantígeno Ku , Neuroblastoma/genética , Neuroblastoma/patologia , Ligação Proteica/efeitos dos fármacosRESUMO
BACKGROUND: Persistent infection with high-risk human papillomavirus (HPV) types is necessary for the development of high-grade cervical dysplasia and cervical carcinoma. The presence of HPV DNA in the blood of cervical cancer patients has been reported; however, whether HPV DNA is detectable in the blood of patients with pre-invasive cervical disease is unclear. OBJECTIVES: The objectives of this study were to determine if HPV 16 and HPV 18 DNA could be detected in the serum of colposcopy clinic patients, and if serum HPV detection was associated with grade of cervical disease and HPV cofactors. STUDY DESIGN: Samples were selected from a biorepository collected from non-pregnant, HIV-negative women ages 18-69 attending colposcopy clinics at two urban public hospitals. Cervical disease status was based on review of colposcopy, biopsy and cytology findings. Serum HPV DNA detection was conducted using a novel PCR and mass spectroscopy-based assay. RESULTS: Of the 116 adequate serum samples, all (100%) were negative for HPV 16 and HPV 18. Over half (51.7%) of participants had cervical HPV 16 and/or HPV 18 infection. Nearly one-third (31.1%) had high grade, 10.3% had low grade, and 50.9% had no cervical disease. Nearly one-third (28.5%) had ever regularly smoked cigarettes, 70.7% had early onset of sexual intercourse, and 75% had ever used oral contraceptives. CONCLUSIONS: In this colposcopy clinic population with a range of clinical characteristics and established HPV cofactors, HPV DNA was undetectable in their serum. Our findings suggest that serum HPV DNA detection is not a cervical cancer screening tool.
Assuntos
DNA Viral/sangue , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/isolamento & purificação , Infecções por Papillomavirus/sangue , Doenças do Colo do Útero/sangue , Adolescente , Adulto , Idoso , Biópsia , Colposcopia/métodos , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase , Doenças do Colo do Útero/diagnóstico , Doenças do Colo do Útero/genética , Doenças do Colo do Útero/patologia , Neoplasias do Colo do Útero/sangue , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Adulto Jovem , Displasia do Colo do Útero/sangue , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/patologia , Displasia do Colo do Útero/virologiaRESUMO
Cells generate adenosine triphosphate (ATP) by glycolysis and by oxidative phosphorylation (OXPHOS). Despite the importance of having sufficient ATP available for the energy-dependent processes involved in immune activation, little is known about the metabolic adaptations that occur in vivo to meet the increased demand for ATP in activated and proliferating lymphocytes. We found that bone marrow (BM) cells proliferating after BM transplantation (BMT) increased aerobic glycolysis but not OXPHOS, whereas T cells proliferating in response to alloantigens during graft-versus-host disease (GVHD) increased both aerobic glycolysis and OXPHOS. Metabolomic analysis of alloreactive T cells showed an accumulation of acylcarnitines consistent with changes in fatty acid oxidation. Alloreactive T cells also exhibited a hyperpolarized mitochondrial membrane potential (ΔΨm), increased superoxide production, and decreased amounts of antioxidants, whereas proliferating BM cells did not. Bz-423, a small-molecule inhibitor of the mitochondrial F(1)F(0) adenosine triphosphate synthase (F(1)F(0)-ATPase), selectively increased superoxide and induced the apoptosis of alloreactive T cells, which arrested established GVHD in several BMT models without affecting hematopoietic engraftment or lymphocyte reconstitution. These findings challenge the current paradigm that activated T cells meet their increased demands for ATP through aerobic glycolysis, and identify the possibility that bioenergetic and redox characteristics can be selectively exploited as a therapeutic strategy for immune disorders.
Assuntos
Apoptose/imunologia , Doença Enxerto-Hospedeiro/imunologia , Isoantígenos/imunologia , Fosforilação Oxidativa , Linfócitos T/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzodiazepinas/farmacologia , Benzodiazepinas/uso terapêutico , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/imunologia , Feminino , Doença Enxerto-Hospedeiro/tratamento farmacológico , Lactatos/metabolismo , Ativação Linfocitária , Metaboloma , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Consumo de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/efeitos dos fármacosRESUMO
Clusterin is a ubiquitously expressed glycoprotein with multiple binding partners including IL-6, Ku70, and Bax. Clusterin blocks apoptosis by binding to activated Bax and sequestering it in the cytoplasm, thereby preventing Bax from entering mitochondria, releasing cytochrome c, and triggering apoptosis. Because increased clusterin expression correlates with aggressive behavior in tumors, clusterin inhibition might be beneficial in cancer treatment. Our recent findings indicated that, in neuroblastoma cells, cytoplasmic Bax also binds to Ku70; when Ku70 is acetylated, Bax is released and can initiate cell death. Therefore, increasing Ku70 acetylation, such as by using histone deacetylase inhibitors, may be therapeutically useful in promoting cell death in neuroblastoma tumors. Since clusterin, Bax, and Ku70 form a complex, it seemed likely that clusterin would mediate its anti-apoptotic effects by inhibiting Ku70 acetylation and blocking Bax release. Our results, however, demonstrate that while clusterin level does indeed determine the sensitivity of neuroblastoma cells to histone deacetylase inhibitor-induced cell death, it does so without affecting histone deacetylase-inhibitor-induced Ku70 acetylation. Our results suggest that in neuroblastoma, clusterin exerts its anti-apoptotic effects downstream of Ku70 acetylation, likely by directly blocking Bax activation.
Assuntos
Apoptose/efeitos dos fármacos , Clusterina/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Neuroblastoma/patologia , Acetilação/efeitos dos fármacos , Antígenos Nucleares/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Clusterina/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Autoantígeno Ku , Neuroblastoma/fisiopatologia , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/fisiologiaRESUMO
BACKGROUND: Chemoresistance is the major factor limiting long-term treatment success in patients with epithelial ovarian cancers. Most cytotoxic drugs kill cells through apoptosis; therefore, defective execution of apoptotic pathways results in a drug-resistant phenotype in many tumor types. METHODS: A panel of ovarian cancer cell lines was screened for expression and function of the apoptosome components Apaf-1 and caspase-9. Expression levels were analyzed by immunohistochemistry and immunoblotting; Apaf-1 function was determined by assessing the ability of endogenous Apaf-1 to cleave caspase-9 in the presence or absence of cytochrome c. The effect of the histone deacetylase inhibitor trichostatin A on Apaf-1 expression and function was evaluated. RESULTS: The authors report here that the resistance of ovarian cancer cells to the proapoptotic effects of chemotherapy is due in part to deficient Apaf-1 activity. Although Apaf-1 is expressed in most ovarian cancers, the functional activity is impaired, as Apaf-1 has a diminished ability to recruit and activate caspase-9. Treatment of ovarian cancer cells with trichostatin A results in restoration of Apaf-1 function independent of alterations in Apaf-1 expression. Furthermore, treating chemoresistant cells with sublethal doses of trichostatin A restores Apaf-1 function and sensitizes cells to cisplatin-induced apoptosis. CONCLUSIONS: Targeting intrinsic pathway defects for therapeutic intervention may result in sensitizing tumors to standard chemotherapy or triggering apoptosis in the absence of other apoptotic signals. The identification of drugs that can use Apaf-1 when it is present, yet can overcome its functional inactivation, may be an important clinical advance.