RESUMO
Epigenomic mechanisms regulate distinct aspects of the inflammatory response in immune cells. Despite the central role for microglia in neuroinflammation and neurodegeneration, little is known about their epigenomic regulation of the inflammatory response. Here, we show that Ten-eleven translocation 2 (TET2) methylcytosine dioxygenase expression is increased in microglia upon stimulation with various inflammogens through a NF-κB-dependent pathway. We found that TET2 regulates early gene transcriptional changes, leading to early metabolic alterations, as well as a later inflammatory response independently of its enzymatic activity. We further show that TET2 regulates the proinflammatory response in microglia of mice intraperitoneally injected with LPS. We observed that microglia associated with amyloid ß plaques expressed TET2 in brain tissue from individuals with Alzheimer's disease (AD) and in 5xFAD mice. Collectively, our findings show that TET2 plays an important role in the microglial inflammatory response and suggest TET2 as a potential target to combat neurodegenerative brain disorders.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Microglia/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/veterinária , Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Dioxigenases , Elementos Facilitadores Genéticos , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Fator de Transcrição RelA/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Glioma cells recruit and exploit microglia (the resident immune cells of the brain) for their proliferation and invasion ability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype has remained elusive. We found that glioma-induced microglia conversion was coupled to a reduction in the basal activity of microglial caspase-3 and increased S-nitrosylation of mitochondria-associated caspase-3 through inhibition of thioredoxin-2 activity, and that inhibition of caspase-3 regulated microglial tumor-supporting function. Furthermore, we identified the activity of nitric oxide synthase 2 (NOS2, also known as iNOS) originating from the glioma cells as a driving stimulus in the control of microglial caspase-3 activity. Repression of glioma NOS2 expression in vivo led to a reduction in both microglia recruitment and tumor expansion, whereas depletion of microglial caspase-3 gene promoted tumor growth. Our results provide evidence that inhibition of the denitrosylation of S-nitrosylated procaspase-3 mediated by the redox protein Trx2 is a part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells.
Assuntos
Caspase 3/metabolismo , Glioma/metabolismo , Glioma/patologia , Microglia/metabolismo , Fenótipo , Animais , Linhagem Celular Tumoral , Movimento Celular , Modelos Animais de Doenças , Ativação Enzimática , Técnicas de Silenciamento de Genes , Glioma/imunologia , Xenoenxertos , Humanos , Masculino , Camundongos , Microglia/imunologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Tiorredoxinas/metabolismo , Carga TumoralRESUMO
The study of monocyte activation and differentiation has great applications in sepsis, chronic inflammatory diseases, and cancer studies. However, despite the existence of well-established protocols for monocyte purification from human blood, the isolation of murine monocytes that can be subsequently activated has not yet been fully optimized. Here we evaluate a recently developed commercial procedure for obtaining monocytes from the bone marrow based on immunomagnetic depletion of non-monocytic cells. Moreover, we compare the advantages and disadvantages of this approach relative to other existing procedures. We found that monocytes isolates generated using this technique had equal purity to those attained via depletion from peripheral blood; however, higher yields were achieved. Furthermore, isolates from this technique have lower levels of macrophage contamination than those reported in samples generated by culturing bone marrow extracts with macrophage colony-stimulating factor (M-CSF). In addition, we demonstrate that the purified monocytes are sensitive to lipopolysaccharide (LPS)-mediated activation and, therefore, are useful for studies aimed at elucidating the molecular mechanisms involved in monocyte activation and differentiation.
Assuntos
Células da Medula Óssea , Separação Celular , Monócitos/citologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacosRESUMO
Research indicates that inflammation and microglial activation are involved in the initiation and progression of Parkinson's disease (PD). Neuroinflammation contributes to the infiltration of peripheral immune cells and blood-brain barrier (BBB) leakage, linking peripheral and central inflammatory events in the pathogenesis of PD. Dopamine (DA) likely plays a role in this process. In the present study, the dopaminergic toxin 6-hydroxydopamine (6-OHDA) was used to damage dopaminergic neurons. Injection of 6-OHDA within the nigrostriatal pathway produced loss of astrocytes, disruption of the BBB, microglia activation and a reduction in osteopontin (OPN) immunoreactivity. Depletion of DA content by alpha-methylparatyrosine (α-MPT, a tyrosine hydroxylase inhibitor) reduced the infiltration of peripheral macrophages as well as the 6-OHDA-induced increase in microglial cells. DA could therefore be relevant in sustaining inflammation and lymphocyte recruitment induced by 6-OHDA, supporting DA implication in the degeneration of dopaminergic neurons induced by inflammatory processes.
Assuntos
Astrócitos/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/metabolismo , Inflamação/imunologia , Linfócitos/fisiologia , Macrófagos/fisiologia , Substância Negra/metabolismo , Animais , Movimento Celular , Corpo Estriado/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inflamação/induzido quimicamente , Inflamação/patologia , Linfócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Atividade Motora/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Osteopontina/metabolismo , Oxidopamina/farmacologia , Ratos , Ratos Wistar , Substância Negra/efeitos dos fármacos , Simpatolíticos/farmacologia , Fatores de Tempo , alfa-Metiltirosina/farmacologiaRESUMO
DNA damage immediate cellular response requires the activation of p53 by kinases. We found that p53 forms a basal stable complex with VRK1, a Ser-Thr kinase that responds to UV-induced DNA damage by specifically phosphorylating p53. This interaction takes place through the p53 DNA binding domain, and frequent DNA-contact mutants of p53, such as R273H, R248H or R280K, do not disrupt the complex. UV-induced DNA damage activates VRK1, and is accompanied by phosphorylation of p53 at Thr-18 before it accumulates. We propose that the VRK1-p53 basal complex is an early-warning system for immediate cellular responses to DNA damage.