RESUMO
APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them histone deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. In this study, to our knowledge we show for the first time that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells results in diminished production of the immunosuppressive cytokine IL-10 and induction of inflammatory APCs that effectively activate Ag-specific naive T cells and restore the responsiveness of anergic CD4(+) T cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain comprising amino acids 503-840 as being required for HDAC6 interaction with STAT3. Furthermore, by re-chromatin immunoprecipitation we confirmed that HDAC6 and STAT3 are both recruited to the same DNA sequence within the Il10 gene promoter. Of note, disruption of this complex by knocking down HDAC6 resulted in decreased STAT3 phosphorylation--but no changes in STAT3 acetylation--as well as diminished recruitment of STAT3 to the Il10 gene promoter region. The additional demonstration that a selective HDAC6 inhibitor disrupts this STAT3/IL-10 tolerogenic axis points to HDAC6 as a novel molecular target in APCs to overcome immune tolerance and tips the balance toward T cell immunity.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Histona Desacetilases/imunologia , Interleucina-10/imunologia , Fator de Transcrição STAT3/imunologia , Acetilação/efeitos dos fármacos , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Expressão Gênica , Regulação da Expressão Gênica , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Histona Desacetilases/genética , Tolerância Imunológica , Inflamação/imunologia , Interleucina-10/biossíntese , Ativação Linfocitária/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Proteínas Recombinantes/genética , Fator de Transcrição STAT3/química , Transcrição GênicaRESUMO
Rhesus TRIM5α (TRIM5α(rh)) is a cytosolic protein that potently restricts HIV-1 at an early postentry stage, prior to reverse transcription. The ability of TRIM5α(rh) to block HIV-1 infection has been correlated with a decrease of pelletable HIV-1 capsid during infection. To genetically dissect the ability of TRIM5α to block reverse transcription, we studied a set of TRIM5α(rh) RING domain mutants that potently restrict HIV-1 but allow the occurrence of reverse transcription. These TRIM5α(rh) RING variants blocked HIV-1 infection after reverse transcription but prior to integration, as suggested by the routing of nuclear viral DNA to circularization in the form of 2-long terminal repeat (2-LTR) circles. The folding of RING domain variants was similar to that of the wild type, as evaluated by nuclear magnetic resonance. RING domain changes that allowed the occurrence of reverse transcription were impaired in their ability to decrease the amount of pelletable capsid compared with wild-type TRIM5α. Similar effects of this particular group of mutations were observed with human TRIM5α inhibition of N-tropic murine leukemia virus (N-MLV). Interestingly, TRIM5α(rh) RING domain variants also prevented the degradation of TRIM5α(rh) that occurs following cell entry of HIV-1. These data correlated the block of reverse transcription with the ability of TRIM5α to accelerate uncoating. Collectively, these results suggest that TRIM5α(rh) blocks HIV-1 reverse transcription by inducing premature viral uncoating in target cells.
Assuntos
Proteínas de Transporte/metabolismo , HIV-1/fisiologia , Mutação , Proteínas/metabolismo , Transcrição Gênica , Animais , Fatores de Restrição Antivirais , Sequência de Bases , Proteínas de Transporte/genética , Linhagem Celular , Sondas de DNA , Cães , Repetição Terminal Longa de HIV , Humanos , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Proteínas/genética , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Reversa , Timócitos/virologia , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Desenvelopamento do VírusRESUMO
TNPO3 is a nuclear importer required for HIV-1 infection. Here, we show that depletion of TNPO3 leads to an HIV-1 block after nuclear import but prior to integration. To investigate the mechanistic requirement of TNPO3 in HIV-1 infection, we tested the binding of TNPO3 to the HIV-1 core and found that TNPO3 binds to the HIV-1 core. Overall, this work suggests that TNPO3 interacts with the incoming HIV-1 core in the cytoplasm to assist a process that is important for HIV-1 infection after nuclear import.
Assuntos
Núcleo Celular/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Integração Viral , Replicação Viral , beta Carioferinas/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/genética , Núcleo Celular/virologia , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/virologia , Infecções por HIV/genética , HIV-1/genética , Humanos , Ligação Proteica , beta Carioferinas/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
TRIM5α(rh) is a cytosolic protein that potently restricts HIV-1 before reverse transcription. TRIM5α(rh) is composed of four different domains: RING, B-box 2, coiled coil, and B30.2(SPRY). The contribution of each of these domains to restriction has been extensively studied, with the exception of the RING domain. The RING domain of TRIM5α exhibits E3-ubiquitin ligase activity, but the contribution of this activity to the restriction of HIV-1 is not known. To test the hypothesis that the E3-ubiquitin ligase activity of the RING domain modulates TRIM5α(rh) restriction of HIV-1, we correlated the E3-ubiquitin ligase activity of a panel of TRIM5α(rh) RING domain variants with the ability of these mutant proteins to restrict HIV-1. For this purpose, we first solved the nuclear magnetic resonance structure of the RING domain of TRIM5α and defined potential functional regions of the RING domain by homology to other RING domains. With this structural information, we performed a systematic mutagenesis of the RING domain regions and tested the TRIM5α RING domain variants for the ability to undergo self-ubiquitylation. Several residues, particularly the ones on the E2-binding region of the RING domain, were defective in their self-ubiquitylation ability. To correlate HIV-1 restriction to self-ubiquitylation, we used RING domain mutant proteins that were defective in self-ubiquitylation but preserve important properties required for potent restriction by TRIM5α(rh), such as capsid binding and higher-order self-association. From these investigations, we found a set of residues that when mutated results in TRIM5α molecules that lost both the ability to potently restrict HIV-1 and their self-ubiquitylation activity. Remarkably, all of these changes were in residues located in the E2-binding region of the RING domain. Overall, these results demonstrate a role for TRIM5α self-ubiquitylation in the ability of TRIM5α to restrict HIV-1.
Assuntos
HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Proteínas/química , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
TRIM5alpha is a tripartite motif (TRIM) protein that consists of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. The TRIM5alpha(rh) protein from rhesus monkeys recognizes the human immunodeficiency virus type 1 (HIV-1) capsid as it enters the host cell and blocks virus infection prior to reverse transcription. HIV-1-restricting ability can be eliminated by disruption of the B-box 2 domain. Changes in the TRIM5alpha(rh) B-box 2 domain have been associated with alterations in TRIM5alpha(rh) turnover, the formation of cytoplasmic bodies and higher-order oligomerization. We present here the nuclear magnetic resonance structure of the TRIM5 B-box 2 domain and identify an unusual hydrophobic patch (cluster 1) on the domain surface. Alteration of cluster 1 or the flanking arginine 121 resulted in various degrees of inactivation of HIV-1 restriction, in some cases depending on compensatory changes in other nearby charged residues. For this panel of TRIM5alpha(rh) B-box 2 mutants, inhibition of HIV-1 infection was strongly correlated with higher-order self-association and binding affinity for capsid complexes but not with TRIM5alpha(rh) half-life or the formation of cytoplasmic bodies. Thus, promoting cooperative TRIM5alpha(rh) interactions with the HIV-1 capsid represents a major mechanism whereby the B-box 2 domain potentiates HIV-1 restriction.
Assuntos
Capsídeo/metabolismo , Proteínas/química , Proteínas/metabolismo , Retroviridae/patogenicidade , Animais , Linhagem Celular , Dimerização , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Macaca mulatta , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/farmacologia , Retroviridae/efeitos dos fármacos , Retroviridae/genética , Retroviridae/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Epigenetic changes in chromatin structure have been recently associated with the deregulated expression of critical genes in normal and malignant processes. HDAC11, the newest member of the HDAC family of enzymes, functions as a negative regulator of IL-10 expression in APCs, as previously described by our lab. However, at the present time, its role in other hematopoietic cells, specifically in neutrophils, has not been fully explored. In this report, for the first time, we present a novel physiologic role for HDAC11 as a multifaceted regulator of neutrophils. Thus far, we have been able to demonstrate a lineage-restricted overexpression of HDAC11 in neutrophils and committed neutrophil precursors (promyelocytes). Additionally, we show that HDAC11 appears to associate with the transcription machinery, possibly regulating the expression of inflammatory and migratory genes in neutrophils. Given the prevalence of neutrophils in the peripheral circulation and their central role in the first line of defense, our results highlight a unique and novel role for HDAC11. With the consideration of the emergence of new, selective HDAC11 inhibitors, we believe that our findings will have significant implications in a wide range of diseases spanning malignancies, autoimmunity, and inflammation.
Assuntos
Regulação da Expressão Gênica/imunologia , Hematopoese/imunologia , Histona Desacetilases/imunologia , Neutrófilos/enzimologia , Animais , Imunoprecipitação da Cromatina , Epigênese Genética , Citometria de Fluxo , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Reação em Cadeia da PolimeraseRESUMO
Histone deacetylase 10 (HDAC10) belongs to the class IIb HDAC family and its biological role remains mostly unidentified. A decreased HDAC10 expression has been reported in patients with aggressive solid tumors (Osada et al. Int J Cancer 112: 26-32, 2004; Jin et al. Int J Clin Exp Pathol 7: 5872-5879, 2014), suggesting that loss of HDAC10 expression might confer a survival advantage to malignant cells. Consequently, results from our lab suggests that overexpression of HDAC10 in aggressive mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) Z138c and MEC1 cells, respectively, resulted in a rapid induction of cell death in vitro with only 5 % of cells being alive at 48 h, cell cycle arrest, and up-regulation of co-stimulatory molecules. Here we present several standard methods to study the function of HDAC10 in B cell malignancies.
Assuntos
Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Animais , Western Blotting , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The physiological role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, remained largely unknown until the discovery of its regulatory function in immune cells. Among them, the regulation of cytokine production by antigen-presenting cells and the modulation of the suppressive ability of myeloid-derived suppressor cells (MDSCs) (Sahakian et al. Mol Immunol 63: 579-585, 2015; Wang et al. J Immunol 186: 3986-3996, 2011; Villagra et al. Nat Immunol 10: 92-100, 2009). Our earlier data has demonstrated that HDAC11, by interacting at the chromatin level with the IL-10 promoter, downregulates il-10 transcription in both murine and human APCs in vitro and ex vivo models (Villagra et al. Nat Immunol 10: 92-100, 2009). However the role of HDAC11 in other cell types still remains unknown. Here we present several methods that can potentially be used to identify the functional role of HDAC11, assigning special attention to the evaluation of immunological parameters.
Assuntos
Imunoprecipitação da Cromatina/métodos , Citocinas/genética , Histona Desacetilases/metabolismo , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Citocinas/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Histona Desacetilases/genética , Camundongos , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/imunologia , Regiões Promotoras GenéticasRESUMO
The anti-inflammatory cytokine IL-10 is a key modulator of immune responses. A better understanding of the regulation of this cytokine offers the possibility of tipping the balance of the immune response toward either tolerance, or enhanced immune responses. Histone deacetylases (HDACs) have been widely described as negative regulators of transcriptional regulation, and in this context, the primarily nuclear protein HDAC11 was shown to repress il-10 gene transcriptional activity in antigen-presenting cells (APCs). Here we report that another HDAC, HDAC6, primarily a cytoplasmic protein, associates with HDAC11 and modulates the expression of IL-10 as a transcriptional activator. To our knowledge, this is the first demonstration of two different HDACs being recruited to the same gene promoter to dictate divergent transcriptional responses. This dynamic interaction results in dynamic changes in the expression of IL-10 and might help to explain the intrinsic plasticity of the APC to determine T-cell activation versus T-cell tolerance.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Histona Desacetilases/imunologia , Interleucina-10/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica , Desacetilase 6 de Histona , Histona Desacetilases/genética , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno , Linfócitos T/imunologia , Transcrição Gênica , Ativação Transcricional/imunologiaRESUMO
Melanoma is the deadliest skin cancer, and its incidence has been increasing faster than any other cancer. Although immunogenic, melanoma is not effectively cleared by host immunity. In this study, we investigate the therapeutic, antimelanoma potential of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) by assessing both its cytotoxic effects on melanoma cells as well as enhancement of immune recognition of melanoma. Utilizing murine and human melanoma cell lines, we analyzed the effects of LBH589 on proliferation and survival. In addition, we analyzed the expression of several immunologically relevant surface markers and melanoma differentiation antigens, and the ability of LBH589-treated melanoma to activate antigen-specific T cells. Finally, we assessed the in-vivo effects of LBH589 in a mouse melanoma model. Low nanomolar concentrations of LBH589 inhibit the growth of all melanoma cell lines tested, but not normal melanocytes. This inhibition is characterized by increased apoptosis as well as a G1 cell cycle arrest. In addition, LBH589 augments the expression of major histocompatibility complex and costimulatory molecules on melanoma cells leading to an increased ability to activate antigen-specific T cells. Treatment also increases expression of melanoma differentiation antigens. In vivo, LBH589 treatment of melanoma-bearing mice results in a significant increase in survival. However, in immunodeficient mice, the therapeutic effect of LBH589 is lost. Taken together, LBH589 exerts a dual effect upon melanoma cells by affecting not only growth/survival but also by increasing melanoma immunogenicity. These effects provide the framework for future evaluation of this HDAC inhibitor in melanoma treatment.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Imunidade Celular/efeitos dos fármacos , Indóis/farmacologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Hospedeiro Imunocomprometido , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/enzimologia , Melanoma/imunologia , Melanoma/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Panobinostat , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: To evaluate how widely Helicobacter pylori (H. pylori) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile. METHODS: H. pylori hopE and hopV genes derived from strain CHCTX-1 were cloned by polymerase chain reaction (PCR), sequenced and expressed in Escherichia coli AD494 (DE3). Gel-purified porins were used to prepare polyclonal antibodies. The presence of both genes was tested by PCR in a collection of H. pylori clinical isolates and their expression was detected in lysates by immunoblotting. Immune responses against HopE, HopV and other H. pylori antigens in sera from infected and non-infected patients were tested by Western blotting using these sera as first antibody on recombinant H. pylori antigens. RESULTS: PCR and Western blotting assays revealed that 60 and 82 out of 130 Chilean isolates carried hopE and hopV genes, respectively, but only 16 and 9, respectively, expressed these porins. IgG serum immunoreactivity evaluation of 69 H. pylori-infected patients revealed that HopE and HopV were infrequently recognized (8.7% and 10.1% respectively) compared to H. pylori VacA (68.1%) and CagA (59.5%) antigens. Similar values were detected for IgA serum immunoreactivity against HopE (11.6%) and HopV (10.5%) although lower values for VacA (42%) and CagA (17.4%) were obtained when compared to the IgG response. CONCLUSION: A scarce expression of HopE and HopV among Chilean isolates was found, in agreement with the infrequent seroconversion against these antigens when tested in infected Chilean patients.