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1.
EMBO Rep ; 19(9)2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30021835

RESUMO

The cytokine interferon-γ (IFNγ) can induce expression of MHC class II (MHCII) on many different cell types, leading to antigen presentation to CD4+ T cells and immune activation. This has also been linked to anti-tumour immunity and graft-versus-host disease. The extent of MHCII upregulation by IFNγ is cell type-dependent and under extensive control of epigenetic regulators and signalling pathways. Here, we identify novel genetic and chemical factors that control this form of MHCII expression. Loss of the oxidative stress sensor Keap1, autophagy adaptor p62/SQSTM1, ubiquitin E3-ligase Cullin-3 and chromatin remodeller BPTF impair IFNγ-mediated MHCII expression. A similar phenotype is observed for arsenite, an oxidative stressor. Effects of the latter can be reversed by the inhibition of HDAC1/2, linking oxidative stress conditions to epigenetic control of MHCII expression. Furthermore, dimethyl fumarate, an antioxidant used for the treatment of several autoimmune diseases, impairs the IFNγ response by manipulating transcriptional control of MHCII We describe novel pathways and drugs related to oxidative conditions in cells impacting on IFNγ-mediated MHCII expression, which provide a molecular basis for the understanding of MHCII-associated diseases.


Assuntos
Arsenitos/farmacologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon gama/metabolismo , Estresse Oxidativo , Imunidade Adaptativa , Apresentação de Antígeno , Antígenos Nucleares/metabolismo , Antioxidantes/farmacologia , Proteínas Culina/metabolismo , Fumarato de Dimetilo/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos
2.
Immunogenetics ; 71(3): 273-282, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30706093

RESUMO

Major histocompatibility complex (MHC) class I and class II molecules play critical roles in the activation of the adaptive immune system by presenting antigens to CD8+ and CD4+ T cells, respectively. Although it has been well known that CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, as a master regulator of MHC class II gene expression, the mechanism of MHC class I gene transactivation was unclear. Recently, another NLR protein, NLRC5 (NLR family, CARD domain-containing 5), was identified as an MHC class I transactivator (CITA). NLRC5 is a critical regulator for the transcriptional activation of MHC class I genes and other genes involved in the MHC class I antigen presentation pathway. CITA/NLRC5 plays a crucial role in human cancer immunity through the recruitment and activation of tumor killing CD8+ T cells. Here, we discuss the molecular function and mechanism of CITA/NLRC5 in the MHC class I pathway and its role in cancer.


Assuntos
Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/imunologia , Transativadores/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/genética , Neoplasias/metabolismo , Transativadores/genética
3.
Clin Immunol ; 194: 80-86, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30006120

RESUMO

HLA-G protein expression could play a role in evasion of tumor immune surveillance. Accumulating evidence demonstrates that HLA-G is expressed in different types of malignancies, including colorectal cancer (CRC). The purpose of the current study was to further unravel whether HLA-G protein expression could play a role in immune evasion of CRC. Therefore, to firmly establish HLA-G protein expression, eight early passage human CRC cell lines and five human rectal cancer tissues were analyzed by western blot analysis. The results obtained by western blot analysis were compared with immunohistochemistry on tumor tissue sections of the same patient. Furthermore, multiple monoclonal antibodies (mAbs), 4H84, MEM-G/1 and 5A6G7, targeting HLA-G were used to unravel staining patterns. We showed that results obtained with immunohistochemistry did not correlate with protein expression detected by western blot analysis, using three different HLA-G targeting mAbs. Furthermore, with respect to the specificity of the mAbs employed, additional immune reactivity was detected using the mAbs MEM-G/1 and 5A6G7 in western blot analysis with K562 control cell lines overexpressing HLA-A2 or HLA-G, all tumor tissues and in two out of eight CRC cell lines. Based on the current study and our previously reported results, we conclude that claiming HLA-G plays a role in immune modulation of CRC seems premature, as results from anti-body based detection of HLA-G protein remain inconclusive. Until the time that detection of HLA-G is sensitive enough to detect all aspects of HLA-G expression in biological samples, rather than transfected cells or long time cultured cell lines, conclusions should be drawn with great care.


Assuntos
Neoplasias Colorretais/metabolismo , Antígenos HLA-G/metabolismo , Anticorpos Monoclonais/metabolismo , Western Blotting/métodos , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica/métodos , Células K562
4.
J Neuroinflammation ; 14(1): 257, 2017 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-29268771

RESUMO

BACKGROUND: Leukocyte infiltration into the central nervous system is an important feature of multiple sclerosis (MS) pathology. Among the infiltrating cells, monocytes comprise the largest population and are considered to play a dual role in the course of the disease. The enzyme tissue transglutaminase (TG2), produced by monocytes, plays a central role in monocyte adhesion/migration in animal models of MS. In the present study, we questioned whether TG2 expression is altered in monocytes from MS patients compared to healthy control (HC) subjects. Moreover, we determined the inflammatory status of these TG2-expressing monocytes, what inflammatory factor regulates TG2 expression, and whether TG2 can functionally contribute to their adhesion/migration processes. METHODS: Primary human monocytes from MS patients and HC subjects were collected, RNA isolated and subjected to qPCR analysis. Human THP-1 monocytes were lentivirally transduced with TG2 siRNA or control and treated with various cytokines. Subsequently, mRNA levels of inflammatory factors, adhesion properties, and activity of RhoA were analyzed in interleukin (IL)-4-treated monocytes. RESULTS: TG2 mRNA levels are significantly increased in monocytes derived from MS patients compared to HC subjects. In addition, correlation analyses indicated that TG2-expressing cells display a more anti-inflammatory, migratory profile in MS patients. Using THP-1 monocytes, we observed that IL-4 is a major trigger of TG2 expression in these cells. Furthermore, knockdown of TG2 expression leads to a pro-inflammatory profile and reduced adhesion/migration properties of IL-4-treated monocytes. CONCLUSIONS: TG2-expressing monocytes in MS patients have a more anti-inflammatory profile. Furthermore, TG2 mediates IL-4-induced anti-inflammatory status in THP-1 monocytes, adhesion, and cytoskeletal rearrangement in vitro. We thus propose that IL-4 upregulates TG2 expression in monocytes of MS patients, driving them into an anti-inflammatory status.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Inflamação/metabolismo , Monócitos/metabolismo , Esclerose Múltipla/metabolismo , Transglutaminases/metabolismo , Adulto , Idoso , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 2 Glutamina gama-Glutamiltransferase , Adulto Jovem
5.
J Immunol ; 193(11): 5480-7, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25355919

RESUMO

The Wnt-responsive transcription factor T cell factor 1 (Tcf1) is well known for its role in thymic T cell development and the formation of memory CD8(+) T cells. However, its role in the initial phases of CD8(+) T effector cell formation has remained unexplored. We report that high levels of Wnt signaling and Tcf1 are operational in naive and memory CD8(+) T cells, whereas Wnt signaling and Tcf1 were low in effector CD8(+) T cells. CD8(+) T cells deficient in Tcf1 produce IFN-γ more rapidly, coinciding with increased demethylation of the IFN-γ enhancer and higher expression of the transcription factors Tbet and Blimp1. Moreover, virus-specific Tcf1(-/-) CD8(+) T cells show accelerated expansion in acute infection, which is associated with increased IFN-γ and TNF production and lower viral load. Genetic complementation experiments with various Tcf1 isoforms indicate that Tcf1 dosage and protein stability are critical in suppressing IFN-γ production. Isoforms lacking the ß-catenin binding domain are equally effective in inhibiting CD8(+) effector T cell formation. Thus, Tcf1 functions as a repressor of CD8(+) effector T cell formation in a ß-catenin/Wnt-independent manner.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Interferon gama/metabolismo , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Células Cultivadas , Citotoxicidade Imunológica , Metilação de DNA , Dosagem de Genes , Fator 1-alfa Nuclear de Hepatócito/genética , Memória Imunológica , Interferon gama/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo , Estabilidade Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Regulação para Cima , Carga Viral , Viroses
6.
Apoptosis ; 19(12): 1769-78, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25292014

RESUMO

The apoptosis pathway of programmed cell death is frequently deregulated in cancer. An intact apoptosis pathway is required for proper response to anti-cancer treatment. We investigated the chromatin status of key apoptosis genes in the apoptosis pathway in colorectal cancer cell lines in relation to apoptosis induced by chemo-, immune- or radiation therapy. Using chromatin immunoprecipitation (ChIP), we measured the presence of transcription-activating histone modifications H3Ac and H3K4me3 and silencing modifications H3K9me3 and H3K27me3 at the gene promoter regions of key apoptosis genes Bax, Bcl2, Caspase-9, Fas (CD95) and p53. Cell lines DLD1, SW620, Colo320, Caco2, Lovo and HT29 were treated with cisplatin, anti-Fas or radiation. The apoptotic response was measured by flow cytometry using propidium iodide and annexin V-FITC. The chromatin status of the apoptosis genes reflected the activation status of the intrinsic (Bax, Bcl2, Caspase-9 and p53) and extrinsic (Fas) pathways. An active intrinsic apoptotic pathway corresponded to sensitivity to cisplatin and radiation treatment of cell lines DLD1, SW620 and Colo320. An active Fas promoter corresponded to an active extrinsic apoptotic pathway in cell line DLD1. mRNA expression data correlated with the chromatin status of the apoptosis genes as measured by ChIP. In conclusion, the results presented in this study indicate that the balance between activating and silencing histone modifications, reflecting the chromatin status of apoptosis genes, can be used to predict the response of tumor cells to different anti-cancer therapies and could provide a novel target to sensitize tumors to obtain adequate treatment responses.


Assuntos
Anticorpos/farmacologia , Antineoplásicos/farmacologia , Apoptose/genética , Cromatina/genética , Neoplasias Colorretais/terapia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Cisplatino/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/radioterapia , Resistencia a Medicamentos Antineoplásicos , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Receptor fas/imunologia
7.
Am J Hum Genet ; 88(6): 796-804, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21596365

RESUMO

Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining patients carry unknown genetic defects (ICF2) but share with ICF1 patients the same immunological and epigenetic features, including hypomethylation of juxtacentromeric repeat sequences. We performed homozygosity mapping in five unrelated ICF2 patients with consanguineous parents and then performed whole-exome sequencing in one of these patients and Sanger sequencing in all to identify mutations in the zinc-finger- and BTB (bric-a-bric, tramtrack, broad complex)-domain-containing 24 (ZBTB24) gene in four consanguineously descended ICF2 patients. Additionally, we found ZBTB24 mutations in an affected sibling pair and in one patient for whom it was not known whether his parents were consanguineous. ZBTB24 belongs to a large family of transcriptional repressors that include members, such as BCL6 and PATZ1, with prominent regulatory roles in hematopoietic development and malignancy. These data thus indicate that ZBTB24 is involved in DNA methylation of juxtacentromeric DNA and in B cell development and/or B and T cell interactions. Because ZBTB24 is a putative DNA-binding protein highly expressed in the lymphoid lineage, we predict that by studying the molecular function of ZBTB24, we will improve our understanding of the molecular pathophysiology of ICF syndrome and of lymphocyte biology in general.


Assuntos
Centrômero/genética , Metilação de DNA/genética , Proteínas Repressoras/genética , Dedos de Zinco , Adolescente , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA , Epigenômica , Face/anormalidades , Feminino , Humanos , Síndromes de Imunodeficiência/genética , Masculino , Mutação , Linhagem , Doenças da Imunodeficiência Primária
8.
Eur J Immunol ; 43(10): 2566-76, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23824566

RESUMO

Hantaviruses are emerging human pathogens. They induce an unusually strong antiviral response of human HLA class I (HLA-I) restricted CD8⁺ T cells that may contribute to tissue damage and hantavirus-associated disease. In this study, we analyzed possible hantaviral mechanisms that enhance the HLA-I antigen presentation machinery. Upon hantavirus infection of various human and primate cell lines, we observed transactivation of promoters controlling classical HLA molecules. Hantavirus-induced HLA-I upregulation required proteasomal activity and was associated with increased TAP expression. Intriguingly, human DCs acquired the capacity to cross-present antigen upon hantavirus infection. Furthermore, knockdown of TIR domain containing adaptor inducing IFN-ß or retinoic acid inducible gene I abolished hantavirus-driven HLA-I induction. In contrast, MyD88-dependent viral sensors were not involved in HLA-I induction. Our results show that hantaviruses strongly boost the HLA-I antigen presentation machinery by mechanisms that are dependent on both retinoic acid inducible gene I and TIR domain containing adaptor inducing IFN-ß.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Apresentação de Antígeno , Células Dendríticas/imunologia , Infecções por Hantavirus/imunologia , Orthohantavírus/imunologia , Receptores do Ácido Retinoico/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Chlorocebus aethiops , Apresentação Cruzada/genética , Células Dendríticas/virologia , Antígenos HLA/genética , Antígenos HLA/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/genética , Receptores do Ácido Retinoico/genética , Regulação para Cima , Células Vero
9.
J Immunol ; 188(10): 4951-8, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22490869

RESUMO

Tight regulation of MHC class I gene expression is critical for CD8 T cell activation and host adaptive-immune responses. The promoters of MHC class I genes contain a well-conserved core module, the W/S-X-Y motif, which assembles a nucleoprotein complex termed MHC enhanceosome. A member of the nucleotide-binding domain, leucine-rich repeat (NLR) protein family, NLRC5, is a newly identified transcriptional regulator of MHC class I genes. NLRC5 associates with and transactivates the proximal promoters of MHC class I genes, although the molecular mechanism of transactivation has not been understood. In this article, we show that NLRC5-mediated MHC class I gene induction requires the W/S and X1, X2 cis-regulatory elements. The transcription factors RFX5, RFXAP, and RFXANK/B, which compose the RFX protein complex and associate with the X1 box, cooperate with NLRC5 for MHC class I expression. Coimmunoprecipitation experiments revealed that NLRC5 specifically interacts with the RFX subunit RFXANK/B via its ankyrin repeats. In addition, we show that NLRC5 can cooperate with ATF1 and the transcriptional coactivators CBP/p300 and general control nonderepressible 5, which display histone acetyltransferase activity. Taken together, our data suggest that NLRC5 participates in an MHC class I-specific enhanceosome, which assembles on the conserved W/S-X-Y core module of the MHC class I proximal promoters, including the RFX factor components and CREB/ATF1 family transcription factors, to promote MHC class I gene expression.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Antígenos HLA-B/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fatores de Transcrição/fisiologia , Fator 1 Ativador da Transcrição/genética , Fator 1 Ativador da Transcrição/fisiologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Células HEK293 , Antígenos HLA-B/biossíntese , Humanos , Família Multigênica , Regiões Promotoras Genéticas , Fatores de Transcrição de Fator Regulador X , Sequências Reguladoras de Ácido Nucleico/imunologia , Fatores de Transcrição/genética , Ativação Transcricional/imunologia
10.
Eur J Immunol ; 42(1): 217-27, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21928277

RESUMO

Induction of experimental autoimmune encephalomyelitis (EAE) has been documented in common marmosets using peptide 34-56 from human myelin/oligodendrocyte glycoprotein (MOG(34-56) ) in incomplete Freund's adjuvant (IFA). Here, we report that this EAE model is associated with widespread demyelination of grey and white matter. We performed an in-depth analysis of the specificity, MHC restriction and functions of the activated T cells in the model, which likely cause EAE in an autoantibody-independent manner. T-cell lines isolated from blood and lymphoid organs of animals immunized with MOG(34-56) displayed high production of IL-17A and specific lysis of MOG(34-56) -pulsed EBV B-lymphoblastoid cells as typical hallmarks. Cytotoxicity was directed at the epitope MOG(40-48) presented by the non-classical MHC class Ib allele Caja-E, which is orthologue to HLA-E and is expressed in non-inflamed brain. In vivo activated T cells identified by flow cytometry in cultures with MOG(34-56,) comprised CD4(+) CD56(+) and CD4(+) CD8(+) CD56(+) T cells. Furthermore, phenotypical analysis showed that CD4(+) CD8(+) CD56(+) T cells also expressed CD27, but CD16, CD45RO, CD28 and CCR7 were absent. These results show that, in the MOG34-56/IFA marmoset EAE model, a Caja-E-restricted population of autoreactive cytotoxic T cells plays a key role in the process of demyelination in the grey and white matter.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Bainha de Mielina/imunologia , Linfócitos T/imunologia , Adjuvantes Imunológicos , Animais , Callithrix , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interleucina-17/imunologia , Células K562 , Proteínas da Mielina , Glicoproteína Mielina-Oligodendrócito , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígenos HLA-E
11.
Proc Natl Acad Sci U S A ; 107(31): 13794-9, 2010 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-20639463

RESUMO

MHC class I plays a critical role in the immune defense against viruses and tumors by presenting antigens to CD8 T cells. An NLR protein, class II transactivator (CIITA), is a key regulator of MHC class II gene expression that associates and cooperates with transcription factors in the MHC class II promoter. Although CIITA also transactivates MHC class I gene promoters, loss of CIITA in humans and mice results in the severe reduction of only MHC class II expression, suggesting that additional mechanisms regulate the expression of MHC class I. Here, we identify another member of the NLR protein family, NLRC5, as a transcriptional regulator of MHC class I genes. Similar to CIITA, NLRC5 is an IFN-gamma-inducible nuclear protein, and the expression of NLRC5 resulted in enhanced MHC class I expression in lymphoid as well as epithelial cell lines. Using chromatin immunoprecipitation and reporter gene assays, we show that NLRC5 associates with and activates the promoters of MHC class I genes. Furthermore, we show that the IFN-gamma-induced up-regulation of MHC class I requires NLRC5, because knockdown of NLRC5 specifically impaired the expression of MHC class I. In addition to MHC class I genes, NLRC5 also induced the expression of beta2-microglobulin, transporter associated with antigen processing, and large multifunctional protease, which are essential for MHC class I antigen presentation. Our results suggest that NLRC5 is a transcriptional regulator, orchestrating the concerted expression of critical components in the MHC class I pathway.


Assuntos
Regulação da Expressão Gênica , Genes MHC Classe I , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transcrição Gênica , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/genética
12.
J Cell Mol Med ; 16(8): 1866-77, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22050776

RESUMO

Previously, we have shown that CCR5 transcription is regulated by CREB-1. However, the ubiquitous pattern of CREB-1 expression suggests the involvement of an additional level of transcriptional control in the cell type-specific expression of CCR5. In this study, we show that epigenetic changes (i.e. DNA methylation and histone modifications) within the context of the CCR5 P1 promoter region correlate with transcript levels of CCR5 in healthy and in malignant CD4(+) T lymphocytes as well as in CD14(+) monocytes. In normal naïve T cells and CD14(+) monocytes the CCR5 P1 promoter resembles a bivalent chromatin state, with both repressive and permissive histone methylation and acetylation marks. The CCR5-expressing CD14(+) monocytes however show much higher levels of acetylated histone H3 (AcH3) compared to the non-CCR5-expressing naïve T cells. Combined with a highly methylated promoter in CD14(+) monocytes, this indicates a dominant role for AcH3 in CCR5 transcription. We also show that pharmacological interference in the epigenetic repressive mechanisms that account for the lack of CCR5 transcription in T leukaemic cell lines results in an increase in CREB-1 association with CCR5 P1 chromatin. Furthermore, RNA polymerase II was also recruited into CCR5 P1 chromatin resulting in CCR5 re-expression. Together, these data indicate that epigenetic modifications of DNA, and of histones, contribute to the control of CCR5 transcription in immune effector cells.


Assuntos
Antagonistas dos Receptores CCR5 , Epigênese Genética/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Linfócitos/metabolismo , Receptores CCR5/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Imunoprecipitação da Cromatina , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Histonas/metabolismo , Humanos , Imunomodulação/genética , Células Jurkat , Linfócitos/efeitos dos fármacos , Modelos Imunológicos , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR5/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Glia ; 60(1): 43-52, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21964955

RESUMO

Statins inhibit the endogenous intracellular mevalonate pathway and exposure to statins affects innate and adaptive immune responses. Different statins are currently under evaluation as (co)therapy in neuro-inflammatory diseases like multiple sclerosis. However, there are important discrepancies in the reported effects of statins on innate immune responses in different cell types. Studies to characterize such responses in clinically relevant primary cells are currently lacking. In this study, we investigated the effect of statins on Toll-like receptor (TLR)-induced responses of microglia, the resident macrophages of the central nervous system (CNS). Exposure of primary microglia from adult rhesus monkeys to different statins strongly amplified pro-inflammatory cytokine protein and mRNA levels in response to myeloid differentiation primary response gene 88-dependent TLR activation in particular. Rather than affecting nuclear facor-κB activation levels, statin exposure affected stress-activated protein/Jun-amino-terminal and p38 kinase signaling pathways. Mechanistic studies using specific pathway inhibitors and rescue experiments show that statin-induced inhibition of cholesterol biosynthesis, rather than inhibition of isoprenylation, was mainly responsible for the amplified TLR responses. Additionally, microglia were more sensitive to statin-mediated effects than bone marrow-derived macrophages of the same donor. This correlated to lower intrinsic microglial expression levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the enzyme targeted by statins. Amplification of TLR-induced responses in microglia by statin exposure might contribute to the generation of a more pro-inflammatory CNS microenvironment which can be of relevance for the pathogenesis of neuroinflammatory disorders.


Assuntos
Anticolesterolemiantes/farmacologia , Colesterol/biossíntese , Ácidos Heptanoicos/farmacologia , Microglia/efeitos dos fármacos , Pirróis/farmacologia , Receptor 2 Toll-Like/metabolismo , Animais , Atorvastatina , Medula Óssea , Encéfalo/citologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Hidroximetilglutaril-CoA Redutases/metabolismo , Macaca mulatta , Macrófagos/efeitos dos fármacos , Microglia/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , RNA Mensageiro , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia
14.
J Neuroinflammation ; 9: 156, 2012 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-22747960

RESUMO

BACKGROUND: In brain tissues from multiple sclerosis (MS) patients, clusters of activated HLA-DR-expressing microglia, also referred to as preactive lesions, are located throughout the normal-appearing white matter. The aim of this study was to gain more insight into the frequency, distribution and cellular architecture of preactive lesions using a large cohort of well-characterized MS brain samples. METHODS: Here, we document the frequency of preactive lesions and their association with distinct white matter lesions in a cohort of 21 MS patients. Immunohistochemistry was used to gain further insight into the cellular and molecular composition of preactive lesions. RESULTS: Preactive lesions were observed in a majority of MS patients (67%) irrespective of disease duration, gender or subtype of disease. Microglial clusters were predominantly observed in the vicinity of active demyelinating lesions and are not associated with T cell infiltrates, axonal alterations, activated astrocytes or blood-brain barrier disruption. Microglia in preactive lesions consistently express interleukin-10 and TNF-α, but not interleukin-4, whereas matrix metalloproteases-2 and -9 are virtually absent in microglial nodules. Interestingly, key subunits of the free-radical-generating enzyme NADPH oxidase-2 were abundantly expressed in microglial clusters. CONCLUSIONS: The high frequency of preactive lesions suggests that it is unlikely that most of them will progress into full-blown demyelinating lesions. Preactive lesions are not associated with blood-brain barrier disruption, suggesting that an intrinsic trigger of innate immune activation, rather than extrinsic factors crossing a damaged blood-brain barrier, induces the formation of clusters of activated microglia.


Assuntos
Encéfalo/imunologia , Encéfalo/metabolismo , Microglia/imunologia , Microglia/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Estudos de Coortes , Humanos , Imunidade Inata , Microglia/citologia , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Fibras Nervosas Mielinizadas/imunologia , Fibras Nervosas Mielinizadas/metabolismo
15.
J Immunol ; 185(6): 3498-503, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20729331

RESUMO

Most cancer cells use anaerobic-like glycolysis to generate energy instead of oxidative phosphorylation. They also avoid recognition by CTLs, which occurs primarily through decreasing the level of MHC class I (MHC-I) at the cell surface. We find that the two phenomena are linked; culture conditions that force respiration in leukemia cells upregulate MHC-I transcription and protein levels at the cell surface, whereas these decrease in cells forced to perform fermentation as well as in leukemia cells lacking a functional mitochondrial respiratory chain. Forced respiration leads to increased expression of the MAPK ERK5, which activates MHC-I gene promoters, and ERK5 accumulation in mitochondria. Respiration-induced MHC-I upregulation is reversed upon short hairpin RNA-mediated ERK5 downregulation and by inactive mutants of ERK5. Moreover, short hairpin RNA for ERK5 leukemia cells do not tolerate forced respiration. Thus, the expression of ERK5 and MHC-I is linked to cell metabolism and notably diminished by the metabolic adaptations found in tumor cells.


Assuntos
Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Leucemia de Células B/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteína Quinase 7 Ativada por Mitógeno/fisiologia , Fosforilação Oxidativa , Trifosfato de Adenosina/biossíntese , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/imunologia , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação para Baixo/imunologia , Glutamina/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Jurkat , Leucemia L1210 , Leucemia de Células B/enzimologia , Leucemia de Células B/patologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/genética , Regulação para Cima/imunologia
16.
J Immunol ; 185(12): 7452-9, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21057081

RESUMO

Nonclassical HLAs, HLA-E and HLA-G, are known to affect clinical outcome in various tumor types. We examined the clinical impact of HLA-E and HLA-G expression in early breast cancer patients, and related the results to tumor expression of classical HLA class I. Our study population (n = 677) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1995. Tissue microarray sections of arrayed tumor and normal control material were immunohistochemically stained for HLA-E and HLA-G. For evaluation of HLA-E and HLA-G and the combined variable, HLA-EG, a binary score was used. Expression of classical HLA class I molecules was determined previously. HLA-E, HLA-G, and HLA-EG on breast tumors were classified as expression in 50, 60, and 23% of patients, respectively. Remarkably, only in patients with loss of classical HLA class I tumor expression, expression of HLA-E (p = 0.027), HLA-G (p = 0.035), or HLA-EG (p = 0.001) resulted in a worse relapse-free period. An interaction was found between classical and nonclassical HLA class I expression (p = 0.002), suggestive for a biological connection. We have demonstrated that, next to expression of classical HLA class I, expression of HLA-E and HLA-G is an important factor in the prediction of outcome of breast cancer patients. These results provide further evidence that breast cancer is immunogenic, but also capable of evading tumor eradication by the host's immune system, by up- or downregulation of HLA class Ia and class Ib loci.


Assuntos
Neoplasias da Mama/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Feminino , Antígenos HLA/biossíntese , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Antígenos HLA-E
17.
Cell Rep ; 41(2): 111485, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36223747

RESUMO

We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides. MHC class II-presented peptide sequences are identified by introducing the MHC class II transactivator (CIITA) in tumor cells that were originally MHC class II negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals oncoviral- and neo-epitopes as well as shared epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses, which helped tumor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes presented by any MHC class II allele that would be otherwise very difficult to predict and reveals important targets for cancer immunotherapy.


Assuntos
Vacinas Anticâncer , Neoplasias , Proteínas Nucleares , Transativadores , Animais , Epitopos de Linfócito T , Antígenos HLA , Antígenos de Histocompatibilidade Classe II , Humanos , Camundongos , Proteínas Nucleares/genética , Peptídeos , Transativadores/genética , Vacinas de Subunidades Antigênicas
18.
Am J Pathol ; 176(2): 542-8, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20035052

RESUMO

We recently demonstrated that neointima formation of adult heterozygous apolipoprotein E (apoE(+/-)) offspring from hypercholesterolemic apoE(-/-) mothers was significantly increased as compared with genetically identical apoE(+/-) offspring from normocholesterolemic wild-type mothers. Since atherosclerosis is the consequence of a complex microenvironment and local cellular interactions, the effects of in utero programming and type of postnatal diet on epigenetic histone modifications in the vasculature were studied in both groups of offspring. An immunohistochemical approach was used to detect cell-specific histone methylation modifications and expression of accompanying lysine methyltransferases in the carotid arteries. Differences in histone triple-methylation modifications in vascular endothelial and smooth muscle cells revealed that the offspring from apoE(-/-) mothers had significantly different responses to a high cholesterol diet when compared with offspring from wild-type mothers. Our results suggest that both in utero programming and postnatal hypercholesterolemia affect epigenetic patterning in the vasculature, thereby providing novel insights regarding initiation and progression of vascular disease in adults.


Assuntos
Apolipoproteínas E/genética , Vasos Sanguíneos/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Hipercolesterolemia/metabolismo , Metiltransferases/metabolismo , Animais , Animais Recém-Nascidos , Apolipoproteínas E/deficiência , Dieta , Epigênese Genética/fisiologia , Feminino , Histona Metiltransferases , Histonas/metabolismo , Hipercolesterolemia/enzimologia , Hipercolesterolemia/genética , Lisina/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/metabolismo
19.
Adv Exp Med Biol ; 711: 36-49, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21627041

RESUMO

This chapter describes recent advances in our understanding how epigenetic events control immune functions with emphasis on transcriptional regulation of major histocompatibility complex ClassI (MIC-I) and Class II (MHC-II) genes. MHC-I and MHC-II molecules play an essential role in the adaptive immune response by virtue of their ability to present peptides, respectively to CD8+ and CD4+ T cells. Central to the onset of an adequate immune response to pathogens is the presentation of pathogen-derived peptides in the context of MHC-II molecules by antigen presenting cells (APCs) to CD4+ T cells of the immune system. In particular dendritic cells are highly specialized APCs that are capable to activate naïve T cells. Given their central role in adaptive immunity, MHC-I and MHC-II genes are regulated in a tight fashion at the transcriptional level to meet with local requirements of an effective antigen-specific immune response. In these regulatory processes the MHC2TA encoded Class II transactivator (CIITA) plays a crucial role. CIITA is essential for transcriptional activation of all MHC-I genes, whereas it plays an ancillary function in the transcriptional control of MHC-I genes. The focus of this chapter therefore will be on the transcription factors that interact with conserved cis-acting promoter elements and epigenetic mechanisms that modulate cell type-specific regulation of MHC-I, MHC-I, and MHC2TA genes. Furthermore, we will also briefly discuss how genetic and epigenetic mechanisms contribute to T helper cell differentiation.


Assuntos
Epigênese Genética , Imunidade , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Animais , Apresentação de Antígeno , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Complexo Principal de Histocompatibilidade , Proteínas Nucleares/genética , Linfócitos T Auxiliares-Indutores/citologia , Transativadores/genética
20.
J Cell Mol Med ; 14(6A): 1225-40, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20132414

RESUMO

Atherosclerosis is a multifactorial disease with a severe burden on western society. Recent insights into the pathogenesis of atherosclerosis underscore the importance of chronic inflammation in both the initiation and progression of vascular remodelling. Expression of immunoregulatory molecules by vascular wall components within the atherosclerotic lesions is accordingly thought to contribute to the ongoing inflammatory process. Besides gene regulatory proteins (transcription factors), epigenetic mechanisms also play an essential and fundamental role in the transcriptional control of gene expression. These epigenetic mechanisms change the accessibility of chromatin by DNA methylation and histone modifications. Epigenetic modulators are thus critically involved in the regulation of vascular, immune and tissue-specific gene expression within the atherosclerotic lesion. Importantly, epigenetic processes are reversible and may provide an excellent therapeutic target. The concept of epigenetic regulation is gradually being recognized as an important factor in the pathogenesis of atherosclerosis. Recent research provides an essential link between inflammation and reprogramming of the epigenome. In this review we therefore discuss the basis of epigenetic regulation - and the contribution thereof in the regulation of inflammatory processes in general and during atherosclerosis in particular. Moreover we highlight potential therapeutic interventions based on epigenetic mechanisms.


Assuntos
Aterosclerose/complicações , Aterosclerose/genética , Epigênese Genética , Inflamação/complicações , Inflamação/genética , Animais , Aterosclerose/patologia , Quimiocinas/genética , Humanos , Inflamação/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Receptores de Quimiocinas/genética
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