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1.
J Biol Chem ; 293(22): 8394-8409, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29618516

RESUMO

High-mobility group box 1 (HMGB1) is a chromatin-associated protein that, in response to stress or injury, translocates from the nucleus to the extracellular milieu, where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and, to a much lesser extent, in healthy subjects. Differential HMGB1c levels were also detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein-cross-linking enzyme transglutaminase-2 (TG2). Cross-link site mapping and MS analysis revealed that HMGB1 can be cross-linked to TG2 as well as a number of additional proteins, including human autoantigens. These findings have significant functional implications for studies of cellular stress responses and innate immunity in SLE and other autoimmune disease.


Assuntos
Autoantígenos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteína HMGB1/metabolismo , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Transglutaminases/metabolismo , Autoantígenos/imunologia , Células Cultivadas , Proteínas de Ligação ao GTP/imunologia , Proteína HMGB1/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Peso Molecular , Proteína 2 Glutamina gama-Glutamiltransferase , Especificidade por Substrato , Transglutaminases/imunologia
2.
J Immunol ; 199(7): 2333-2342, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28842466

RESUMO

Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-κB pathway, which is activated by IL-18 signaling. We found that the NF-κB complex signaling component, p65, binds to the proximal region of the IL22 promoter and promotes transcriptional activity. Finally, we observed that CD11c+ dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-κB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL-18 and/or IL-22.


Assuntos
Proliferação de Células , Interleucina-18/metabolismo , Interleucinas/biossíntese , Linfócitos/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais , Células Dendríticas/fisiologia , Humanos , Imunidade Inata , Interleucina-15/imunologia , Interleucinas/genética , Interleucinas/imunologia , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Regiões Promotoras Genéticas , Transdução de Sinais/imunologia , Fator de Transcrição RelA/metabolismo , Interleucina 22
3.
Clin Immunol ; 176: 12-22, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28039018

RESUMO

Recent studies implicate innate immunity to systemic lupus erythematosus (SLE) pathogenesis. Toll-like receptor (TLR)8 is estrogen-regulated and binds viral ssRNA to stimulate innate immune responses, but recent work indicates that microRNA (miR)-21 within extracellular vesicles (EVs) can also trigger this receptor. Our objective was to examine TLR8 expression/activation to better understand sex-biased responses involving TLR8 in SLE. Our data identify an estrogen response element that promotes STAT1 expression and demonstrate STAT1-dependent transcriptional activation of TLR8 with estrogen stimulation. In lieu of viral ssRNA activation, we explored EV-encapsulated miR-21 as an endogenous ligand and observed induction of both TLR8 and cytokine expression in vitro. Moreover, extracellular miR detection was found predominantly within EVs. Thus, just as a cytokine or chemokine, EV-encapsulated miR-21 can act as an inflammatory signaling molecule, or miRokine, by virtue of being an endogenous ligand of TLR8. Collectively, our data elucidates a novel innate inflammatory pathway in SLE.


Assuntos
Estrogênios/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , MicroRNAs/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/fisiologia , Receptor 8 Toll-Like/metabolismo , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Humanos , Imunidade Inata/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Ligantes , Lúpus Eritematoso Sistêmico/imunologia , Células MCF-7
4.
Biochim Biophys Acta ; 1848(2): 742-51, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25445671

RESUMO

Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a Vmax of 12 µmol/min/mg protein and a Km of 45 µM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5-7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.


Assuntos
Proteínas de Bactérias/química , Hidrolases/química , Legionella pneumophila/química , Lisofosfolipídeos/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cromatografia de Afinidade , Clonagem Molecular , Sequência Conservada , Endopeptidases/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Hidrolases/genética , Hidrolases/metabolismo , Hidrólise , Cinética , Legionella pneumophila/enzimologia , Lisofosfolipídeos/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato
5.
J Immunol ; 193(6): 2994-3002, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25122922

RESUMO

Natural products are a major source for cancer drug development. NK cells are a critical component of innate immunity with the capacity to destroy cancer cells, cancer-initiating cells, and clear viral infections. However, few reports describe a natural product that stimulates NK cell IFN-γ production and unravel a mechanism of action. In this study, through screening, we found that a natural product, phyllanthusmin C (PL-C), alone enhanced IFN-γ production by human NK cells. PL-C also synergized with IL-12, even at the low cytokine concentration of 0.1 ng/ml, and stimulated IFN-γ production in both human CD56(bright) and CD56(dim) NK cell subsets. Mechanistically, TLR1 and/or TLR6 mediated PL-C's activation of the NF-κB p65 subunit that in turn bound to the proximal promoter of IFNG and subsequently resulted in increased IFN-γ production in NK cells. However, IL-12 and IL-15Rs and their related STAT signaling pathways were not responsible for the enhanced IFN-γ secretion by PL-C. PL-C induced little or no T cell IFN-γ production or NK cell cytotoxicity. Collectively, we identify a natural product with the capacity to selectively enhance human NK cell IFN-γ production. Given the role of IFN-γ in immune surveillance, additional studies to understand the role of this natural product in prevention of cancer or infection in select populations are warranted.


Assuntos
Benzodioxóis/farmacologia , Glicosídeos/farmacologia , Interferon gama/biossíntese , Células Matadoras Naturais/imunologia , Fator de Transcrição RelA/imunologia , Antígeno CD56/biossíntese , Antígeno CD56/genética , Células Cultivadas , Células HEK293 , Humanos , Interleucina-12/farmacologia , Interleucina-15/farmacologia , Ativação Linfocitária/imunologia , Interferência de RNA , RNA Interferente Pequeno , Receptores de Interleucina-15 , Transdução de Sinais/imunologia , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/imunologia , Receptor 6 Toll-Like/imunologia , Fator de Transcrição RelA/biossíntese , Regulação para Cima
6.
Clin Immunol ; 156(1): 1-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25451161

RESUMO

Despite recent advances in the understanding of Sjögren's Syndrome (SjS), the pathogenic mechanisms remain elusive and an ideal model for early drug discovery is not yet available. To establish a humanized mouse model of SjS, peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with SjS were transferred into immunodeficient NOD-scid IL-2rγ(null) mouse recipients to produce chimeric mice. While no difference was observed in the distribution of cells, chimeric mice transferred with PBMCs from SjS patients produced enhanced cytokine levels, most significantly IFN-γ and IL-10. Histological examination revealed enhanced inflammatory responses in the lacrimal and salivary glands of SjS chimeras, as measured by digital image analysis and blinded histopathological scoring. Infiltrates were primarily CD4+, with minimal detection of CD8+ T-cells and B-cells. These results demonstrate a novel chimeric mouse model of human SjS that provides a unique in vivo environment to test experimental therapeutics and investigate T-cell disease pathology.


Assuntos
Quimera , Modelos Animais de Doenças , Síndrome de Sjogren , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Camundongos , Síndrome de Sjogren/imunologia
7.
Blood ; 121(23): 4663-71, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23580661

RESUMO

MicroRNAs (miRNAs) bind to complementary sequences of target mRNAs, resulting in translational repression or target degradation and thus gene silencing. miRNAs are abundant in circulating blood, yet it is not known whether, as a class of regulatory molecules, they interact with human natural killer (NK) cells. Here we found that the treatment of human NK cells with several mature miRNAs in the presence of a low concentration of interleukin-12 induced CD69 expression, interferon-γ production, and degranulation marker CD107a expression. In vivo, infusion of several miRNAs alone in murine peripheral blood also resulted in comparable NK-cell activation, but not T-cell activation. Furthermore, miRNA administration significantly protected mice from tumor development in an NK cell-dependent manner. Mechanistically, we found that miRNA stimulation led to downstream activation of nuclear factor κB (NF-κB), an effect that was blunted by a block in Toll-like receptor 1(TLR1) signaling and attenuated in lymphoma patients. Knockdown of TLR1 resulted in less activation by miRNAs. Collectively, we show that miRNAs have a capacity to selectively activate innate immune effector cells that is, at least in part, via the TLR1-NF-κB signaling pathway. This may be important in the normal host defense against infection and/or malignant transformation.


Assuntos
Células Matadoras Naturais/imunologia , Linfoma/prevenção & controle , MicroRNAs/genética , Baço/imunologia , Receptores Toll-Like/metabolismo , Animais , Western Blotting , Células Cultivadas , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Ativação Linfocitária , Linfoma/genética , Linfoma/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismo , Baço/patologia , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/genética
8.
Blood ; 121(1): 159-69, 2013 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-23100311

RESUMO

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , MicroRNAs/biossíntese , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/biossíntese , Talidomida/análogos & derivados , Adulto , Animais , Antimetabólitos Antineoplásicos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/transplante , Citarabina/farmacologia , Mutação da Fase de Leitura , Humanos , Fatores Imunológicos/uso terapêutico , Células K562 , Lenalidomida , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , Proteínas de Neoplasias/genética , Mutação Puntual , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Estrutura Terciária de Proteína , RNA Neoplásico/genética , Proteínas Recombinantes de Fusão/fisiologia , Talidomida/farmacologia , Talidomida/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Clin Immunol ; 151(1): 66-77, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24525049

RESUMO

Females of child-bearing age are more resistant to infectious disease and have an increased risk of systemic lupus erythematosus (SLE). We hypothesized that estrogen-induced gene expression could establish an immunoactivated state which would render enhanced defense against infection, but may be deleterious in autoimmune development. Using peripheral blood mononuclear cells (PBMCs), we demonstrate enhanced responses with immunogen stimulation in the presence of 17ß-estradiol (E2) and gene array analyses reveal toll-like receptor 8 (TLR8) as an E2-responsive candidate gene. TLR8 expression levels are up-regulated in SLE and PBMCs stimulated with TLR8 agonist display a female sex-biased, E2-sensitive response. Moreover, we identify a putative ERα-binding region near the TLR8 locus and blocking ERα expression significantly decreases E2-mediated TLR8 induction. Our findings characterize TLR8 as a novel estrogen target gene that can lower the inflammatory threshold and implicate an IFNα-independent inflammatory mechanism that could contribute to higher SLE incidence in women.


Assuntos
Endossomos/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/imunologia , Receptor 8 Toll-Like/imunologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Endossomos/imunologia , Endossomos/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Regulação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Fatores Imunológicos/farmacologia , Interferon-alfa/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Fatores Sexuais , Transdução de Sinais , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/genética
10.
Eur J Immunol ; 43(2): 468-79, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23225163

RESUMO

IL-27 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p40-related protein subunit, EBV-induced gene 3, and a p35-related subunit, p28. IL-27 functions through IL-27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8(+) T-cell responses. However, the exact mechanisms of how IL-27 enhances antitumor CD8(+) T-cell responses remain unclear. Here we show that IL-27 significantly enhances the survival of activated tumor antigen-specific CD8(+) T cells in vitro and in vivo, and programs tumor antigen-specific CD8(+) T cells into memory precursor-like effector cells, characterized by upregulation of Bcl-6, SOCS3, Sca-1, and IL-10. While STAT3 activation and the CTL survival-enhancing effects can be independent of CTL IL-10 production, we show here that IL-27-induced CTL IL-10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL-27 enhances antitumor CTL responses via programming tumor antigen-specific CD8(+) T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Interleucina-10/imunologia , Interleucinas/imunologia , Animais , Antígenos Ly/imunologia , Antígenos Ly/metabolismo , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Interleucina-10/metabolismo , Interleucinas/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Regulação para Cima/imunologia
11.
Front Immunol ; 14: 1090177, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38939646

RESUMO

Introduction: Distinct, disease-associated intracellular miRNA (miR) expression profiles have been observed in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematous (SLE) patients. Additionally, we have identified novel estrogenic responses in PBMCs from SLE patients and demonstrated that estrogen upregulates toll-like receptor (TLR)7 and TLR8 expression. TLR7 and TLR8 bind viral-derived single-stranded RNA to stimulate innate inflammatory responses, but recent studies have shown that miR-21, mir-29a, and miR-29b can also bind and activate these receptors when packaged and secreted in extracellular vesicles (EVs). The objective of this study was to evaluate the association of EV-encapsulated small RNA species in SLE and examine the therapeutic approach of miR inhibition in humanized mice. Methods: Plasma-derived EVs were isolated from SLE patients and quantified. RNA was then isolated and bulk RNA-sequencing reads were analyzed. Also, PBMCs from active SLE patients were injected into immunodeficient mice to produce chimeras. Prior to transfer, the PBMCs were incubated with liposomal EVs containing locked nucleic acid (LNA) antagonists to miR-21, mir-29a, and miR-29b. After three weeks, blood was collected for both immunophenotyping and cytokine analysis; tissue was harvested for histopathological examination. Results: EVs were significantly increased in the plasma of SLE patients and differentially expressed EV-derived small RNA profiles were detected compared to healthy controls, including miR-21, mir-29a, and miR-29b. LNA antagonists significantly reduced proinflammatory cytokines and histopathological infiltrates in the small intestine, liver, and kidney, as demonstrated by H&E-stained tissue sections and immunohistochemistry measuring human CD3. Discussion: These data demonstrate distinct EV-derived small RNA signatures representing SLE-associated biomarkers. Moreover, targeting upregulated EV-encapsulated miR signaling by antagonizing miRs that may bind to TLR7 and TLR8 reveals a novel therapeutic opportunity to suppress autoimmune-mediated inflammation and pathogenesis in SLE.


Assuntos
Modelos Animais de Doenças , Vesículas Extracelulares , Leucócitos Mononucleares , Lúpus Eritematoso Sistêmico , MicroRNAs , Receptor 7 Toll-Like , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Humanos , Animais , MicroRNAs/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Camundongos , Feminino , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/imunologia , Receptor 7 Toll-Like/metabolismo , Receptor 7 Toll-Like/genética , Inflamação/imunologia , Receptor 8 Toll-Like/metabolismo , Receptor 8 Toll-Like/genética , Adulto , Masculino , Pessoa de Meia-Idade , Camundongos SCID
12.
J Biol Chem ; 286(28): 24916-30, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21515882

RESUMO

Lysoplasmalogenase (EC 3.3.2.2 and EC 3.3.2.5) is an enzyme that catalyzes hydrolytic cleavage of the vinyl ether bond of lysoplasmalogen, forming fatty aldehyde and glycerophosphoethanolamine or glycerophosphocholine and is specific for the sn-2-deacylated form of plasmalogen. Here we report the purification, characterization, identification, and cloning of lysoplasmalogenase. Rat liver microsomal lysoplasmalogenase was solubilized with octyl glucoside and purified 500-fold to near homogeneity using four chromatography steps. The purified enzyme has apparent K(m) values of ∼50 µm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent V(m) values of 24.5 and 17.5 µmol/min/mg protein for the two substrates, respectively. The pH optimum was 7.0. Lysoplasmalogenase was competitively inhibited by lysophosphatidic acid (K(i) ∼20 µm). The predominant band on a gel at ∼19 kDa was subjected to trypsinolysis, and the peptides were identified by mass spectrometry as Tmem86b, a protein of unknown function. Transient transfection of human embryonic kidney (HEK) 293T cells showed that TMEM86b cDNA yielded lysoplasmalogenase activity, and Western blot analyses confirmed the synthesis of TMEM86b protein. The protein was localized in the membrane fractions. The TMEM86b gene was also transformed into Escherichia coli, and its expression was verified by Western blot and activity analyses. Tmem86b is a hydrophobic transmembrane protein of the YhhN family. Northern blot analyses demonstrated that liver expressed the highest level of Tmem86b, which agreed with tissue distribution of activity. Overexpression of TMEM86b in HEK 293T cells resulted in decreased levels of plasmalogens, suggesting that the enzyme may be important in regulating plasmalogen levels in animal cells.


Assuntos
Hidrolases , Fígado/enzimologia , Lisofosfolipídeos/metabolismo , Proteínas de Membrana , Microssomos Hepáticos/enzimologia , Plasmalogênios/metabolismo , Animais , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Hidrolases/química , Hidrolases/genética , Hidrolases/metabolismo , Lisofosfolipídeos/genética , Masculino , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Plasmalogênios/genética , Ratos , Ratos Sprague-Dawley
13.
Nutr Cancer ; 64(8): 1228-35, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23145775

RESUMO

Reactivation of tumor suppressor genes (TSGs) involved in carcinogenesis by nontoxic bioactive food component represents a promising strategy for cancer chemoprevention. Recently, curcumin has been demonstrated to inhibit a bacterial DNA methyltransferase (M. Sss I) activity, induce global DNA hypomethylation in leukemia cells, and reactivate several hypermethylation silenced genes in lung and prostate cancer cells. Herein, we demonstrated that curcumin can enhance the mRNA and protein levels of ras-association domain family protein 1A (RASSF1A), 1 hypermethylation-silenced TSG, and decrease its promoter methylation in breast cancer cells. Mechanistic study demonstrated that curcumin can decrease DNA methylation activity of nuclear extract and downregulate the mRNA and protein levels of DNMT1 in MCF-7 cells, which may be associated with curcumin-induced disruption of NF-κB/Sp1 complex bound to the promoter region of DNMT1. Altogether, this study reveals a novel molecular mechanism of curcumin as a chemo-preventive agent for breast cancer through hypomethylation reactivation of RASSF1A.


Assuntos
Neoplasias da Mama/prevenção & controle , Curcumina/farmacologia , Ativação Transcricional/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Animais , Neoplasias da Mama/química , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , Proteínas Repressoras/análise , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/análise , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Arthritis Rheum ; 63(6): 1613-25, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21400474

RESUMO

OBJECTIVE: Physiotherapies are the most widely recommended conservative treatment for arthritic diseases. The present study was undertaken to examine the molecular mechanisms underlying the effects of gentle treadmill walking (GTW) on various stages of monoiodoacetate-induced arthritis (MIA) to elucidate the basis for the success or failure of such therapies in joint damage. METHODS: Knees were obtained from untreated control rats, rats with MIA that did not undergo GTW, rats with MIA in which GTW regimens were started 1 day post-MIA induction, and rats with MIA in which GTW regimens were started after cartilage damage had progressed to grade 1 or grade 2. The cartilage was examined macroscopically, microscopically, and by microfocal computed tomography imaging. Transcriptome-wide gene expression analysis was performed, and microarray data were assessed by Ingenuity Pathways Analysis to identify molecular functional networks regulated by GTW. RESULTS: GTW intervention started on day 1 post-MIA induction significantly prevented the progression of MIA, but its efficacy was reduced when implemented on knees exhibiting close to grade 1 cartilage damage. GTW accelerated cartilage damage in knees with close to grade 2 damage. Transcriptome-wide gene expression analysis revealed that GTW intervention started 1 day post-MIA inception significantly suppressed inflammation-associated genes and up-regulated matrix-associated gene networks. However, delayed GTW intervention after grade 1 damage had occurred was less effective in suppressing proinflammatory genes or up-regulating matrix synthesis. CONCLUSION: The present findings suggest that GTW suppresses proinflammatory gene networks and up-regulates matrix synthesis to prevent progression of cartilage damage in MIA-affected knees. However, the extent of cartilage damage at the initiation of GTW may be an important determinant of the success or failure of such therapies.


Assuntos
Artrite Experimental/patologia , Artrite Experimental/terapia , Terapia por Exercício , Caminhada , Animais , Artrite Experimental/induzido quimicamente , Cartilagem/metabolismo , Cartilagem/patologia , Progressão da Doença , Teste de Esforço , Feminino , Perfilação da Expressão Gênica , Ácido Iodoacético/farmacologia , Articulação do Joelho/patologia , Ratos , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Regulação para Cima
15.
J Immunol ; 184(10): 5435-43, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20404277

RESUMO

Activation-induced cytidine deaminase (AID) is an enzyme essential for the generation of Ab diversity in B cells and is considered to be a general gene mutator. In addition, AID expression was also implicated in the pathogenesis of human B cell malignancies and associated with poor prognosis. In this study, we report that small interfering RNA silencing of AID in plasmacytoma dramatically increased its susceptibility to immunotherapy by CTLs. AID silencing did not decrease the mutation frequencies of tumor Ag gene P1A. Gene-array analysis showed dramatically altered expression of a number of genes in AID-silenced plasmacytoma cells, and upregulation of CD200 was shown to be in favor of tumor eradication by CTLs. Taken together, we demonstrate a novel function of AID in tumor evasion of CTL therapy and that targeting AID should be beneficial in the immunotherapy of AID-positive tumors.


Assuntos
Citidina Desaminase/metabolismo , Marcação de Genes , Imunoterapia Adotiva , Linfócitos T Citotóxicos/imunologia , Evasão Tumoral/imunologia , Animais , Linhagem Celular Tumoral , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/genética , Citidina Desaminase/fisiologia , Citotoxicidade Imunológica/genética , Marcação de Genes/métodos , Imunoterapia Adotiva/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Plasmocitoma/enzimologia , Plasmocitoma/genética , Plasmocitoma/imunologia , RNA Interferente Pequeno/fisiologia , Linfócitos T Citotóxicos/metabolismo , Evasão Tumoral/genética
16.
Pharmaceuticals (Basel) ; 15(12)2022 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-36559029

RESUMO

Aromatase Inhibitors (AIs) block estrogen production and improve survival in patients with hormone-receptor-positive breast cancer. However, half of patients develop aromatase-inhibitor-induced arthralgia (AIIA), which is characterized by inflammation of the joints and the surrounding musculoskeletal tissue. To create a platform for future interventional strategies, our objective was to characterize a novel animal model of AIIA. Female BALB/C-Tg(NFκB-RE-luc)-Xen mice, which have a firefly luciferase NFκB reporter gene, were oophorectomized and treated with an AI (letrozole). Bioluminescent imaging showed significantly enhanced NFκB activation with AI treatment in the hind limbs. Moreover, an analysis of the knee joints and legs via MRI showed enhanced signal detection in the joint space and the surrounding tissue. Surprisingly, the responses observed with AI treatment were independent of oophorectomy, indicating that inflammation is not mediated by physiological estrogen levels. Histopathological and pro-inflammatory cytokine analyses further demonstrated the same trend, as tenosynovitis and musculoskeletal infiltrates were detected in all mice receiving AI, and serum cytokines were significantly upregulated. Human PBMCs treated with letrozole/estrogen combinations did not demonstrate an AI-specific gene expression pattern, suggesting AIIA-mediated pathogenesis through other cell types. Collectively, these data identify an AI-induced stimulation of disease pathology and suggest that AIIA pathogenesis may not be mediated by estrogen deficiency, as previously hypothesized.

17.
Sci Rep ; 12(1): 152, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34996983

RESUMO

The gut microbiota (GM) exerts a strong influence over the host immune system and dysbiosis of this microbial community can affect the clinical phenotype in chronic inflammatory conditions. To explore the role of the GM in lupus nephritis, we colonized NZM2410 mice with Segmented Filamentous Bacteria (SFB). Gut colonization with SFB was associated with worsening glomerulonephritis, glomerular and tubular immune complex deposition and interstitial inflammation compared to NZM2410 mice free of SFB. With SFB colonization mice experienced an increase in small intestinal lamina propria Th17 cells and group 3 innate lymphoid cells (ILC3s). However, although serum IL-17A expression was elevated in these mice, Th17 cells and ILC3s were not detected in the inflammatory infiltrate in the kidney. In contrast, serum and kidney tissue expression of the macrophage chemoattractants MCP-1 and CXCL1 were significantly elevated in SFB colonized mice. Furthermore, kidney infiltrating F4/80+CD206+M2-like macrophages were significantly increased in these mice. Evidence of increased gut permeability or "leakiness" was also detected in SFB colonized mice. Finally, the intestinal microbiome of SFB colonized mice at 15 and 30 weeks of age exhibited dysbiosis when compared to uncolonized mice at the same time points. Both microbial relative abundance as well as biodiversity of colonized mice was found to be altered. Collectively, SFB gut colonization in the NZM2410 mouse exacerbates kidney disease, promotes kidney M2-like macrophage infiltration and overall intestinal microbiota dysbiosis.


Assuntos
Bactérias/crescimento & desenvolvimento , Microbioma Gastrointestinal , Intestinos/microbiologia , Rim/imunologia , Nefrite Lúpica/microbiologia , Macrófagos/imunologia , Animais , Bactérias/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Disbiose , Feminino , Imunidade Inata , Mediadores da Inflamação/metabolismo , Intestinos/imunologia , Intestinos/metabolismo , Intestinos/patologia , Rim/metabolismo , Rim/patologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Fenótipo , Células Th17/imunologia , Células Th17/metabolismo
18.
Blood ; 113(25): 6411-8, 2009 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-19211935

RESUMO

Aberrant DNA hypermethylation contributes to myeloid leukemogenesis by silencing structurally normal genes involved in hematopoiesis. MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression by targeting protein-coding mRNAs. Recently, miRNAs have been shown to play a role as both targets and effectors in gene hypermethylation and silencing in malignant cells. In the current study, we showed that enforced expression of miR-29b in acute myeloid leukemia cells resulted in marked reduction of the expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B at both RNA and protein levels. This in turn led to decrease in global DNA methylation and reexpression of p15(INK4b) and ESR1 via promoter DNA hypomethylation. Although down-regulation of DNMT3A and DNMT3B was the result of a direct interaction of miR-29b with the 3' untranslated regions of these genes, no predicted miR-29b interaction sites were found in the DNMT1 3' untranslated regions. Further experiments revealed that miR-29b down-regulates DNMT1 indirectly by targeting Sp1, a transactivator of the DNMT1 gene. Altogether, these data provide novel functional links between miRNAs and aberrant DNA hypermethylation in acute myeloid leukemia and suggest a potentially therapeutic use of synthetic miR-29b oligonucleotides as effective hypomethylating compounds.


Assuntos
DNA (Citosina-5-)-Metiltransferases/biossíntese , Metilação de DNA/genética , Regulação Leucêmica da Expressão Gênica , Genes Supressores de Tumor , Leucemia Mieloide/genética , MicroRNAs/genética , RNA Neoplásico/genética , Regiões 3' não Traduzidas/genética , Doença Aguda , Diferenciação Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p15/biossíntese , Inibidor de Quinase Dependente de Ciclina p15/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Regulação para Baixo/genética , Indução Enzimática/genética , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/genética , Vetores Genéticos/genética , Humanos , Vírus da Imunodeficiência Felina/genética , Leucemia Mieloide/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , RNA Neoplásico/biossíntese , Fator de Transcrição Sp1/antagonistas & inibidores , DNA Metiltransferase 3B
19.
Bio Protoc ; 10(12): e3657, 2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-33659327

RESUMO

Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient identification and characterization of candidate substrates are an important first-step to uncovering the function of crosslinked proteins. Herein we describe an optimized and flexible protocol for in vitro TG2 crosslink reactions and substrate incorporation assays. We have previously employed these techniques in the identification of the protein high mobility group box 1 (HMGB1) as a TG2 substrate. However, the protocol can be adapted for identification of any candidate transamidation substrate.

20.
J Pharmacol Exp Ther ; 329(2): 505-14, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19201992

RESUMO

Hypermethylation of 5'-cytosine-guanosine islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys(38) of p65 to inhibit nuclear factor-kappaB activation and exhibit anti-tumor activity in human malignancies. In this article, we report that parthenolide 1) inhibits DNA methyltransferase 1 (DNMT1) with an IC(50) of 3.5 microM, possibly through alkylation of the proximal thiolate of Cys(1226) of the catalytic domain by its gamma-methylene lactone, and 2) down-regulates DNMT1 expression possibly associated with its SubG(1) cell-cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in vitro and in vivo global DNA hypomethylation. Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.


Assuntos
Antineoplásicos/farmacologia , Metilação de DNA/efeitos dos fármacos , Lactonas/farmacologia , Sesquiterpenos/farmacologia , Alquilação , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Domínio Catalítico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Cisteína/metabolismo , Citocinas/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Humanos , Immunoblotting , Lactonas/química , Lactonas/uso terapêutico , Camundongos , Camundongos Nus , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Sesquiterpenos/química , Sesquiterpenos/uso terapêutico , Fator de Transcrição Sp1/metabolismo , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
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