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1.
Sci Rep ; 9(1): 14363, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591456

RESUMO

Endothelial cell motility has fundamental role in vasculogenesis and angiogenesis during developmental or pathological processes. Tks4 is a scaffold protein known to organize the cytoskeleton of lamellipodia and podosomes, and thus modulating cell motility and invasion. In particular, Tks4 is required for the localization and activity of membrane type 1-matrix metalloproteinase, a key factor for extracellular matrix (ECM) cleavage during cell migration. While its role in transformed cells is well established, little is known about the function of Tks4 under physiological conditions. In this study we examined the impact of Tks4 gene silencing on the functional activity of primary human umbilical vein endothelial cells (HUVEC) and used time-lapse videomicrosopy and quantitative image analysis to characterize cell motility phenotypes in culture. We demonstrate that the absence of Tks4 in endothelial cells leads to impaired ECM cleavage and decreased motility within a 3-dimensional ECM environment. Furthermore, absence of Tks4 also decreases the ability of HUVEC cells to form multicellular sprouts, a key requirement for angiogenesis. To establish the involvement of Tks4 in vascular development in vivo, we show that loss of Tks4 leads sparser vasculature in the fetal chorion in the Tks4-deficient 'nee' mouse strain.

2.
Clin Immunol ; 204: 43-49, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30522694

RESUMO

CD84 (SLAMF5) is a member of the SLAM family of cell-surface immunoreceptors. Broadly expressed on most immune cell subsets, CD84 functions as a homophilic adhesion molecule, whose signaling can activate or inhibit leukocyte function depending on the cell type and its stage of activation or differentiation. CD84-mediated signaling regulates diverse immunological processes, including T cell cytokine secretion, natural killer cell cytotoxicity, monocyte activation, autophagy, cognate T:B interactions, and B cell tolerance at the germinal center checkpoint. Recently, alterations in CD84 have been related to autoimmune and lymphoproliferative disorders. Specific allelic variations in CD84 are associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. In chronic lymphocytic leukemia, CD84 mediates intrinsic and stroma-induced survival of malignant cells. In this review, we describe our current understanding of the structure and function of CD84 and its potential role as a therapeutic target and biomarker in inflammatory autoimmune disorders and cancer.

3.
Front Immunol ; 9: 62, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29434592

RESUMO

Signaling lymphocyte activation molecule family (SLAMF) receptors are essential regulators of innate and adaptive immune responses. The function of SLAMF5/CD84, a family member with almost ubiquitous expression within the hematopoietic lineage is poorly defined. In this article, we provide evidence that in human monocyte-derived dendritic cells (moDCs) SLAMF5 increases autophagy, a degradative pathway, which is highly active in dendritic cells (DCs) and plays a critical role in orchestration of the immune response. While investigating the underlying mechanism, we found that SLAMF5 inhibited proteolytic degradation of interferon regulatory factor 8 (IRF8) a master regulator of the autophagy process by a mechanism dependent on the E3-ubiquitin ligase tripartite motif-containing protein 21 (TRIM21). Furthermore, we demonstrate that SLAMF5 influences the ratio of CD1a+ cells in differentiating DCs and partakes in the regulation of IL-1ß, IL-23, and IL-12 production in LPS/IFNγ-activated moDCs in a manner that is consistent with its effect on IRF8 stability. In summary, our experiments identified SLAMF5 as a novel cell surface receptor modulator of autophagy and revealed an unexpected link between the SLAMF and IRF8 signaling pathways, both implicated in multiple human pathologies.


Assuntos
Autofagia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Fatores Reguladores de Interferon/metabolismo , Transdução de Sinais , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Autofagia/efeitos dos fármacos , Diferenciação Celular , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Sirolimo/farmacologia
4.
Redox Biol ; 13: 633-645, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28818792

RESUMO

Mitochondrial reactive oxygen species (mtROS) generated continuously under physiological conditions have recently emerged as critical players in the regulation of immune signaling pathways. In this study we have investigated the regulation of antiviral signaling by increased mtROS production in plasmacytoid dendritic cells (pDCs), which, as major producers of type I interferons (IFN), are the key coordinators of antiviral immunity. The early phase of type I IFN production in pDCs is mediated by endosomal Toll-like receptors (TLRs), whereas the late phase of IFN response can also be triggered by cytosolic retinoic acid-inducible gene-I (RIG-I), expression of which is induced upon TLR stimulation. Therefore, pDCs provide an ideal model to study the impact of elevated mtROS on the antiviral signaling pathways initiated by receptors with distinct subcellular localization. We found that elevated level of mtROS alone did not change the phenotype and the baseline cytokine profile of resting pDCs. Nevertheless increased mtROS levels in pDCs lowered the TLR9-induced secretion of pro-inflammatory mediators slightly, whereas reduced type I IFN production markedly via blocking phosphorylation of interferon regulatory factor 7 (IRF7), the key transcription factor of the TLR9 signaling pathway. The TLR9-induced expression of RIG-I in pDCs was also negatively regulated by enhanced mtROS production. On the contrary, elevated mtROS significantly augmented the RIG-I-stimulated expression of type I IFNs, as well as the expression of mitochondrial antiviral-signaling (MAVS) protein and the phosphorylation of Akt and IRF3 that are essential components of RIG-I signaling. Collectively, our data suggest that increased mtROS exert diverse immunoregulatory functions in pDCs both in the early and late phase of type I IFN responses depending on which type of viral sensing pathway is stimulated.


Assuntos
Células Dendríticas/metabolismo , Interferon Tipo I/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Células Cultivadas , Proteína DEAD-box 58/metabolismo , Humanos , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/genética , Transdução de Sinais , Receptor Toll-Like 9/metabolismo
5.
Sci Rep ; 6: 34280, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27711054

RESUMO

The commitment steps of mesenchymal stromal cells (MSCs) to adipogenic and other lineages have been widely studied but not fully understood. Therefore, it is critical to understand which molecules contribute to the conversion of stem cells into differentiated cells. The scaffold protein Tks4 plays a role in podosome formation, EGFR signaling and ROS production. Dysfunction of Tks4 causes a hereditary disease called Frank-ter Haar syndrome with a variety of defects concerning certain mesenchymal tissues (bone, fat and cartilage) throughout embryogenic and postnatal development. In this study, we aimed to analyze how the mutation of Tks4 affects the differentiation potential of multipotent bone marrow MSCs (BM-MSCs). We generated a Tks4 knock-out mouse strain on C57Bl/6 background, and characterized BM-MSCs isolated from wild type and Tks4-/- mice to evaluate their differentiation. Tks4-/- BM-MSCs had reduced ability to differentiate into osteogenic and adipogenic lineages compared to wild type. Studying the expression profile of a panel of lipid-regulated genes during adipogenic induction revealed that the expression of adipogenic transcription factors, genes responsible for lipid droplet formation, sterol and fatty acid metabolism was delayed or reduced in Tks4-/- BM-MSCs. Taken together, these results establish a novel function for Tks4 in the regulation of MSC differentiation.


Assuntos
Adipogenia , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Fosfoproteínas/metabolismo , Transdução de Sinais , Animais , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Camundongos , Camundongos Knockout , Osteocondrodisplasias/congênito , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Fosfoproteínas/genética
6.
Cell Signal ; 28(5): 335-347, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26829212

RESUMO

BACKGROUND: BRAF-mutant melanoma is characterized by aggressive metastatic potential and therapeutic resistance. The innate immune receptor RIG-I has emerged as a potential target in melanoma therapies but the contributing pathways involved in anti-cancer activity are poorly characterized. METHODS: Baseline and ATRA-induced expression of RIG-I in nine (3 wild type and 6 BRAF-mutant) melanoma cell lines was measured with Q-PCR and Western blot. Ligand-specific stimulation of RIG-I was detected by Q-PCR and ELISA. Activation of the RIG-I-coupled IRF3, NF-κB and MAPK pathways was tested with protein array and Western blot. Cell proliferation and apoptosis was monitored by flow cytometry and cell counting. Down modulation of MKP-1 expression in melanoma cells was performed by specific siRNA. RESULTS: Short-term ATRA pre-treatment increases the expression of RIG-I in BRAF-mutant melanoma cells. Specific activation of RIG-I by 5'ppp-dsRNA leads to increased activity of the IRF3-IFNß pathway but does not influence NF-κB signaling. RIG-I mediates the targeted dephosphorylation of several MAPKs (p38, RSK1, GSK-3α/ß, HSP27) via the endogenous regulator MKP-1 resulting in decreased melanoma cell proliferation. CONCLUSION: RIG-I has the potential to exert anticancer activity in BRAF-mutant melanoma via controlling IFNß production and MAPK signaling. This is the first study showing that RIG-I activation results in MKP-1-mediated inhibition of cell proliferation via controlling the p38-HSP27, c-Jun and rpS6 pathways thus identifying RIG-I and MKP-1 as novel and promising therapeutical targets.


Assuntos
Proteína DEAD-box 58/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Sistema de Sinalização das MAP Quinases , Melanoma/enzimologia , Proteínas Proto-Oncogênicas B-raf/genética , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Melanoma/genética , Melanoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Tretinoína/farmacologia
7.
J Leukoc Biol ; 97(6): 1133-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25821233

RESUMO

Regulated production of ROS is mainly attributed to Nox family enzymes. In neutrophil granulocytes and macrophages, Nox2 has a crucial role in bacterial killing, and the absence of phagocytic ROS production leads to the development of CGD. Expression of Nox2 was also described in B lymphocytes, where the role of the enzyme is still poorly understood. Here, we show that peritoneal B cells, which were shown recently to possess phagocytic activity, have a high capacity to produce ROS in a Nox2-dependent manner. In phagocytosing B cells, intense intraphagosomal ROS production is detected. Finally, by studying 2 animal models of CGD, we demonstrate that phagocyte oxidase-deficient B cells have a reduced capacity to kill bacteria. Our observations extend the number of immune cell types that produce ROS to kill pathogens.


Assuntos
Linfócitos B/imunologia , Doença Granulomatosa Crônica/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/imunologia , NADPH Oxidases/imunologia , Fagossomos/imunologia , Infecções Estafilocócicas/imunologia , Animais , Linfócitos B/metabolismo , Linfócitos B/microbiologia , Linfócitos B/patologia , Regulação da Expressão Gênica , Doença Granulomatosa Crônica/metabolismo , Doença Granulomatosa Crônica/microbiologia , Doença Granulomatosa Crônica/patologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/genética , Fagocitose , Fagossomos/metabolismo , Fagossomos/microbiologia , Fagossomos/patologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/imunologia
8.
Free Radic Biol Med ; 77: 281-90, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25301097

RESUMO

Inflammation is associated with oxidative stress and characterized by elevated levels of damage-associated molecular pattern (DAMP) molecules released from injured or even living cells into the surrounding microenvironment. One of these endogenous danger signals is the extracellular mitochondrial DNA (mtDNA) containing evolutionary conserved unmethylated CpG repeats. Increased levels of reactive oxygen species (ROS) generated by recruited inflammatory cells modify mtDNA oxidatively, resulting primarily in accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) lesions. In this study, we examined the impact of native and oxidatively modified mtDNAs on the phenotypic and functional properties of plasmacytoid dendritic cells (pDCs), which possess a fundamental role in the regulation of inflammation and T cell immunity. Treatment of human primary pDCs with native mtDNA up-regulated the expression of a costimulatory molecule (CD86), a specific maturation marker (CD83), and a main antigen-presenting molecule (HLA-DQ) on the cell surface, as well as increased TNF-α and IL-8 production from the cells. These effects were more apparent when pDCs were exposed to oxidatively modified mtDNA. Neither native nor oxidized mtDNA molecules were able to induce interferon (IFN)-α secretion from pDCs unless they formed a complex with human cathelicidin LL-37, an antimicrobial peptide. Interestingly, simultaneous administration of a Toll-like receptor (TLR)9 antagonist abrogated the effects of both native and oxidized mtDNAs on human pDCs. In a murine model, oxidized mtDNA also proved a more potent activator of pDCs compared to the native form, except for induction of IFN-α production. Collectively, we demonstrate here for the first time that elevated levels of 8-oxoG bases in the extracellular mtDNA induced by oxidative stress increase the immunostimulatory capacity of mtDNA on pDCs.


Assuntos
DNA Mitocondrial/fisiologia , Células Dendríticas/imunologia , Animais , Antimicina A/farmacologia , Linhagem Celular Tumoral , Quimiocinas/sangue , Desoxiadenosinas/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Imunomodulação , Camundongos da Linhagem 129 , Oxirredução , Estresse Oxidativo
9.
PLoS One ; 7(12): e52085, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23251688

RESUMO

Ragweed (Ambrosia artemisiifolia) pollen grains, which are generally considered too large to reach the lower respiratory tract, release subpollen particles (SPPs) of respirable size upon hydration. These SPPs contain allergenic proteins and functional NAD(P)H oxidases. In this study, we examined whether exposure to SPPs initiates the activation of human monocyte-derived dendritic cells (moDCs). We found that treatment with freshly isolated ragweed SPPs increased the intracellular levels of reactive oxygen species (ROS) in moDCs. Phagocytosis of SPPs by moDCs, as demonstrated by confocal laser-scanning microscopy, led to an up-regulation of the cell surface expression of CD40, CD80, CD86, and HLA-DQ and an increase in the production of IL-6, TNF-α, IL-8, and IL-10. Furthermore, SPP-treated moDCs had an increased capacity to stimulate the proliferation of naïve T cells. Co-culture of SPP-treated moDCs with allogeneic CD3(+) pan-T cells resulted in increased secretion of IFN-γ and IL-17 by T cells of both allergic and non-allergic subjects, but induced the production of IL-4 exclusively from the T cells of allergic individuals. Addition of exogenous NADPH further increased, while heat-inactivation or pre-treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, strongly diminished, the ability of SPPs to induce phenotypic and functional changes in moDCs, indicating that these processes were mediated, at least partly, by the intrinsic NAD(P)H oxidase activity of SPPs. Collectively, our data suggest that inhaled ragweed SPPs are fully capable of activating dendritic cells (DCs) in the airways and SPPs' NAD(P)H oxidase activity is involved in initiation of adaptive immune responses against innocuous pollen proteins.


Assuntos
Alérgenos/imunologia , Ambrosia/imunologia , Células Dendríticas/imunologia , Pólen/imunologia , Sistema Respiratório/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Proliferação de Células , Técnicas de Cocultura , Células Dendríticas/metabolismo , Antígenos HLA-DQ/imunologia , Antígenos HLA-DQ/metabolismo , Humanos , Interleucinas/imunologia , Interleucinas/metabolismo , NADP/imunologia , NADP/metabolismo , NADPH Oxidases/imunologia , NADPH Oxidases/metabolismo , Fagocitose/imunologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Sistema Respiratório/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/imunologia
10.
Melanoma Res ; 22(5): 351-61, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22797253

RESUMO

In the last three decades, the incidence of melanoma has increased worldwide and no effective treatment modalities have been developed yet. All-trans retinoic acid (ATRA) and polyinosinic:polycytidylic acid (polyI:C) are strong inducers of toll-like receptor 3 (TLR3) and MDA5 expression, and polyI:C-induced TLR3 and MDA5 signaling specifically causes cell death in melanoma cells in vitro. We addressed the question of whether ATRA pretreatment could enhance the efficacy of polyI:C and, if so, would ATRA have any additional effects on this process. We found that the combined treatment of human melanoma cells with ATRA and polyI:C strongly increased the expression of TLR3 and MDA5 in both WM35 and WM983A cells associated with significantly higher mRNA and secreted levels of interferon ß (IFNß), CXCL1, CXCL8/IL-8, CXCL9, and CXCL10 than cells treated with either ATRA or polyI:C. Silencing of MDA5 by siRNA moderately affected IFNß secretion, whereas TLR3 knockdown interfered with both CXCL chemokine and IFNß production. Furthermore, the supernatants of ATRA+polyI:C-activated cultures increased the migration of both human monocyte-derived macrophages and CD1a dendritic cells significantly as compared with the supernatants of cells treated with either ATRA or polyI:C, and this effect occurred in a TLR3-dependent manner. In conclusion, consecutive treatment with ATRA and polyI:C results in strong, TLR3/MDA5-mediated chemokine and IFN responses in cultured human melanoma cells, which triggers a functional migratory response in professional antigen-presenting cells. This novel mode of concomitant activation may represent a more efficient treatment option for future melanoma therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Interferon beta/metabolismo , Melanoma/tratamento farmacológico , Poli I-C/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Receptor 3 Toll-Like/metabolismo , Tretinoína/farmacologia , Linhagem Celular Tumoral , Quimiocinas/genética , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Humanos , Indutores de Interferon/farmacologia , Interferon beta/genética , Macrófagos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Poli I-C/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Receptor 3 Toll-Like/genética , Transcriptoma , Tretinoína/administração & dosagem
11.
J Biol Chem ; 287(37): 31321-9, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22829589

RESUMO

Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Anormalidades Craniofaciais/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Cardiopatias Congênitas/metabolismo , Osteocondrodisplasias/congênito , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Cromonas/farmacologia , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/mortalidade , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/mortalidade , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Inativação Gênica , Células HeLa , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/mortalidade , Humanos , Morfolinas/farmacologia , Mutação , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/mortalidade , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Estrutura Terciária de Proteína , Quinases da Família src/genética , Quinases da Família src/metabolismo
12.
J Leukoc Biol ; 92(1): 159-69, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22517920

RESUMO

Cytosolic RIG-I-like helicases (RLR) are PRRs involved in type I IFN production and antiviral immunity. This study focuses to the comparison of the expression, function, and signaling cascades associated to RLR in the previously identified CD14(-)DC-SIGN(+)PPARγ(low)CD1a(+) and CD14(low)DC-SIGN(+)PPARγ(high)CD1a(-) human moDC subsets. Our results revealed that the expression of RLR genes and proteins as well as the activity of the coupled signaling pathways are significantly higher in the CD1a(+) subset than in its phenotypically and functionally distinct counterpart. Specific activation of RLR in moDCs by poly(I:C) or influenza virus was shown to induce the secretion of IFN-ß via IRF3, whereas induction of proinflammatory cytokine responses were predominantly controlled by TLR3. The requirement of RLR-mediated signaling in CD1a(+) moDCs for priming naïve CD8(+) T lymphocytes and inducing influenza virus-specific cellular immune responses was confirmed by RIG-I/MDA5 silencing, which abrogated these functions. Our results demonstrate the subset-specific activation of RLR and the underlying mechanisms behind its cytokine secretion profile and identify CD1a(+) moDCs as an inflammatory subset with specialized functional activities. We also provide evidence that this migratory DC subset can be detected in human tonsil and reactive LNs.


Assuntos
RNA Helicases DEAD-box/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/virologia , Imunidade Inata , Influenza Humana/imunologia , Interferon beta/metabolismo , Orthomyxoviridae/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos CD1/genética , Antígenos CD1/metabolismo , Western Blotting , Comunicação Celular , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , RNA Helicases DEAD-box/genética , Células Dendríticas/citologia , Humanos , Técnicas Imunoenzimáticas , Influenza Humana/metabolismo , Influenza Humana/virologia , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Linfonodos/metabolismo , Tonsila Palatina/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/virologia
13.
Eur J Immunol ; 42(2): 458-69, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22057588

RESUMO

The activation of TLRs expressed by macrophages or DCs, in the long run, leads to persistently impaired functionality. TLR signals activate a wide range of negative feedback mechanisms; it is not known, however, which of these can lead to long-lasting tolerance for further stimulatory signals. In addition, it is not yet understood how the functionality of monocyte-derived DCs (MoDCs) is influenced in inflamed tissues by the continuous presence of stimulatory signals during their differentiation. Here we studied the role of a wide range of DC-inhibitory mechanisms in a simple and robust model of MoDC inactivation induced by early TLR signals during differentiation. We show that the activation-induced suppressor of cytokine signaling 1 (SOCS1), IL-10, STAT3, miR146a and CD150 (SLAM) molecules possessed short-term inhibitory effects on cytokine production but did not induce persistent DC inactivation. On the contrary, the LPS-induced IRAK-1 downregulation could alone lead to persistent MoDC inactivation. Studying cellular functions in line with the activation-induced negative feedback mechanisms, we show that early activation of developing MoDCs allowed only a transient cytokine production that was followed by the downregulation of effector functions and the preservation of a tissue-resident non-migratory phenotype.


Assuntos
Citocinas/metabolismo , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Diferenciação Celular , Células Cultivadas , Citocinas/genética , Células Dendríticas/imunologia , Células Dendríticas/patologia , Retroalimentação Fisiológica , Regulação da Expressão Gênica/imunologia , Humanos , Tolerância Imunológica , Inflamação , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Interleucina-10/genética , Interleucina-10/metabolismo , Lipopolissacarídeos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Monócitos/patologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptores Toll-Like/metabolismo
14.
PLoS One ; 6(8): e23653, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21886807

RESUMO

Motility of normal and transformed cells within and across tissues requires specialized subcellular structures, e.g. membrane ruffles, lamellipodia and podosomes, which are generated by dynamic rearrangements of the actin cytoskeleton. Because the formation of these sub-cellular structures is complex and relatively poorly understood, we evaluated the role of the adapter protein SH3PXD2B [HOFI, fad49, Tks4], which plays a role in the development of the eye, skeleton and adipose tissue. Surprisingly, we find that SH3PXD2B is requisite for the development of EGF-induced membrane ruffles and lamellipodia, as well as for efficient cellular attachment and spreading of HeLa cells. Furthermore, SH3PXD2B is present in a complex with the non-receptor protein tyrosine kinase Src, phosphorylated by Src, which is consistent with SH3PXD2B accumulating in Src-induced podosomes. Furthermore, SH3PXD2B closely follows the subcellular relocalization of cortactin to Src-induced podosomes, EGF-induced membrane ruffles and lamellipodia. Because SH3PXD2B also forms a complex with the C-terminal region of cortactin, we propose that SH3PXD2B is a scaffold protein that plays a key role in regulating the actin cytoskeleton via Src and cortactin.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Pseudópodes/metabolismo , Homologia de Sequência de Aminoácidos , Domínios de Homologia de src , Actinas/metabolismo , Cortactina/metabolismo , Receptores ErbB/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Fosfatidilinositóis/metabolismo , Ligação Proteica , Transporte Proteico
15.
FEBS Lett ; 585(8): 1191-6, 2011 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-21439283

RESUMO

Despite intensive efforts to improve therapies, small cell lung cancer (SCLC) still has a dismal median survival of 18 months. Since miR-126 is under-expressed in the majority of SCLC tumors, we investigated the effect of miR-126 overexpression on the proliferation and cell cycle distribution of H69 cells. Our results demonstrate that miR-126 inhibits proliferation of H69 cells, by delaying the cells in the G1 phase. Short interfering RNA (siRNA) mediated suppression of SLC7A5, a predicted target of mir-126, has the same effect on H69 cells. We also show for the first time that SLC7A5 is a direct target of miR-126.


Assuntos
Proliferação de Células , Transportador 1 de Aminoácidos Neutros Grandes/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Carcinoma de Pequenas Células do Pulmão/genética , Western Blotting , Linhagem Celular Tumoral , Fase G1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Fatores de Tempo , Análise Serial de Tecidos
16.
PLoS One ; 5(11): e14081, 2010 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-21124855

RESUMO

Voltage-gated proton current (I(Hv)) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of I(Hv) in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently H(v)1, a voltage-gated proton channel, was cloned, and leukocytes from H(v)1 knockout mice display impaired respiratory burst. On the other hand, hardly anything is known about H(v)1 in human granulocytes. Using qPCR and a self made antibody, we detected a significant amount of H(v)1 in human eosinophil and neutrophil granulocytes and in PLB-985 leukemia cells. Using different crosslinking agents and detergents in reducing and non-reducing PAGE, significant expression of H(v)1 homodimers, but not that of higher-order multimers, could be detected in granulocytes. Results of subcellular fractionation and confocal imaging indicate that H(v)1 is resident in both plasmalemmal and granular membrane compartments of resting neutrophils. Furthermore, it is also demonstrated that H(v)1 accumulates in phagosome wall during zymosan engulfment together with, but independently of Nox2. During granulocytic differentiation early and parallel upregulation of H(v)1 and Nox2 expression was observed in PLB-985 cells. The upregulation of H(v)1 or Nox2 expression did not require the normal expression of the other molecule. Using RNA interference, we obtained strong correlation between H(v)1 expression and I(Hv) density in PLB-985 cells. It is also demonstrated that a massive reduction in H(v)1 expression can limit the Nox2 mediated superoxide production of PLB-985 granulocytes. In summary, beside monomers native H(v)1 forms stable proton channel dimer in resting and activated human granulocytes. The expression pattern of H(v)1 in granulocytes is optimized to support intense NADPH oxidase activity.


Assuntos
Granulócitos/metabolismo , Canais Iônicos/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Animais , Western Blotting , Células COS , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Eosinófilos/citologia , Eosinófilos/metabolismo , Expressão Gênica , Granulócitos/citologia , Humanos , Membranas Intracelulares/metabolismo , Canais Iônicos/química , Canais Iônicos/genética , Células Jurkat , Glicoproteínas de Membrana/genética , Microscopia Confocal , NADPH Oxidase 2 , NADPH Oxidases/genética , Neutrófilos/citologia , Neutrófilos/metabolismo , Fagossomos/metabolismo , Multimerização Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxidos/metabolismo
17.
Immunome Res ; 6: 10, 2010 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-21092113

RESUMO

BACKGROUND: The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs). RESULTS: Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules. CONCLUSIONS: The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies.

18.
J Immunol ; 184(10): 5456-65, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20410489

RESUMO

Dendritic cells (DCs) respond to changes in their lipid environment by altering gene expression and immunophenotype. Some of these alterations are mediated via the nuclear receptor superfamily. However, little is known about the contribution of liver X receptor (LXR) to DC biology. In this study, we present a systematic analysis of LXR, activated by synthetic ligands or naturally occurring oxysterols in developing human monocyte-derived DCs. We found that LXRs are present and can be activated throughout DC differentiation in monocyte- and blood-derived DCs. Administration of LXR-specific natural or synthetic activators induced target gene expression accompanied by increased expression of DC maturation markers, such as CD80 and CD86. In mature DCs, LXR activation augmented the production of inflammatory cytokines IL-12, TNF-alpha, IL-6, and IL-8 and resulted in an increased capacity to activate CD4+ T cell proliferation upon ligation with TLR4 or TLR3 ligands. These effects appear to be underpinned by prolonged NF-kappaB signaling. Supporting such an inflammatory role, we found that LXR positive DCs are present in reactive lymph nodes in vivo. We propose that activation of LXR represents a novel lipid-signaling paradigm that alters the inflammatory response of human DCs.


Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Mediadores da Inflamação/fisiologia , Receptores Nucleares Órfãos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Proliferação de Células , Células Cultivadas , Citocinas/biossíntese , Células Dendríticas/patologia , Humanos , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/imunologia , Receptores X do Fígado , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , NF-kappa B/fisiologia , Receptores Nucleares Órfãos/fisiologia , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Transdução de Sinais/imunologia , Regulação para Cima/imunologia
19.
Adv Immunol ; 97: 177-250, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18501771

RESUMO

The nine SLAM-family genes, SLAMF1-9, a subfamily of the immunoglobulin superfamily, encode differentially expressed cell-surface receptors of hematopoietic cells. Engagement with their ligands, which are predominantly homotypic, leads to distinct signal transduction events, for instance those that occur in the T or NK cell immune synapse. Upon phosphorylation of one or more copies of a unique tyrosine-based signaling motif in their cytoplasmic tails, six of the SLAM receptors recruit the highly specific single SH2-domain adapters SLAM-associated protein (SAP), EAT-2A, and/or EAT-2B. These adapters in turn bind to the tyrosine kinase Fyn and/or other protein tyrosine kinases connecting the receptors to signal transduction networks. Individuals deficient in the SAP gene, SH2D1A, develop an immunodeficiency syndrome: X-linked lympho-proliferative disease. In addition to operating in the immune synapse, SLAM receptors initiate or partake in multiple effector functions of hematopoietic cells, for example, neutrophil and macrophage killing and platelet aggregation. Here we discuss the current understanding of the structure and function of these recently discovered receptors and adapter molecules in the regulation of adaptive and innate immune responses.


Assuntos
Imunidade/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Receptores Imunológicos/fisiologia , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/fisiopatologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/fisiopatologia , Modelos Biológicos , Receptores Imunológicos/genética , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária
20.
Blood ; 109(2): 643-52, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-16968896

RESUMO

Accumulating data have shown that the microenvironment of dendritic cells modulates subtype differentiation and CD1 expression, but the mechanisms by which exogenous factors confer these effects are poorly understood. Here we describe the dependence of CD1a- monocyte-derived dendritic cell (moDC) development on lipids associated with the expression of peroxisome proliferator-activated receptor-gamma (PPARgamma). We also show the consecutive differentiation of immature CD1a-PPARgamma+ moDCs to CD1a+PPARgamma- cells limited by serum lipoproteins and terminated by proinflammatory cytokines. Immature CD1a- moDCs possess higher internalizing capacity than CD1a+ cells, whereas both activated subtypes have similar migratory potential but differ in their cytokine and chemokine profiles, which translates to distinct T-lymphocyte-polarizing capacities. CD1a+ moDCs stand out by their capability to secrete high amounts of IL-12p70 and CCL1. As lipoproteins skew moDC differentiation toward the generation of CD1a-PPARgamma+ cells and inhibit the development of CD1a+PPARgamma- cells, we suggest that the uptake of lipids results in endogenous PPARgamma agonists that induce a cascade of gene transcription coordinating lipid metabolism, the expression of lipid-presenting CD1 molecules, subtype dichotomy, and function. The presence of CD1a-PPARgamma+ and CD1a+PPARgamma- DCs in lymph nodes and in pulmonary Langerhans cell histiocytosis confirms the functional relevance of these DC subsets in vivo.


Assuntos
Antígenos CD1/biossíntese , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Lipídeos/fisiologia , Monócitos/imunologia , PPAR gama/fisiologia , Quimiocinas/biossíntese , Citocinas/biossíntese , Células Dendríticas/citologia , Humanos , Monócitos/citologia , Fenótipo
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