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1.
Free Radic Biol Med ; 143: 209-220, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31408726

RESUMO

A growing body of evidence suggests that elevated levels of reactive oxygen species (ROS) in the airways caused by exposure to gas phase pollutants or particulate matter are able to activate dendritic cells (DCs); however, the exact mechanisms are still unclear. When present in excess, ROS can modify macromolecules including DNA. One of the most abundant DNA base lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which is repaired by the 8-oxoguanine DNA glycosylase 1 (OGG1)-initiated base excision repair (BER) (OGG1-BER) pathway. Studies have also demonstrated that in addition to its role in repairing oxidized purines, OGG1 has guanine nucleotide exchange factor activity when bound to 8-oxoG. In the present study, we tested the hypothesis that exposure to 8-oxoG, the specific product of OGG1-BER, induces functional changes of DCs. Supporting our hypothesis, transcriptome analysis revealed that in mouse lungs, out of 95 genes associated with DCs' function, 22 or 42 were significantly upregulated after a single or multiple intranasal 8-oxoG challenges, respectively. In a murine model of allergic airway inflammation, significantly increased serum levels of ovalbumin (OVA)-specific IgE antibodies were detected in mice sensitized via nasal challenges with OVA+8-oxoG compared to those challenged with OVA alone. Furthermore, exposure of primary human monocyte-derived DCs (moDC) to 8-oxoG base resulted in significantly enhanced expression of cell surface molecules (CD40, CD86, CD83, HLA-DQ) and augmented the secretion of pro-inflammatory mediators IL-6, TNF and IL-8, whereas it did not considerably influence the production of the anti-inflammatory cytokine IL-10. The stimulatory effects of 8-oxoG on human moDCs were abolished upon siRNA-mediated OGG1 depletion. Collectively, these data suggest that OGG1-BER-generated 8-oxoG base-driven cell signaling activates DCs, which may contribute to initiation of both the innate and adaptive immune responses under conditions of oxidative stress.

2.
Front Immunol ; 9: 2314, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30344524

RESUMO

Unique members of the nucleotide-binding domain leucine-rich repeat (NLR) family have been found to regulate intracellular signaling pathways initiated by other families of pattern recognition receptors (PRR) such as Toll-like receptors (TLRs) and retinoic-acid inducible gene I (RIG-I)-like receptors (RLRs). Plasmacytoid dendritic cells (pDCs), the most powerful type I interferon (IFN) producing cells, preferentially employ endosomal TLRs to elicit antiviral IFN responses. By contrast, conventional DCs (cDCs) predominantly use cytosolic RLRs, which are constitutively expressed in them, to sense foreign nucleic acids. Previously we have reported that, though RIG-I is absent from resting pDCs, it is inducible upon TLR stimulation. In the recent study we investigated the regulatory ability of NLRs, namely NLRC5 and NLRX1 directly associated with the RLR-mediated signaling pathway in DC subtypes showing different RLR expression, particularly in pDCs, and monocyte-derived DCs (moDCs). Here we demonstrate that similarly to RLRs, NLRC5 is also inducible upon TLR9 stimulation, whereas NLRX1 is constitutively expressed in pDCs. Inhibition of NLRC5 and NLRX1 expression in pDCs augmented the RLR-stimulated expression of type I IFNs but did not affect the production of the pro-inflammatory cytokines TNF, IL-6, and the chemokine IL-8. Further we show that immature moDCs constantly express RLRs, NLRX1 and NLRC5 that are gradually upregulated during their differentiation. Similarly to pDCs, NLRX1 suppression increased the RLR-induced production of type I IFNs in moDCs. Interestingly, RLR stimulation of NLRX1-silenced moDCs leads to a significant increase in pro-inflammatory cytokine production and IκBα degradation, suggesting increased NF-κB activity. On the contrary, NLRC5 does not seem to have any effect on the RLR-mediated cytokine responses in moDCs. In summary, our results indicate that NLRX1 negatively regulates the RLR-mediated type I IFN production both in pDCs and moDCs. Further we show that NLRX1 inhibits pro-inflammatory cytokine secretion in moDCs but not in pDCs following RLR stimulation. Interestingly, NLRC5 suppresses the RLR-induced type I IFN secretion in pDCs but does not appear to have any regulatory function on the RLR pathway in moDCs. Collectively, our work demonstrates that RLR-mediated innate immune responses are primarily regulated by NLRX1 and partly controlled by NLRC5 in human DCs.


Assuntos
RNA Helicases DEAD-box/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Interferon Tipo I/metabolismo , Proteínas NLR/metabolismo , Biomarcadores , Linhagem Celular , RNA Helicases DEAD-box/genética , Inativação Gênica , Interações Hospedeiro-Patógeno , Humanos , Inflamação/patologia , Proteínas NLR/genética , Transdução de Sinais , Receptores Toll-Like/metabolismo
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.
Sci Rep ; 8(1): 1765, 2018 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-29379077

RESUMO

Serotonin is a monoamine neurotransmitter that signals through a wide array of receptors (5-HT1-7) many of which are also involved in immune processes. Dendritic cells (DCs) are crucial players in immune defense by bridging innate and adaptive immune responses via their vast repertoire of pattern recognition receptors and antigen-presenting capability. Although serotonin is known to influence immunity at many levels, cell type-specific expression and function of its receptors remains poorly understood. Here we aimed to study 5-HT1-7 expression and function in CD1a- and CD1a+ human monocyte-derived DCs (moDCs). We found that the 5-HT2B receptor-subtype is solely expressed by the inflammatory CD1a+ moDC subset. Specific 5-HT2B activation potently inhibited TLR2, TLR3, and TLR7/8-induced proinflammatory cytokine and chemokine (TNF-α, IL-6, IL-8, IP-10, IL-12) but not type I interferon-ß responses. 5-HT2B agonism also interfered with the polarization of CD1a+ moDC-primed CD4+ T cells towards inflammatory Th1 and Th17 effector lymphocytes. Here we report the subset-specific expression and immunomodulatory function of 5-HT2B in human moDCs. Our results expand the biological role of 5-HT2B which may act not only as a neurotransmitter receptor, but also as an important modulator of both innate and adaptive immune responses.


Assuntos
Células Dendríticas/imunologia , Fatores Imunológicos/imunologia , Receptor 5-HT2B de Serotonina/imunologia , Antígenos CD1/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/imunologia , Humanos , Monócitos/imunologia , Transdução de Sinais/imunologia , Células Th17/imunologia
5.
Front Immunol ; 9: 3070, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30622542

RESUMO

Recent advances reveal that metabolic reprogramming is required for adequate antiviral responses of dendritic cells (DCs) that possess the capacity to initiate innate and adaptive immune responses. Several reports indicate that Toll-like receptor (TLR) stimulation of DCs is accompanied by a rapid induction of glycolysis; however, the metabolic requirements of retinoic-acid inducible gene I (RIG-I)-like receptor (RLR) activation have not defined either in conventional DCs (cDCs) or in plasmacytoid DCs (pDCs) that are the major producers of type I interferons (IFN) upon viral infections. To sense viruses and trigger an early type I IFN response, pDCs rely on endosomal TLRs, whereas cDCs employ cytosolic RIG-I, which is constitutively present in their cytoplasm. We previously found that RIG-I is upregulated in pDCs upon endosomal TLR activation and contributes to the late phase of type I IFN responses. Here we report that TLR9-driven activation of human pDCs leads to a metabolic transition to glycolysis supporting the production of type I IFNs, whereas RIG-I-mediated antiviral responses of pDCs do not require glycolysis and rather rely on oxidative phosphorylation (OXPHOS) activity. In particular, TLR9-activated pDCs show increased extracellular acidification rate (ECAR), lactate production, and upregulation of key glycolytic genes indicating an elevation in glycolytic flux. Furthermore, administration of 2-deoxy-D-glucose (2-DG), an inhibitor of glycolysis, significantly impairs the TLR9-induced secretion of type I IFNs by human pDCs. In contrast, RIG-I stimulation of pDCs does not result in any alterations of ECAR, and type I IFN production is not inhibited but rather promoted by 2-DG treatment. Moreover, pDCs activated via TLR9 but not RIG-I in the presence of 2-DG are impaired in their capacity to prime allogeneic naïve CD8+ T cell proliferation. Interestingly, human monocyte-derived DCs (moDC) triggered via RIG-I show a commitment to glycolysis to promote type I IFN production and T cell priming in contrast to pDCs. Our findings reveal for the first time, that pDCs display a unique metabolic profile; TLR9-driven but not RIG-I-mediated activation of pDCs requires glycolytic reprogramming. Nevertheless, the metabolic signature of RIG-I-stimulated moDCs is characterized by glycolysis suggesting that RIG-I-induced metabolic alterations are rather cell type-specific and not receptor-specific.


Assuntos
Reprogramação Celular/imunologia , Proteína DEAD-box 58/metabolismo , Células Dendríticas/imunologia , Metaboloma/imunologia , Monócitos/imunologia , Antimetabólitos/farmacologia , Buffy Coat , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Proliferação de Células , Proteína DEAD-box 58/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Desoxiglucose/farmacologia , Glicólise/efeitos dos fármacos , Glicólise/imunologia , Voluntários Saudáveis , Humanos , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Metaboloma/efeitos dos fármacos , Monócitos/metabolismo , Fosforilação Oxidativa , Cultura Primária de Células , Transdução de Sinais/imunologia , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo , Regulação para Cima
6.
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
7.
Mol Nutr Food Res ; 61(11)2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28731263

RESUMO

SCOPE: Saccharomyces cerevisiae is one of the most important microbes in food industry, but there is growing evidence on its potential pathogenicity as well. Its status as a member of human mycobiome is still not fully understood. METHODS AND RESULTS: In this study, we characterize clinical S. cerevisiae isolates from Hungarian hospitals along with commercial baking and probiotic strains, and determine their phenotypic parameters, virulence factors, interactions with human macrophages, and pathogenicity. Four of the clinical isolates could be traced back to commercial strains based on genetic fingerprinting. Our observations indicate that the commercial-derived clinical isolates have evolved new phenotypes and show similar, or in two cases, significantly decreased pathogenicity. Furthermore, immunological experiments revealed that the variability in human primary macrophage activation after coincubation with yeasts is largely donor and not isolate dependent. CONCLUSION: Isolates in this study offer an interesting insight into the potential microevolution of probiotic and food strains in human hosts. These commensal yeasts display various changes in their phenotypes, indicating that the colonization of the host does not necessarily impose a selective pressure toward higher virulence/pathogenicity.


Assuntos
Evolução Molecular , Microbiologia de Alimentos , Probióticos , Saccharomyces cerevisiae/fisiologia , Animais , Células Cultivadas , Culinária , Marcadores Genéticos , Interações Hospedeiro-Patógeno , Humanos , Hungria , Larva/crescimento & desenvolvimento , Larva/microbiologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/microbiologia , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , Micoses/microbiologia , Mapeamento de Peptídeos , Fagocitose , Probióticos/efeitos adversos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/patogenicidade , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Virulência/metabolismo
8.
Immunol Lett ; 189: 109-113, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28414181

RESUMO

Psoriasis is a common inflammatory skin disease and dendritic cells (DCs) play crucial role in the development of skin inflammation. Although the characteristics of skin DCs in psoriasis are well defined, less is known about their peripheral blood precursors. Our aim was to characterize the phenotypic features as well as the cytokine and chemokine production of CD1c+ myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in the blood samples of psoriatic patients. Blood DCs were isolated by using a magnetic separation kit, and their intracytoplasmic cytokine production and CD83/CD86 maturation/activation marker expression were investigated by 8-colour flow cytometry. In CD1c+ mDCs the intracellular productions of Th1, Th2, Th17, Th22 and Treg polarizing cytokines were examined simultaneously, whereas in pDCs the amounts of IFNα as well as IL-12, IL-23 and IL-6 were investigated. The chemokine production of both DC populations was investigated by flow-cytometry and ELISA. According to our results psoriatic CD1c+ mDCs were in a premature state since their CD83/CD86 maturation/activation marker expression, IL-12 cytokine, CXCL9 and CCL20 chemokine production was significantly higher compared to control cells. On the other hand, blood pDCs neither produced any of the investigated cytokines and chemokines nor expressed CD83/CD86 maturation/activation markers. Our results indicate that in psoriasis not only skin but also blood mDCs perform Th1 polarizing and Th1/Th17 recruiting capacity, while pDCs function only in the skin milieu.


Assuntos
Células Dendríticas/fisiologia , Células Mieloides/fisiologia , Psoríase/imunologia , Pele/imunologia , Células Th1/imunologia , Células Th17/imunologia , Adulto , Idoso , Antígenos CD1/metabolismo , Diferenciação Celular , Células Cultivadas , Quimiocina CCL20/metabolismo , Quimiocina CXCL9/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
9.
Headache ; 57(3): 441-454, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28133727

RESUMO

OBJECTIVE: Exploring the pathophysiological changes in transient receptor potential vanilloid 1 (TRPV1) receptor of the trigeminovascular system in high-fat, high-sucrose (HFHS) diet-induced obesity of experimental animals. BACKGROUND: Clinical and experimental observations suggest a link between obesity and migraine. Accumulating evidence indicates that metabolic and immunological alterations associated with obesity may potentially modulate trigeminovascular functions. A possible target for obesity-induced pathophysiological changes is the TRPV1/capsaicin receptor which is implicated in the pathomechanism of headaches in a complex way. METHODS: Male Sprague-Dawley rats were fed a regular (n = 25) or HFHS diet (n = 26) for 20 weeks. At the end of the dietary period, body weight of the animals was normally distributed in both groups and it was significantly higher in animals on HFHS diet. Therefore, experimental groups were regarded as control and HFHS diet-induced obese groups. Capsaicin-induced changes in meningeal blood flow and release of calcitonin gene-related peptide (CGRP) from dural trigeminal afferents were measured in control and obese rats. The distribution of TRPV1- and CGRP-immunoreactive meningeal sensory nerves was also compared in whole mount preparations of the dura mater. Metabolic parameters of the animals were assessed by examining glucose and insulin homeostasis as well as plasma cytokine concentrations. RESULTS: HFHS diet was accompanied by reduced food consumption and greater fluid and energy intakes in addition to increased body weight of the animals. HFHS diet increased fasting blood glucose and insulin concentrations as well as levels of circulating proinflammatory cytokines interleukin-1ß and interleukin-6. In obese animals, dural application of the archetypal TRPV1 agonist capsaicin resulted in significantly augmented vasodilatory and vasoconstrictor responses as compared to controls. Diet-induced obesity was also associated with enhanced basal and capsaicin-induced CGRP release from meningeal afferents ex vivo. Except for minor morphological changes, the distribution of dural TRPV1- and CGRP-immunoreactive afferents was similar in control and obese animals. CONCLUSIONS: Our results suggest that obesity induced by long-term HFHS diet results in sensitization of the trigeminovascular system. Changes in TRPV1-mediated vascular reactions and CGRP release are pathophysiological alterations that may be of relevance to the enhanced headache susceptibility of obese individuals.


Assuntos
Dieta/efeitos adversos , Dura-Máter/metabolismo , Obesidade/etiologia , Obesidade/patologia , Canais de Cátion TRPV/metabolismo , Análise de Variância , Animais , Glicemia/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/farmacologia , Circulação Cerebrovascular/efeitos dos fármacos , Modelos Animais de Doenças , Ingestão de Alimentos/fisiologia , Jejum/sangue , Insulina/sangue , Interleucina-1beta/sangue , Interleucina-6/sangue , Masculino , Meninges/irrigação sanguínea , Obesidade/sangue , Ratos , Ratos Sprague-Dawley , Estatísticas não Paramétricas
10.
Cephalalgia ; 37(6): 581-591, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27301459

RESUMO

Background Clinical studies suggest a link between obesity and the primary headache disorder migraine. In our study we aimed to reveal the effect of obesity on meningeal nociceptor function in rats receiving a high-fat, high-sucrose diet. Methods Transient receptor potential ankyrin 1 (TRPA1) receptor activation-induced changes in meningeal blood flow, release of calcitonin gene-related peptide (CGRP) from trigeminal afferents and TRPA1 protein expression in the trigeminal ganglia were measured in control and obese rats. Metabolic parameters of the animals were assessed by measuring glucose and insulin homeostasis as well as plasma cytokine concentrations. Results The present experiments revealed an enhanced basal and TRPA1 receptor agonist-induced CGRP release from meningeal afferents of obese insulin-resistant rats and an attenuated CGRP release to potassium chloride. Obesity was also associated with an augmented vasodilatation in meningeal arteries after dural application of the TRPA1 agonist acrolein, a reduction in TRPA1 protein expression in the trigeminal ganglia and elevations in circulating proinflammatory cytokines IL-1ß and IL-6 in addition to increased fasting blood glucose and insulin concentrations. Conclusions Our results suggest trigeminal sensitisation as a mechanism for enhanced headache susceptibility in obese individuals after chemical exposure of trigeminal nociceptors.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Dieta Hiperlipídica/efeitos adversos , Sacarose na Dieta/efeitos adversos , Obesidade/metabolismo , Canal de Cátion TRPA1/fisiologia , Gânglio Trigeminal/metabolismo , Cefaleias Vasculares/metabolismo , Animais , Glicemia/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Obesidade/complicações , Ratos , Ratos Sprague-Dawley , Cefaleias Vasculares/etiologia
11.
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
12.
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
13.
J Leukoc Biol ; 96(4): 579-89, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25001862

RESUMO

Type I and III IFNs are crucial, soluble components of potent antiviral responses. It has been explored recently that mTOR is involved in the regulation of IFN-α/ß production by pDCs, albeit its role in the induction of IFN responses in cDCs remained unrevealed. In this study, we demonstrate that the PI3K/mTOR pathway is indispensable for eliciting intact type I and III IFN responses in moDCs stimulated with polyI:C. The inhibition of mTOR functionality by rapamycin impairs the pIRF3 and also a few members of the MAPK family, suggesting that mTOR contributes to the activation of multiple signaling pathways in the presence of viral antigens. Furthermore, rapamycin-treated moDCs show decreased capacity to prime IFN-γ secretion by naive CD8(+) T-lymphocytes. As in moDCs, mTOR-mediated regulation is also essential for the production of type I and III IFNs in circulating CD1c(+) DCs. To our best knowledge, these results demonstrate for the first time that mTOR has an impact on the functional activities of cDCs via modulating the outcome of IFN secretion.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Antígenos CD1/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Células Dendríticas/efeitos dos fármacos , Expressão Gênica , Regulação da Expressão Gênica , Glicoproteínas/metabolismo , Humanos , Fator Regulador 3 de Interferon/genética , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Poli I-C/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Ativação Transcricional
14.
Immunol Cell Biol ; 92(8): 671-8, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24839978

RESUMO

Plasmacytoid dendritic cells (pDCs) are professional type I interferon (IFN)-producing cells that play an essential role in antiviral immunity. In many cell types, detection of intracellular pathogens is mostly dependent on endosomal Toll-like receptors (TLRs) and cytosolic sensors, such as retinoic acid-inducible gene I (RIG-I). However, the possible interplay between these two systems has not yet been elucidated. Here we aimed to study the collaboration of endosomal TLRs and RIG-I in primary human pDCs. We found that under steady-state conditions, pDCs express RIG-I at very low level, but the expression of this receptor is rapidly and dramatically upregulated upon stimulation by the TLR7 ligand imiquimod or the TLR9 ligand type A CpG. We also demonstrated that pDCs are able to sense and respond to 5'-triphosphate double-stranded RNA (5'-ppp-dsRNA) only following activation by endosomal TLRs. Experiments on primary pDCs with functionally blocked IFN-α/ß receptor 1 (IFNAR1) and those on human pDC leukemia (pDC-L) cells defective in type I IFN secretion indicated that the upregulation of RIG-I expression in pDCs upon stimulation by endosomal TLR occurs in a type I IFN-independent manner. Selective phosphorylation of signal transducer and activator of transcription 1 (STAT1) on tyrosine 701 could be identified as an early signaling event in this process. Our results show that in contrast to many other cell types, where RIG-I expression is induced by type I IFN, in pDCs a disparate mechanism is responsible for the upregulation of RIG-I. Our findings also indicate that along with autophagy, an additional mechanism is operating in pDCs to promote the detection of replicating viruses.


Assuntos
RNA Helicases DEAD-box/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Interferon Tipo I/metabolismo , Receptores Toll-Like/metabolismo , Aminoquinolinas/farmacologia , Células Cultivadas , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Células Dendríticas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imiquimode , Interferon Tipo I/farmacologia , Ligantes , Fosforilação , RNA de Cadeia Dupla , Fator de Transcrição STAT1/metabolismo , Regulação para Cima
15.
J R Soc Interface ; 11(95): 20140097, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24647908

RESUMO

Previous observations suggest that static magnetic field (SMF)-exposure acts on living organisms partly through reactive oxygen species (ROS) reactions. In this study, we aimed to define the impact of SMF-exposure on ragweed pollen extract (RWPE)-induced allergic inflammation closely associated with oxidative stress. Inhomogeneous SMF was generated with an apparatus validated previously providing a peak-to-peak magnetic induction of the dominant SMF component 389 mT by 39 T m(-1) lateral gradient in the in vivo and in vitro experiments, and 192 mT by 19 T m(-1) in the human study at the 3 mm target distance. Effects of SMF-exposure were studied in a murine model of allergic inflammation and also in human provoked skin allergy. We found that even a single 30-min exposure of mice to SMF immediately following intranasal RWPE challenge significantly lowered the increase in the total antioxidant capacity of the airways and decreased allergic inflammation. Repeated (on 3 consecutive days) or prolonged (60 min) exposure to SMF after RWPE challenge decreased the severity of allergic responses more efficiently than a single 30-min treatment. SMF-exposure did not alter ROS production by RWPE under cell-free conditions, while diminished RWPE-induced increase in the ROS levels in A549 epithelial cells. Results of the human skin prick tests indicated that SMF-exposure had no significant direct effect on provoked mast cell degranulation. The observed beneficial effects of SMF are likely owing to the mobilization of cellular ROS-eliminating mechanisms rather than direct modulation of ROS production by pollen NAD(P)H oxidases.


Assuntos
Asma , Dermatite Atópica , Terapia de Campo Magnético/métodos , Campos Magnéticos , Rinite Alérgica Sazonal , Adulto , Animais , Asma/metabolismo , Asma/patologia , Asma/terapia , Linhagem Celular , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Dermatite Atópica/terapia , Modelos Animais de Doenças , Método Duplo-Cego , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Inflamação/terapia , Terapia de Campo Magnético/instrumentação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio , Rinite Alérgica Sazonal/metabolismo , Rinite Alérgica Sazonal/patologia , Rinite Alérgica Sazonal/terapia
16.
PLoS One ; 8(2): e55264, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23405128

RESUMO

The aim of this study was to characterize and identify the mode of action of IC31®, a two-component vaccine adjuvant. We found that IC31® was accumulated in human peripheral blood monocytes, MHC class II positive cells and monocyte-derived DCs (moDCs) but not in plasmacytoid DCs (pDCs). In the presence of IC31® the differentiation of inflammatory CD1a(+) moDCs and the secretion of chemokines, TNF-α and IL-6 cytokines was inhibited but the production of IFNß was increased. Sustained addition of IC31® to differentiating moDCs interfered with IκBα phosphorylation, while the level of phospho-IRF3 increased. We also showed that both IC31® and its KLK component exhibited a booster effect on type I IFN responses induced by the specific ligands of TLR3 or TLR7/8, whereas TLR9 ligand induces type I IFN production only in the presence of IC31® or ODN1. Furthermore, long term incubation of moDCs with IC31® caused significantly higher expression of IRF and IFN genes than a single 24 hr treatment. The adjuvant activity of IC31® on the IFN response was shown to be exerted through TLRs residing in the vesicular compartment of moDCs. Based on these results IC31® was identified as a moDC modulatory adjuvant that sets the balance of the NF-κB and IRF3 mediated signaling pathways to the production of IFNß. Thus IC31® is emerging as a potent adjuvant to increase immune responses against intracellular pathogens and cancer in future vaccination strategies.


Assuntos
Células Dendríticas/efeitos dos fármacos , Endossomos/imunologia , Interferon Tipo I/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Oligopeptídeos/farmacologia , Receptores Toll-Like/imunologia , Adjuvantes Imunológicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Quimiocinas/imunologia , Células Dendríticas/imunologia , Combinação de Medicamentos , Endossomos/efeitos dos fármacos , Humanos , Proteínas I-kappa B/biossíntese , Proteínas I-kappa B/imunologia , Fator Regulador 3 de Interferon/imunologia , Interferon Tipo I/imunologia , Interferon beta/imunologia , Interleucina-6/imunologia , Leucócitos Mononucleares/imunologia , Ligadura , Inibidor de NF-kappaB alfa , NF-kappa B/imunologia , Oligodesoxirribonucleotídeos/imunologia , Oligopeptídeos/imunologia , Fosforilação , Fator de Necrose Tumoral alfa/imunologia
17.
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
18.
Free Radic Biol Med ; 52(3): 635-645, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22178414

RESUMO

Under normal conditions, plasmacytoid dendritic cells (pDCs) are located in peripheral lymphoid organs or circulate in the blood, from where they can migrate to sites of infection or inflammation. In inflamed tissues, pDCs can be exposed to elevated levels of reactive oxygen species produced by inflammatory cells and we presume that oxidative stress could affect the cellular responses of pDCs to microenvironmental stimuli. To explore this possibility, human pDCs isolated from peripheral blood of healthy donors were treated with H(2)O(2) and R837 (a Toll-like receptor 7 ligand), separately and in combination. Our results demonstrate that treatment with a low concentration (0.01 µM) of H(2)O(2) resulted in only slight changes in the expression of CD40, CD80, CD86, and CD83; however, low-dose H(2)O(2) markedly decreased the expression of HLA-DQ on pDCs. Exposure to H(2)O(2) did not trigger the release of IL-6, TNF-α, IL-8, or IFN-α from pDCs. Although addition of H(2)O(2) did not modify the capacity of pDCs to activate allogeneic IL-17- or IFN-γ-producing T cells, it significantly increased the ability of pDCs to stimulate IL-4-secreting T cells. Exposure of pDCs to H(2)O(2) before cocultivation with naïve autologous T cells significantly lowered IL-10 production by T cells, but did not affect IL-17 release. It was also observed that H(2)O(2)-exposed pDCs provided stronger stimuli for Th2 than for Th1 differentiation upon autologous activation, compared to untreated pDCs, possibly because of elevated surface expression of OX40-L. Most importantly, when pDCs were stimulated with R837 in the presence of H(2)O(2), decreased phenotypic activation, decreased chemokine and cytokine release, and impaired allo- and autostimulatory functions of pDCs were detected, indicating that pDCs exposed to oxidative stress in vivo may have an anti-inflammatory or tolerogenic role in regulating adaptive immune responses.


Assuntos
Células Dendríticas/fisiologia , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Imunidade Adaptativa , Antígenos CD/metabolismo , Polaridade Celular , Sobrevivência Celular , Células Cultivadas , Quimiocinas/metabolismo , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Ativação Linfocitária , Estresse Oxidativo , Fenótipo , Quinolinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/metabolismo
19.
J Immunol ; 184(5): 2377-85, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20118277

RESUMO

It has been demonstrated that pollen grains contain NAD(P)H oxidases that induce oxidative stress in the airways, and this oxidative insult is critical for the development of allergic inflammation in sensitized mice. On the basis of this observation, we have examined whether pollen grain exposure triggers oxidative stress in dendritic cells (DCs), altering their functions. To test this hypothesis, human monocyte-derived DCs were treated with ragweed pollen grains. Our findings show that exposure to pollen grains induces an increase in the intracellular levels of reactive oxygen species in DCs. Our data also indicate that besides the NAD(P)H oxidases, other component(s) of pollen grains contributes to this phenomenon. Elevated levels of intracellular reactive oxygen species triggered the production of IL-8 as well as proinflammatory cytokines, such as TNF-alpha and IL-6. Treatment with pollen grains initiated the maturation of DCs, strongly upregulated the membrane expression of CD80, CD86, CD83, and HLA-DR, and caused only a slight increase in the expression of CD40. The pollen-treated DCs induced the development of naive T lymphocytes toward effector T cells with a mixed profile of cytokine production. Antioxidant inhibited both the phenotypic and functional changes of DCs, underlining the importance of oxidative stress in these processes. Collectively, these data show that pollen exposure-induced oxidative stress may contribute to local innate immunity and participate in the initiation of adaptive immune responses to pollen Ags.


Assuntos
Células Dendríticas/imunologia , Estresse Oxidativo , Pólen/imunologia , Espécies Reativas de Oxigênio/metabolismo , Ambrosia/imunologia , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD40/metabolismo , Proliferação de Células , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Antígenos HLA-DR/metabolismo , Humanos , Imunoglobulinas/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
Cytometry A ; 73(3): 254-8, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18205196

RESUMO

Plasmacytoid pre-dendritic cells (pDCs) are able to prime and polarize naive T-cells, while also having an important effector function in antiviral immunity through the rapid and robust production of interferon-alpha. The main setback of pDCs investigation is the rarity and ex vivo fragility of these cells. Relative simple, reliable, and accurate methods for phenotypic analysis and functional studies of pDCs without isolation would be a great deal of interest. Fresh whole blood samples were analyzed by two-color and one-color flow cytometric pDC-identification assays. The changes in the surface expression of CD62L and HLA-DQ on pDCs in whole blood samples after 24-h treatment with imiquimod, a toll-like receptor 7 agonist, were analyzed. Our data demonstrate that the identification of pDCs in peripheral blood samples can be achieved by using only one fluorescent channel for blood dendritic cell antigen (BDCA)-4 staining combined with the light scatter parameters, thus leaving the other channels open for further phenotypic and/or functional analysis. Recently, several lines of evidence supported the involvement of pDCs in the development of several human diseases, so our new one-color identification approach may provide a useful tool for investigation of the pathomechanism of the relevant diseases by using common, 2-laser benchtop cytometers.


Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Células-Tronco/citologia , Células-Tronco/imunologia , Adulto , Cor , Células Dendríticas/classificação , Humanos , Células-Tronco/classificação
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