RESUMO
Hypoxia represents an inadequate oxygen supply to tissues, which can modulate cell functions, primarily through the hypoxia-inducible transcription factor HIF-1α. Dendritic cells (DC) are professional antigen-presenting cells and their migration maybe affected by hypoxia, since the local microenvironment in lymphoid organs, as well as in inflamed and tumor tissues, is characterized by low oxygen tensions. In this study we observed an enhanced migratory capability of human monocyte-derived DC, using in vitro migration assays performed under hypoxic conditions. Such enhancement was independent on either the chemoattractants involved or the maturation level of DC. However, HIF-1α appeared to be crucial for the migration only of immature DC and not for mature DC under hypoxia, as indicated by HIF-1α siRNA approaches. Furthermore, we observed that while Akt phosphorylation was enhanced in both immature and mature DC exposed to hypoxia, other signaling pathways, such as p38 and p42/p44 MAPK, were differently affected during hypoxic treatment. More interestingly, aspecific and specific inhibition of PI3K/Akt indicated that such pathway was relevant for the migration of both immature and matured DC under hypoxia, even when DC were transfected with HIF-1α siRNA. Our results indicate that, besides HIF-1α, several other pathways, including PI3K/Akt, may be involved in the response to hypoxia of immature and, more specifically, of mature DC to sustain their trafficking and functions within hypoxic microenvironments.
Assuntos
Células Dendríticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Apresentadoras de Antígenos , Movimento Celular/genética , Células Dendríticas/imunologia , Células Dendríticas/patologia , Humanos , Hipóxia/genética , Sistema de Sinalização das MAP Quinases/genética , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , RNA Interferente Pequeno , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
Dendritic cells (DC) are the most potent antigen-presenting cells and during their life cycle they are exposed to different oxygen tensions. Similarly to inflamed and tumor tissues, lymphoid organs are characterized by a hypoxic microenvironment; thus, the modality by which hypoxia may affect DC is important for regulating both the quality and the intensity of the immune response. Here, we show that human monocyte-derived DC, exposed to hypoxia, expressed high levels of the hypoxia-inducible factor (HIF)-1α, associated with upregulation of BNIP3 and BAX expression. This was paralleled with downregulation of the anti-apoptotic molecule Bcl-2, enhanced caspase-3 activity and poly (ADP-ribose) polymerase cleavage, along with cell death. Transfection of HIF-1α siRNA protected DC from the effects of hypoxia. Of interest, when hypoxic DC were maturated with lipopolysaccharide (LPS), we did not observe an increased cell death, while HIF-1α accumulation and BNIP3 expression were still significantly upregulated. In contrast with immature DC, mature DC expressed higher levels of Bcl-2, and, more importantly, of phosphorylated Akt. Transfection of HIF-1α siRNA to mature DC resulted in a significant upregulation of Akt phosphorylation as well. Moreover, inhibition of PI3K/Akt pathway resulted in an increased cell death of hypoxic mature DC. We may conclude that a prolonged exposure to hypoxia induces a cell death program which could be prevented by HIF-1α inhibition and/or LPS maturation. Our results may contribute to further understand the physiology of DC and the molecular mechanisms involved in the survival of DC, with important implications in the regulation of the immune response.
Assuntos
Células Dendríticas/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia , Lipopolissacarídeos/toxicidade , Oxigênio/metabolismo , Morte Celular , Sobrevivência Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
The potential therapeutic value and versatility of platelet-derived products has recently stimulated the research and interest in the field of regenerative medicine. Platelet gels (PG), generated by thrombin-activated platelets, represent a new biotechnology for stimulation and acceleration of tissue healing and regeneration. However, despite the diffused and successful use of PG in clinical practice, a more detailed knowledge of the cellular and molecular mechanisms involved is required. In the present study, we show that human peripheral blood mononuclear cells (PBMC) co-cultured with PG, in the presence of the inflammatory activator lipopolysaccharide, secreted higher amounts of pro-inflammatory and pro-angiogenic cytokines, such as interleukin (IL)-1beta, IL-6 and IL-8. In contrast, the release of the anti-angiogenic cytokines interferon-gamma and IL-12 was significantly reduced. In addition the production of the anti-inflammatory cytokine IL-10 was not affected by PG. Finally, hypoxia, a common feature of the healing tissue, potentiated the effects exerted by PG on the release of IL-1beta by PBMC. In conclusion, PG treatment reveals a unique capacity of articulating a pro-inflammatory and pro-angiogenic cytokine profile in human PBMC, which may partially explain the clinical success of PG application in a wide range of diseases.
Assuntos
Terapia Biológica/métodos , Plaquetas , Citocinas/metabolismo , Leucócitos Mononucleares/imunologia , Técnicas de Cocultura , Humanos , Hipóxia , Inflamação , Lipopolissacarídeos/farmacologia , Neovascularização Fisiológica , Regeneração , CicatrizaçãoRESUMO
Thrombin, the key enzyme of the coagulation cascade, is involved in inflammation. It was proposed recently that thrombin activity may play an important role in allergic inflammation. Interferon-gamma (IFN-gamma) is a potent Th1-related cytokine secreted by activated T cells and is usually downregulated in allergic inflammation. We recently demonstrated that thrombin enhances interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC). Thus, we hypothesized that thrombin may promote a Th2 profile. We here report that human alpha- thrombin downregulates IFN-gamma expression at both protein and mRNA levels in activated PBMCs. The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, shows that this downregulation is thrombin specific and requires thrombin proteolytic activity. The addition of an anti- IL-10 monoclonal antibody (mAb) to thrombin-treated PBMCs abolishes IFN-gamma downregulation, suggesting that thrombin exerts its effect through IL-10, a Th2-related cytokine. Furthermore, IFN-gamma reduction was accompanied by increased IL-4 release, as well as by an increase in the proinflammatory cytokine IL-1. In conclusion, the observation that thrombin affects the production of IFN-gamma (Th1 profile) and IL-4 (Th2 profile) provides further evidence for the role played by thrombin in modulating Th1/Th2 cytokine balance, which could be particularly relevant in allergic inflammation.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Hemostáticos/farmacologia , Interferon gama/biossíntese , Células Th2/imunologia , Trombina/farmacologia , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Regulação para Baixo/imunologia , Hemostáticos/imunologia , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/imunologia , Peptídeos/imunologia , Peptídeos/farmacologia , Receptores de Trombina/antagonistas & inibidores , Receptores de Trombina/imunologia , Células Th2/metabolismo , Trombina/imunologiaRESUMO
BACKGROUND: Huntington's disease is a neurodegenerative disorder characterized by transcriptional alterations both in central and peripheral tissues. Therefore, the identification of a transcriptional signature in an accessible tissue can meaningfully complement current efforts in clinical biomarker development. Gene expression normalization represents an essential step in transcriptional signatures identification, and since many reference genes show altered expressions in several pathologies, the definition of stable genes in the desired tissue is required to allow correct result interpretations. OBJECTIVE: The present work aimed at identifying a set of suitable reference genes for expression normalization in blood of HD patients and R6/2 mice. METHODS: By crossing literature investigation and analysis of microarrays performed on blood of HD patients and healthy subjects, a set of genes was selected and tested by RT-qPCR. Employment of statistical algorithms allowed the identification of the most stable genes in human samples that were than confirmed in R6/2. RESULTS: PPIB, PGK1, ACTB and YWHAZ represent the best possible genes combination, useful to normalize blood transcriptional analysis. To link clinical and preclinical studies, the identified genes were investigated also in blood of R6/2 and wild type mice, confirming that Ppib, Actb and Ywhaz were appropriate for expression normalization. Selected references were subsequently applied to evaluate expression of genes known to be involved in Huntington's pathological progression. CONCLUSIONS: This work highlights the importance for correct data normalization to avoid misinterpretation of results, while providing a suitable method to support quantitative gene expression analysis in preclinical and clinical investigations.
Assuntos
Perfilação da Expressão Gênica/métodos , Expressão Gênica/genética , Doença de Huntington/genética , Animais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Blood coagulation cascades can be activated by different mechanisms and to different levels in cancer patients. In a study conducted on the transcriptional profile of epithelial ovarian cancer patients a number of possible links between coagulation and inflammation have been suggested and we and others have reported that, in addition to its central role in blood coagulation and haemostasis, thrombin is a powerful regulator of inflammatory responses. Here, we report that thrombin- like activities were present in the malignant ascites of patients with ovarian carcinoma. Malignant ascites significantly enhanced the release of cytokines/chemokines, which have been previously shown to support tumour progression, such as interleukin (IL)-6, IL-1ß, CCL2 and CXCL8, in human peripheral blood mononuclear cells of healthy volunteers. Interestingly, ascites enhanced the release of the anti-inflammatory cytokine IL-10 and inhibited the production of interferon-γ and IL-12. The presence of the anticoagulant antithrombin reversed IL-12 inhibition induced by ascites in human monocytes. Finally, the use of thrombin and of the specific thrombin receptor (PAR) agonist peptides, TFLLRN and AYGPK, suggests that IL-12 inhibition is thrombin-specific and related to PAR-1, but not to PAR-4. These findings underline the tight relationship between the coagulation pathway, where thrombin is the key enzyme, and cytokine modulation, including IL-12 inhibition, which is a critical feature of the tumour microenvironment, and may represent a powerful strategy used by tumours to escape immune surveillance.
Assuntos
Líquido Ascítico/metabolismo , Carcinoma/imunologia , Citocinas/metabolismo , Leucócitos Mononucleares/metabolismo , Neoplasias Ovarianas/imunologia , Trombina/metabolismo , Ascite , Líquido Ascítico/imunologia , Coagulação Sanguínea/imunologia , Carcinoma/patologia , Carcinoma/fisiopatologia , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Imunomodulação , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Oligopeptídeos/farmacologia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/fisiopatologia , Comunicação Parácrina , Receptor PAR-1/agonistas , Trombina/imunologia , Evasão Tumoral , Microambiente TumoralRESUMO
Immune cells play an important role in the onset of angiogenesis. Here, we report that VEGF represents the major proangiogenic factor expressed by T cells exposed to hypoxia, a common feature of inflammation and tumor microenvironment. The supernatants of hypoxic T cells were highly angiogenic when delivered on the chick embryo CAM. The angiogenic response was abrogated by a neutralizing anti-VEGF antibody and mimicked by rVEGF. Interestingly, VEGF induction by hypoxia was up-regulated in Jurkat T cells overexpressing the adaptor protein p66Shc but not the inactive S36 p66Shc mutant, and it was abolished in p66Shc-/- mouse splenocytes. Accordingly, the angiogenic response induced by the supernatants from hypoxic p66Shc-/- splenocytes was reduced dramatically when compared with the wild-type controls. In conclusion, hypoxic T cells may contribute to the onset of angiogenesis through a novel VEGF-mediated mechanism, where p66Shc acts as a positive regulator.
Assuntos
Neovascularização Fisiológica , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Animais , Hipóxia Celular , Embrião de Galinha , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Jurkat , Leucócitos Mononucleares/metabolismo , Camundongos , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Inflammation plays an important role in the onset of angiogenesis. In the present study, we show that osteopontin (OPN), a proinflammatory mediator involved in tissue repair, induces IL-1beta up-regulation in human monocytes. This was accompanied by the enhanced production of TNF-alpha, IL-8, and IL-6, a decreased release of IL-10, and increased p38 phosphorylation. The supernatants of OPN-treated monocytes were highly angiogenic when delivered on the chick embryo chorioallantoic membrane. The angiogenic response was completely abrogated by a neutralizing anti-IL-1 Ab, thus indicating that this cytokine represents the major proangiogenic factor expressed by OPN-activated monocytes. Accordingly, rIL-1beta mimicked the proangiogenic activity of OPN-treated monocyte supernatants, and IL-1R (type I) was found to be expressed in the chorioallantoic membrane. In conclusion, OPN-activated monocytes may contribute to the onset of angiogenesis through a mechanism mediated by IL-1beta.