RESUMEN
Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover, Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands, such as HIV and CpG respectively, turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions, and pDC production of IFN-α as well as cell-cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection, but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here, we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α, TNF-α, IFN-γ and IL-12, and CCR5-interacting chemokines (MIP-1α and MIP-1ß) in NK-pDCs co-cultures. At high HIV-1BaL concentrations, the addition of NK cells did not promote the release of these mediators, suggesting that once efficiently triggered by the virus, pDCs could not integrate new activating signals delivered by NK cells. However, high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly, we identified the alarmin HMGB1, released at pDC-NK cell synapse, as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover, HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1, HMGB1-specific antibodies, sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether, these findings identify HMGB1 as a trigger for IFN-α-mediated TRAIL expression at the surface of pDCs and NK cells, and they suggest a novel mechanism of innate control of HIV-1 infection.
Asunto(s)
Células Dendríticas/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Proteína HMGB1/inmunología , Interferón-alfa/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Adulto , Membrana Celular/metabolismo , Quimiocinas/inmunología , Quimiocinas/metabolismo , Estudios de Cohortes , Citocinas/inmunología , Citocinas/metabolismo , Citoplasma/metabolismo , Células Dendríticas/virología , Infecciones por VIH/tratamiento farmacológico , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Inmunidad Innata , Interferón-alfa/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Persona de Mediana Edad , Transporte de Proteínas , Linfocitos T/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Adulto JovenAsunto(s)
Transmisión de Enfermedad Infecciosa/prevención & control , Infecciones por VIH/epidemiología , Infecciones por VIH/prevención & control , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/prevención & control , Infecciones por VIH/transmisión , Fiebre Hemorrágica Ebola/transmisión , HumanosAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/epidemiología , Síndrome de Inmunodeficiencia Adquirida/prevención & control , Antirretrovirales/uso terapéutico , Terapia Antirretroviral Altamente Activa/métodos , Control de Enfermedades Transmisibles/métodos , Transmisión de Enfermedad Infecciosa/prevención & control , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Femenino , Salud Global , Política de Salud , Humanos , MasculinoRESUMEN
Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches.
Asunto(s)
Adenosina Trifosfato/metabolismo , Membrana Celular/metabolismo , Infecciones por VIH/metabolismo , VIH-1/fisiología , Mutación , Receptores Purinérgicos P2Y2/metabolismo , Adenosina Trifosfato/genética , Adulto , Terapia Antirretroviral Altamente Activa/métodos , Membrana Celular/genética , Conexinas/genética , Conexinas/metabolismo , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Quinasa 2 de Adhesión Focal/genética , Quinasa 2 de Adhesión Focal/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Humanos , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores Purinérgicos P2Y2/genética , Transducción de Señal , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
PURPOSE OF REVIEW: Natural killer (NK) cells promote antiviral immunity by producing proinflammatory cytokines and by lysing infected cells. In addition, NK cells can modulate dendritic cell functions. NK-dendritic cell crosstalk results in activation of both cell types, with dendritic cells promoting NK-cell activity and NK cells inducing further maturation of dendritic cells. Here we review the recent evidence suggesting that NK-dendritic cell crosstalk is disrupted during HIV-1 infection and we discuss the consequences on HIV persistence in dendritic cells. RECENT FINDINGS: NK cell-mediated dendritic cell editing is compromised during HIV-1 infection, and NK cells from viremic individuals show a decreased ability to kill immature dendritic cells. The defect is associated with impaired NKp30 function. Moreover, the resistance of HIV-1-infected dendritic cells to NK-mediated lysis is associated with the upregulation of apoptosis inhibitors, thus protecting infected dendritic cells from TRAIL-dependent apoptosis. These inhibitors are upregulated by the high-mobility group box 1 protein (HMGB1), an alarmin produced at NK-dendritic cell synapse that is essential for NK-dependent dendritic cell maturation, but also promotes viral replication in infected dendritic cells. SUMMARY: HIV-1-induced impairment of NK-dendritic cell crosstalk may significantly alter both innate and adaptive immunity. It may also contribute to HIV persistence in dendritic cells through an HMGB1-dependent mechanism.
Asunto(s)
Células Dendríticas/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , VIH-1/patogenicidad , Proteína HMGB1/metabolismo , Células Asesinas Naturales/inmunología , Inmunidad Adaptativa , Infecciones por VIH/virología , Proteína HMGB1/inmunología , Humanos , Evasión Inmune , Inmunidad InnataRESUMEN
BACKGROUND: Programmed cell death has been traditionally related with caspase activation. However, it is now accepted that caspase-independent forms of programmed cell death also regulate cell death. In chronic lymphocytic leukemia, CD47 ligation induces one of these alternative forms of cell death: type III programmed cell death. This poorly understood process is characterized by cytoplasmic hallmarks, such as mitochondrial damage. To gain insights into the molecular pathways regulating type III programmed cell death in chronic lymphocytic leukemia, we performed extensive biochemical and cell biology assessments. DESIGN AND METHODS: After CD47 triggering, purified B-cells from 20 patients with chronic lymphocytic leukemia were studied by flow cytometry, immunofluorescence and three-dimensional imaging, immunoblotting, electron microscopy, and fibrillar/globular actin measurements. Finally, we subjected CD47-treated chronic lymphocytic leukemia cells to a phagocytosis assay. RESULTS: We first confirmed that induction of type III programmed cell death is an efficient means of triggering cell death in chronic lymphocytic leukemia. Further, we demonstrated that the signaling events induced by CD47 ligation provoked a reduction in cell size. This alteration is related to F-actin disruption, as the two other cytoskeleton networks, microtubules and intermediate filaments, remain undisturbed in type III programmed cell death. Strikingly, we revealed that the pharmacological modulation of F-actin dynamics regulated this type of death. Finally, our data delineated a new programmed cell death pathway in chronic lymphocytic leukemia initiated by CD47 triggering, and followed by serine protease activation, F-actin rearrangement, mitochondrial damage, phosphatidylserine exposure, and cell clearance. CONCLUSIONS: Our work reveals a key molecular tool in the modulation of cell death in chronic lymphocytic leukemia: F-actin. By assessing the regulation of F-actin and type III programmed cell death, this analysis provides new options for destroying chronic lymphocytic leukemia cells, such as a combination of therapies based on apoptosis regulators (e.g., caspases, Bcl-2, Bax) along with alternative therapies based on type III death effectors (e.g., F-actin).
Asunto(s)
Actinas/metabolismo , Apoptosis/inmunología , Citoesqueleto/patología , Leucemia Linfocítica Crónica de Células B/patología , Linfocitos B/patología , Caspasas , Citoesqueleto/inmunología , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , Células Tumorales CultivadasRESUMEN
Ligation of CD47 triggers caspase-independent programmed cell death (PCD) in normal and leukemic cells. Here, we characterize the morphological and biochemical features of this type of death and show that it displays the hallmarks of type III PCD. A molecular and biochemical approach has led us to identify a key mediator of this type of death, dynamin-related protein 1 (Drp1). CD47 ligation induces Drp1 translocation from cytosol to mitochondria, a process controlled by chymotrypsin-like serine proteases. Once in mitochondria, Drp1 provokes an impairment of the mitochondrial electron transport chain, which results in dissipation of mitochondrial transmembrane potential, reactive oxygen species generation, and a drop in ATP levels. Surprisingly, neither the activation of the most representative proapoptotic members of the Bcl-2 family, such as Bax or Bak, nor the release of apoptogenic proteins AIF (apoptosis-inducing factor), cytochrome c, endonuclease G (EndoG), Omi/HtrA2, or Smac/DIABLO from mitochondria to cytosol is observed. Responsiveness of cells to CD47 ligation increases following Drp1 overexpression, while Drp1 downregulation confers resistance to CD47-mediated death. Importantly, in B-cell chronic lymphocytic leukemia cells, mRNA levels of Drp1 strongly correlate with death sensitivity. Thus, this previously unknown mechanism controlling caspase-independent type III PCD may provide the basis for novel therapeutic approaches to overcome apoptotic avoidance in malignant cells.
Asunto(s)
Caspasas/metabolismo , Muerte Celular/fisiología , GTP Fosfohidrolasas/metabolismo , Leucemia/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Antígeno CD47/genética , Antígeno CD47/metabolismo , Forma de la Célula , Células Cultivadas , Dinaminas , Transporte de Electrón/fisiología , GTP Fosfohidrolasas/genética , Humanos , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Serina Endopeptidasas/metabolismo , Linfocitos T/citología , Linfocitos T/fisiologíaRESUMEN
PURPOSE: Gamma-irradiation leads to activation of p53 tumour suppressor gene and to p53-dependant stimulation of a large panel of cellular genes including proapoptotic genes involved in intrinsic and extrinsic pathways. Most in vivo published data referred to high (lethal) irradiation doses. The present study was performed to analyse the p53-dependent response to more relevant low irradiation doses. MATERIALS AND METHODS: Mice were whole body exposed to irradiation doses decreasing from 5 - 0.05 Gy. Gene expression was estimated by real time reverse transcriptase polymerase chain reaction measurements on RNA extracted from thymus and spleen. Apoptosis was evaluated by the percentage of either annexin V positive or sub-G1 cells. RESULTS: A 0.1 Gy irradiation dose already gives a significant stimulation of Puma (p53 up-regulated modulator of apoptosis), and 0.2 Gy of Bax (Bcl-2-associated X protein) and Killer/DR5 (Death Receptor 5). The expression of genes involved in the two apoptotic pathways was induced as soon as 1 h post-irradiation and reached a maximum at 3 h, the induction level depending on both the gene and the organ. A significant increase in the number of apoptotic cells is already detectable at 0.5 Gy with a maximum of induction at 6 h. CONCLUSIONS: Our results reveal the high in vivo sensitivity of p53-dependent transcriptional activation of genes involved in the two main apoptotic pathways, their stimulation preceding the induction of apoptosis.
Asunto(s)
Apoptosis/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Bazo/efectos de la radiación , Timo/efectos de la radiación , Proteína p53 Supresora de Tumor/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Mitocondrias/fisiología , ARN Mensajero/análisis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Bazo/metabolismo , Bazo/patología , Timo/metabolismo , Timo/patología , Proteínas Supresoras de Tumor/genética , Irradiación Corporal Total , Proteína bcl-X/genéticaRESUMEN
Pretende-se traçar um panorama sobre a Bioética, campo de conhecimento assim chamado desde 1970, por Potter. E também assinalar suas diferenças em relação à Ética Profissional e ao Direito. O crescimento vigoroso da bioética, suas correntes filosóficas, escolas e ferramentas são descritas de modo a introduzir o leitor não familiarizado com esta disciplina e despertar o interesse pelas discussões suscitadas pelos avanços tecnológicos, pelo incremento de pesquisas clínicas e pelo respeito aos direitos humanos tão arduamente conseguidos após séculos de submissão e desrespeito aos indivíduos. Apresenta-se, ainda, a relação existenteentre Direitos Humanos, Bioética, Saúde Pública e Cultura de Paz.
Asunto(s)
Bioética , Derechos Humanos , Salud Pública , Calidad de VidaRESUMEN
Apoptosis-inducing factor (AIF) is a bifunctional NADH oxidase involved in mitochondrial respiration and caspase-independent apoptosis. Three alternatively spliced mRNA isoforms of AIF have been identified previously: AIF, AIF-exB, and AIFsh. Here, we report the cloning and the biochemical characterization of a new isoform named AIF short 2 (AIFsh2). AIFsh2 transcript includes a previously unknown exon placed between exons 9 and 10 of AIF. The resulting AIFsh2 protein, which localizes in mitochondria, corresponds to the oxidoreductase domain of AIF. In this way, AIFsh2 exhibits similar NADH oxidase activity to AIF and generates reactive oxygen species. Like AIF, AIFsh2 is released from mitochondria to cytosol after an apoptotic insult in a calpain or cathepsin-dependent manner. However, in contrast to AIF, AIFsh2 does not induce nuclear apoptosis. Thus, it seems that the reactive oxygen species produced by the oxidoreductase domain of AIF/AIFsh2 are not important for AIF-dependent nuclear apoptosis. In addition, we demonstrate that the AIFsh2 mRNA is absent in normal brain tissue, whereas it is expressed in neuroblastoma-derived cells, suggesting a different regulation in normal and transformed cells from the brain lineage. Together, our results reveal that AIF yields an original and independent genetic regulation of the two AIF functions. This is an important issue to understand the physiological role of this protein.
Asunto(s)
Factor Inductor de la Apoptosis/genética , Exones/genética , Mitocondrias/enzimología , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Apoptosis , Factor Inductor de la Apoptosis/metabolismo , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Mitocondrias Hepáticas/enzimología , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , NADH NADPH Oxidorreductasas/genética , Especificidad de Órganos , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Alineación de Secuencia , Transcripción GenéticaRESUMEN
IGF-II and type I-IGF receptor (IGF-IR) gene expression is increased in primary liver tumors, and transgenic mice overexpressing IGF-II in the liver develop hepatocellular carcinoma (HCC) spontaneously, suggesting that alterations of IGF-IR signaling in vivo may play a role in the auto/paracrine control of hepatocarcinogenesis. We have addressed the contribution of PI-3'K/Akt signaling on the proliferation of HepG2 human hepatoma cells and on their protection against doxorubicin-induced apoptosis. Both basal HepG2 cell DNA replication and that stimulated by IGF-IR signaling were inhibited by the specific PI-3'K inhibitor Ly294002 (Ly). In the former case, PI-3'K signaling overcame cell cycle arrest in G1 via increased cyclin D1 protein and decreased p27kip1 gene expression. Doxorubicin treatment induced apoptosis in HepG2 cells and was concomitant with the proteolytic cleavage of Akt-1 and -2. Drug-induced apoptosis was reversed by IGF-I and this effect was (i) dependent on Akt-1 and -2 phosphorylation and (ii) accompanied by the inhibition of initiator caspase-9 activity, suggesting that IGF-IR signaling interferes with mitochondria-dependent apoptosis. Accordingly, Ly enhanced doxorubicin-induced apoptosis and suppressed its reversal by IGF-I. Altogether, the data emphasize the crucial role of PI-3'K/Akt signaling (i) in basal as well as IGF-IR-stimulated HepG2 cell proliferation and (ii) in controlling both doxorubicin-induced apoptosis (e.g., drug-induced cleavage of Akt) and its reversal by IGF-I (protection against apoptosis parallels the extent of Akt phosphorylation). They suggest that targeting Akt activity or downstream Akt effectors (e.g., GSK3-beta, FOXO transcription factors) may help define novel therapeutic strategies of increased efficacy in the treatment of HCC-bearing patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Doxorrubicina/farmacología , Fase G1 , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Receptor IGF Tipo 1RESUMEN
AIF is a main mediator of caspase-independent cell death. It is encoded by a single gene located on chromosome X, region q25-26 and A6 in humans and mice, respectively. Previous studies established that AIF codes for two isoforms of the protein, AIF and AIF-exB. Here, we identify a third AIF isoform resulting from an alternate transcriptional start site located at intron 9 of AIF. The resulting mRNA encodes a cytosolic protein that corresponds to the C-terminal domain of AIF (amino acids 353-613). We named this new isoform AIFshort (AIFsh). AIFsh overexpression in HeLa cells results in nuclear translocation and caspase-independent cell death. Once in the nucleus, AIFsh provokes the same effects than AIF, namely chromatin condensation and large scale (50 kb) DNA fragmentation. In contrast, these apoptogenic effects are not precluded by the AIF-inhibiting protein Hsp70. These findings identify AIFsh as a new pro-apoptotic isoform of AIF, and also reveal that the first N-terminal 352 amino acids of AIF are not required for its apoptotic activity. In addition, we demonstrate that AIFsh is strongly down-regulated in tumor cells derived from kidney, vulva, skin, thyroid, and pancreas, whereas, gamma-irradiation treatment provokes AIFsh up-regulation. Overall, our results identify a novel member of the AIF-dependent pathway and shed new light on the role of caspase-independent cell death in tumor formation/suppression.