RESUMEN
INTRODUCTION: The formation of neutrophil extracellular traps (NETs) is a process in which several kinds of enzymes participate generating posttranslational modifications of proteins. NETs have been associated with infectious, autoimmune, and inflammatory diseases. Inhibition of several proteases reduces the formation of NETs. In the present work, we analyzed the role of several broad-acting and specific inhibitors of proteases in the formation of NETs. METHODS: Neutrophils were isolated from peripheral blood of healthy individuals by density gradient. The neutrophils were quantified and seeded into cell culture plates. Phorbol myristate acetate and A23187 were used as NETs inducers, and several specific inhibitors of proteases were used. The cells were stained for cytoskeleton or DNA. The cell-free supernatants were used to assess DNA release. Statistical analysis was carried out by a Kruskal-Wallis or ANOVA test. RESULTS: We observed marked changes in actin organization after the induction of NETs, suggesting that the cytoskeleton is being actively regulated. When we used protease inhibitors, the release of DNA was reduced, suggesting the participation of actin remodeling in the process. Further characterization of the specific proteases revealed that calpain modulates the reorganization of actin cytoskeleton and DNA release. Preservation of part of the actin cytoskeleton suggests that DNA release is not only a mechanic process associated to the chromatin decondensation; rather the process is highly regulated by active proteases that promote cytoskeleton reorganization and chromatin decondensation that culminates in DNA release. CONCLUSION: Calpain mediates the DNA release in the NET formation process by the modification of cortical actin cytoskeleton in a calcium-dependent manner.
Asunto(s)
Calpaína/metabolismo , Citoesqueleto/metabolismo , ADN/metabolismo , Trampas Extracelulares/inmunología , Neutrófilos/metabolismo , Actinas/metabolismo , Calcio/metabolismo , Células Cultivadas , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Inhibidores de Proteasas/farmacologíaRESUMEN
The first degree relatives of rheumatoid arthritis (RA) patients have a higher risk of developing RA, which is related to the expression of autoantibodies against citrullinated proteins (ACPA). Remarkably, prior to the onset of RA, cartilage damage is already initiated, whereas ACPA autoantibodies are already expressed. Here we show that both TNF-α and IL-6 are also increased prior to the onset of RA. Furthermore, when the levels of DKK1 and Sclerostin were evaluated in first degree relatives of RA patients, we found that the serum levels of TNF- α correlate with the expression levels of both DKK1 and Sclerostin. Interestingly, when the disease is already established, the correlation of TNF- α with DKK1 is lost in RA patients, whereas the correlation of Sclerostin with both TNF- α and IL-6 is further increased. Our data suggest a subclinical inflammation in patients at high risk of developing RA, which might lead to an increase in the levels of both DKK1 and Sclerostin, contributing to joint damage in the preclinical phase of the disease linked to the expression of ACPA autoantibodies.
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Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Enfermedades Asintomáticas , Cartílago Articular/inmunología , Cartílago Articular/patología , Familia , Proteínas Adaptadoras Transductoras de Señales/sangre , Adulto , Anticuerpos Antiproteína Citrulinada/sangre , Artritis Reumatoide/sangre , Artritis Reumatoide/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intercelular/sangre , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/sangreRESUMEN
INTRODUCTION: AIM2 inflammasome activation leads to the release of IL-ß, which plays an important role in rheumatoid arthritis pathogenesis. In this work, we evaluated AIM2 expression and activity in RA patients and healthy controls. METHODS: AIM2 and RANKL expression were evaluated by flow cytometry. Inflammasome activity was determined in monocyte cultures stimulated with synthetic DNA by measuring IL-1ß levels in supernatants using an ELISA assay. The caspase-1 expression in monocytes was measured by western blot, the POP3 expression was analysed by qPCR, and serum levels of IFN-γ were evaluated using ELISA assay. RESULTS: We observed a diminution of CD14+AIM2+ cells in RA patients, associated with disease activity and evolution. Likewise, the levels of IL-1ß were increased in monocyte cultures un-stimulated and stimulated with LPS from RA patients with DAS28 ≥ 4. The Caspase-1 activity and RANKL + monocytes in RA patients were slightly increased. Finally, augmented POP3 expression and diminished IFN-γ serum levels were detected in RA patients. CONCLUSION: Our results showed that the monocytes from RA patients were prone to release IL-1ß in the absence of the AIM2 inflammasome signal. The down-regulation of AIM2 to a systemic level in RA patients might be a consequence of augmented POP3 expression and might imply the survival of pro-inflammatory cells contributing to the inflammation process.
Asunto(s)
Artritis Reumatoide/metabolismo , Proteínas de Unión al ADN/metabolismo , Inflamasomas/metabolismo , Adulto , Caspasa 1/metabolismo , Células Cultivadas , Femenino , Humanos , Inflamación/metabolismo , Interferón gamma/metabolismo , Interleucina-1beta/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Masculino , Monocitos/metabolismo , Homóloga LST8 de la Proteína Asociada al mTOR/metabolismoRESUMEN
Dopaminergic neurons have the ability to release Dopamine from their axons as well as from their soma and dendrites. This somatodendritically-released Dopamine induces an autoinhibition of Dopaminergic neurons mediated by D2 autoreceptors, and the stimulation of neighbor GABAergic neurons mediated by D1 receptors (D1r). Here, our results suggest that the somatodendritic release of Dopamine in the substantia nigra (SN) may stimulate GABAergic neurons that project their axons into the hippocampus. Using semiquantitative multiplex RT-PCR we show that chronic blockade of the Dopaminergic neurotransmission with both AMPT and reserpine specifically decreases the expression levels of D1r, remarkably this may be the result of an antagonistic effect between AMPT and reserpine, as they induced the expression of a different set of genes when treated by separate. Furthermore, using anterograde and retrograde tracing techniques, we found that the GABAergic neurons that express D1r also project their axons in to the CA1 region of the hippocampus. Finally, we also found that the same treatment that decreases the expression levels of D1r in SN, also induces an impairment in the performance in an appetitive learning task that requires the coding of reward as well as navigational skills. Overall, our findings show the presence of a GABAergic interconnection between the SNr and the hippocampus mediated by D1r.
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Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Receptores de Dopamina D1/biosíntesis , Reserpina/farmacología , Sustancia Negra/metabolismo , alfa-Metiltirosina/farmacología , Inhibidores de Captación Adrenérgica/farmacología , Animales , Antagonistas de los Receptores de Dopamina D2/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/biosíntesis , Neuronas Dopaminérgicas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Expresión Génica , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos BALB C , Fenotipo , Receptores de Dopamina D1/antagonistas & inhibidores , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/biosíntesis , Receptores de Dopamina D2/genética , Sustancia Negra/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptors have been shown to modulate the morphology of the lamelar processes of Bergmann glia cells in the molecular layer of the cerebellum. Here we suggest that reorganization of F-actin may underlay the changes in the morphology of the lamelar processes. Using the fluorescent staining of F-actin with Phalloidin and the quantification of RhoA activation through immunoprecipitation or pull-down assays, we show that RhoA is activated after stimulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors and leads to the reorganization of the actin cytoskeleton of Bergmann fibers. This reorganization of the actin cytoskeleton is reflected in the form of an increase in the intensity of the F-actin staining as well as in the loss of the number of Bergmann fibers stained with Phalloidin. Moreover, using a pharmacological approach, we show that activation of RhoA and the change in the intensity of the F-actin staining depends on the activation of PI3-K, focal adhesion kinase, and protein kinase C, whereas changes in the number of Bergmann fibers depend on external calcium in a RhoA independent manner. Our findings show that glutamate may induce a form of structural plasticity in Bergmann glia cells through the reorganization of the actin cytoskeleton. This may have implications in the way the synaptic transmission is processed in the cerebellum.
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Actinas/metabolismo , Ácido Glutámico/metabolismo , Neuroglía/metabolismo , Receptores AMPA/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Cerebelo/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Transducción de Señal/fisiologíaRESUMEN
The physiopathology of rheumatoid arthritis (RA) is mediated by proinflammatory cytokines, some of which are regulated by the JAK/STAT pathway. Tofacitinib is a JAK inhibitor, but its role in the regulation of microRNAs (miRNAs) is unknown. There is also no information regarding the role of miRNAs in the clinical relapse/remission of RA. The present project aims to identify a signature profile of miRNA expression in a subgroup of RA patients who had to discontinue tofacitinib treatment (because of the ending of a 5-year open-label clinical trial) and to describe the expression of miRNAs during RA remission or flare-up. The relative expression of 61 miRNAs was determined in serum samples with the Firefly™ BioWorks assay. Statistical analysis was performed by means of Student's t-test and heatmap analysis was performed with Firefly™ Analysis Workbench software and in the software GraphPad® Prism v5.0. Target prediction and Gene Ontology analysis were carried out using bioinformatic tools. We found a distinctive signature of miRNA expression associated with relapse, featuring upregulated expression of hsamiR4325p (pâ¯<â¯0.05). We also found upregulation of hsamiR1945p (pâ¯<â¯0.05) in samples of patients with RA flare-up. Gene Ontology analysis of the target genes for hsamiR4325p was performed to identify relevant pathways associated with relapse; the implications of these pathways in the physiopathology of RA are discussed. Tofacitinib treatment does not have a direct effect on the expression of measured miRNAs. The changes in hsamiR4325p and hsamiR1945p are associated with the regulation of proinflammatory pathways and RA flare-up.
Asunto(s)
Antirreumáticos/farmacología , Artritis Reumatoide/genética , MicroARNs/sangre , Piperidinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Adulto , Antirreumáticos/uso terapéutico , Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Piperidinas/uso terapéutico , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , RecurrenciaRESUMEN
A role for sperm-specific proteins during the early embryonic development has been suggested by a number of recent studies. However, little is known about the participation of transcription factors in that stage. Here, we show that the signal transducer and activator of transcription 1 (Stat1), but not Stat4, was phosphorylated in response to capacitation and the acrosomal reaction (AR). Moreover, Stat1 phosphorylation correlated with changes in its localization: during capacitation, Stat1 moved from the cytoplasm to the theca/flagellum fraction. During AR, Stat1 phosphorylation increased again. In addition, blocking protein kinase A (PKA) and PKC during capacitation abolished both phosphorylation and migration of Stat1. Our results show tight spatio-temporal rearrangements of Stat1, suggesting that after fertilization Stat1 participates in the first rounds of transcription within the male pronucleus.