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Osteoclasts (OCs) are bone-resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild-type (WT) mice, Ly6Chi and Ly6Cint monocytes are the primary source of OC formation when compared to Ly6C- monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of preestablished transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of interferon regulatory factor 8 (IRF8), a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8-deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis-regulatory elements that regulate OC differentiation. © 2020 American Society for Bone and Mineral Research (ASBMR).
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Monocitos , Osteogénesis , Animales , Diferenciación Celular , Epigénesis Genética , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Ratones , Monocitos/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Osteogénesis/genética , Ligando RANK/metabolismoRESUMEN
Background & objectives: The National AIDS Control Organisation (NACO) and the ICMR-National Institute of Medical Statistics, the nodal agency for conducting HIV estimations in India, have been generating HIV estimates regularly since 2003. The objective of this study was to describe India's biennial HIV estimation 2017 process, data inputs, tool, methodology and epidemiological assumptions used to generate the HIV estimates and trends of key indicators for 2010-2017 at national and State/Union Territory levels. Methods: Demographic Projection (DemProj) and AIDS Impact Modules (AIM) of Spectrum 5.63 software recommended by the United Nations Programme on HIV and AIDS Global Reference Group on HIV Estimates, Modelling and Projections, were used for generating HIV estimations on key indicators. HIV sentinel surveillance, epidemiological and programme data were entered into Estimation Projection Package (EPP), and curve fitting was done using EPP classic model. Finally, calibration was done using the State HIV prevalence of two rounds of National Family Health Survey (NFHS) -3 and -4 and Integrated Biological and Behavioural Surveillance (IBBS), 2014-2015. Results: The national adult prevalence of HIV was estimated to be 0.22 per cent in 2017. Mizoram, Manipur and Nagaland had the highest prevalence over one per cent. An estimated 2.1 million people were living with HIV in 2017, with Maharashtra estimated to have the highest number. Of the 88 thousand annual new HIV infections estimated nationally in 2017, Telangana accounted for the largest share. HIV incidence was found to be higher among key population groups, especially people who inject drugs. The annual AIDS-related deaths were estimated to be 69 thousand nationally. For all indicators, geographic variation in levels and trends between States existed. Interpretation & conclusions: With a slow decline in annual new HIV infections by only 27 per cent from 2010 to 2017 against the national target of 75 per cent by 2020, the national target to end AIDS by 2030 may be missed; although at the sub-national level some States have made better progress to reduce new HIV infection. It calls for reinforcement of HIV prevention, diagnosis and treatment efforts by geographical regions and population groups.
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Infecciones por VIH , Trabajadores Sexuales , Adulto , Femenino , Infecciones por VIH/diagnóstico , Infecciones por VIH/epidemiología , Infecciones por VIH/transmisión , Humanos , Incidencia , India/epidemiología , Transmisión Vertical de Enfermedad Infecciosa , Masculino , Embarazo , PrevalenciaRESUMEN
Mesenchymal stromal cells (MSCs) can potently regulate the functions of immune cells and are being investigated for the management of inflammatory diseases. Toll-like receptor 3 (TLR3)-stimulated human MSCs (hMSCs) exhibit increased migration and chemotaxis within and toward damaged tissues. However, the regulatory mechanisms underlying these migratory activities are unclear. Therefore, we analyzed the migration capability and gene expression profiles of TLR3-stimulated hMSCs using RNA-Seq, wound healing, and transwell cell migration assay. Along with increased cell migration, the TLR3 stimulation also increased the expression of cytokines, chemokines, and cell migration-related genes. The promoter regions of the latter showed an enrichment of putative motifs for binding the transcription factors forkhead box O1 (FOXO1), FOXO3, NF-κB (NF-κB1), and RELA proto-oncogene and NF-κB subunit. Of note, FOXO1 inhibition by the FOXO1-selective inhibitor AS1842856 significantly reduced both migration and the expression of migration-related genes. In summary, our results indicate that TLR3 stimulation induces hMSC migration through the expression of FOXO1-activated genes.
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Movimiento Celular , Proteína Forkhead Box O1/metabolismo , Regulación de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Receptor Toll-Like 3/metabolismo , Adulto , Femenino , Proteína Forkhead Box O1/antagonistas & inhibidores , Proteína Forkhead Box O1/genética , Humanos , Masculino , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Proto-Oncogenes Mas , Quinolonas/farmacología , Receptor Toll-Like 3/agonistas , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
Electronic structure of Pr2CoFeO6 (at 300 K) was investigated by x-ray photoemission spectroscopy (XPS) and x-ray absorption spectroscopy techniques. All three cations, i.e. Pr, Co and Fe were found to be trivalent in nature. XPS valance band analysis suggested the system to be insulating in nature. The analysis suggested that Co3+ ions exist in low spin state in the system. Moreover, Raman spectroscopy study indicated the random distribution of the B-site ions (Co/Fe) triggered by same charge states. In temperature-dependent Raman study, the relative heights of the two observed phonon modes exhibited anomalous behaviour near magnetic transition temperature T N ~ 270 K, thus indicating towards interplay between spin and phonon degrees of freedom in the system. Furthermore, clear anomalous softening was observed below T N which confirmed the existence of strong spin-phonon coupling occurring for at least two phonon modes of the system. The line width analysis of the phonon modes essentially ruled out the role of magnetostriction effect in the observed phonon anomaly. The investigation of the lattice parameter variation across T N (obtained from the temperature-dependent neutron diffraction measurements) further confirmed the existence of the spin-phonon coupling.
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Macrophages are the prime innate immune cells of the inflammatory response, and the combination of multiple signaling inputs derived from the recognition of host factors [e.g., interferon-g (IFN-γ)] and invading pathogen products (e.g., toll-like receptors (TLRs) agonists) are required to maintain essential macrophage function. The profound effects on biological outcomes of inflammation associated with IFN-γ pretreatment ("priming") and TLR4 ligand bacterial lipopolysaccharide (LPS)-induced macrophage activation (M1 or classical activation) have long been recognized, but the underlying mechanisms are not well defined. Therefore, we analyzed gene expression profiles of macrophages and identified genes, transcription factors (TFs), and transcription co-factors (TcoFs) that are uniquely or highly expressed in IFN-γ-mediated TLR4 ligand LPS-inducible versus only TLR4 ligand LPS-inducible primary macrophages. This macrophage gene expression has not been observed in macrophage cell lines. We also showed that interleukin (IL)-4 and IL-13 (M2 or alternative activation) elicited the induction of a distinct subset of genes related to M2 macrophage polarization. Importantly, this macrophage gene expression was also associated with promoter conservation. In particular, our approach revealed novel roles for the TFs and TcoFs in response to inflammation. We believe that the systematic approach presented herein is an important framework to better understand the transcriptional machinery of different macrophage subtypes.
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Células de la Médula Ósea/inmunología , Lipopolisacáridos/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Línea Celular , Perfilación de la Expresión Génica , Interferón gamma/inmunología , Interleucina-13/inmunología , Ratones , Ratones Endogámicos C57BL , Células RAW 264.7 , Receptor Toll-Like 4/inmunologíaRESUMEN
Double perovskite antimonates of the type BaLaMSbO6 (M=Mg, Ca) were synthesized by a standard solid-state route. The compounds were characterized by X-ray crystallography and the structures were refined using Rietveld method. BaLaMgSbO6 and BaLaCaSbO6 crystallized in monoclinic space groups (I2/m) and (P21 /n), respectively. In both compounds, La occupied the A-site of perovskite, which is 12-coordinated as compared to Ba2 LaSbO6 where La ion shifts to the B-site octahedral coordination due to the larger size of Ba as compared with Mg and Ca. The samples were further characterized using FTIR and the frequency of the octahedral vibration is correlated to the electronegativity of the B-site ions. Photoluminescence study of the title compounds and Ba2 LaSbO6 was carried out upon doping with 2â atom% Eu3+ ion, which confirmed that Eu3+ occupies distorted 12-coordinated A-site in BaLaMSbO6 (M=Mg, Ca) and symmetrical octahedral B-site in Ba2 LaSbO6 . Furthermore, the emission spectrum corresponding to each Eu3+ ion at different crystal site was successfully isolated through a TRES study. This site selective occupancy of Eu3+ ion also has a direct impact on the light emission, which was found to change from orange to red in a dark room in the order Ba2 LaSbO6 : EuâBaLaCaSbO6 : EuâBaLaMgSbO6 : Eu. Such an outcome will have high impact in designing new commercial Eu3+ ion doped phosphor materials and tailoring of their optical properties.
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Persistent microglial activation is associated with the production and secretion of various pro-inflammatory genes, cytokines and chemokines, which may initiate or amplify neurodegenerative diseases. A novel synthetic histone 3 lysine 27 (H3K27) demethylase JMJD3 inhibitor, GSK-J4, was proven to exert immunosuppressive activities in macrophages. However, a genome-wide search for GSK-J4 molecular targets has not been undertaken in microglia. To study the immuno-modulatory effects of GSK-J4 at the transcriptomic level, triplicate RNA sequencing and quantitative real-time PCR analyses were performed with resting, GSK-J4-, LPS- and LPS + GSK-J4-challenged primary microglial (PM) and BV-2 microglial cells. Among the annotated genes, the transcriptional sequencing of microglia that were treated with GSK-J4 revealed a selective effect on LPS-induced gene expression, in which the induction of cytokines/chemokines, interferon-stimulated genes, and prominent transcription factors TFs, as well as previously unidentified genes that are important in inflammation was suppressed. Furthermore, we showed that GSK-J4 controls are important inflammatory gene targets by modulating STAT1, IRF7, and H3K27me3 levels at their promoter sites. These unprecedented results demonstrate that the histone demethylase inhibitor GSK-J4 could have therapeutic applications for neuroinflammatory diseases.
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Benzazepinas/metabolismo , Inhibidores Enzimáticos/metabolismo , Inflamación/patología , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Microglía/efectos de los fármacos , Pirimidinas/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Perfilación de la Expresión Génica , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARNRESUMEN
Although microglial cells have an essential role in the host defense of the brain, the abnormal activation of microglia can lead to devastating outcomes, such as neuroinflammation and neurodegeneration. Emerging evidence indicates that FTY720 (fingolimod), an FDA-approved drug, has beneficial effects on brain cells in the central nervous system (CNS) and, more recently, immunosuppressive activities in microglia via modulation of the sphingosine 1 phosphate (S1P) 1 receptor. However, the exact molecular aspects of FTY720 contribution in microglia remain largely unaddressed. To understand the molecular mechanisms underlying the roles of FTY720 in microglia, we performed gene expression profiling in resting, FTY720, LPS and LPS + FTY720 challenged primary microglial (PM) cells isolated from 3-day-old ICR mice, and we identified FTY720 target genes and co-regulated modules that were critical in inflammation. By examining RNA sequencing and binding motif datasets from FTY720 suppressed LPS-induced inflammatory mediators, we also identified unexpected relationships between the inducible transcription factors (TFs), motif strength, and the transcription of key inflammatory mediators. Furthermore, we showed that FTY720 controls important inflammatory genes targets by modulating STAT1 and IRF8 levels at their promoter site. Our unprecedented findings demonstrate that FTY720 could be a useful therapeutic application for neuroinflammatory diseases associated with microglia activation, as well as provide a rich resource and framework for future analyses of FTY720 effects on microglia interaction.
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Clorhidrato de Fingolimod/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Inmunosupresores/farmacología , Microglía/efectos de los fármacos , Factores de Transcripción/genética , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos ICR , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismoRESUMEN
Multiple studies have documented that Enhancer of zeste homolog 2 (EZH2) could play a role in inflammation and a wide range of malignancies; however, the underlying mechanisms remain largely unaddressed. Microglial activation is a key process in the production and release of numerous pro-inflammatory mediators that play important roles in inflammation and neurodegeneration in the central nervous system (CNS). Therefore, our aim was to investigate whether inhibition of EZH2 with the selective small molecule inhibitor EPZ-6438 protects against neonatal microglial activation. First, in mouse primary microglial cells and a microglial cell line, we found that LPS can rapidly increase EZH2 mRNA level and we subsequently performed gene expression profiling and constructed networks in resting, EPZ-6438-treated, LPS-treated and LPS+EPZ-6438-treated primary microglial cells and a microglial cell line using transcriptome RNA sequencing and bioinformatics analyses. By examining the RNA sequencing, we identified EPZ-6438 target genes and co-regulated modules that were critical for inflammation. We also identified unexpected relationships between the inducible transcription factors (TFs), motif strength, and the transcription of key inflammatory mediators. Furthermore, we showed that EPZ-6438 controls important inflammatory gene targets by modulating interferon regulatory factor (IRF) 1, IRF8, and signal transducer and activator of transcription (STAT) 1 levels at their promoter sites. Our unprecedented findings demonstrate that pharmacological interventions built upon EZH2 inhibition by EPZ-6438 could be a useful therapeutic approach for the treatment of neuroinflammatory diseases associated with microglial activation.
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Benzamidas/farmacología , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/biosíntesis , Mediadores de Inflamación/antagonistas & inhibidores , Microglía/efectos de los fármacos , Piridonas/farmacología , Animales , Compuestos de Bifenilo , Línea Celular , Células Cultivadas , Proteína Potenciadora del Homólogo Zeste 2/genética , Expresión Génica , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/fisiología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos ICR , Microglía/metabolismo , MorfolinasRESUMEN
A one-third lanthanum deficiency was created in Ba2LaM5+O6 compounds (LaM compounds) to form Ba2La2/3M5+O5.5 compounds (La2/3M compounds) for M = Nb, Sb, and Bi. The compounds were prepared by a gel-combustion method using citric acid as a fuel. All the compounds were characterized by powder X-ray diffraction (XRD). The XRD analysis showed that the space group of the La2/3M compounds remains the same for the Bi and Sb samples when compared to the reported LaM compounds, except for the Nb sample. La2/3Nb and La2/3Sb adopt a rhombohedral structure with the space group R3[combining macron], whereas La2/3Bi adopts a monoclinic structure with the space group I2/m. As the positron annihilation spectroscopy (PALS) technique is sensitive to cation deficiency, it was used to detect the presence of cation vacancies in the samples, which are formed due to the decrease in the lanthanum concentration. The PALS analyses indicated that the absence of cation deficiency in the La2/3M compounds is similar to that observed in the LaM compound. Thus, the crystal structure of the La2/3M compound was modeled, such that the cation deficiency at the La site is filled by Ba2+ and M5+ ions, and the crystal structure formula is given as Ba2(La0.727Ba0.182M0.091)MO6. This model was confirmed by Rietveld refinement of the XRD data. The emission spectra of Eu3+ showed a strong dependence on its local site symmetry in the host material, in which it is being doped and this can be used as a spectroscopic probe for detecting any differences in the symmetry. Comparison of the local symmetry around La3+ cation was studied using photoluminescence (PL) by doping 2 atom% Eu3+ in LaM and La2/3M compounds. Infrared spectroscopy (IRS) analyses were also carried out for LaM and La2/3M compounds. There was complete agreement between the PL and IRS results and they were also in concordance with the predicted crystal structure model. Interestingly in these La2/3M compounds, the equilibrium structure prefers large Ba2+ ion to occupy the octahedral B-site rather than forming an octahedral vacancy at that site, making these perovskite compounds rare and novel in their class.
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BACKGROUND: Microglia are resident myeloid cells in the CNS that are activated by infection, neuronal injury, and inflammation. Established BV2 microglial cell lines have been the primary in vitro models used to study neuroinflammation for more than a decade because they reduce the requirement of continuously maintaining cell preparations and animal experimentation models. However, doubt has recently been raised regarding the value of BV2 cell lines as a model system. METHODS: We used triplicate RNA sequencing (RNA-seq) to investigate the molecular signature of primary and BV2 microglial cell lines using two transcriptomic techniques: global transcriptomic biological triplicate RNA-seq and quantitative real-time PCR. We analyzed differentially expressed genes (DEGs) to identify transcription factor (TF) motifs (-950 to +50 bp of the 5' upstream promoters) and epigenetic mechanisms. RESULTS: Sequencing assessment and quality evaluation revealed that primary microglia have a distinct transcriptomic signature and express a unique cluster of transcripts in response to lipopolysaccharide. This microglial signature was not observed in BV2 microglial cell lines. Importantly, we observed that previously unidentified TFs (i.e., IRF2, IRF5, IRF8, STAT1, STAT2, and STAT5A) and the epigenetic regulators KDM1A, NSD3, and SETDB2 were significantly and selectively expressed in primary microglia (PM). Although transcriptomic alterations known to occur in BV2 microglial cell lines were identified in PM, we also observed several novel transcriptomic alterations in PM that are not frequently observed in BV2 microglial cell lines. CONCLUSIONS: Collectively, these unprecedented findings demonstrate that established BV2 microglial cell lines are probably a poor representation of PM, and we establish a resource for future studies of neuroinflammation.
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Regulación de la Expresión Génica/efectos de los fármacos , Microglía/efectos de los fármacos , Factores de Transcripción/metabolismo , Transcriptoma/efectos de los fármacos , Análisis de Varianza , Animales , Animales Recién Nacidos , Encéfalo/citología , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Lipopolisacáridos/farmacología , Ratones , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Transcriptoma/fisiologíaRESUMEN
Due to their multipotentiality and immunomodulation, human mesenchymal stem cells (hMSCs) are widely studied for the treatment of degenerative and inflammatory diseases. Transplantation of hMSCs to damaged tissue is a promising approach for tissue regeneration. However, the physiological mechanisms and regulatory processes of MSC trafficking to injured tissue are largely unexplored. Here, we evaluated the gene expression profile and migratory potential of hMSCs upon stimulation with the TLR4 ligand lipopolysaccharide (LPS). Using RNA sequencing, we identified unique induction patterns of interferon stimulated genes, cytokines and chemokines involved in chemotaxis and homing. The -950 to +50 bp regions of many of these LPS-responsive genes were enriched with putative binding motifs for the transcription factors (TFs) interferon regulatory factor (IRF1) and nuclear factor kappa B (NF-κB1, REL), which were also induced by LPS along with other TFs. Chromatin immunoprecipitation assays showed that IRF1 bound within their target genes promoter region. In addition, IRF1 attenuation significantly down-regulated interferon stimulated genes as well as key cytokines. Furthermore, using pharmacological inhibitors, we showed that the NF-κB and phosphatidylinositol 3-kinase (PI3K) pathways regulate the migratory and cytokines/chemokines response to LPS. These unprecedented data suggest that IRF1 and NF-κB orchestrate the TLR4-primed immunomodulatory response of hMSCs and that this response also involves the PI3K pathway.
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Factor 1 Regulador del Interferón/genética , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Receptor Toll-Like 4/genética , Regulación del Desarrollo de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ligandos , Lipopolisacáridos/química , Células Madre Mesenquimatosas/citología , Fosfatidilinositol 3-Quinasas/genética , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Factor de Transcripción ReIA/genética , Transcriptoma/genéticaRESUMEN
In human mesenchymal stem cells (hMSCs), toll-like receptor 3 (TLR3) and TLR4 act as key players in the tissue repair process by recognizing their ligands and stimulating downstream processes including cytokine release. The mechanisms of TLR3- and TLR4-mediated cytokine releases from hMSCs remain uncertain. Here, we show that exposure to the TLR3 agonist polyinosinic-polycytidylic acid (poly(I:C)) or incubation with the TLR4 agonist lipopolysaccharide (LPS) increased the mRNA expression levels of TLR3, TLR4 and cytokines in hMSCs. Poly(I:C) exposure rather than LPS incubation not only elevated inositol 1,4,5-triphosphate receptor (IP3R) expression and IP3R-mediated Ca(2+) release, but also promoted Orai and STIM expression as well as store-operated Ca(2+) entry into hMSCs. In addition, we also observed that 21 Ca(2+) signaling genes were significantly up-regulated in response to TLR3 priming of hMSCs by RNA sequencing analysis. Both poly(I:C) and LPS exposure enhanced cytokine release from hMSCs. The enhanced cytokine release vanished upon siRNA knockdown and chelation of intracellular Ca(2+). These data demonstrate that TLR3- and TLR4-priming differentially enhance Ca(2+) signaling and cytokine expression, and Ca(2+) -dependently potentiates cytokine release in hMSCs.
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Señalización del Calcio/genética , Citocinas/genética , Células Madre Mesenquimatosas/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 4/genética , Western Blotting , Calcio/metabolismo , Células Cultivadas , Citocinas/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Lipopolisacáridos/farmacología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Poli I-C/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/metabolismo , Adulto JovenRESUMEN
Persistent macrophage activation is associated with the expression of various pro-inflammatory genes, cytokines and chemokines, which may initiate or amplify inflammatory disorders. A novel synthetic BET inhibitor, JQ1, was proven to exert immunosuppressive activities in macrophages. However, a genome-wide search for JQ1 molecular targets has not been undertaken. The present study aimed at evaluating the anti-inflammatory function and underlying genes that are targeted by JQ1 in LPS-stimulated primary bone marrow-derived macrophages (BMDMs) using global transcriptomic RNA sequencing and quantitative real-time PCR. Among the annotated genes, transcriptional sequencing of BMDMs that were treated with JQ1 revealed a selective effect on LPS-induced gene expression in which the induction of cytokines/chemokines, interferon-stimulated genes, and prominent (transcription factors) TFs was suppressed. Additionally, we found that JQ1 reduced the expression of previously unidentified genes that are important in inflammation. Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Furthermore, we confirmed that JQ1 reduced cytokines/chemokines in the supernatants of LPS treated BMDMs. Moreover, the biological pathways and gene ontology of the differentially expressed genes were determined in the JQ1 treatment of BMDMs. These unprecedented results suggest that the BET inhibitor JQ1 is a candidate for the prevention or therapeutic treatment of inflammatory disorders.
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Azepinas/farmacología , Células de la Médula Ósea/patología , Inflamación/patología , Macrófagos/patología , Proteínas Nucleares/antagonistas & inhibidores , Análisis de Secuencia de ARN , Receptor Toll-Like 4/metabolismo , Transcriptoma/genética , Triazoles/farmacología , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Ligandos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Anotación de Secuencia Molecular , Proteínas Nucleares/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Resident macrophages in the CNS microglia become activated and produce proinflammatory molecules upon encountering bacteria or viruses. TLRs are a phylogenetically conserved diverse family of sensors that drive innate immune responses following interactions with PAMPs. TLR3 and TLR4 recognize viral dsRNA Poly (I:C) and bacterial endotoxin LPS, respectively. Importantly, these receptors differ in their downstream adaptor molecules. Thus far, only a few studies have investigated the effects of TLR3 and TLR4 in macrophages. However, a genome-wide search for the effects of these TLRs has not been performed in microglia using RNA-seq. Gene expression patterns were determined for the BV-2 microglial cell line when stimulated with viral dsRNA Poly (I:C) or bacterial endotoxin LPS to identify novel transcribed genes, as well as investigate how differences in downstream signaling could influence gene expression in innate immunity. RESULTS: Sequencing assessment and quality evaluation revealed that common and unique patterns of proinflammatory genes were significantly up-regulated in response to TLR3 and TLR4 stimulation. However, the IFN/viral response gene showed a stronger response to TLR3 stimulation than to TLR4 stimulation. Unexpectedly, TLR3 and TLR4 stimulation did not activate IFN-ß and IRF3 in BV-2 microglia. Most importantly, we observed that previously unidentified transcription factors (TFs) (i.e., IRF1, IRF7, and IRF9) and the epigenetic regulators KDM4A and DNMT3L were significantly up-regulated in both TLR3- and TLR4-stimulated microglia. We also identified 29 previously unidentified genes that are important in immune regulation. In addition, we confirmed the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in TLR3-and TLR4-stimulated primary microglial cells. Moreover, transcriptional start sites (TSSs) and isoforms, as well as differential promoter usage, revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with TLR3 and TLR4. Furthermore, TF motif analysis (-950 to +50 bp of the 5' upstream promoters) revealed that the DNA sequences for NF-κB, IRF1, and STAT1 were significantly enriched in TLR3- and TLR4-stimulated microglia. CONCLUSIONS: These unprecedented findings not only permit a comparison of TLR3-and TLR4-stimulated genes but also identify new genes that have not been previously implicated in innate immunity.
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Microglía/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 4/genética , Transcriptoma/genética , Animales , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Mediadores de Inflamación/metabolismo , Factor 3 Regulador del Interferón/genética , Interferón beta/genética , Ligandos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Microglía/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Procesamiento Postranscripcional del ARN/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Sitio de Iniciación de la Transcripción/fisiología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Transcriptoma/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Microglial cells become rapidly activated through interaction with pathogens, and their persistent activation is associated with the production and secretion of various pro-inflammatory genes, cytokines, and chemokines, which may initiate or amplify neurodegenerative diseases. Bromodomain and extraterminal domain (BET) proteins are a group of epigenetic regulators that associate with acetylated histones and facilitate the transcription of target genes. A novel synthetic BET inhibitor, JQ1, was proven to exert immunosuppressive activities by inhibiting the expression of IL-6 and Tnf-α in macrophages. However, a genome-wide search for JQ1 molecular targets is largely unexplored in microglia. METHODS: The present study was aimed at evaluating the anti-inflammatory function and underlying genes targeted by JQ1 in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells using two transcriptomic techniques: global transcriptomic biological duplicate RNA sequencing and quantitative real-time PCR. Associated biological pathways and functional gene ontology were also evaluated. RESULTS: With a cutoff value of P ≤ 0.01 and fold change ≥1.5 log2, the expression level of 214 and 301 genes, including pro-inflammatory cytokine, chemokine, and transcription factors, was found to be upregulated in BV-2 cells stimulated with LPS for 2 and 4 h, respectively. Among these annotated genes, we found that JQ1 selectively reduced the expression of 78 and 118 genes (P ≤ 0.01, and fold change ≥ 1.5, respectively). Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Furthermore, we confirmed that JQ1 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines in the supernatants of LPS-treated primary microglial cells isolated from 3-day-old ICR mice. Utilizing functional group analysis, the genes affected by JQ1 were classified into four categories related to biological regulation, immune system processes, and response to stimuli. Moreover, the biological pathways and functional genomics obtained in this study may facilitate the suppression of different key inflammatory genes through JQ1-treated BV-2 microglial cells. CONCLUSIONS: These unprecedented results suggest the BET inhibitor JQ1 as a candidate for the prevention or therapeutic treatment of inflammation-mediated neurodegenerative diseases.
Asunto(s)
Azepinas/farmacología , Secuencia de Bases , Citocinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inmunosupresores/farmacología , Triazoles/farmacología , Animales , Línea Celular Transformada , Citocinas/genética , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Lipopolisacáridos/farmacología , Ratones , Microglía/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
Microglial cells become rapidly activated through interactions with pathogens, and the persistent activation of these cells is associated with various neurodegenerative diseases. Previous studies have investigated the transcriptomic signatures in microglia or macrophages using microarray technologies. However, this method has numerous restrictions, such as spatial biases, uneven probe properties, low sensitivity, and dependency on the probes spotted. To overcome this limitation and identify novel transcribed genes in response to LPS, we used RNA Sequencing (RNA-Seq) to determine the novel transcriptomic signatures in BV-2 microglial cells. Sequencing assessment and quality evaluation showed that approximately 263 and 319 genes (≥ 1.5 log2-fold), such as cytokines and chemokines, were strongly induced after 2 and 4 h, respectively, and the induction of several genes with unknown immunological functions was also observed. Importantly, we observed that previously unidentified transcription factors (TFs) (irf1, irf7, and irf9), histone demethylases (kdm4a) and DNA methyltransferases (dnmt3l) were significantly and selectively expressed in BV-2 microglial cells. The gene expression levels, transcription start sites (TSS), isoforms, and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with LPS. In addition, gene ontology, molecular networks and pathway analyses identified the top significantly regulated functional classification, canonical pathways and network functions at each activation status. Moreover, we further analyzed differentially expressed genes to identify transcription factor (TF) motifs (-950 to +50 bp of the 5' upstream promoters) and epigenetic mechanisms. Furthermore, we confirmed that the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in LPS treated primary microglial cells. This transcriptomic analysis is the first to show a comparison of the family-wide differential expression of most known immune genes and also reveal transcription evidence of multiple gene families in BV-2 microglial cells. Collectively, these findings reveal unique transcriptomic signatures in BV-2 microglial cells required for homeostasis and effective immune responses.
Asunto(s)
Perfilación de la Expresión Génica , Lipopolisacáridos/farmacología , Microglía/metabolismo , Análisis de Secuencia de ARN/métodos , Transcriptoma/genética , Péptidos beta-Amiloides/metabolismo , Animales , Citocinas/genética , Citocinas/metabolismo , Epigénesis Genética/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Inflamación/genética , Mediadores de Inflamación/metabolismo , Ratones , Microglía/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
JMJD2A is a lysine trimethyl-specific histone demethylase that is highly expressed in a variety of tumours. The role of JMJD2A in tumour progression remains unclear. The objectives of this study were to identify JMJD2A-regulated genes and understand the function of JMJD2A in p53-null neuroectodermal stem cells (p53(-/-) NE-4Cs). We determined the effect of LPS as a model of inflammation in p53(-/-) NE-4Cs and investigated whether the epigenetic modifier JMJD2A alter the expression of tumourigenic inflammatory genes. Global gene expression was measured in JMJD2A knockdown (kd) p53(-/-) NE-4Cs and in LPS-stimulated JMJD2A-kd p53(-/-) NE-4C cells. JMJD2A attenuation significantly down-regulated genes were Cdca2, Ccnd2, Ccnd1, Crebbp, IL6rα, and Stat3 related with cell cycle, proliferation, and inflammatory-disease responses. Importantly, some tumour-suppressor genes including Dapk3, Timp2 and TFPI were significantly up-regulated but were not affected by silencing of the JMJD2B. Furthermore, we confirmed the attenuation of JMJD2A also down-regulated Cdca2, Ccnd2, Crebbp, and Rest in primary NSCs isolated from the forebrains of E15 embryos of C57/BL6J mice with effective p53 inhibitor pifithrin-α (PFT-α). Transcription factor (TF) motif analysis revealed known binding patterns for CDC5, MYC, and CREB, as well as three novel motifs in JMJD2A-regulated genes. IPA established molecular networks. The molecular network signatures and functional gene-expression profiling data from this study warrants further investigation as an effective therapeutic target, and studies to elucidate the molecular mechanism of JMJD2A-kd-dependent effects in neuroectodermal stem cells should be performed.
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Carcinogénesis/genética , Ciclo Celular/genética , Histona Demetilasas/genética , Inflamación/genética , Lipopolisacáridos/farmacología , Placa Neural/metabolismo , Células Madre/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular/genética , Supervivencia Celular/genética , Regulación hacia Abajo/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Genes Supresores de Tumor , Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Placa Neural/efectos de los fármacos , Células Madre/efectos de los fármacos , Factores de Transcripción/genética , Transcriptoma/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
JMJD3, a Jumonji C family histone demethylase, plays an important role in the regulation of inflammation induced by the transcription factor nuclear factor-kappa B (NF-κB) in response to various stimuli. JMJD3 is a histone-3 lysine-27 trimethylation (H3K27me3) demethylase, a histone mark associated with transcriptional repression and activation of a diverse set of genes. The present study assessed stable JMJD3 knockdown (KD)-dependent proteomic profiling in human leukemia monocyte (THP-1) cells to analyze the JMJD3-mediated differential changes of marker expression in inflammatory cells. To analyze the protein expression profile of tumor necrosis factor-alpha (TNF-α)-stimulated JMJD3-kd THP-1 cells, we employed matrix-assisted-laser-desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Additionally, Ingenuity Pathways Analysis (IPA) was applied to establish the molecular networks. A comparative proteomic profile was determined in TNF-α-treated both of JMJD3-kd THP-1 cells and THP-1 scrambled (sc) cells. The expression of tripartite motif protein (TRIM5), glutathione peroxidase (GPx), glia maturation factor-γ (GMFG), caspase recruitment domain family, member 14 (CARMA2), and dUTP pyrophosphatase were significantly down-regulated, whereas heat shock protein beta-1 (HspB1) and prohibition were significantly up-regulated in JMJD3-kd THP-1 cells. The molecular and signaling networks of the differentially expressed proteins in JMJD3-kd THP-1 cells were determined by IPA. The molecular network signatures and functional proteomics obtained in this study may facilitate the suppression of different key inflammatory regulators through JMJD3-attenuation, which would be crucial to evaluate potential therapeutic targets and to elucidate the molecular mechanism of JMJD3-kd dependent effects in THP-1 cells.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji/metabolismo , Monocitos/efectos de los fármacos , Proteómica/métodos , Factor de Necrosis Tumoral alfa/farmacología , Factores de Restricción Antivirales , Western Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Regulación Neoplásica de la Expresión Génica , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Monocitos/metabolismo , Monocitos/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteoma/genética , Proteoma/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
Neural stem cell (NSC) neurogenesis is the formation of new neurons by which the brain maintains its lifelong plasticity in response to extrinsic and intrinsic changes. Here, we show the effect of lipopolysaccharides (LPS) as an in vitro model of inflammation on NSCs to determine whether the inflammatory mediators can epigenetically affect NSCs and alter their proliferation and differentiation abilities. To study the effect of LPS on NSCs, we used an immortalized mouse neuroectodermal stem cell line, NE-4C. We found that Jmjd2b, histone-3 lysine-9 di-/tri-methyl (H3K9me2/3) demethylase, is functional following LPS treatment and is crucial in multiple signaling pathways and biological processes. The global gene expression levels were detected in Jmjd2b-knockdown (kd) NE-4C cells and in LPS-stimulated Jmjd2b-kd NE-4C cells using an Affymetrix GeneChip(®) Mouse Gene 1.0 ST Array. In addition, the datasets were analyzed using MetaCore Pathway Analysis (GeneGo). The attenuation of Jmjd2b in NE-4C cells significantly affected the p65, iNOS, Bcl2, and TGF-ß expression levels and had downstream effects on related signaling pathways. In addition, chromatin immunoprecipitation revealed that Jmjd2b-kd could inhibit the Notch1, IL-1ß, and IL-2 genes by recruiting repressive H3K9me3 to their promoters. Moreover, this study highlights Jmjd2b role in LPS-mediated inflammation, which suggests an epigenetic regulation in NE-4C cells. Finally, this study establishes novel Jmjd2b targets that potentiate a biological rationale involving Jmjd2b in NSC inflammation.