RESUMEN
Innate immune cells, such as monocytes, can adopt a long-lasting pro-inflammatory phenotype, a phenomenon called 'trained immunity'. In trained immunity, increased cytokine levels of genes, like interleukin (IL)-6 and tumor necrosis factor (TNF)-α, are observed, which are associated with increased histone 3 lysine 4 trimethylation (H3K4me3) in the promoter region. As systemic IL6 and TNFα levels are increased in rheumatoid arthritis (RA) patients and monocytes are known to be the primary producers of TNFα and IL6, we hypothesized that 'trained immunity' signals may be observed at these genes in monocytes from RA patients. CD14+ monocytes were isolated from untreated RA patients and paired age-matched healthy controls. H3K4me3, mRNA, protein and serum levels of IL6 and TNFα were evaluated by chromatin immunoprecipitation, reverse-transcription quantitative PCR and enzyme-linked immunosorbent assays. Despite elevated serum levels of TNFα and IL6 in the tested RA patients (P<0.05), ex vivo isolated monocytes displayed similar H3K4me3 levels to healthy controls in the promoter region of TNFα and IL6. Concordantly, mRNA and protein levels of IL6 and TNFα were similar before and after lipopolysaccharide stimulation between patients and controls. Together, with the current number of individuals tested we have not detected enhanced trained immunity signals in circulating monocytes from untreated RA patients, despite increased IL6 and TNFα serum levels.
Asunto(s)
Artritis Reumatoide/genética , Histonas/genética , Interleucina-6/genética , Factor de Necrosis Tumoral alfa/genética , Adulto , Anciano , Artritis Reumatoide/sangre , Estudios de Casos y Controles , Femenino , Humanos , Interleucina-6/sangre , Interleucina-6/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Long non-coding RNAs (lncRNAs) can regulate the transcript levels of genes in the same genomic region. These locally acting lncRNAs have been found deregulated in human disease and some have been shown to harbour quantitative trait loci (eQTLs) in autoimmune diseases. However, lncRNAs linked to the transcription of candidate risk genes in loci associated to rheumatoid arthritis (RA) have not yet been identified. The TRAF1 and C5 risk locus shows evidence of multiple eQTLs and transcription of intergenic non-coding sequences. Here, we identified a non-coding transcript (C5T1lncRNA) starting in the 3' untranslated region (UTR) of C5. RA-relevant cell types express C5T1lncRNA and RNA levels are further enhanced by specific immune stimuli. C5T1lncRNA is expressed predominantly in the nucleus and its expression correlates positively with C5 mRNA in various tissues (P=0.001) and in peripheral blood mononuclear cells (P=0.02) indicating transcriptional co-regulation. Knockdown results in a concurrent decrease in C5 mRNA levels but not of other neighbouring genes. Overall, our data show the identification of a novel lncRNA C5T1lncRNA that is fully located in the associated region and influences transcript levels of C5, a gene previously linked to RA pathogenesis.
Asunto(s)
Artritis Reumatoide/genética , ADN Intergénico/genética , Fibroblastos/metabolismo , Predisposición Genética a la Enfermedad , ARN Largo no Codificante/genética , ARN Mensajero/genética , Alfa-Amanitina/farmacología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Línea Celular Tumoral , ADN Intergénico/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Sitios Genéticos , Genotipo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Polimorfismo de Nucleótido Simple , Cultivo Primario de Células , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/metabolismo , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Membrana Sinovial/citología , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
We previously generated a doxycycline-inducible H2B-mTurq2 reporter in hiPSCs to track cells and study cell division and apoptosis. To improve visualization of cycling cells, we introduced a ubiquitously transcribed mScarletI-Geminin (GMMN) (1-110) into the previously untargeted second AAVS1 allele. Fusion to the N-terminal part of GMNN provided tightly controlled mScarletI expression during the cell cycle. mScarletI fluorescence increased gradually from the S-phase through the M-phase of the cell cycle and was lost at the metaphase-anaphase transition. The resulting hiPSC reporter line generated, which we named ProLiving, is a valuable tool to study cell division and cell cycle characteristics in living hiPSC-derived cells.
Asunto(s)
Células Madre Pluripotentes Inducidas , Geminina/genética , Geminina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ciclo Celular , División Celular , Proteínas de Ciclo Celular/genéticaRESUMEN
An induced pluripotent stem cell (iPSC) line, in which a H2B-fluorescent protein fusion is temporally expressed, is a valuable tool to track cells and study cell divisions and apoptosis. To this end we introduced a 3rd generation "all-in-one" doxycycline-inducible H2B-mTurquoise2 vector into the AAVS1 locus of PAX3-Venus iPSCs via CRISPR/Cas9. H2B-mTurquoise2 expression is absent but readily induced by doxycycline allowing quantification of cell divisions and imaging of living cells. Besides being a universal reporter in iPSC-based differentiation and toxicity assays, the generated pluripotent and genomically normal LUMCi041-A-2 line is particularly suited to study PAX3-positive stages of development.