RESUMEN
Interleukin-21 (IL-21) is a cytokine with potent regulatory effects on different immune cells. Recently, IL-21 has been contemplated for use in the treatment of cancers. However, the molecular mechanisms regulating human IL-21 gene expression has not yet been described. In this study, we initially studied the promoter region and identified the transcription start site. We thereafter described the essential region upstream of the transcription start site and showed the in vivo binding of NFATc2 and SP1 transcription factors to this region, in addition to their positive role in IL-21 expression. We also studied the role of microRNAs (miRNAs) in regulating IL-21 expression. We, thus, established the miRNA profile of CD4+CD45RO+ versus CD4+CD45RA+ isolated from healthy volunteers and identified a signature composed of 12 differentially expressed miRNAs. We showed that miR-302c is able to negatively regulate IL-21 expression by binding directly to its target site in the 3'-untranslated region. Moreover, after using fresh human CD4-positive T cells, we observed the high acetylation level of histone H4, an observation well in line with the already described high expression of IL-21 in CD4+CD45RO+ versus CD4+CD45RA+ T cells. Altogether, our data identified different molecular mechanisms regulating IL-21 expression.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Interleucinas/metabolismo , Antígenos Comunes de Leucocito/metabolismo , MicroARNs/metabolismo , Factores de Transcripción NFATC/metabolismo , Factor de Transcripción Sp1/metabolismo , Regiones no Traducidas 3' , Acetilación , Sitios de Unión , Linfocitos T CD4-Positivos/inmunología , Células HEK293 , Células HeLa , Voluntarios Sanos , Histonas/metabolismo , Humanos , Interleucinas/genética , Células Jurkat , Antígenos Comunes de Leucocito/inmunología , MicroARNs/genética , Factores de Transcripción NFATC/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/genética , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Sangre Fetal/inmunología , Factores de Transcripción Forkhead/genética , Histona Desacetilasas/genética , Ácido Valproico/farmacología , Regiones no Traducidas 5'/genética , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Secuencia Conservada , Cartilla de ADN , Histona Desacetilasas/inmunología , Humanos , Recién Nacido , MicroARNs/genética , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , TransfecciónRESUMEN
Treg are the main mediators of dominant tolerance. Their mechanisms of action and applications are subjects of considerable debate currently. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Treg and identified a signature composed of five miR (21, 31, 125a, 181c and 374). Among those, two were considerably under-expressed (miR-31 and miR-125a). We identified a functional target sequence for miR-31 in the 3' untranslated region (3' UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-21 had an effect on FOXP3 expression levels. We showed that miR-31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3' UTR of FOXP3 mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator of FOXP3 expression. Transduction of the remaining three miR had no direct effect on FOXP3 expression or on the phenotype and will remain the subject of future investigations. In conclusion, not only have we identified and validated a miR signature for human natural Treg, but also unveiled some of the mechanisms by which this signature was related to the control of FOXP3 expression in these cells.