RESUMEN
Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients' primary T cells might be feasible.
Asunto(s)
Sistemas CRISPR-Cas/genética , Metilación de ADN/genética , Factores de Transcripción Forkhead/metabolismo , Edición Génica/métodos , Oxigenasas de Función Mixta/genética , Proteínas Proto-Oncogénicas/genética , Linfocitos T Reguladores/citología , Proteína 9 Asociada a CRISPR/genética , Diferenciación Celular/inmunología , Línea Celular Tumoral , Desmetilación , Factores de Transcripción Forkhead/genética , Factor de Transcripción GATA3/genética , Humanos , Células Jurkat , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , ARN Guía de Kinetoplastida/genética , ARN Mensajero/genética , Linfocitos T Reguladores/inmunologíaRESUMEN
A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild-type littermates at different time-points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time-points after Treg cell depletion in DEREG mice from samples of early time-points before Treg cell depletion in these mice and from gut microbiota samples of wild-type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies.
Asunto(s)
Colon/microbiología , Firmicutes/crecimiento & desarrollo , Factores de Transcripción Forkhead/inmunología , Microbioma Gastrointestinal , Depleción Linfocítica , Linfocitos T Reguladores/inmunología , Animales , Cruzamiento , Colitis/inmunología , Colitis/metabolismo , Colitis/microbiología , Colon/inmunología , Colon/metabolismo , Disbiosis , Heces/microbiología , Femenino , Firmicutes/inmunología , Factores de Transcripción Forkhead/metabolismo , Interacciones Huésped-Patógeno , Vivienda para Animales , Masculino , Ratones , Factores Sexuales , Linfocitos T Reguladores/metabolismo , Factores de TiempoRESUMEN
About 8 % of the human genome consists of human endogenous retroviruses (HERVs), which are relicts of ancient exogenous retroviral infections incurred during evolution. Although the majority of HERVs have functional gene defects or epigenetic modifications, many of them are still able to produce retroviral proteins that have been proposed to be involved in cellular transformation and cancer development. We found that, in chemo-resistant U87RETO glioblastoma cells, cytotoxic stress induced by etoposide promotes accumulation and large-scale fission of mitochondria, associated with the detection of HERV-WE1 (syncytin-1) and HERV-FRD1 (syncytin-2) in these organelles. In addition, mitochondrial preparations also contained the corresponding receptors, i.e. ASCT2 and MFSD2. We clearly demonstrated that mitochondria associated with HERV-proteins were shuttled between adjacent cancer cells not only via tunneling tubes, but also by direct cellular uptake across the cell membrane. Furthermore, anti-syncytin-1 and anti-syncytin-2 antibodies were able to specifically block this direct cellular uptake of mitochondria even more than antibodies targeting the cognate receptors. Here, we suggest that the association of mitochondria with syncytin-1/syncytin-2 together with their respective receptors could represent a novel mechanism of cell-to-cell transfer. In chemotherapy-refractory cancer cells, this might open up attractive avenues to novel mitochondria-targeting therapies.
