RESUMEN
Maintaining the identity of Foxp3(+) regulatory T cells (Tregs) is critical for controlling immune responses in the gut, where an imbalance between Tregs and T effector cells has been linked to inflammatory bowel disease. Accumulating evidence suggests that Tregs can convert into Th17 cells and acquire an inflammatory phenotype. In this study, we used an adoptive transfer model of Ag-specific T cells to study the contribution of different factors to the reprogramming of in vitro-generated Treg cells (iTreg) into IL-17-producing cells in a mouse model of gut inflammation in vivo. Our results show that intestinal inflammation induces the reprogramming of iTreg cells into IL-17-producing cells and that vitamin A restrains reprogramming in the gut. We also demonstrate that the presence of IL-2 during the in vitro generation of iTreg cells confers resistance to Th17 conversion but that IL-2 and retinoic acid (RA) cooperate to maintain Foxp3 expression following stimulation under Th17-polarizing conditions. Additionally, although IL-2 and RA differentially regulate the expression of different Treg cell suppressive markers, Treg cells generated under different polarizing conditions present similar suppressive capacity.
Asunto(s)
Inflamación/genética , Interleucina-17/biosíntesis , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Vitamina A/administración & dosificación , Animales , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/inmunología , Reprogramación Celular/genética , Reprogramación Celular/inmunología , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunidad Celular/genética , Inflamación/inmunología , Interleucina-17/inmunología , Interleucina-2/inmunología , Mucosa Intestinal/metabolismo , Intestinos/patología , Ratones , Linfocitos T Reguladores/efectos de los fármacos , Células Th17/patología , Tretinoina/administración & dosificaciónRESUMEN
The acquired immune response begins with Ag presentation by dendritic cells (DCs) to naive T cells in a heterocellular cell-cell contact-dependent process. Although both DCs and T cells are known to express connexin43, a gap junction protein subunit, the role of connexin43 on the initiation of T cell responses remains to be elucidated. In the present work, we report the formation of gap junctions between DCs and T cells and their role on T cell activation during Ag presentation by DCs. In cocultures of DCs and T cells, Lucifer yellow microinjected into DCs is transferred to adjacent transgenic CD4(+) T cells, only if the specific antigenic peptide was present at least during the first 24 h of cocultures. This dye transfer was sensitive to gap junction blockers, such as oleamide, and small peptides containing the extracellular loop sequences of conexin. Furthermore, in this system, gap junction blockers drastically reduced T cell activation as reflected by lower proliferation, CD69 expression, and IL-2 secretion. This lower T cell activation produced by gap junction blockers was not due to a lower expression of CD80, CD86, CD40, and MHC-II on DCs. Furthermore, gap junction blocker did not affect polyclonal activation of T cell induced with anti-CD3 plus anti-CD28 Abs in the absence of DCs. These results strongly suggest that functional gap junctions assemble at the interface between DCs and T cells during Ag presentation and that they play an essential role in T cell activation.
Asunto(s)
Comunicación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Epítopos de Linfocito T/fisiología , Uniones Comunicantes/inmunología , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Biomarcadores/metabolismo , Antígenos CD28/fisiología , Complejo CD3/fisiología , Comunicación Celular/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Uniones Comunicantes/genética , Uniones Comunicantes/metabolismo , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Bazo/citología , Bazo/inmunología , Bazo/metabolismoRESUMEN
The neural crest is induced at the border of the neural plate in a multistep process by signals emanated from the epidermis, neural plate and mesoderm. In this work we show for the first time the existence of a neural crest maintenance step which is dependent on signals released from the mesoderm. We identified Endothelin-1 (Edn1) and its receptor (Ednra) as key players of this signal and we show that Edn1/Ednra signaling is required for maintenance of the neural crest by a dual mechanism of cell specification and cell survival. We show that: (i) Ednra is expressed in prospective neural crest; (ii) loss-of-function experiments with antisense morpholino or with specific chemical inhibitor suppress the expression of early neural crest markers; (iii) gain-of-function experiments expand the neural crest territory; (iv) epistatic experiments show that Ednra/Edn1 is downstream of the early neural crest gene Msx1 and upstream of the late genes Sox9 and Sox10; and (v) Edn1/Ednra signaling inhibits apoptosis and controls cell specification of the neural crest. Together, our results provide insight on a new role of Edn1/Ednra cell signaling pathway during early neural crest development.
Asunto(s)
Inducción Embrionaria/genética , Endotelina-1/metabolismo , Cresta Neural/fisiología , Receptor de Endotelina A/metabolismo , Transducción de Señal/fisiología , Animales , Embrión no Mamífero/metabolismo , Embrión no Mamífero/fisiología , Endotelina-1/genética , Inmunohistoquímica , Hibridación in Situ , Modelos Biológicos , Cresta Neural/metabolismo , Receptor de Endotelina A/genética , Transducción de Señal/genética , Xenopus/embriología , Xenopus/genética , Xenopus/metabolismoRESUMEN
In recent years, research on neural crest induction has allowed the identification of several molecules as candidates for neural crest inducers. Although many of these molecules have the ability to induce neural crest in different assays, a general mechanism of neural crest induction that includes a description of the tissues that produce the inductive signals and the time and steps in which this process takes place remains elusive. To better understand the mechanism of neural crest induction, we developed an assay that has been used previously by Nieuwkoop to study anterior-posterior pattern of the neural plate. Folds of competent ectoderm were implanted in different positions of a young neurula embryo, and the induction of neural crest was analyzed using the expression of the neural crest marker Xslug. We identified a very localized region of the early neurula where it is possible to get neural crest induction, whereas all of the regions tested showed a clear induction of the neural plate marker Xsox2. These results indicate that there is a region in the embryo with the appropriate combination of signals needed to induce neural crest cells; we called this region the neural crest competence territory. In addition, our results show that neural crest induction is always accompanied by neural plate induction, but there are many cases where neural plate was induced without neural crest. These results support the model in which the neural crest is induced by an interaction between neural plate and epidermis, but they also suggest that additional signals are required. By making grafts of different sizes and implanting them in the epidermis or the neural plate, we concluded that one of the inductive signals is produced in the dorsal region of the embryo and travels into the ectoderm. Finally, by performing gain- and loss-of-function of Wnt signaling experiments, we show that this pathway plays an important role not only in neural crest induction but also in the specification of the neural crest competence territory. Developmental Dynamics 229:109-117, 2004.