RESUMEN
IL-17-producing CD8+ (Tc17) cells are enriched in active lesions of patients with multiple sclerosis (MS), suggesting a role in the pathogenesis of autoimmunity. Here we show that amelioration of MS by dimethyl fumarate (DMF), a mechanistically elusive drug, associates with suppression of Tc17 cells. DMF treatment results in reduced frequency of Tc17, contrary to Th17 cells, and in a decreased ratio of the regulators RORC-to-TBX21, along with a shift towards cytotoxic T lymphocyte gene expression signature in CD8+ T cells from MS patients. Mechanistically, DMF potentiates the PI3K-AKT-FOXO1-T-BET pathway, thereby limiting IL-17 and RORγt expression as well as STAT5-signaling in a glutathione-dependent manner. This results in chromatin remodeling at the Il17 locus. Consequently, T-BET-deficiency in mice or inhibition of PI3K-AKT, STAT5 or reactive oxygen species prevents DMF-mediated Tc17 suppression. Overall, our data disclose a DMF-AKT-T-BET driven immune modulation and suggest putative therapy targets in MS and beyond.
Asunto(s)
Linfocitos T CD8-positivos/efectos de los fármacos , Dimetilfumarato/farmacología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Esclerosis Múltiple/tratamiento farmacológico , Adolescente , Adulto , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Dimetilfumarato/uso terapéutico , Encefalomielitis Autoinmune Experimental/sangre , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Humanos , Inmunosupresores , Interleucina-17/inmunología , Interleucina-17/metabolismo , Estudios Longitudinales , Masculino , Ratones , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Resultado del Tratamiento , Adulto JovenRESUMEN
The T helper 9 (Th9) cell transcriptional network is formed by an equilibrium of signals induced by cytokines and antigen presentation. Here we show that, within this network, two interferon regulatory factors (IRF), IRF1 and IRF4, display opposing effects on Th9 differentiation. IRF4 dose-dependently promotes, whereas IRF1 inhibits, IL-9 production. Likewise, IRF1 inhibits IL-9 production by human Th9 cells. IRF1 counteracts IRF4-driven Il9 promoter activity, and IRF1 and IRF4 have opposing function on activating histone modifications, thus modulating RNA polymerase II recruitment. IRF1 occupancy correlates with decreased IRF4 abundance, suggesting an IRF1-IRF4-binding competition at the Il9 locus. Furthermore, IRF1 shapes Th9 cells with an interferon/Th1 gene signature. Consistently, IRF1 restricts the IL-9-dependent pathogenicity of Th9 cells in a mouse model of allergic asthma. Thus our study reveals that the molecular ratio between IRF4 and IRF1 balances Th9 fate, thus providing new possibilities for manipulation of Th9 differentiation.
Asunto(s)
Factor 1 Regulador del Interferón/metabolismo , Factores Reguladores del Interferón/metabolismo , Interleucina-9/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/genética , Perfilación de la Expresión Génica , Humanos , Factor 1 Regulador del Interferón/genética , Factores Reguladores del Interferón/genética , Interleucina-9/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
Robust cytotoxic CD8(+) T-cell response is important for immunity to intracellular pathogens. Here, we show that the transcription factor IFN Regulatory Factor 4 (IRF4) is crucial for the protective CD8(+) T-cell response to the intracellular bacterium Listeria monocytogenes. IRF4-deficient (Irf4(-/-)) mice could not clear L. monocytogenes infection and generated decreased numbers of L. monocytogenes-specific CD8(+) T cells with impaired effector phenotype and function. Transfer of wild-type CD8(+) T cells into Irf4(-/-) mice improved bacterial clearance, suggesting an intrinsic defect of CD8(+) T cells in Irf4(-/-) mice. Following transfer into wild-type recipients, Irf4(-/-) CD8(+) T cells became activated and showed initial proliferation upon L. monocytogenes infection. However, these cells could not sustain proliferation, produced reduced amounts of IFN-γ and TNF-α, and failed to acquire cytotoxic function. Forced IRF4 expression in Irf4(-/-) CD8(+) T cells rescued the defect. During acute infection, Irf4(-/-) CD8(+) T cells demonstrated diminished expression of B lymphocyte-induced maturation protein-1 (Blimp-1), inhibitor of DNA binding (Id)2, and T-box expressed in T cells (T-bet), transcription factors programming effector-cell generation. IRF4 was essential for expression of Blimp-1, suggesting that altered regulation of Blimp-1 contributes to the defects of Irf4(-/-) CD8(+) T cells. Despite increased levels of B-cell lymphoma 6 (BCL-6), Eomesodermin, and Id3, Irf4(-/-) CD8(+) T cells showed impaired memory-cell formation, indicating additional functions for IRF4 in this process. As IRF4 governs B-cell and CD4(+) T-cell differentiation, the identification of its decisive role in peripheral CD8(+) T-cell differentiation, suggests a common regulatory function for IRF4 in adaptive lymphocytes fate decision.
Asunto(s)
Factores Reguladores del Interferón/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Diferenciación Celular , Proliferación Celular , Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/genética , Listeria monocytogenes/inmunología , Listeriosis/genética , Listeriosis/inmunología , Listeriosis/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/patología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Similar to T-helper (Th) cells, CD8(+) T cells also differentiate into distinct subpopulations. However, the existence of IL-9-producing CD8(+) T (Tc9) cells has not been elucidated so far. We show that murine CD8(+) T cells activated in the presence of IL-4 plus TGF-ß develop into transient IL-9 producers characterized by specific IFN-γ and IL-10 expression patterns as well as by low cytotoxic function along with diminished expression of the CTL-associated transcription factors T-bet and Eomesodermin. Similarly to the CD4(+) counterpart, Tc9 cells required for their differentiation STAT6 and IRF4. Tc9 cells deficient for these master regulators displayed increased levels of Foxp3 that in turn suppressed IL-9 production. In an allergic airway disease model, Tc9 cells promoted the onset of airway inflammation, mediated by subpathogenic numbers of Th2 cells. This support was specific for Tc9 cells because CTLs failed to exert this function. We detected increased Tc9 frequency in the periphery in mice and humans with atopic dermatitis, a Th2-associated skin disease that often precedes asthma. Thus, our data point to the existence of Tc9 cells and to their supportive function in Th2-dependent airway inflammation, suggesting that these cells might be a therapeutic target in allergic disorders.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucina-9/biosíntesis , Hipersensibilidad Respiratoria/inmunología , Subgrupos de Linfocitos T/inmunología , Células Th2/inmunología , Animales , Linfocitos T CD8-positivos/clasificación , Linfocitos T CD8-positivos/metabolismo , Citocinas/genética , Citocinas/metabolismo , Citotoxicidad Inmunológica , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Ratones , Ratones Noqueados , Hipersensibilidad Respiratoria/genética , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Células Th2/metabolismoRESUMEN
IL-17-producing CD8+ T (Tc17) cells are detectible in multiple sclerosis (MS) lesions; however, their contribution to the disease is unknown. To identify functions of Tc17 cells, we induced EAE, a murine model of MS, in mice lacking IFN regulatory factor 4 (IRF4). IRF4-deficient mice failed to generate Tc17 and Th17 cells and were resistant to EAE. After adoptive transfer of WT CD8+ T cells and subsequent immunization for EAE induction in these mice, the CD8+ T cells developed a Tc17 phenotype in the periphery but could not infiltrate the CNS. Similarly, transfer of small numbers of WT CD4+ T cells alone did not evoke EAE, but when transferred together with CD8+ T cells, IL-17-producing CD4+ (Th17) T cells accumulated in the CNS and mice developed severe disease. Th17 accumulation and development of EAE required IL-17A production by CD8+ T cells, suggesting that Tc17 cells are required to promote CD4+ T cell-mediated induction of EAE. Accordingly, patients with early-stage MS harbored a greater number of Tc17 cells in the cerebrospinal fluid than in peripheral blood. Our results reveal that Tc17 cells contribute to the initiation of CNS autoimmunity in mice and humans by supporting Th17 cell pathogenicity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Interleucina-17/inmunología , Células Th17/inmunología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/inmunología , Factores Reguladores del Interferón/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Ratones , Ratones Noqueados , Células Th17/metabolismo , Células Th17/patología , Células Th17/trasplanteRESUMEN
Apart from conventional CD4(+) Th17 cells, the cytokines IL-17A and IL-22 can also be produced by γδ T cells, NK cells and lymphoid tissue inducer (LTi) cells. Th17 cells develop from precursor cells after T-cell receptor stimulation in the presence of TGF-ß, IL-6 and IL-23. In contrast, a subset of γδ T cells ("γδT17") is committed for fast IL-17 production already in the thymus; however, γδ T cells can also produce IL-17 after prolonged in vitro stimulation via their γδ T-cell receptor plus IL-23. Here, we show that γδ T-, LTi- and NKT cells differ extensively from Th17 cells in their signalling requirements for the generation of IL-17A and IL-22. While production of these cytokines by Th17 cells totally depends on the transcription factor interferon regulatory factor 4 (IRF4), IRF4 is irrelevant in the other cell types. As for γδ T cells, this finding pertains to both thymic commitment and prolonged in vitro culture. Furthermore, IL-17A-producing γδ T cells accumulate in the central nervous system of IRF4 deficient (Irf4(-/-)) mice during experimental autoimmune encephalomyelitis. IL-17A-producing WT and Irf4(-/-) γδ T cells equally express CCR6 and lack CD27. The underlying IRF4-independent pathway partially involves STAT3 during in vitro stimulation.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Factores Reguladores del Interferón/inmunología , Interleucina-17/inmunología , Interleucinas/inmunología , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental , Regulación de la Expresión Génica/genética , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Interleucina-17/biosíntesis , Interleucina-17/genética , Interleucinas/biosíntesis , Interleucinas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Células T Asesinas Naturales/metabolismo , Células T Asesinas Naturales/patología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores CCR6/biosíntesis , Receptores CCR6/genética , Receptores CCR6/inmunología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT3/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Células Th17/patología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/biosíntesis , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Interleucina-22RESUMEN
Activation of naive CD8(+) T cells with antigen in the absence of skewing cytokines triggers their differentiation into effector CTL, which induces death of target cells. We show that CD8(+) T cells activated in the presence of the cytokines IL-6 or IL-21 plus TGF-beta similar to CD4(+) T cells, develop into IL-17-producing (Tc17) cells. These cells display greatly suppressed cytotoxic function along with low levels of the CTL markers: T-box transcription factor Eomesodermin, granzyme B and IFN-gamma. Instead, these cells express hallmark molecules of Th17 program including retinoic acid receptor-related orphan receptor (ROR)gammat, RORalpha, IL-21 and IL-23R. The expression of the type 17 master regulator RORgammat is causally linked to Tc17 generation, because its overexpression stimulates production of IL-17 in the presence of IL-6 or IL-21. Both, upregulation of the type 17 program as well as suppression of CTL differentiation are STAT3 dependent. Furthermore, Tc17 cells producing IL-17 but not granzyme B are also detectable in EAE, a mouse model for multiple sclerosis. Our data point to the existence of mutually exclusive CTL and Tc17 developmental pathways in vitro and in vivo.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Interleucina-17/metabolismo , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica/inmunología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Granzimas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Interferón gamma/metabolismo , Interleucina-6/farmacología , Interleucinas/farmacología , Ratones , Ratones Endogámicos C57BL , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Differentiation of murine T-helper (Th) 17 cells is induced by antigenic stimulation and the sequential action of the cytokines IL-6, IL-21, and IL-23, along with TGFbeta. Current dogma proposes that IL-6 induces IL-21, which, in a STAT3-dependent manner, amplifies its own transcription, contributes to IL-17 production, and, moreover, promotes the expression of the IL-23 receptor. This, in turn, prepares cells for IL-23-mediated stabilization of the Th17 phenotype. Here we demonstrate that these effects of IL-21 on Th17 differentiation are completely dependent on IFN regulatory factor 4 (IRF4). After culturing in the presence of IL-21 plus TGFbeta, IRF4-deficient (Irf4(-/-)) Th cells showed a profound intrinsic defect in IL-17 production and in the autocrine IL-21 loop. Likewise, the levels of IL-23 receptor and the lineage-specific orphan nuclear receptors RORalpha and RORgammat were diminished, whereas the T regulatory (Treg) transcription factor forkhead box P3 (Foxp3) was strongly up-regulated, consistent with the reciprocal relationship between Th17 and Treg development. Despite this loss of IL-21 functions, IL-21-induced STAT3 activation was unimpaired and induced normal Socs3 expression. Forced expression of Foxp3 in WT cells inhibited IL-21-mediated IL-17 production, suggesting that the increase in Foxp3 contributes to the Irf4(-/-) phenotype. Additionally, the low levels of RORalpha and RORgammat are also partially responsible, because simultaneous overexpression of both proteins restored IL-17 production in Irf4(-/-) cells to some extent. These data highlight IRF4 as a decisive factor during the IL-21-mediated steps of Th17 development by influencing the balance of Foxp3, RORalpha, and RORgammat.
Asunto(s)
Comunicación Autocrina/inmunología , Factores Reguladores del Interferón/inmunología , Interleucinas/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores Reguladores del Interferón/genética , Interleucinas/genética , Ratones , Ratones Noqueados , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Fenotipo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/inmunología , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/inmunología , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/inmunología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Transactivadores/genética , Transactivadores/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Regulatory CD4+ T cells are important for the homeostasis of the immune system and their absence correlates with autoimmune disorders. Here, we investigate the capacity of IL-27, a cytokine with pro- and anti-inflammatory properties, to regulate the generation of transforming growth factor beta (TGFbeta)-inducible forkhead box P3 (Foxp3)-positive regulatory T (Treg) cells. Our results demonstrate that IL-27 inhibits the acquisition of the Treg phenotype at the level of Foxp3, CD25 and CTLA-4 (CD152) expression as well as the suppressive function. In contrast to TGFbeta-induced Treg cells, the cells generated after differentiation in the presence of TGFbeta and IL-27 maintained the ability for IL-2 and tumour necrosis factor alpha (TNFalpha) production. The inhibitory effect of IL-27 on Treg generation was at least partially signal transducer and activator of transcription 3 (STAT3) dependent as examined by targeted STAT3 protein inhibition using small interfering RNA (siRNA), while STAT1-dependent signals seemed to oppose the STAT3 signals. In turn, TGFbeta blocked IL-27-induced T(h)1 differentiation. Thus, IL-27 and TGFbeta mutually control their effects on CD4+ T-cell differentiation, whereby IL-27 favours inflammatory conditions through a STAT3-dependent inhibition of Treg generation.