RESUMEN
Steroid resistance poses a major challenge for the management of autoimmune neuroinflammation. T helper 17 (TH17) cells are widely implicated in the pathology of steroid resistance; however, the underlying mechanisms are unknown. In this study, we identified that interleukin-1 receptor (IL-1R) blockade rendered experimental autoimmune encephalomyelitis (EAE) mice sensitive to dexamethasone (Dex) treatment. Interleukin-1ß (IL-1ß) induced a signal transducer and activator of transcription 5 (STAT5)-mediated steroid-resistant transcriptional program in TH17 cells, which promoted inflammatory cytokine production and suppressed Dex-induced anti-inflammatory genes. TH17-specific deletion of STAT5 ablated the IL-1ß-induced steroid-resistant transcriptional program and rendered EAE mice sensitive to Dex treatment. IL-1ß synergized with Dex to promote the STAT5-dependent expression of CD69 and the development of central nervous system (CNS)-resident CD69+ TH17 cells. Combined IL-1R blockade and Dex treatment ablated CNS-resident TH17 cells, reduced EAE severity, and prevented relapse. CD69+ tissue-resident TH17 cells were also detected in brain lesions of patients with multiple sclerosis. These findings (i) demonstrate that IL-1ß-STAT5 signaling in TH17 cells mediates steroid resistance and (ii) identify a therapeutic strategy for reversing steroid resistance in TH17-mediated CNS autoimmunity.
Asunto(s)
Dexametasona , Encefalomielitis Autoinmune Experimental , Interleucina-1beta , Factor de Transcripción STAT5 , Células Th17 , Animales , Células Th17/inmunología , Factor de Transcripción STAT5/metabolismo , Factor de Transcripción STAT5/inmunología , Ratones , Interleucina-1beta/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Dexametasona/farmacología , Dexametasona/uso terapéutico , Ratones Endogámicos C57BL , Resistencia a Medicamentos , Transducción de Señal/inmunología , Ratones Noqueados , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Femenino , HumanosRESUMEN
Here, we show that the capacity to manufacture IL-2 identifies constituents of the expanded CD8 T cell effector pool that display stem-like features, preferentially survive, rapidly attain memory traits, resist exhaustion, and control chronic viral challenges. The cell-intrinsic synthesis of IL-2 by CD8 T cells attenuates the ability to receive IL-2-dependent STAT5 signals, thereby limiting terminal effector formation, endowing the IL-2-producing effector subset with superior protective powers. In contrast, the non-IL-2-producing effector cells respond to IL-2 signals and gain effector traits at the expense of memory formation. Despite having distinct properties during the effector phase, IL-2-producing and nonproducing CD8 T cells appear to converge transcriptionally as memory matures to form populations with equal recall abilities. Therefore, the potential to produce IL-2 during the effector, but not memory stage, is a consequential feature that dictates the protective capabilities of the response.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucina-2/biosíntesis , Factor de Transcripción STAT5/inmunología , Animales , Interleucina-2/inmunología , Ratones , Ratones Congénicos , Ratones Transgénicos , Transducción de Señal/inmunologíaRESUMEN
Programmed cell death-1 (PD-1) is a co-inhibitory receptor that dampens immune responses upon interaction with PD-L1 and PD-L2. Although PD-1 expression on T cells is known to be activation-dependent, how cytokines modify its regulation is not fully resolved. Using polyclonal T-cell activation to study cytokine-dependent PD-1 regulation, we found that IL-2 inhibited transcriptional up-regulation of PD-1 despite the promotion of T-cell activation. The IL-2-mediated reduction in PD-1 expression augmented CD8+ T-cell activities against PD-L1-expressing target cells. To study the mechanism of PD-1 reduction, we focused on STAT5 activation in the IL-2 signaling pathway. Bioinformatic analysis suggested a novel conserved PD-1 promoter domain where NFAT and STAT5 can potentially compete with each other for binding. NFAT1 interaction with this domain revealed substantial potency in PD-1 transcription compared to STAT5A, and STAT5A overexpression could quench NFAT1-dependent PD-1 up-regulation in a sequence-specific manner. Chromatin immunoprecipitation analysis of activated T cells showed that IL-2 treatment significantly diminished the binding of NFAT1 and NFAT2 in the hypothesized competition site, while STAT5 binding to the same region was increased. These results raise the possibility that the competition of transcriptional factors might be involved in the fine-tuning of PD-1 expression by cytokines such as IL-2.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Factor de Transcripción STAT5/inmunología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Muerte Celular Programada 1/deficiencia , Receptor de Muerte Celular Programada 1/genéticaRESUMEN
Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly (p < 0.0001) higher amounts of IL-6 and IL-10 than in the patients' blood, as well as significantly (p < 0.05) increased expression of checkpoint inhibitory receptors (programmed death protein-1, PD-1) and ectonucleotidase (CD39) on T lymphocytes. However, NK lymphocytes from EOC-associated ascites showed higher (p < 0.05) pS6 phosphorylation compared with NK from blood. Additionally, in vitro treatment of lymphocytes with IL-2 or IL-15 elicited significantly (p < 0.001) phosphorylation of the STAT5 protein in NK, CD3 and CD8 lymphocytes, both from blood and ascites. EOC-associated ascites had a significantly (p < 0.0001) higher proportion of NK CD56bright lymphocytes than blood, which, in addition, were more responsive (p < 0.05) to stimulation by IL-2 than CD56dim NK. EOC-associated ascites allow studies on lymphocyte phenotype modulation in the tumor environment, where inflammatory profile contrasts with the presence of immunosuppressive elements and development of cellular self-regulating mechanisms.
Asunto(s)
Ascitis/inmunología , Antígeno CD56/inmunología , Cistadenocarcinoma Seroso/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Ováricas/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Apirasa/genética , Apirasa/inmunología , Ascitis/genética , Ascitis/patología , Antígeno CD56/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunofenotipificación , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-15/genética , Interleucina-15/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Células K562 , Células Asesinas Naturales/patología , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/patología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Heart transplantation is the optimal therapy for patients with end-stage heart disease, but its long-term outcome remains inadequate. Recent studies have highlighted the importance of the melanocortin receptors (MCRs) in inflammation, but how MCRs regulate the balance between alloreactive T cells and Tregs, and whether they impact chronic heart transplant rejection, is unknown. Here, we found that Tregs express MC2R, and MC2R expression was highest among all MCRs by Tregs. Our data indicate that adrenocorticotropic hormone (ACTH), the sole ligand for MC2R, promoted the formation of Tregs by increasing the expression of IL-2Rα (CD25) in CD4+ T cells and activation of STAT5 in CD4+CD25+ T cells. ACTH treatment also improved the survival of heart allografts and increased the formation of Tregs in CD28KO mice. ACTH treatment synergized with the tolerogenic effect of CTLA-4-Ig, resulting in long-term survival of heart allografts and an increase in intragraft Tregs. ACTH administration also demonstrated higher prolongation of heart allograft survival in transgenic mouse recipients with both complete KO and conditional KO of PI3Kγ in T cells. Finally, ACTH treatment reduced chronic rejection markedly. These data demonstrate that ACTH treatment improved heart transplant outcomes, and this effect correlated with an increase in Tregs.
Asunto(s)
Hormona Adrenocorticotrópica , Rechazo de Injerto , Trasplante de Corazón/efectos adversos , Proteínas de la Membrana/inmunología , Linfocitos T Reguladores/inmunología , Hormona Adrenocorticotrópica/inmunología , Hormona Adrenocorticotrópica/farmacología , Animales , Antígenos CD4/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Hormonas/farmacología , Inflamación/inmunología , Inflamación/patología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Ratones , Factor de Transcripción STAT5/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Tolerancia al Trasplante/efectos de los fármacos , Tolerancia al Trasplante/inmunologíaRESUMEN
Water-soluble ions (WSI) and organic extract (OE) in traffic-related particulate matter with aerodynamic diameters ≤ 2.5 µm (TRPM2.5) are potential risk factors for asthma exacerbation. Although CD4+ T lymphocytes mediated immune response is involved in the pathogenesis of asthma, the effect of WSI-TRPM2.5 and OE-TRPM2.5 on the balance of Th17/Treg cells in asthma remains poorly understood. In this study, the ovalbumin (OVA)-sensitized rats were repeatedly exposure to TRPM2.5 (3 mg/kg·bw), WSI-TRPM2.5 (1.8 mg/kg·bw, 7.2 mg/kg·bw) and OE-TRPM2.5 (0.6 mg/kg·bw, 2.4 mg/kg·bw) every three days for five times. The inflammation response and hyperemia edema were observed in the lung and trachea tissues. DNA methylation levels of STAT3 and RORγt genes in rats with WSI-TRPM2.5 and OE-TRPM2.5 treatment were decreased. DNA methylation level in STAT5 gene tended to decrease, with no change observed on Foxp3 expression. WSI-TRPM2.5 and OE-TRPM2.5 enhanced the mRNA and protein expression of STAT3 and RORγt while inhibited the expression of STAT5 and Foxp3, which may contribute to the imbalance of Th17/Treg cells (P < 0.05). More importantly, recovered balance of Th17/Treg cell subsets, upregulated p-STAT5 and Foxp3 expression and reduced p-STAT3 and RORγt levels were observed after 5-Aza treatment. Our results demonstrate that the STAT3/RORγt-STAT5/Foxp3 signaling pathway is involved in asthma exacerbation induced by WSI-TRPM2.5 and OE-TRPM2.5 through disrupting the balance of Th17/Treg cells. The alteration of DNA methylation of STAT3, STAT5, and RORγt genes may be involved in asthma exacerbation as well.
Asunto(s)
Asma/inmunología , Material Particulado/toxicidad , Linfocitos T Reguladores/efectos de los fármacos , Células Th17/efectos de los fármacos , Emisiones de Vehículos/toxicidad , Animales , Asma/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/inmunología , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Masculino , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Ratas Sprague-Dawley , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT5/inmunología , Transducción de Señal/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Tráquea/efectos de los fármacos , Tráquea/patologíaRESUMEN
The small molecule cyclotriazadisulfonamide (CADA) down-modulates the human CD4 receptor, an important factor in T cell activation. Here, we addressed the immunosuppressive potential of CADA using different activation models. CADA inhibited lymphocyte proliferation with low cellular toxicity in a mixed lymphocyte reaction, and when human PBMCs were stimulated with CD3/CD28 beads, phytohemagglutinin or anti-CD3 antibodies. The immunosuppressive effect of CADA involved both CD4+ and CD8+ T cells but was, surprisingly, most prominent in the CD8+ T cell subpopulation where it inhibited cell-mediated lympholysis. Immunosuppression by CADA was characterized by suppressed secretion of various cytokines, and reduced CD25, phosphoSTAT5 and CTPS-1 levels. We discovered a direct down-modulatory effect of CADA on 4-1BB (CD137) expression, a survival factor for activated CD8+ T cells. More specifically, CADA blocked 41BB protein biosynthesis by inhibition of its co-translational translocation into the ER in a signal peptide-dependent way. Taken together, this study demonstrates that CADA, as potent down-modulator of human CD4 and 41BB receptor, has promising immunomodulatory characteristics. This would open up new avenues toward chemotherapeutics that act as selective protein down-modulators to treat various human immunological disorders.
Asunto(s)
Antígenos CD4/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonamidas/farmacología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Células Jurkat , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Transporte de Proteínas/efectos de los fármacos , Factor de Transcripción STAT5/inmunología , Factor de Transcripción STAT5/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Sulfonamidas/químicaRESUMEN
Activation of the STAT5 transcription factor downstream of the Interleukin-2 receptor (IL-2R) induces expression of Foxp3, a critical step in the differentiation of regulatory T (Treg) cells. Due to the pleiotropic effects of IL-2R signaling, it is unclear how STAT5 acts directly on the Foxp3 locus to promote its expression. Here, we report that IL-2 - STAT5 signaling converged on an enhancer (CNS0) during Foxp3 induction. CNS0 facilitated the IL-2 dependent CD25+Foxp3- precursor to Treg cell transition in the thymus. Its deficiency resulted in impaired Treg cell generation in neonates, which was partially mitigated with age. While the thymic Treg cell paucity caused by CNS0 deficiency did not result in autoimmunity on its own, it exacerbated autoimmune manifestations caused by disruption of the Aire gene. Thus, CNS0 enhancer activity ensures robust Treg cell differentiation early in postnatal life and cooperatively with other tolerance mechanisms minimizes autoimmunity.
Asunto(s)
Linaje de la Célula/inmunología , Factores de Transcripción Forkhead/inmunología , Tolerancia Inmunológica/inmunología , Interleucina-2/inmunología , Linfocitos T Reguladores/inmunología , Animales , Autoinmunidad/inmunología , Diferenciación Celular/inmunología , Elementos de Facilitación Genéticos/inmunología , Femenino , Humanos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Masculino , Ratones , Receptores de Interleucina-2/inmunología , Factor de Transcripción STAT5/inmunología , Transducción de Señal/inmunologíaRESUMEN
T regulatory type 1 (Tr1) cells act as a key regulator in maintaining peripheral immune tolerance. Several costimulatory molecules for T cells have been identified in Tr1 cells, but their intrinsic functions are still unclear. Here we showed CD226 was highly expressed in Tr1 cells. CD226-deficient Tr1 cells were defective in proliferation and sensitive to apoptosis. In addition, CD226-deficient Tr1 cells showed lower inhibitory capacity of T cell proliferation and reduced IL-10 production. CD226 deficiency also inhibited Tr1 cell differentiation in vitro. When stimulated with IL-2, CD226-deficient Tr1 cells showed impaired STAT5 signaling. Therefore, our data suggest CD226 might play an important role in Tr1 cell homeostasis, function and differentiation. This study facilitates further biological characterization of this regulatory T cell subset.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Homeostasis/inmunología , Linfocitos T Reguladores/inmunología , Animales , Apoptosis/inmunología , Diferenciación Celular/inmunología , Proliferación Celular/fisiología , Interleucina-10/inmunología , Interleucina-2/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción STAT5/inmunología , Transducción de Señal/inmunologíaRESUMEN
Posttranslational modification with SUMO is known to regulate the activity of transcription factors, but how SUMOylation of individual proteins might influence immunity is largely unexplored. The NFAT transcription factors play an essential role in antigen receptor-mediated gene regulation. SUMOylation of NFATc1 represses IL-2 in vitro, but its role in T cell-mediated immune responses in vivo is unclear. To this end, we generated a novel transgenic mouse in which SUMO modification of NFATc1 is prevented. Avoidance of NFATc1 SUMOylation ameliorated experimental autoimmune encephalomyelitis as well as graft-versus-host disease. Elevated IL-2 production in T cells promoted T reg expansion and suppressed autoreactive or alloreactive immune responses. Mechanistically, increased IL-2 secretion counteracted IL-17 and IFN-γ expression through STAT5 and Blimp-1 induction. Then, Blimp-1 repressed IL-2 itself, as well as the induced, proliferation-associated survival factor Bcl2A1. Collectively, these data demonstrate that prevention of NFATc1 SUMOylation fine-tunes T cell responses toward lasting tolerance. Thus, targeting NFATc1 SUMOylation presents a novel and promising strategy to treat T cell-mediated inflammatory diseases.
Asunto(s)
Autoinmunidad , Encefalomielitis Autoinmune Experimental/inmunología , Factores de Transcripción NFATC/inmunología , Sumoilación/inmunología , Linfocitos T Reguladores/inmunología , Animales , Citocinas/genética , Citocinas/inmunología , Encefalomielitis Autoinmune Experimental/genética , Ratones , Ratones Noqueados , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/inmunología , Factores de Transcripción NFATC/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Sumoilación/genéticaRESUMEN
IL-13 plays a critical role in mediating many biological processes responsible for allergic inflammation. Mast cells express Il13 mRNA and produce IL-13 protein in response to antigenic stimulation. Enhancers are essential in promoting gene transcription and are thought to activate transcription by delivering essential accessory cofactors to the promoter to potentiate gene transcription. However, enhancers mediating Il13 have not been identified. Furthermore, which Il13 enhancers detect signals triggered by antigenic stimulation have not yet been defined. In this study, we identified potential mouse Il13 enhancers using histone modification monomethylation at lysine residue 4 on histone 3 (H3K4me1) chromatin immunoprecipitation sequencing and acetylation at lysine residue 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing. We used Omni-assay for transposase-accessible chromatin sequencing to determine which accessible regions within the potential Il13 enhancers that responded to IgE receptor crosslinking. We also demonstrated that the transcription factor cluster consisting of the NFATC2, STAT5, GATA2, AP1, and RUNX1 binding sites at the proximal Il13 enhancer and the transcription factor cluster consisting of the EGR2 binding site at the distal Il13 E+6.5 enhancer are critical in sensing the signals triggered by antigenic stimulation. Those enhancers, which are responsive to antigenic stimulation and are constitutively active, cooperate to generate greater transcriptional outputs. Our study reveals a novel mechanism underlying how antigenic stimulation induces robust Il13 mRNA expression in mouse mast cells.
Asunto(s)
Antígenos/inmunología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/inmunología , Proteína 2 de la Respuesta de Crecimiento Precoz/inmunología , Factor de Transcripción GATA2/inmunología , Interleucina-13/inmunología , Mastocitos/inmunología , Factores de Transcripción NFATC/inmunología , Elementos de Respuesta/inmunología , Factor de Transcripción STAT5/inmunología , Factor de Transcripción AP-1/inmunología , Transcripción Genética/inmunología , Animales , Línea Celular , Mastocitos/citología , RatonesRESUMEN
MicroRNAs regulate gene expression of transcriptional factors, which influence Th17/Treg (regulatory T cells) balance, establishing the molecular mechanism of genetic and epigenetic regulation of Treg and Th17 cells is crucial for understanding rheumatoid arthritis (RA) pathogenesis. The study goal was to understand the potential impact of the selected microRNAs expression profiles on Treg/Th17 cells frequency, RA phenotype, the expression profile of selected microRNAs, and their correlation with the expression profiles of selected transcriptional factors: SOCS1, SMAD3, SMAD4, STAT3, STAT5 in RA; we used osteoarthritis (OA) and healthy controls (HCs) as controls. The study was conducted on 14 RA and 11 OA patients, and 15 HCs. Treg/Th17 frequency was established by flow cytometry. Gene expression analysis was estimated by qPCR. We noticed correlations in RA Th17 cells between miR-26 and SMAD3, STAT3, SOCS1; and miR-155 and STAT3-and in RA Treg cells between miR-26 and SOCS1; miR-31, -155 and SMAD3; and miR-155 and SMAD4. In RA Tregs, we found a negative correlation between miR-26, -126 and STAT5a. The expression level of miR-31 in Th17 cells from RA patients with DAS28 ≤ 5.1 is higher and that for miR-24 is greater in Tregs from patients with DAS28 > 5.1. MiR-146a in Tregs is higher in rheumatoid factor (RF) positive RA patients.
Asunto(s)
Artritis Reumatoide/genética , MicroARNs/genética , Osteoartritis/genética , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , MicroARNs/inmunología , Persona de Mediana Edad , Osteoartritis/inmunología , Osteoartritis/patología , Fenotipo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Índice de Severidad de la Enfermedad , Proteína smad3/genética , Proteína smad3/inmunología , Proteína Smad4/genética , Proteína Smad4/inmunología , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 1 Supresora de la Señalización de Citocinas/inmunología , Linfocitos T Reguladores/patología , Células Th17/patologíaRESUMEN
Previous studies suggest that the presence of antigen-specific polyfunctional T cells is correlated with improved pathogen clearance, disease control, and clinical outcomes; however, the molecular mechanisms responsible for the generation, function, and survival of polyfunctional T cells remain unknown. The study of polyfunctional T cells has been, in part, limited by the need for intracellular cytokine staining (ICS), necessitating fixation and cell membrane permeabilization that leads to unacceptable degradation of RNA. Adopting elements from prior research efforts, we developed and optimized a modified protocol for the isolation of high-quality RNA (i.e., RIN > 7) from primary human T cells following aldehyde-fixation, detergent-based permeabilization, intracellular cytokines staining, and sorting. Additionally, this method also demonstrated utility preserving RNA when staining for transcription factors. This modified protocol utilizes an optimized combination of an RNase inhibitor and high-salt buffer that is cost-effective while maintaining the ability to identify and resolve cell populations for sorting. Overall, this protocol resulted in minimal loss of RNA integrity, quality, and quantity during cytoplasmic staining of cytokines and subsequent flourescence-activated cell sorting. Using this technique, we obtained the transcriptional profiles of functional subsets (i.e., non-functional, monofunctional, bifunctional, polyfunctional) of CMV-specific CD8+T cells. Our analyses demonstrated that these functional subsets are molecularly distinct, and that polyfunctional T cells are uniquely enriched for transcripts involved in viral response, inflammation, cell survival, proliferation, and metabolism when compared to monofunctional cells. Polyfunctional T cells demonstrate reduced activation-induced cell death and increased proliferation after antigen re-challenge. Further in silico analysis of transcriptional data suggested a critical role for STAT5 transcriptional activity in polyfunctional cell activation. Pharmacologic inhibition of STAT5 was associated with a significant reduction in polyfunctional cell cytokine expression and proliferation, demonstrating the requirement of STAT5 activity not only for proliferation and cell survival, but also cytokine expression. Finally, we confirmed this association between CMV-specific CD8+ polyfunctionality with STAT5 signaling also exists in immunosuppressed transplant recipients using single cell transcriptomics, indicating that results from this study may translate to this vulnerable patient population. Collectively, these results shed light on the mechanisms governing polyfunctional T cell function and survival and may ultimately inform multiple areas of immunology, including but not limited to the development of new vaccines, CAR-T cell therapies, and adoptive T cell transfer.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/aislamiento & purificación , Análisis de Secuencia de ARN/métodos , Fijación del Tejido/métodos , Linfocitos T CD8-positivos/virología , Infecciones por Citomegalovirus/inmunología , Citometría de Flujo/métodos , Perfilación de la Expresión Génica/métodos , Humanos , Factor de Transcripción STAT5/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/virologíaRESUMEN
T helper cell differentiation requires lineage-defining transcription factors and factors that have shared expression among multiple subsets. BATF is required for development of multiple Th subsets but functions in a lineage-specific manner. BATF is required for IL-9 production in Th9 cells but in contrast to its function as a pioneer factor in Th17 cells, BATF is neither sufficient nor required for accessibility at the Il9 locus. Here we show that STAT5 is the earliest factor binding and remodeling the Il9 locus to allow BATF binding in both mouse and human Th9 cultures. The ability of STAT5 to mediate accessibility for BATF is observed in other Th lineages and allows acquisition of the IL-9-secreting phenotype. STAT5 and BATF convert Th17 cells into cells that mediate IL-9-dependent effects in allergic airway inflammation and anti-tumor immunity. Thus, BATF requires the STAT5 signal to mediate plasticity at the Il9 locus.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Interleucina-9/inmunología , Factor de Transcripción STAT5/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Diferenciación Celular , Femenino , Humanos , Interleucina-9/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción STAT5/genética , Linfocitos T Colaboradores-Inductores/citología , Células Th17/inmunologíaRESUMEN
Mast cells, well established effectors in allergic disease, can be activated by numerous stimuli. We previously found that the Fyn-Stat5B pathway is critical for FcεRI-stimulated mast cell function. Because IgG receptors employ similar signaling pathways, we investigated Fyn-Stat5B function downstream of FcγR. We report that FcγR elicits Fyn-dependent Stat5B tyrosine phosphorylation in mast cells. As we previously found for Fyn kinase, Stat5B is indispensable for IgG-mediated mast cell cytokine expression and secretion. However, Stat5B KO macrophages responded normally to FcγR signaling, indicating a lineage-restricted role for Stat5B. This was consistent in vivo, since passive FcγR activation induced anaphylaxis in a macrophage-dominated response even when Stat5B was deleted. We further investigated this lineage restriction using the K/BxN model of inflammatory arthritis. This model exhibits a rapid and transient mast cell-dependent joint inflammation followed days later by a macrophage- and neutrophil-dependent response. Consistent with our hypothesis, Fyn or Stat5B deficiency did not protect mice from late joint swelling, but greatly reduced the early mast cell-dependent response. This was associated with decreased joint and plasma histamine. We conclude that Fyn-Stat5B is a linage-restricted pathway critical for IgG-mediated mast cell responses.
Asunto(s)
Mastocitos/fisiología , Receptores de IgG/metabolismo , Factor de Transcripción STAT5/metabolismo , Anafilaxia/inmunología , Animales , Degranulación de la Célula/fisiología , Femenino , Humanos , Masculino , Mastocitos/citología , Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Receptores de IgE/metabolismo , Receptores de IgG/inmunología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transducción de Señal , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
There is increasing appreciation of the critical pathogenic role of IL-17 in inflammation and autoimmune diseases, which could be produced from both adaptive Th17 cells and innate γδ T cells. Existing evidences suggest that IL-2 is important for in vivo accumulation of IL-17+ γδ T cells, leaving the mechanisms still elusive. Herein, using lupus-prone MRL/lpr mice, we demonstrated that splenic γδ T cells were potent IL-17 producers at the onset of lupus, which could be diminished by in vivo IL-2 neutralization. Additional in vivo results showed that neutralization of IL-2 also significantly deleted the IL-17-producing γδ T cells in ovalbumin (OVA) /CFA-immunized B6 mice. Using splenic γδ T cells from OVA/CFA-immunized B6 mice, we further demonstrated that IL-2 could induce IL-17 production alone or together with IL-1ß or IL-23 or anti-TCRγδ. Mechanism studies demonstrated that IL-2 could support the survival of γδ T cells, rather than induce the proliferation. Through specific pharmacologic inhibitor, we demonstrated that IL-2 could maintain that RORγt expression of γδ T cells in a STAT5-dependent manner. Collectively, this study suggested that the interplay between IL and 2 and other pro-inflammatory cytokines could trigger the rapid IL-17 production from innate γδ T cells, thus to orchestrate an inflammatory response before the development of adaptive Th17 cells.
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Enfermedades Autoinmunes/inmunología , Inflamación/inmunología , Interleucina-2/inmunología , Linfocitos Intraepiteliales/inmunología , Animales , Supervivencia Celular/inmunología , Femenino , Interleucina-17/inmunología , Interleucina-17/metabolismo , Interleucina-1beta/inmunología , Interleucina-2/antagonistas & inhibidores , Interleucina-23/inmunología , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Pruebas de Neutralización , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Ovalbúmina/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/antagonistas & inhibidores , Factor de Transcripción STAT5/inmunología , Bazo/inmunología , Células Th17/inmunologíaRESUMEN
BACKGROUND: RelB, a member of the NF-κB family, plays a critical role in the development of T cells. However, the role of RelB in Foxp3+ regulatory T cells (Tregs) remains controversial. RESULTS: Using a bone marrow chimeric mouse model, we demonstrated that the expansion of Foxp3+ Tregs in vivo could be mediated by extrinsic mechanisms. RelB plays an important role in inhibiting the homeostatic proliferation of Tregs, but not their survival. Even with the heightened expansion, RelB-/- Treg cells displayed normal suppressive function in vitro. Among the expanded populations of Treg cells, most were nTreg cells; however, the population of iTregs did not increase. Mechanistically, RelB seems to regulate Treg proliferation independently of the signal transducer and activator of transcription 5 (STAT5) pathway. CONCLUSIONS: These data suggest that RelB regulates Treg proliferation independently of the STAT5 pathway, but does not alter the function of Tregs. Further studies are warranted to uncover such mechanisms.
Asunto(s)
Proliferación Celular/fisiología , Linfocitos T Reguladores/citología , Factor de Transcripción ReIB/inmunología , Animales , Células Cultivadas , Femenino , Factores de Transcripción Forkhead/inmunología , Homeostasis/inmunología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/inmunología , Factor de Transcripción STAT5/inmunología , Transducción de Señal/inmunologíaRESUMEN
The disturbance of the immune homeostasis caused by infection is decisive for multiple organ dysfunction caused by sepsis. Both the th17 cell and the regulatory cell(Tregs) are important components of the immune system and play a crucial role in maintaining immune homeostasis. In this study, we explored the effect of Maresin1, an emerging specific pro-inflammatory mediator, on the balance of Th17/Treg in sepsis, and investigated the underlying mechanism. We used the male C57BL/6 mice to establish the model of sepsis-induced lung injury by cecal ligation and puncture to verify the protective effect of Maresin1. Our study showed that Maresin1 could significantly inhibit the excessive inflammatory response and promote the inflammation regression in the process of sepsis-induced acute lung injury, thereby reducing lung damage and improving lung function. These effects were accompanied with the regulation of Maresin1 on the Th17/Treg balance in the early stages of sepsis. We demonstrated that Maresin1 has a certain effect on increasing the number of Treg and decreasing the number of Th17 cells in the early stages of sepsis, which is consistent with its effect on STAT3/RORγt and STAT5/Foxp3 signal pathways. Our study elucidated for the first time the relationship between Maresin1 and Th17/Treg balance in sepsis-induced acute lung injury.
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Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/inmunología , Ácidos Docosahexaenoicos/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Células Th17/efectos de los fármacos , Animales , Citocinas/inmunología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/inmunología , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/patología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Distribución Aleatoria , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT5/inmunología , Sepsis/tratamiento farmacológico , Sepsis/inmunología , Transducción de Señal/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Células Th17/inmunologíaRESUMEN
Signal transducer and activator of transcription (STAT) proteins have critical roles in the development and function of immune cells. STAT signaling is often dysregulated in patients with inflammatory bowel disease (IBD), suggesting the importance of STAT regulation during the disease process. Moreover, genetic alterations in STAT3 and STAT5 (e.g., deletions, mutations, and single-nucleotide polymorphisms) are associated with an increased risk for IBD. In this study, we elucidated the precise roles of STAT5 signaling in group 3 innate lymphoid cells (ILC3s), a key subset of immune cells involved in the maintenance of gut barrier integrity. We show that mice lacking either STAT5a or STAT5b are more susceptible to Citrobacter rodentium-mediated colitis and that interleukin-2 (IL-2)- and IL-23-induced STAT5 drives IL-22 production in both mouse and human colonic lamina propria ILC3s. Mechanistically, IL-23 induces a STAT3-STAT5 complex that binds IL-22 promoter DNA elements in ILC3s. Our data suggest that STAT5a/b signaling in ILC3s maintains gut epithelial integrity during pathogen-induced intestinal disease.
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Colitis/inmunología , Interleucina-23/inmunología , Interleucina-2/inmunología , Interleucinas/biosíntesis , Factor de Transcripción STAT5/inmunología , Animales , Interleucinas/inmunología , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción STAT3/inmunología , Interleucina-22RESUMEN
IL-17-producing RORγt+ γδ T cells (γδT17 cells) are innate lymphocytes that participate in type 3 immune responses during infection and inflammation. Herein, we show that γδT17 cells rapidly proliferate within neonatal lymph nodes and gut, where, upon entry, they upregulate T-bet and coexpress IL-17, IL-22, and IFN-γ in a STAT3- and retinoic acid-dependent manner. Neonatal expansion was halted in mice conditionally deficient in STAT5, and its loss resulted in γδT17 cell depletion from all adult organs. Hyperactive STAT5 mutant mice showed that the STAT5A homolog had a dominant role over STAT5B in promoting γδT17 cell expansion and downregulating gut-associated T-bet. In contrast, STAT5B preferentially expanded IFN-γ-producing γδ populations, implying a previously unknown differential role of STAT5 gene products in lymphocyte lineage regulation. Importantly, mice lacking γδT17 cells as a result of STAT5 deficiency displayed a profound resistance to experimental autoimmune encephalomyelitis. Our data identify that the neonatal microenvironment in combination with STAT5 is critical for post-thymic γδT17 development and tissue-specific imprinting, which is essential for infection and autoimmunity.