RESUMEN
Stable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function.
Asunto(s)
Colitis/inmunología , Factores de Transcripción Forkhead/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Traslado Adoptivo , Animales , Diferenciación Celular/inmunología , Línea Celular , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Endopeptidasas/metabolismo , Células HEK293 , Proteínas de Homeodominio/genética , Humanos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Interferencia de ARN , ARN Interferente Pequeño , Ubiquitina Tiolesterasa/genética , Peptidasa Específica de Ubiquitina 7 , UbiquitinaciónRESUMEN
Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that T cell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.
Asunto(s)
Artritis/inmunología , Colitis/inmunología , Factores de Transcripción Forkhead/metabolismo , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Proliferación Celular , Células Cultivadas , Factores de Transcripción Forkhead/genética , Células HEK293 , Factor Nuclear 1-alfa del Hepatocito/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Líquido Sinovial/citología , Linfocitos T Reguladores/metabolismo , Vía de Señalización Wnt , Proteína Wnt3A/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Autologous hematopoietic stem cell transplantation (HSCT) is increasingly considered for patients with severe autoimmune diseases whose prognosis is poor with standard treatments. Regulatory T cells (Tregs) are thought to be important for disease remission after HSCT. However, eliciting the role of donor and host Tregs in autologous HSCT is not possible in humans due to the autologous nature of the intervention. Therefore, we investigated their role during immune reconstitution and re-establishment of immune tolerance and their therapeutic potential following congenic bone marrow transplantation (BMT) in a proteoglycan-induced arthritis (PGIA) mouse model. In addition, we determined Treg T-cell receptor (TCR) CDR3 diversity before and after HSCT in patients with juvenile idiopathic arthritis and juvenile dermatomyositis. In the PGIA BMT model, after an initial predominance of host Tregs, graft-derived Tregs started dominating and displayed a more stable phenotype with better suppressive capacity. Patient samples revealed a striking lack of diversity of the Treg repertoire before HSCT. This ameliorated after HSCT, confirming reset of the Treg compartment following HSCT. In the mouse model, a therapeutic approach was initiated by infusing extra Foxp3(GFP+) Tregs during BMT. Infusion of Foxp3(GFP+) Tregs did not elicit additional clinical improvement but conversely delayed reconstitution of the graft-derived T-cell compartment. These data indicate that HSCT-mediated amelioration of autoimmune disease involves renewal of the Treg pool. In addition, infusion of extra Tregs during BMT results in a delayed reconstitution of T-cell compartments. Therefore, Treg therapy may hamper development of long-term tolerance and should be approached with caution in the clinical autologous setting.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Trasplante de Médula Ósea , Factores de Transcripción Forkhead/fisiología , Inflamación/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Western Blotting , Células Cultivadas , Femenino , Citometría de Flujo , Humanos , Tolerancia Inmunológica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante AutólogoRESUMEN
Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune-monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal. We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze-thaw cycles are performed of this in-house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants. Intra-assay variance of each mediator was <10%. Inter-assay variance ranged between 1.6 and 37% with an average of 12.2%. Recoveries were similar for all mediators (mean 99.8 ± 2.6%) with a range between 89-107%. Next we measured all mediators in serum, EDTA plasma and sodium heparin plasma of 43 healthy control donors. Of these markers only 19 showed similar expression profiles in the 3 different matrixes. Only 5 mediators were effected by multiple freeze-thawing cycles. Principal component analysis revealed different coagulants cluster separately and that sodium heparin shows the most consistent profile.
Asunto(s)
Anticoagulantes/farmacología , Voluntarios Sanos , Inmunoproteínas/metabolismo , Adulto , Ácido Edético/farmacología , Femenino , Congelación , Heparina/farmacología , Humanos , Inmunoensayo , Límite de Detección , Masculino , Persona de Mediana Edad , Estabilidad Proteica , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: To determine whether therapeutic strategies that block interleukin-6 (IL-6) or tumor necrosis factor α (TNFα) can improve the responsiveness of Teff cells to suppression in patients with juvenile idiopathic arthritis (JIA). METHODS: Synovial fluid mononuclear cells (SFMCs) from the inflamed joints of patients with JIA were cultured in the presence of etanercept or anti-IL-6 in vitro, and protein kinase B (PKB)/c-Akt activation and responsiveness to suppression were measured. In addition, the in vivo effects of TNFα blockade were investigated using peripheral blood mononuclear cells obtained from patients before and after the start of etanercept therapy. RESULTS: In vitro treatment of SFMCs with anti-IL-6 led to improved Treg cell-mediated suppression of cell proliferation in some but not all patients. Blocking TNFα with etanercept, however, clearly enhanced suppression, especially that of CD8+ T cells. In the presence of etanercept, PKB/c-Akt activation of Teff cells was reduced, and cells became more susceptible to transforming growth factor ß-mediated suppression, indicating that anti-TNFα directly targets resistant Teff cells. CONCLUSION: This study is the first to show that anti-TNFα targets the resistance of Teff cells to suppression, resulting in improved regulation of inflammatory effector cells.
Asunto(s)
Antirreumáticos/farmacología , Artritis Juvenil/metabolismo , Inmunoglobulina G/farmacología , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adolescente , Antirreumáticos/uso terapéutico , Artritis Juvenil/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Niño , Etanercept , Humanos , Inmunoglobulina G/uso terapéutico , Interleucina-6/antagonistas & inhibidores , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Transducción de Señal/fisiología , Líquido Sinovial/efectos de los fármacos , Líquido Sinovial/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismoRESUMEN
Cytokines are key components of the innate and adaptive immune system. As pivotal players in the progression or regression of a pathological process, these molecules provide a window through which diseases can be monitored and can thus act as biomarkers. In order to measure cytokine levels, a plethora of protocols can be applied. These methods include bioassays, protein microarrays, high-performance liquid chromatography (HPLC), sandwich enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence and bead based multiplex immunoassays (MIA). Due to the interaction and activity of cytokines, multiplex immunoassays are at the forefront of cytokine analysis by allowing multiple cytokines to be measured in parallel. However, even with optimized protocols, sample standardization needs to occur before these proteins can optimally act as biomarkers. This review describes various factors influencing the levels of cytokines measured in plasma, serum, dried blood spots and tissue biopsies, focusing on sample collection and handling, long term storage and the repetitive use of samples. By analyzing how each of these factors influences protein levels, it is concluded that samples should be stored at low temperatures in order to maintain cytokine stability. In addition, within a study, sample manipulations should be kept the same, with measurement protocols being chosen for their compatibility with the research in question. By having a clear understanding of what factors influence cytokine levels and how to overcome these technical issues, minimally confounded data can be obtained and cytokines can achieve optimal biomarker activity.
Asunto(s)
Citocinas/sangre , Inmunoensayo/normas , Manejo de Especímenes/normas , Biomarcadores/sangre , Pruebas con Sangre Seca , Humanos , Análisis por Matrices de Proteínas , Estándares de Referencia , Temperatura , Factores de TiempoRESUMEN
BACKGROUND: A robust inflammatory response occurs in the hours and days following cerebral ischemia. However, little is known about the immediate innate immune response in the first minutes after an ischemic insult in humans. We utilized the use of circulatory arrest during cardiac surgery to assess this. METHODS: Twelve neonates diagnosed with an aortic arch obstruction underwent cardiac surgery with cardiopulmonary bypass and approximately 30 minutes of deep hypothermic circulatory arrest (DHCA, representing cerebral ischemia). Blood samples were drawn from the vena cava superior immediately after DHCA and at various other time points from preoperatively to 24 hours after surgery. The innate immune response was assessed by neutrophil and monocyte count and phenotype using FACS, and concentrations of cytokines IL-1ß, IL-6, IL-8, IL-10, TNFα, sVCAM-1 and MCP-1 were assessed using multiplex immunoassay. Results were compared to a simultaneously drawn sample from the arterial cannula. Twelve other neonates were randomly allocated to undergo the same procedure but with continuous antegrade cerebral perfusion (ACP). RESULTS: Immediately after cerebral ischemia (DHCA), neutrophil and monocyte counts were higher in venous blood than arterial (P = 0.03 and P = 0.02 respectively). The phenotypes of these cells showed an activated state (both P <0.01). Most striking was the increase in the 'non-classical' monocyte subpopulations (CD16(intermediate); arterial 6.6% vs. venous 14%; CD16+ 13% vs. 22%, both P <0.01). Also, higher IL-6 and lower sVCAM-1 concentrations were found in venous blood (both P = 0.03). In contrast, in the ACP group, all inflammatory parameters remained stable. CONCLUSIONS: In neonates, approximately 30 minutes of cerebral ischemia during deep hypothermia elicits an immediate innate immune response, especially of the monocyte compartment. This phenomenon may hold important clues for the understanding of the inflammatory response to stroke and its potentially detrimental consequences. TRIAL REGISTRATION: ClinicalTrial.gov: NCT01032876.
Asunto(s)
Aorta Torácica/inmunología , Aorta Torácica/cirugía , Isquemia Encefálica/inmunología , Procedimientos Quirúrgicos Cardíacos , Inmunidad Innata/fisiología , Monitoreo Intraoperatorio , Aorta Torácica/metabolismo , Isquemia Encefálica/metabolismo , Procedimientos Quirúrgicos Cardíacos/métodos , Femenino , Humanos , Recién Nacido , Masculino , Monitoreo Intraoperatorio/métodos , Estudios ProspectivosRESUMEN
During the last decade research has focused on the application of FOXP3(+) regulatory T cells (Tregs) in the treatment of autoimmune disease. However, thorough functional characterization of these cells in patients with chronic autoimmune disease, especially at the site of inflammation, is still missing. Here we studied Treg function in patients with juvenile idiopathic arthritis (JIA) and observed that Tregs from the peripheral blood as well as the inflamed joints are fully functional. Nevertheless, Treg-mediated suppression of cell proliferation and cytokine production by effector cells from the site of inflammation was severely impaired, because of resistance to suppression. This resistance to suppression was not caused by a memory phenotype of effector T cells or activation status of antigen presenting cells. Instead, activation of protein kinase B (PKB)/c-akt was enhanced in inflammatory effector cells, at least partially in response to TNFα and IL-6, and inhibition of this kinase restored responsiveness to suppression. We are the first to show that PKB/c-akt hyperactivation causes resistance of effector cells to suppression in human autoimmune disease. Furthermore, these findings suggest that for a Treg enhancing strategy to be successful in the treatment of autoimmune inflammation, resistance because of PKB/c-akt hyperactivation should be targeted as well.
Asunto(s)
Subgrupos de Linfocitos B/metabolismo , Inflamación/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Linfocitos T Reguladores/fisiología , Adolescente , Artritis Juvenil/inmunología , Artritis Juvenil/metabolismo , Artritis Juvenil/patología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/terapia , Subgrupos de Linfocitos B/inmunología , Células Cultivadas , Niño , Preescolar , Activación Enzimática , Humanos , Terapia de Inmunosupresión , Inmunoterapia Adoptiva , Inflamación/patología , Inflamación/terapia , Masculino , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Insuficiencia del Tratamiento , Regulación hacia ArribaRESUMEN
OBJECTIVES: Peptide-based immune tolerance induction is considered an attractive treatment option for autoimmune diseases. The authors have developed a novel method that can enhance the induction of protective peptide-specific T-cell responses, using a rat arthritis model. The authors focused on the Toll-like receptor 9 ligand CpG, which was shown to stimulate regulatory T-cell proliferation when added to plasmacytoid dendritic cells (pDC) using in-vitro cultures. METHODS: The peptide used is a heat shock protein 60 epitope (p1) that elicits tolerogenic peptide-specific immune responses in human arthritis patients and was recently shown to have protective capacity as a bystander antigen in the rat adjuvant arthritis model. Rats were treated with three nasal doses of p1, CpG or a combination of p1 and CpG. Antigen-presenting cells were studied in nose-draining lymph nodes (mandibular lymph nodes; MLN) after nasal treatment, and T-cell responses were analysed in joint-draining lymph nodes after arthritis induction. RESULTS: Nasal co-administration of p1/CpG significantly augmented the arthritis-protective effect of p1, while CpG treatment alone did not. Co-treatment of p1/CpG increased both the number and activation status of pDC in draining MLN, which was accompanied by amplified p1-specific T-cell proliferation and interleukin (IL)-10 production. During early arthritis, p1-specific IL-10 production was identified at the site of inflammation. P1 and p1/CpG-treated rats showed a greater amount of CD4+FoxP3+ regulatory T cells in the joint-draining lymph nodes, which correlated with lower arthritis scores. CONCLUSIONS: These clinical and immunological data suggest the use of CpG as a potent adjuvant for mucosal peptide-specific immune therapy in arthritis.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Artritis Reumatoide/inmunología , Chaperonina 60/inmunología , Oligodesoxirribonucleótidos/inmunología , Vacunas de Subunidad/inmunología , Administración Intranasal , Animales , Artritis Experimental/inmunología , Chaperonina 60/administración & dosificación , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Activación de Linfocitos/inmunología , Masculino , Oligodesoxirribonucleótidos/administración & dosificación , Ratas , Ratas Endogámicas Lew , Linfocitos T Reguladores/inmunología , Receptor Toll-Like 9/agonistas , Vacunas de Subunidad/administración & dosificaciónRESUMEN
In many animal models, the manifestations of inflammatory diseases can be prevented by the adoptive transfer of CD4(+)FOXP3(+) regulatory T cells (Tregs). CD4(+)FOXP3(+) Tregs can be obtained by isolation and expansion of polyclonal naturally occurring Tregs or by Ag-specific activation of CD4(+)CD25(-)FOXP3(-) T cells. Two major obstacles are hampering the translation of this latter protocol into therapeutic application. First, there is a lack of knowledge on relevant autoantigens. Second, the resulting population is contaminated with activated CD4(+) T cells that transiently express Forkhead box P3 but gain no regulatory function. Therefore, these cells may not be safe for clinical application. In this study, we demonstrate that highly suppressive FOXP3(+) Tregs can be induced in vitro by the activation of CD4(+)CD25(-) T cells with the self-Ag human 60-kDa heat shock protein (HSP60). The activation induced suppressive FOXP3(+) Tregs can be distinguished by surface expression of CD30 from nonsuppressive FOXP3(+) effector cells. We confirm that the induced CD30(+)FOXP3(+) Tregs recognize HSP60 epitopes and that the induction of Tregs by HSP60 is enhanced by signaling via TLR4 on APCs. These findings have implications for the generation and isolation of pure populations of Ag-specific Tregs, with the potential to prevent and treat human inflammatory diseases.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Chaperonina 60/farmacología , Factores de Transcripción Forkhead/inmunología , Antígeno Ki-1/inmunología , Linfocitos T Reguladores/inmunología , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Chaperonina 60/inmunología , Citocinas/metabolismo , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Inmunofenotipificación , Antígeno Ki-1/genética , Antígeno Ki-1/metabolismo , Activación de Linfocitos/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismoRESUMEN
BACKGROUND: Adipose tissue secretory proteins, called adipokines, play pivotal roles in the pathophysiology of obesity and its associated disorders such as metabolic syndrome, type 2 diabetes, and cardiovascular disease. Because methods for comprehensive adipokine profiling in patient plasma and other biological samples are currently limited, we developed a multiplex immunoassay for rapid and high-throughput measurement of 25 adipokines in only 50 microL of sample. METHODS: (Pre)adipocyte and ex vivo cultured adipose tissue supernatants were generated and together with plasma from 5 morbidly obese patients and 5 healthy and normal weight controls used to develop the adipokine multiplex immunoassay and test its usefulness in biological samples. We assessed adipokine dynamic ranges, lower limits of detection and quantification, cross-reactivity, intra- and interassay variation, and correlation with adipokine ELISAs. RESULTS: The limits of quantification and broad dynamic ranges enabled measurement of all 25 adipokines in supernatants and patient plasmas, with the exception of TNF-alpha in plasma samples. Intraassay variation was <10% for all adipokines; interassay variation was < 15%. The multiplex immunoassay results correlated significantly with ELISA measurements. Plasma adipokine profiling showed significantly higher concentrations of the novel adipokines cathepsin S (5.1 x 10(4) vs 4.3 x 10(4) ng/L, P = 0.003) and chemerin (4.1 x 10(5) vs 2.7 x 10(5) ng/L, P = 0.0008) in morbidly obese patients than normal weight controls, besides the established differences in adiponectin and leptin concentrations. CONCLUSIONS: Our findings underscore the relevance of the novel adipokines cathepsin S and chemerin, but foremost the potential of this novel method for both comprehensive adipokine profiling in large patient cohorts and for biological discovery.
Asunto(s)
Adipoquinas/análisis , Adipocitos/química , Adipoquinas/sangre , Tejido Adiposo/citología , Adulto , Línea Celular , Reacciones Cruzadas , Femenino , Humanos , Inmunoensayo , Persona de Mediana Edad , Obesidad Mórbida/sangre , Reproducibilidad de los Resultados , Adulto JovenAsunto(s)
Asma/sangre , Quimiocinas/sangre , Ruidos Respiratorios/fisiología , Factores de Edad , Asma/fisiopatología , Biomarcadores/sangre , Quimiocinas/fisiología , Niño , Preescolar , Femenino , Humanos , Hipersensibilidad/sangre , Hipersensibilidad/fisiopatología , Masculino , Valor Predictivo de las PruebasAsunto(s)
Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos/inmunología , Cisteína Endopeptidasas/inmunología , Factores de Transcripción Forkhead/metabolismo , Factor de Transcripción GATA3/metabolismo , Hipersensibilidad/inmunología , Linfocitos T Reguladores/inmunología , Adolescente , Animales , Antígenos CD4/metabolismo , Diferenciación Celular , Células Cultivadas , Niño , Citocinas/inmunología , Femenino , Factores de Transcripción Forkhead/genética , Factor de Transcripción GATA3/genética , Humanos , Terapia de Inmunosupresión , Masculino , Pyroglyphidae , Balance Th1 - Th2Asunto(s)
Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Adulto , Citocinas/genética , Citocinas/inmunología , Sangre Fetal/citología , Sangre Fetal/inmunología , Factores de Transcripción Forkhead/inmunología , Humanos , Recién Nacido , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , ARN Mensajero/metabolismo , Adulto JovenAsunto(s)
Antígeno CTLA-4/metabolismo , Factores de Transcripción Forkhead/metabolismo , Inmunoterapia Adoptiva , Subgrupos de Linfocitos T/trasplante , Linfocitos T Reguladores/trasplante , Animales , Biomarcadores/metabolismo , Biotecnología/métodos , Antígenos CD4/metabolismo , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Proliferación Celular , Células Cultivadas , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Proteína Relacionada con TNFR Inducida por Glucocorticoide/genética , Proteína Relacionada con TNFR Inducida por Glucocorticoide/inmunología , Proteína Relacionada con TNFR Inducida por Glucocorticoide/metabolismo , Humanos , Terapia de Inmunosupresión , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Ratones SCID , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
OBJECTIVE: Neutrophils are the most abundant innate immune cells in the blood, but little is known about their role in (acquired) chronic autoinflammatory diseases. This study was undertaken to investigate the role of neutrophils in systemic-onset juvenile idiopathic arthritis (JIA), a prototypical multifactorial autoinflammatory disease that is characterized by arthritis and severe systemic inflammation. METHODS: Fifty patients with systemic-onset JIA who were receiving treatment with recombinant interleukin-1 receptor antagonist (rIL-1Ra; anakinra) were analyzed at disease onset and during remission. RNA sequencing was performed on fluorescence-activated cell-sorted neutrophils from 3 patients with active systemic-onset JIA and 3 healthy controls. Expression of activation markers, apoptosis, production of reactive oxygen species (ROS), and degranulation of secretory vesicles from neutrophils were assessed by flow cytometry in serum samples from 17 patients with systemic-onset JIA and 15 healthy controls. RESULTS: Neutrophil counts were markedly increased at disease onset, and this correlated with the levels of inflammatory mediators. The neutrophil counts normalized within days after the initiation of rIL-1Ra therapy. RNA-sequencing analysis revealed a substantial up-regulation of inflammatory processes in neutrophils from patients with active systemic-onset JIA, significantly overlapping with the transcriptome of sepsis. Correspondingly, neutrophils from patients with active systemic-onset JIA displayed a primed phenotype that was characterized by increased ROS production, CD62L shedding, and secretory vesicle degranulation, which was reversed by rIL-1Ra treatment in patients who had achieved clinical remission. Patients with a short disease duration had high neutrophil counts, more immature neutrophils, and a complete response to rIL-1Ra, whereas patients with symptoms for >1 month had normal neutrophil counts and an unsatisfactory response to rIL-1Ra. In vitro, rIL-1Ra antagonized the priming effect of IL-1ß on neutrophils from healthy subjects. CONCLUSION: These results strongly support the notion that neutrophils play an important role in systemic-onset JIA, especially in the early inflammatory phase of the disease. The findings also demonstrate that neutrophil numbers and the inflammatory activity of systemic-onset JIA are both susceptible to IL-1 blockade.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Juvenil/tratamiento farmacológico , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Neutrófilos/efectos de los fármacos , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Artritis Juvenil/inmunología , Niño , Femenino , Humanos , Masculino , Países Bajos , Estudios Prospectivos , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: Islet cell transplantation holds a potential cure for type 1 diabetes, but many islet recipients do not reach long-lasting insulin independence. In this exploratory study, we investigated whether serum cytokines, chemokines and adipokines are associated with the clinical outcome of islet transplantation. METHODS: Thirteen islet transplant patients were selected on basis of good graft function (reaching insulin independence) or insufficient engraftment (insulin requiring) from our cohort receiving standardized grafts and immune suppressive therapy. Patients reaching insulin independence were divided in those with continued (>12 months) versus transient (<6 months) insulin independence. A panel of 94 proteins including cytokines and adipokines was measured in sera taken before and at one year after transplantation using a validated multiplex immunoassay platform. RESULTS: Ninety serum proteins were detectable in concentrations varying markedly among patients at either time point. Thirteen markers changed after transplantation, while another seven markers changed in a clinical subpopulation. All other markers remained unaffected after transplantation under generalized immunosuppression. Patterns of cytokines could distinguish good graft function from insufficient function including IFN-α, LIF, SCF and IL-1RII before and after transplantation, by IL-16, CCL3, BDNF and M-CSF only before and by IL-22, IL-33, KIM-1, S100A12 and sCD14 after transplantation. Three other proteins (Leptin, Cathepsin L and S100A12) associated with loss of temporary graft function before or after transplantation. CONCLUSIONS: Distinct cytokine signatures could be identified in serum that predict or associate with clinical outcome. These serum markers may help guiding patient selection and choice of immunotherapy, or act as novel drug targets in islet transplantation.
Asunto(s)
Biomarcadores/sangre , Citocinas/sangre , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/terapia , Trasplante de Islotes Pancreáticos , Adipoquinas/metabolismo , Adulto , Quimiocinas/metabolismo , Estudios de Cohortes , Femenino , Humanos , Terapia de Inmunosupresión , Inmunosupresores , Insulina/metabolismo , Islotes Pancreáticos/citología , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Juvenile dermatomyositis (JDM) is an immune-mediated inflammatory disease affecting the microvasculature of skin and muscle. CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) are key regulators of immune homeostasis. A role for Tregs in JDM pathogenesis has not yet been established. Here, we explored Treg presence and function in peripheral blood and muscle of JDM patients. We analyzed number, phenotype and function of Tregs in blood from JDM patients by flow cytometry and in vitro suppression assays, in comparison to healthy controls and disease controls (Duchenne's Muscular Dystrophy). Presence of Tregs in muscle was analyzed by immunohistochemistry. Overall, Treg percentages in peripheral blood of JDM patients were similar compared to both control groups. Muscle biopsies of new onset JDM patients showed increased infiltration of numbers of T cells compared to Duchenne's muscular dystrophy. Both in JDM and Duchenne's muscular dystrophy the proportion of FOXP3+ T cells in muscles were increased compared to JDM peripheral blood. Interestingly, JDM is not a self-remitting disease, suggesting that the high proportion of Tregs in inflamed muscle do not suppress inflammation. In line with this, peripheral blood Tregs of active JDM patients were less capable of suppressing effector T cell activation in vitro, compared to Tregs of JDM in clinical remission. These data show a functional impairment of Tregs in a proportion of patients with active disease, and suggest a regulatory role for Tregs in JDM inflammation.
Asunto(s)
Dermatomiositis/inmunología , Factores de Transcripción Forkhead/análisis , Músculos/patología , Linfocitos T Reguladores/inmunología , Adolescente , Niño , Preescolar , Dermatomiositis/sangre , Dermatomiositis/patología , Femenino , Factores de Transcripción Forkhead/inmunología , Humanos , Lactante , Masculino , Músculos/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Respiratory syncytial virus (RSV) bronchiolitis triggers a strong innate immune response characterized by excessive neutrophil infiltration which contributes to RSV induced pathology. The cytokine IL-17A enhances neutrophil infiltration into virus infected lungs. IL-17A is however best known as an effector of adaptive immune responses. The role of IL-17A in early immune modulation in RSV infection is unknown. We aimed to elucidate whether local IL-17A facilitates the innate neutrophil infiltration into RSV infected lungs prior to adaptive immunity. To this end, we studied IL-17A production in newborns that were hospitalized for severe RSV bronchiolitis. In tracheal aspirates we measured IL-17A concentration and neutrophil counts. We utilized cultured human epithelial cells to test if IL-17A regulates RSV infection-induced IL-8 release as mediator of neutrophil recruitment. In mice we investigated the cell types that are responsible for early innate IL-17A production during RSV infection. Using IL-17A neutralizing antibodies we tested if IL-17A is responsible for innate neutrophil infiltration in mice. Our data show that increased IL-17A production in newborn RSV patient lungs correlates with subsequent neutrophil counts recruited to the lungs. IL-17A potentiates RSV-induced production of the neutrophil-attracting chemokine IL-8 by airway epithelial cells in vitro. Various lung-resident lymphocytes produced IL-17A during early RSV infection in Balb/c mice, of which a local population of CD4 T cells stood out as the predominant RSV-induced cell type. By removing IL-17A during early RSV infection in mice we showed that IL-17A is responsible for enhanced innate neutrophil infiltration in vivo. Using patient material, in vitro studies, and an animal model of RSV infection, we thus show that early local IL-17A production in the airways during RSV bronchiolitis facilitates neutrophil recruitment with pathologic consequences to infant lungs.