RESUMEN
×Ö¹ndoleamine-2,3-dioxygenase (IDO) plays a role in immune regulation. Increased IDO activity was reported in systemic lupus erythematosus (SLE). We investigated the effects of the tolerogenic peptide hCDR1, shown to ameliorate lupus manifestations, on IDO gene expression. mRNA was prepared from splenocytes of hCDR1- treated SLE-afflicted (NZBxNZW)F1 mice, from blood samples of lupus patients, collected before and after their in vivo treatment with hCDR1 and from peripheral blood mononuclear cells (PBMC) of patients incubated with hCDR1. IDO gene expression was determined by real-time RT-PCR. hCDR1 significantly down-regulated IDO expression in SLE-affected mice and in lupus patients (treated in vivo and in vitro). No effects were observed in healthy donors or following treatment with a control peptide. Diminished IDO gene expression was associated with hCDR1 beneficial effects. Our results suggest that the hCDR1-induced FOXP3 expressing regulatory T cells in lupus are not driven by IDO but rather by other hCDR1 regulated pathways.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/efectos de los fármacos , Lupus Eritematoso Sistémico/genética , Fragmentos de Péptidos/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Adulto , Anciano , Animales , Anticuerpos Monoclonales/uso terapéutico , Ensayos Clínicos Fase II como Asunto , Regulación hacia Abajo , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Fragmentos de Péptidos/uso terapéutico , Reacción en Cadena en Tiempo Real de la Polimerasa , Bazo/citología , Linfocitos T Reguladores/metabolismoRESUMEN
Primary Sjogren's syndrome (pSS) is an autoimmune disease characterized by lymphocytic infiltration of exocrine glands. We investigated whether the tolerogenic peptide, hCDR1, that ameliorates lupus manifestations would have beneficial effects on pSS as well. The in vitro effects of hCDR1 on gene expression of pro-inflammatory cytokines and regulatory molecules were tested in peripheral blood mononuclear cells (PBMC) of 16 pSS patients. hCDR1, but not a control peptide, significantly reduced gene expression of IL-1ß, TNF-α, MX-1 and BlyS and up-regulated immunosuppressive (TGF-ß, FOXP3) molecules in PBMC of pSS patients. hCDR1 did not affect gene expression in patients with rheumatoid arthritis and anti-phospholipid syndrome. Further, hCDR1 up-regulated the expression of Indoleamine 2,3-dioxygenase (IDO) via elevation of TGF-ß. IDO inhibition led to a significant decrease in the expression of FOXP3 which is crucial for the induction of T regulatory cells. Thus, hCDR1 is potential candidate for the specific treatment of pSS patients.
Asunto(s)
Citocinas/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Péptidos/farmacología , Síndrome de Sjögren/inmunología , Adulto , Anciano , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Citocinas/genética , Citocinas/metabolismo , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Factores Inmunológicos/farmacología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Péptidos/genética , Síndrome de Sjögren/sangre , Síndrome de Sjögren/genética , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVE: Anticitrullinated protein antibodies (ACPA) have major diagnostic significance in rheumatoid arthritis (RA). ACPA are directed against different citrullinated antigens, including filaggrin, fibrinogen, vimentin, and collagen. The presence of ACPA is associated with joint damage and extraarticular manifestations, suggesting that ACPA may have a significant role in the pathogenesis of RA. METHODS: To verify the effect of ACPA on RA-immune cells, peripheral blood mononuclear cells (PBMC) from cyclic citrullinated peptide (CCP)-positive patients with RA and healthy controls were cocultured in vitro with ACPA. ACPA-positive stained cells were analyzed by flow cytometry and the effect of ACPA on mRNA expression levels was evaluated by real-time PCR. We tested whether the stimulatory effects induced by ACPA could be inhibited by the addition of a new multiepitope citrullinated peptide (Cit-ME). RESULTS: We found that ACPA bind specifically to PBMC from CCP-positive patients with RA through the Fab portion. ACPA induce upregulation of pathogenic cytokine expression (4- to 13-fold increase) in PBMC derived from CCP-positive patients with RA. Moreover, ACPA upregulated IL-1ß and IL-6 mRNA expression levels by 10- and 6-fold, respectively, compared to control IgG. Cit-ME, a genuine ligand of ACPA, inhibited the ACPA-induced upregulation of IL-1ß and IL-6 by 30%. CONCLUSION: ACPA bind to a limited percentage of PBMC and upregulate inflammatory cytokine expression, suggesting that ACPA is involved in RA pathogenesis. Targeting ACPA to decrease their pathogenic effects might provide a novel direction in developing therapeutic strategies for RA.
Asunto(s)
Artritis Reumatoide/sangre , Autoanticuerpos/sangre , Citrulina/inmunología , Leucocitos Mononucleares/metabolismo , Péptidos Cíclicos/metabolismo , Transcriptoma/inmunología , Afinidad de Anticuerpos/inmunología , Citrulina/síntesis química , Proteínas Filagrina , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Estadísticas no ParamétricasRESUMEN
Systemic lupus erythematosus (SLE) is a chronic multisystem disease in which various cell types and immunological pathways are dysregulated. Current therapies for SLE are based mainly on the use of non-specific immunosuppressive drugs that cause serious side effects. There is, therefore, an unmet need for novel therapeutic means with improved efficacy and lower toxicity. Based on recent better understanding of the pathogenesis of SLE, targeted biological therapies are under different stages of development. The latter include B-cell targeted treatments, agents directed against the B lymphocyte stimulator (BLyS), inhibitors of T cell activation as well as cytokine blocking means. Out of the latter, Belimumab was the first drug approved by the FDA for the treatment of SLE patients. In addition to the non-antigen specific agents that may affect the normal immune system as well, SLE-specific therapeutic means are under development. These are synthetic peptides (e.g. pConsensus, nucleosomal peptides, P140 and hCDR1) that are sequences of conserved regions of molecules involved in the pathogenesis of lupus. The peptides are tolerogenic T-cell epitopes that immunomodulate only cell types and pathways that play a role in the pathogenesis of SLE without interfering with normal immune functions. Two of the peptides (P140 and hCDR1) were tested in clinical trials and were reported to be safe and well tolerated. Thus, synthetic peptides are attractive potential means for the specific treatment of lupus patients. In this review we discuss the various biological treatments that have been developed for lupus with a special focus on the tolerogenic peptides.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Sistemas de Liberación de Medicamentos , Epítopos de Linfocito T/uso terapéutico , Inmunosupresores/uso terapéutico , Lupus Eritematoso Sistémico , Péptidos/uso terapéutico , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Ensayos Clínicos como Asunto , Epítopos de Linfocito T/inmunología , Humanos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
BACKGROUND: The tolerogenic peptide, hCDR1, ameliorated manifestations of systemic lupus erythematosus (SLE) via the immunomodulation of pro-inflammatory and immunosuppressive cytokines and the induction of regulatory T cells. Because type I interferon (IFN-α) has been implicated to play a role in SLE pathogenesis, we investigated the effects of hCDR1 on IFN-α in a murine model of SLE and in human lupus. METHODOLOGY PRINCIPAL FINDINGS: (NZBxNZW)F1 mice with established SLE were treated with hCDR1 (10 weekly injections). Splenocytes were obtained for gene expression studies by real-time RT-PCR. hCDR1 down-regulated significantly IFN-α gene expression (73% inhibition compared to vehicle treated mice, p = 0.002) in association with diminished clinical manifestations. Further, hCDR1 reduced, in vitro, IFN-α gene expression in peripheral blood mononuclear cells (PBMC) of 10 lupus patients (74% inhibition compared to medium, p = 0.002) but had no significant effects on the expression levels of IFN-α in PBMC of primary anti-phospholipid syndrome patients or of healthy controls. Lupus patients were treated for 24 weeks with hCDR1 (5) or placebo (4) by weekly subcutaneous injections. Blood samples collected, before and after treatment, were frozen until mRNA isolation. A significant reduction in IFN-α was determined in hCDR1 treated patients (64.4% inhibition compared to pretreatment expression levels, p = 0.015). No inhibition was observed in the placebo treated patients. In agreement, treatment with hCDR1 resulted in a significant decrease of disease activity. IFN-α appears to play a role in the mechanism of action of hCDR1 since recombinant IFN-α diminished the immunomodulating effects of hCDR1 on IL-1ß, TGFß and FoxP3 gene expression. CONCLUSIONS SIGNIFICANCE: We reported previously that hCDR1 affected various cell types and immune pathways in correlation to disease amelioration. The present studies demonstrate that hCDR1 is also capable of down-regulating significantly (and specifically to lupus) IFN-α gene expression. Thus, hCDR1 has a potential role as a novel, disease specific treatment for lupus.
Asunto(s)
Autoantígenos/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Factores Inmunológicos/uso terapéutico , Interferón-alfa/genética , Lupus Eritematoso Sistémico/tratamiento farmacológico , Proteínas del Tejido Nervioso/uso terapéutico , Péptidos/uso terapéutico , Secuencia de Aminoácidos , Animales , Autoantígenos/química , Células Cultivadas , Femenino , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/genética , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Péptidos/químicaRESUMEN
BACKGROUND: The 16/6-idiotype (16/6-Id) of the human anti-DNA antibody was found to induce experimental lupus in naïve mice, manifested by production of autoantibodies, leukopenia and elevated inflammatory markers, as well as kidney and brain involvement. We assessed behavior and brain pathology of naive mice injected intra-cerebra-ventricularly (ICV) with the 16/6-Id antibody. METHODS: C3H female mice were injected ICV to the right hemisphere with the human 16/6-Id antibody or commercial human IgG antibodies (control). The mice were tested for depression by the forced swimming test (FST), locomotor and explorative activity by the staircase test, and cognitive functions were examined by the novel object recognition and Y-maze tests. Brain slices were stained for inflammatory processes. RESULTS: 16/6-Id injected mice were cognitively impaired as shown by significant differences in the preference for a new object in the novel object recognition test compared to controls (P = 0.012). Similarly, the preference for spatial novelty in the Y-maze test was significantly higher in the control group compared to the 16/6-Id-injected mice (42% vs. 9%, respectively, P = 0.065). Depression-like behavior and locomotor activity were not significantly different between the16/6-Id-injected and the control mice. Immunohistochemistry analysis revealed an increase in astrocytes and microglial activation in the hippocampus and amygdala, in the 16/6-Id injected group compared to the control. CONCLUSIONS: Passive transfer of 16/6-Id antibodies directly into mice brain resulted in cognitive impairments and histological evidence for brain inflammation. These findings shed additional light on the diverse mosaic pathophysiology of neuropsychiatric lupus.See related Commentary article: http://www.biomedcentral.com/1741-7015/11/91.
Asunto(s)
Anticuerpos/administración & dosificación , Anticuerpos/toxicidad , Disfunción Cognitiva/inducido químicamente , Encefalitis/inducido químicamente , Animales , Encéfalo/patología , Femenino , Histocitoquímica , Humanos , Ratones , Ratones Endogámicos C3HRESUMEN
To determine the effect of the tolerogenic peptide hCDR1 on hippocampal neurogenesis, we treated SLE-afflicted (NZBxNZW)F1 mice with hCDR1 (once a week for 10weeks). The treatment resulted in the up-regulation of neurogenesis in the dentate gyrus and restored the NeuN immunoreactivity in brain hippocampi of the mice in association with increased gene expression of IGF-1, NGF and BDNF. Furthermore, hCDR1 treatment significantly up-regulated p-ERK and p-Akt that are suggested to be key components in mediating growth factor-induced neurogenesis. The observed effects of hCDR1 on hippocampal-neurogenesis and on associated signaling pathways suggest a potential role for hCDR1 in CNS lupus.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Hipocampo/efectos de los fármacos , Vasculitis por Lupus del Sistema Nervioso Central/tratamiento farmacológico , Neurogénesis/efectos de los fármacos , Fragmentos de Péptidos/uso terapéutico , Animales , Western Blotting , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Inmunohistoquímica , Vasculitis por Lupus del Sistema Nervioso Central/metabolismo , Vasculitis por Lupus del Sistema Nervioso Central/patología , Ratones , Factores de Crecimiento Nervioso/efectos de los fármacos , Proteínas Quinasas/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Systemic lupus erythematosus (SLE) is an autoimmune disease that involves dysregulation of B and T cells. A tolerogenic peptide, designated hCDR1, ameliorates disease manifestations in SLE-afflicted mice. In the present study, the effect of treatment with hCDR1 on the CD74/macrophage migration inhibitory factor (MIF) pathway was studied. We report here that B lymphocytes from SLE-afflicted mice express relatively elevated levels of CD74, compared with B cells from healthy mice. CD74 is a receptor found in complex with CD44, and it binds the pro-inflammatory cytokine MIF. The latter components were also up-regulated in B cells from the diseased mice, and treatment with hCDR1 resulted in their down-regulation and in reduced B-cell survival. Furthermore, up-regulation of CD74 and CD44 expression was detected in brain hippocampi and kidneys, two target organs in SLE. Treatment with hCDR1 diminished the expression of those molecules to the levels determined for young healthy mice. These results suggest that the CD74/MIF pathway plays an important role in lupus pathology.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/inmunología , Autoantígenos/inmunología , Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Oxidorreductasas Intramoleculares/inmunología , Lupus Eritematoso Sistémico/inmunología , Factores Inhibidores de la Migración de Macrófagos/inmunología , Proteínas del Tejido Nervioso/inmunología , Péptidos/inmunología , Animales , Antígenos de Diferenciación de Linfocitos B/biosíntesis , Antígenos de Diferenciación de Linfocitos B/genética , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Autoantígenos/química , Linfocitos B/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Inmunomodulación , Oxidorreductasas Intramoleculares/biosíntesis , Oxidorreductasas Intramoleculares/genética , Lupus Eritematoso Sistémico/patología , Factores Inhibidores de la Migración de Macrófagos/biosíntesis , Factores Inhibidores de la Migración de Macrófagos/genética , Ratones , Ratones Endogámicos NZB , Proteínas del Tejido Nervioso/química , Péptidos/química , Péptidos/farmacologíaRESUMEN
Treatment with a tolerogenic peptide, hCDR1, designed for the specific treatment of systemic lupus erythematosus (SLE) ameliorated the serological and kidney-related clinical manifestations in murine models of induced and spontaneous lupus. Furthermore, hCDR1 reduced brain pathology and improved behavior parameters in mice with central nervous system manifestations. The beneficial effects were associated with a diminished production of pathogenic cytokines (e.g. IFN-γ, IL-10, and IL-1ß) and with increased production of the immunosuppressive cytokine, TGF-ß. Treatment with hCDR1 up-regulated CD4 and CD8 regulatory T cells (Tregs) that played a key role in the ameliorating effects of hCDR1. Reduction of T cell apoptosis by hCDR1 contributed to the beneficial effects of hCDR. Moreover, treatment with hCDR1 down-regulated B cell maturation and autoreactive B cell survival by diminishing the B cell activating factor (BAFF/BLyS). Finally, hCDR1 suppressed in vivo gene expression of pathogenic cytokines, apoptosis and BLyS and up-regulated immunosuppressive molecules in peripheral blood lymphocytes of SLE patients. The latter was associated with clinical amelioration. Thus, treatment with hCDR1 leads to a cascade of events that culminate in the down-regulation of SLE-associated autoreactive T and B cells and in the clinical amelioration of lupus. hCDR1 is therefore a candidate for the specific treatment of SLE patients.
Asunto(s)
Autoantígenos/uso terapéutico , Tolerancia Inmunológica , Lupus Eritematoso Sistémico/terapia , Proteínas del Tejido Nervioso/uso terapéutico , Péptidos/uso terapéutico , Animales , Autoantígenos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Lupus Eritematoso Sistémico/inmunología , Ratones , Proteínas del Tejido Nervioso/genética , Péptidos/genética , Péptidos/farmacología , PorcinosRESUMEN
Dysregulated expression of Bcl-xL and Bcl-2 may initiate the development of autoimmune diseases including systemic lupus erythematosus (SLE). A tolerogenic peptide designated hCDR1 was shown to ameliorate manifestations of spontaneous and induced murine SLE. Recently, we demonstrated that Bcl-xL plays a critical role in the modulating effects of hCDR1, as manifested by reducing the state of activation of lymphocytes and by down-regulating the secretion of the pathogenic cytokines, IFN-gamma and IL-10. Here we studied the role of Bcl-xL in the development and function of CD4 regulatory T-cells (Treg) from hCDR1-treated, SLE-afflicted (New-Zealand-Black x New-Zealand-White) F1 mice. We report that Bcl-xL was up-regulated in CD4 Treg of tolerized mice, where it played a role in inducing the regulatory/inhibitory molecules Foxp3, CTLA-4, and TGF-beta and in repressing PD-1. Further, Bcl-xL mediated the induction of CTLA-4 and TGF-beta in effector T cells (Teff) by CD4 Treg of the tolerized mice. The induction of Bcl-xL in Teff by Treg was TGF-beta dependent and CTLA-4 independent, leading to inhibition of proliferation and to a decrease in activated Teff. We conclude that Bcl-xL is required for the development and function of CD4 Treg, which ameliorate lupus following treatment with a tolerogenic peptide.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Lupus Eritematoso Sistémico/inmunología , Fragmentos de Péptidos/administración & dosificación , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo , Proteína bcl-X/metabolismo , Animales , Antígenos CD/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Factores de Transcripción Forkhead/biosíntesis , Humanos , Tolerancia Inmunológica , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Lupus Eritematoso Sistémico/terapia , Activación de Linfocitos/efectos de los fármacos , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba , Proteína bcl-X/genéticaRESUMEN
INTRODUCTION: Systemic lupus erythematosus (SLE) is characterized by a variety of autoantibodies and systemic clinical manifestations. A tolerogenic peptide, hCDR1, ameliorated lupus manifestations in mice models. The objectives of this study were to induce experimental SLE in pigs and to determine the ability of hCDR1 to immunomodulate the disease manifestations. RESULTS AND DISCUSSION: We report here the successful induction, by a monoclonal anti-DNA antibody, of an SLE-like disease in pigs, manifested by autoantibody production and glomerular immune complex deposits. Treatment of pigs with hCDR1 ameliorated the lupus-related manifestations. Furthermore, the treatment downregulated the gene expression of the pathogenic cytokines, interleukin (IL)-1beta, tumor necrosis factor alpha, interferon gamma, and IL-10, and upregulated the expression of the immunosuppressive cytokine transforming growth factor beta, the antiapoptotic molecule Bcl-xL, and the suppressive master gene, Foxp3, hence restoring the expression of the latter to normal levels. Thus, hCDR1 is capable of ameliorating lupus in large animals and is a potential candidate for the treatment of SLE patients.
Asunto(s)
Anticuerpos Antinucleares/administración & dosificación , Regiones Determinantes de Complementariedad/administración & dosificación , Riñón/metabolismo , Lupus Eritematoso Sistémico/inmunología , Fragmentos de Péptidos/administración & dosificación , Animales , Anticuerpos Antinucleares/química , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Regiones Determinantes de Complementariedad/química , Citocinas/biosíntesis , Citocinas/genética , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Tolerancia Inmunológica , Inmunomodulación , Riñón/inmunología , Lupus Eritematoso Sistémico/fisiopatología , Lupus Eritematoso Sistémico/terapia , Ratones , Fragmentos de Péptidos/química , Porcinos/inmunología , Proteína bcl-X/biosíntesis , Proteína bcl-X/genéticaRESUMEN
OBJECTIVE: Central nervous system (CNS) involvement in systemic lupus erythematosus (SLE) is manifested by neurologic deficits and psychiatric disorders. The aim of this study was to examine SLE-associated CNS pathology in lupus-prone (NZBxNZW)F1 (NZB/NZW) mice, and to evaluate the ameliorating effects of treatment with a tolerogenic peptide, hCDR1 (human first complementarity-determining region), on these manifestations. METHODS: Histopathologic analyses of brains from lupus-prone NZB/NZW mice treated with vehicle, hCDR1, or a control scrambled peptide were performed. The messenger RNA expression of SLE-associated cytokines and apoptosis-related molecules from the hippocampi was determined. Anxiety-like behavior was assessed by open-field tests and dark/light transfer tests, and memory deficit was assessed using a novel object recognition test. RESULTS: Infiltration was evident in the hippocampi of the lupus-afflicted mice, and the presence of CD3+ T cells as well as IgG and complement C3 complex deposition was observed. Furthermore, elevated levels of gliosis and loss of neuronal nuclei immunoreactivity were also observed in the hippocampi of the mice with lupus. Treatment with hCDR1 ameliorated the histopathologic changes. Treatment with hCDR1 down-regulated the high expression of interleukin-1beta (IL-1beta), IL-6, IL-10, interferon-gamma, transforming growth factor beta, and the proapoptotic molecule caspase 8 in the hippocampi of the mice with lupus, and up-regulated expression of the antiapoptotic bcl-xL gene. Diseased mice exhibited increased anxiety-like behavior and memory deficit. Treatment with hCDR1 improved these parameters, as assessed by behavior tests. CONCLUSION: Treatment with hCDR1 ameliorated CNS pathology and improved the tested cognitive and mood-related behavior of the mice with lupus. Thus, hCDR1 is a novel candidate for the treatment of CNS lupus.
Asunto(s)
Ansiedad/inmunología , Regiones Determinantes de Complementariedad/inmunología , Hipocampo/efectos de los fármacos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Animales , Complejo Antígeno-Anticuerpo/efectos de los fármacos , Complejo Antígeno-Anticuerpo/inmunología , Ansiedad/genética , Ansiedad/metabolismo , Apoptosis/efectos de los fármacos , Conducta Animal/fisiología , Complejo CD3/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Inmunización , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/inmunología , Trastornos de la Memoria/patología , Ratones , Ratones Endogámicos NZB , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/inmunología , ARN Mensajero/metabolismo , Linfocitos T/metabolismo , Linfocitos T/patología , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
BACKGROUND: Regulatory T cells (Tregs) were shown to be central in maintaining immunological homeostasis and preventing the development of autoimmune diseases. Several subsets of Tregs have been identified to date; however, the dynamics of the interactions between these subsets, and their implications on their regulatory functions are yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We employed a combination of mathematical modeling and frequent in vivo measurements of several T cell subsets. Healthy BALB/c mice received a single injection of either hCDR1--a tolerogenic peptide previously shown to induce Tregs, a control peptide or vehicle alone, and were monitored for 16 days. During this period, splenocytes from the treated mice were analyzed for the levels of CD4, CD25, CD8, CD28 and Foxp3. The collected data were then fitted to mathematical models, in order to test competing hypotheses regarding the interactions between the followed T cell subsets. In all 3 treatment groups, a significant, lasting, non-random perturbation of the immune system could be observed. Our analysis predicted the emergence of functional CD4 Tregs based on inverse oscillations of the latter and CD4(+)CD25(-) cells. Furthermore, CD4 Tregs seemed to require a sufficiently high level of CD8 Tregs in order to become functional, while conversion was unlikely to be their major source. Our results indicated in addition that Foxp3 is not a sufficient marker for regulatory activity. CONCLUSIONS/SIGNIFICANCE: In this work, we unraveled the dynamics of the interplay between CD4, CD8 Tregs and effector T cells, using, for the first time, a mathematical-mechanistic perspective in the analysis of Treg kinetics. Furthermore, the results obtained from this interdisciplinary approach supported the notion that CD4 Tregs need to interact with CD8 Tregs in order to become functional. Finally, we generated predictions regarding the time-dependent function of Tregs, which can be further tested empirically in future work.
Asunto(s)
Antígenos CD4/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Comunicación Celular , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD28/metabolismo , Antígenos CD8/metabolismo , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Cinética , Subgrupos Linfocitarios/citología , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos Inmunológicos , Bazo/citologíaRESUMEN
CONTEXT: Autoimmune Addison's disease is thought to result from T cell mediated autoimmunity. Autoantibodies against the steroidogenic cytochrome P450 enzyme 21-hydroxylase (21OH) are found in most patients, and 21OH is therefore a likely target for antigen-specific T cells. OBJECTIVE: The aim was to study cellular immunity to 21OH and its associations with 21OH autoantibodies and human leukocyte antigen alleles in autoimmune Addison's disease. DESIGN/PATIENTS: Peripheral blood mononuclear cells were collected from 33 patients with autoimmune Addison's disease and 21 controls. Cellular proliferation and production of cytokines in response to stimulation with 21OH or 21OH-derived peptides were tested. RESULTS: Cellular proliferation (P = 0.0009) and secretion of interferon-gamma (P < 0.0001) in response to 21OH was significantly higher in patients compared to healthy controls and associated with the presence of 21OH autoantibodies (P = 0.0052). Furthermore, the 21OH-specific production of interferon-gamma was enhanced in the presence of 21OH autoantibodies. This effect was partially inhibited by antibodies against the Fc receptor for IgG, CD32. Moreover, mature dendritic cells proved superior to the other antigen-presenting cells in invoking cellular responses to 21OH. An association between cellular immunity to 21OH and the high-risk HLA genotype for Addison's disease, DRB1*0301-DQ2/DRB1*0404-DQ8, was observed (P = 0.0089). Finally, a significant association between the DRB1*0404-DQ8 haplotype and cellular responses to a 21OH-derived peptide predicted to bind to DRB1*0404 was detected (P = 0.0055). CONCLUSION: Patients with autoimmune Addison's disease have circulating 21OH-specific T cells, with amino acids 342-361 of 21OH possibly constituting a disease-specific epitope presented by HLA-DRB1*0404.
Asunto(s)
Insuficiencia Suprarrenal/enzimología , Enfermedades Autoinmunes/enzimología , Esteroide 21-Hidroxilasa/metabolismo , Linfocitos T/enzimología , Insuficiencia Suprarrenal/inmunología , Adulto , Anciano , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Autoanticuerpos/metabolismo , Adhesión Celular/fisiología , Proliferación Celular/efectos de los fármacos , Citocinas/biosíntesis , Células Dendríticas/metabolismo , Femenino , Antígenos HLA/genética , Humanos , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Receptores Fc/metabolismo , Esteroide 21-Hidroxilasa/inmunología , Adulto JovenRESUMEN
Systemic lupus erythematosus (SLE) is an autoimmune disease mediated by T and B cells. It is characterized by a variety of autoantibodies and systemic clinical manifestations. A tolerogenic peptide, designated hCDR1, ameliorated the serological and clinical manifestations of SLE in both spontaneous and induced models of lupus. In the present study, we evaluated the status of mature B cells in the bone marrow (BM) of SLE-afflicted mice, and determined the effect of treatment with the tolerogenic peptide hCDR1 on these cells. We demonstrate herein that mature B cells of the BM of SLE-afflicted (New Zealand Black x New Zealand White)F(1) mice were largely expanded, and that treatment with hCDR1 down-regulated this population. Moreover, treatment with hCDR1 inhibited the expression of the pathogenic cytokines [interferon-gamma and interleukin (IL)-10], whereas it up-regulated the expression of transforming growth factor-beta in the BM. Treatment with hCDR1 up-regulated the rates of apoptosis of mature B cells. The latter was associated with inhibited expression of the survival Bcl-xL gene and of IL-7 by BM cells. Furthermore, the addition of recombinant IL-7 abrogated the suppressive effects of hCDR1 on Bcl-xL in the BM cells and resulted in elevated levels of apoptosis. Hence, the down-regulated production of IL-7 contributes to the hCDR1-mediated apoptosis of mature B cells in the BM of SLE-afflicted mice.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Subgrupos de Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Interleucina-7/antagonistas & inhibidores , Lupus Eritematoso Sistémico/inmunología , Fragmentos de Péptidos/inmunología , Animales , Apoptosis/inmunología , Modelos Animales de Enfermedad , Regulación hacia Abajo/inmunología , Femenino , Tolerancia Inmunológica/inmunología , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-7/inmunología , Ratones , Ratones Endogámicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Bazo/inmunología , Factor de Crecimiento Transformador beta/biosíntesis , Regulación hacia Arriba/inmunologíaRESUMEN
Interferon-gamma (IFN-gamma) plays a pathogenic role in systemic lupus erythematosus (SLE). Uncontrolled IFN-gamma signaling may result from a deficiency in the negative regulator, namely, suppressor of cytokine signaling-1 (SOCS-1). We investigated the activation status of IFN-gamma signaling pathway in SLE-afflicted (New-Zealand-BlackxNew-Zealand-White)F1 mice and determined its responsiveness when treating with a tolerogenic peptide, hCDR1, which ameliorates SLE. SOCS-1 was suppressed and pSTAT1 was enhanced in spleen-derived cells from SLE-affected mice as compared with healthy controls. Treatment with hCDR1 reversed the expression of these two molecules in association with clinical amelioration. In vitro stimulation with IFN-gamma resulted in elevated levels of SOCS-1 in cells from both vehicle and hCDR1-treated mice but this effect reached significance only in cells of the latter group, which also exhibited reduced levels of pSTAT1. Thus, SOCS-1 is diminished in SLE-affected mice, and treatment with hCDR1 results in its up-regulation thereby restoring control of IFN-gamma signaling pathway.
Asunto(s)
Autoantígenos/uso terapéutico , Terapia de Inmunosupresión , Interferón gamma/inmunología , Lupus Eritematoso Sistémico/terapia , Proteínas del Tejido Nervioso/uso terapéutico , Factor de Transcripción STAT1/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Femenino , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/inmunología , Lupus Eritematoso Sistémico/inmunología , Ratones , Péptidos/uso terapéutico , Factor de Transcripción STAT1/agonistas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/agonistas , Proteínas Supresoras de la Señalización de Citocinas/antagonistas & inhibidores , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by dysregulation of cytokines, apoptosis, and B- and T-cell functions. The tolerogenic peptide, hCDR1 (Edratide), ameliorated the clinical manifestations of murine lupus via down-regulation of pro-inflammatory cytokines and apoptosis, up-regulation of the immunosuppressive cytokine TGF-beta, and the induction of regulatory T-cells. In the present study, gene expression was determined in peripheral blood mononuclear cells of 9 lupus patients that were treated for 26 weeks with either hCDR1 (five patients), or placebo (four patients). Disease activity was assessed by SLEDAI-2K and the BILAG scores. Treatment with hCDR1 significantly down-regulated the mRNA expression of the pathogenic cytokines IL-1beta, TNF-alpha, IFN-gamma, and IL-10, of BLyS (B-lymphocyte stimulator) and of the pro-apoptotic molecules caspase-3 and caspase-8. In contrast, the treatment up-regulated in vivo gene expression of both TGF-beta and FoxP3. Furthermore, hCDR1 treatment resulted in a significant decrease in SLEDAI-2K (from 8.0+/-2.45 to 4.4+/-1.67; P=0.02) and BILAG (from 8.2+/-2.7 to 3.6+/-2.9; P=0.03) scores. Thus, the tolerogenic peptide hCDR1, immunomodulates, in vivo, the expression of genes that play a role in SLE, consequently restoring the global immune dysregulation of lupus patients. Hence, hCDR1 has a potential role as a novel disease-specific treatment for lupus patients.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Factores de Transcripción Forkhead/metabolismo , Inmunoterapia , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/inmunología , Fragmentos de Péptidos/administración & dosificación , Adulto , Apoptosis/efectos de los fármacos , Caspasas/genética , Caspasas/inmunología , Caspasas/metabolismo , Regiones Determinantes de Complementariedad/inmunología , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Progresión de la Enfermedad , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/fisiopatología , Lupus Eritematoso Sistémico/terapia , Masculino , Persona de Mediana Edad , Autotolerancia/inmunología , Índice de Severidad de la Enfermedad , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Resultado del TratamientoRESUMEN
A tolerogenic peptide, hCDR1, ameliorated murine lupus via the upregulation of functional regulatory cells and by immunomodulating cytokine production. In the present study we analyzed the ability of hCDR1 to similarly affect gene expression and regulatory T cells when incubated with peripheral blood mononuclear cells (PBMC) of lupus patients. To this end, peripheral blood mononuclear cells (PBMC) of 11 lupus patients and five gender- and age-matched healthy controls were cultured with hCDR1 or a control peptide. Gene expression and regulatory T-cells were assessed. hCDR1 significantly downregulated interleukin (IL)-1beta, interferon (IFN)-gamma, and IL-10 gene expression. Furthermore, hCDR1 upregulated the expression of the anti-apoptotic Bcl-xL molecule and downregulated the pro-apoptotic caspase-3, resulting in reduced rates of apoptosis. hCDR1 increased the expression of transforming growth factor (TGF)-beta, FoxP3 and the negative regulators Foxj1 and Foxo3a. No significant effects were observed using a control peptide or when PBMC of healthy donors were incubated with hCDR1. The elevated gene expression of FoxP3 was due to hCDR1-induced upregulation of TGF-beta, resulting in an increase of CD4+CD25+FoxP3+ functional, regulatory cells. The ability of the regulatory cells to diminish IFN-gamma expression and to upregulate TGF-beta was abrogated after the addition of a neutralizing anti-CD25 antibody, confirming their role in the beneficial effects of hCDR1.