RESUMEN
Tolerance induction is central to the suppression of autoimmunity. Here, we engineered the preferential uptake of nano-conjugated autoantigens by spleen-resident macrophages to re-introduce self-tolerance and suppress autoimmunity. The brain autoantigen, myelin oligodendrocyte glycoprotein (MOG), was conjugated to 200 or 500â¯nm silica nanoparticles (SNP) and delivered to the spleen and liver-resident macrophages of experimental autoimmune encephalomyelitis (EAE) mice, used as a model of multiple sclerosis. MOG-SNP conjugates significantly reduced signs of EAE at a very low dose (50⯵g) compared to the higher dose (>800⯵g) of free-MOG. This was associated with reduced proliferation of splenocytes and pro-inflammatory cytokines secretion, decreased spinal cord inflammation, demyelination and axonal damage. Notably, biodegradable porous SNP showed an enhanced disease suppression assisted by elevated levels of regulatory T cells and programmed-death ligands (PD-L1/2) in splenic and lymph node cells. Our results demonstrate that targeting nano-conjugated autoantigens to tissue-resident macrophages in lymphoid organs can effectively suppress autoimmunity.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Nanopartículas , Animales , Autoinmunidad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patología , Glicoproteína Mielina-Oligodendrócito/uso terapéuticoRESUMEN
The anti-CD20 antibody ocrelizumab, approved for treatment of multiple sclerosis, leads to rapid elimination of B cells from the blood. The extent of B cell depletion and kinetics of their recovery in different immune compartments is largely unknown. Here, we studied how anti-CD20 treatment influences B cells in bone marrow, blood, lymph nodes, and spleen in models of experimental autoimmune encephalomyelitis (EAE). Anti-CD20 reduced mature B cells in all compartments examined, although a subpopulation of antigen-experienced B cells persisted in splenic follicles. Upon treatment cessation, CD20+ B cells simultaneously repopulated in bone marrow and spleen before their reappearance in blood. In EAE induced by native myelin oligodendrocyte glycoprotein (MOG), a model in which B cells are activated, B cell recovery was characterized by expansion of mature, differentiated cells containing a high frequency of myelin-reactive B cells with restricted B cell receptor gene diversity. Those B cells served as efficient antigen-presenting cells (APCs) for activation of myelin-specific T cells. In MOG peptide-induced EAE, a purely T cell-mediated model that does not require B cells, in contrast, reconstituting B cells exhibited a naive phenotype without efficient APC capacity. Our results demonstrate that distinct subpopulations of B cells differ in their sensitivity to anti-CD20 treatment and suggest that differentiated B cells persisting in secondary lymphoid organs contribute to the recovering B cell pool.
Asunto(s)
Antígenos CD20/inmunología , Linfocitos B/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Células de la Médula Ósea/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina/inmunología , Bazo/citología , Bazo/inmunologíaRESUMEN
Natalizumab, which binds very late antigen-4 (VLA-4), is a potent therapy for multiple sclerosis (MS). Studies have focused primarily upon its capacity to interfere with T-cell migration into the central nervous system (CNS). B cells are important in MS pathogenesis and express high levels of VLA-4. Here, we report that the selective inhibition of VLA-4 expression on B cells impedes CNS accumulation of B cells, and recruitment of Th17 cells and macrophages, and reduces susceptibility to experimental autoimmune encephalomyelitis. These results underscore the importance of B-cell VLA-4 expression in the pathogenesis of CNS autoimmunity and provide insight regarding mechanisms that may contribute to the benefit of natalizumab in MS, as well as candidate therapeutics that selectively target B cells.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos B/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Integrina alfa4beta1/deficiencia , Animales , Linfocitos B/metabolismo , Susceptibilidad a Enfermedades , Encefalomielitis Autoinmune Experimental/metabolismo , Humanos , Leucocitos/inmunología , Leucocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Th17/inmunología , Células Th17/metabolismoRESUMEN
BACKGROUND: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). In recent years, it has been found that cells such as human amnion epithelial cells (hAECs) have the ability to modulate immune responses in vitro and in vivo and can differentiate into multiple cell lineages. Accordingly, we investigated the immunoregulatory effects of hAECs as a potential therapy in an MS-like disease, EAE (experimental autoimmune encephalomyelitis), in mice. METHODS: Using flow cytometry, the phenotypic profile of hAECs from different donors was assessed. The immunomodulatory properties of hAECs were examined in vitro using antigen-specific and one-way mixed lymphocyte proliferation assays. The therapeutic efficacy of hAECs was examined using a relapsing-remitting model of EAE in NOD/Lt mice. T cell responsiveness, cytokine secretion, T regulatory, and T helper cell phenotype were determined in the peripheral lymphoid organs and CNS of these animals. RESULTS: In vitro, hAECs suppressed both specific and non-specific T cell proliferation, decreased pro-inflammatory cytokine production, and inhibited the activation of stimulated T cells. Furthermore, T cells retained their naïve phenotype when co-cultured with hAECs. In vivo studies revealed that hAECs not only suppressed the development of EAE but also prevented disease relapse in these mice. T cell responses and production of the pro-inflammatory cytokine interleukin (IL)-17A were reduced in hAEC-treated mice, and this was coupled with a significant increase in the number of peripheral T regulatory cells and naïve CD4+ T cells. Furthermore, increased proportions of Th2 cells in the peripheral lymphoid organs and within the CNS were observed. CONCLUSION: The therapeutic effect of hAECs is in part mediated by inducing an anti-inflammatory response within the CNS, demonstrating that hAECs hold promise for the treatment of autoimmune diseases like MS.
Asunto(s)
Amnios/citología , Amnios/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Células Epiteliales/citología , Células Epiteliales/inmunología , Terapia de Inmunosupresión/métodos , Amnios/trasplante , Animales , Proliferación Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Cultivadas , Sistema Nervioso Central/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/fisiopatología , Células Epiteliales/trasplante , Femenino , Humanos , Técnicas In Vitro , Tejido Linfoide/patología , Ratones , Ratones Endogámicos NOD , Fenotipo , Linfocitos T/patología , Linfocitos T Reguladores/patologíaRESUMEN
Induced pluripotent stem cell (iPSC)-derived neurospheres, which consist mainly of neural progenitors, are considered to be a good source of neural cells for transplantation in regenerative medicine. In this study, we have used lithium chloride, which is known to be a neuroprotective agent, in an iPSC-derived neurosphere model, and examined both the formation rate and size of the neurospheres as well as the proliferative and apoptotic status of their contents. Our results showed that lithium enhanced the formation and the sizes of the iPSC-derived neurospheres, increased the number of Ki67-positive proliferating cells, but reduced the number of the TUNEL-positive apoptotic cells. This increased number of Ki67 proliferating cells was secondary to the decreased apoptosis and not to the stimulation of cell cycle entry, as the expression of the proliferation marker cyclin D1 mRNA did not change after lithium treatment. Altogether, we suggest that lithium enhances the survival of neural progenitors and thus the quality of the iPSC-derived neurospheres, which may strengthen the prospect of using lithium-treated pluripotent cells and their derivatives in a clinical setting.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Cloruro de Litio/farmacología , Neuronas/efectos de los fármacos , Apoptosis/efectos de los fármacos , Células Cultivadas , Ciclina D1/genética , Humanos , Etiquetado Corte-Fin in Situ , Neuronas/citología , Neuronas/metabolismo , ARN Mensajero/genéticaRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is a murine model of multiple sclerosis, a chronic neurodegenerative and inflammatory autoimmune condition of the central nervous system (CNS). Pathology is driven by the infiltration of autoreactive CD4(+) lymphocytes into the CNS, where they attack neuronal sheaths causing ascending paralysis. We used an isotope-coded protein labeling approach to investigate the proteome of CD4(+) cells isolated from the spinal cord and brain of mice at various stages of EAE progression in two EAE disease models: PLP139-151-induced relapsing-remitting EAE and MOG35-55-induced chronic EAE, which emulate the two forms of human multiple sclerosis. A total of 1120 proteins were quantified across disease onset, peak-disease, and remission phases of disease, and of these 13 up-regulated proteins of interest were identified with functions relating to the regulation of inflammation, leukocyte adhesion and migration, tissue repair, and the regulation of transcription/translation. Proteins implicated in processes such as inflammation (S100A4 and S100A9) and tissue repair (annexin A1), which represent key events during EAE progression, were validated by quantitative PCR. This is the first targeted analysis of autoreactive cells purified from the CNS during EAE, highlighting fundamental CD4(+) cell-driven processes that occur during the initiation of relapse and remission stages of disease.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Sistema Nervioso Central/citología , Encefalomielitis Autoinmune Experimental/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Proteoma/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Adhesión Celular/genética , Movimiento Celular/genética , Sistema Nervioso Central/metabolismo , Cromatografía Líquida de Alta Presión , Femenino , Citometría de Flujo , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Proteína Proteolipídica de la Mielina/genética , Proteína Proteolipídica de la Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Toxina del Pertussis , Proteoma/genéticaRESUMEN
Interleukin (IL)-10 is an important immunoregulatory cytokine shown to impact inflammatory processes as manifested in patients with multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). Several lines of evidence indicate that the effectiveness of IL-10-based therapies may be dependent on the timing and mode of delivery. In the present study we engineered the expression of IL-10 in human adipose-derived mesenchymal stem cells (Adi-IL-10-MSCs) and transplanted these cells early in the disease course to mice with EAE. Adi-IL-10-MSCs transplanted via the intraperitoneal route prevented or delayed the development of EAE. This protective effect was associated with several anti-inflammatory response mechanisms, including a reduction in peripheral T-cell proliferative responses, a decrease in pro-inflammatory cytokine secretion as well as a preferential inhibition of Th17-mediated neuroinflammation. In vitro analyses revealed that Adi-IL-10-MSCs inhibited the phenotypic maturation, cytokine production and antigen presenting capacity of bone marrow-derived myeloid dendritic cells, suggesting that the mechanism of action may involve an indirect effect on pathogenic T-cells via the modulation of antigen presenting cell function. Collectively, these results suggest that early intervention with gene modified Adi-MSCs may be beneficial for the treatment of autoimmune diseases such as MS.
Asunto(s)
Adipocitos/metabolismo , Encefalomielitis Autoinmune Experimental/terapia , Interleucina-10/metabolismo , Células Madre Mesenquimatosas/metabolismo , Adipocitos/trasplante , Animales , Autoinmunidad/inmunología , Diferenciación Celular/inmunología , Proliferación Celular , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Humanos , Trasplante de Células Madre Mesenquimatosas/métodos , Ratones , Linfocitos T/inmunologíaRESUMEN
Multiple sclerosis involves demyelination and axonal degeneration of the central nervous system. The molecular mechanisms of axonal degeneration are relatively unexplored in both multiple sclerosis and its mouse model, experimental autoimmune encephalomyelitis. We previously reported that targeting the axonal growth inhibitor, Nogo-A, may protect against neurodegeneration in experimental autoimmune encephalomyelitis; however, the mechanism by which this occurs is unclear. We now show that the collapsin response mediator protein 2 (CRMP-2), an important tubulin-associated protein that regulates axonal growth, is phosphorylated and hence inhibited during the progression of experimental autoimmune encephalomyelitis in degenerating axons. The phosphorylated form of CRMP-2 (pThr555CRMP-2) is localized to spinal cord neurons and axons in chronic-active multiple sclerosis lesions. Specifically, pThr555CRMP-2 is implicated to be Nogo-66 receptor 1 (NgR1)-dependent, since myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced NgR1 knock-out (ngr1(-)(/)(-)) mice display a reduced experimental autoimmune encephalomyelitis disease progression, without a deregulation of ngr1(-)(/)(-) MOG(35-55)-reactive lymphocytes and monocytes. The limitation of axonal degeneration/loss in experimental autoimmune encephalomyelitis-induced ngr1(-)(/)(-) mice is associated with lower levels of pThr555CRMP-2 in the spinal cord and optic nerve during experimental autoimmune encephalomyelitis. Furthermore, transduction of retinal ganglion cells with an adeno-associated viral vector encoding a site-specific mutant T555ACRMP-2 construct, limits optic nerve axonal degeneration occurring at peak stage of experimental autoimmune encephalomyelitis. Therapeutic administration of the anti-Nogo(623-640) antibody during the course of experimental autoimmune encephalomyelitis, associated with an improved clinical outcome, is demonstrated to abrogate the protein levels of pThr555CRMP-2 in the spinal cord and improve pathological outcome. We conclude that phosphorylation of CRMP-2 may be downstream of NgR1 activation and play a role in axonal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis. Blockade of Nogo-A/NgR1 interaction may serve as a viable therapeutic target in multiple sclerosis.
Asunto(s)
Axones/metabolismo , Encefalomielitis Autoinmune Experimental/complicaciones , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Esclerosis Múltiple/patología , Degeneración Nerviosa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Adulto , Análisis de Varianza , Animales , Anticuerpos/uso terapéutico , Axones/patología , Axones/ultraestructura , Complejo CD3/metabolismo , Línea Celular Tumoral , Enfermedades Desmielinizantes/etiología , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/deficiencia , Proteínas Ligadas a GPI/inmunología , Regulación de la Expresión Génica/genética , Glicoproteínas/efectos adversos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Esclerosis Múltiple/complicaciones , Mutación/genética , Proteínas de la Mielina/antagonistas & inhibidores , Proteínas de la Mielina/deficiencia , Proteínas de la Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , Degeneración Nerviosa/etiología , Proteínas del Tejido Nervioso/genética , Neuroblastoma/patología , Proteínas de Neurofilamentos/metabolismo , Receptor Nogo 1 , Nervio Óptico/metabolismo , Nervio Óptico/patología , Fragmentos de Péptidos/efectos adversos , Fosforilación , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/inmunología , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patologíaRESUMEN
Neurodegenerative diseases, such as multiple sclerosis represent global health issues. Accordingly, there is an urgent need to understand the pathogenesis of this and other central nervous system disorders, so that more effective therapeutics can be developed. Cerebrospinal fluid is a potential source of important reporter molecules released from various cell types as a result of central nervous system pathology. Here, we report the development of an unbiased approach for the detection of reactive cerebrospinal fluid molecules and target brain proteins from patients with multiple sclerosis. To help identify molecules that may serve as clinical biomarkers for multiple sclerosis, we have biotinylated proteins present in the cerebrospinal fluid and tested their reactivity against brain homogenate as well as myelin and myelin-axolemmal complexes. Proteins were separated by two-dimensional gel electrophoresis, blotted onto membranes and probed separately with biotinylated unprocessed cerebrospinal fluid samples. Protein spots that reacted to two or more multiple sclerosis-cerebrospinal fluids were further analyzed by matrix assisted laser desorption ionization-time-of-flight time-of-flight mass spectrometry. In addition to previously reported proteins found in multiple sclerosis cerebrospinal fluid, such as αß crystallin, enolase, and 14-3-3-protein, we have identified several additional molecules involved in mitochondrial and energy metabolism, myelin gene expression and/or cytoskeletal organization. These include aspartate aminotransferase, cyclophilin-A, quaking protein, collapsin response mediator protein-2, ubiquitin carboxy-terminal hydrolase L1, and cofilin. To further validate these findings, the cellular expression pattern of collapsin response mediator protein-2 and ubiquitin carboxy-terminal hydrolase L1 were investigated in human chronic-active MS lesions by immunohistochemistry. The observation that in multiple sclerosis lesions phosphorylated collapsin response mediator protein-2 was increased, whereas Ubiquitin carboxy-terminal hydrolase L1 was down-regulated, not only highlights the importance of these molecules in the pathology of this disease, but also illustrates the use of our approach in attempting to decipher the complex pathological processes leading to multiple sclerosis and other neurodegenerative diseases.
Asunto(s)
Proteínas del Líquido Cefalorraquídeo/química , Esclerosis Múltiple/líquido cefalorraquídeo , Adulto , Anciano , Axones/metabolismo , Biotinilación , Western Blotting , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Proteínas del Líquido Cefalorraquídeo/inmunología , Proteínas del Líquido Cefalorraquídeo/metabolismo , Electroforesis en Gel Bidimensional , Metabolismo Energético , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Vaina de Mielina/enzimología , Vaina de Mielina/inmunología , Vaina de Mielina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteómica , Ubiquitina Tiolesterasa/metabolismoRESUMEN
In the last two decades the field of infrared spectroscopy has seen enormous advances in both instrumentation and the development of bioinformatic methods for spectral analysis, allowing the examination of a large variety of healthy and diseased samples, including biological fluids, isolated cells, whole tissues, and tissue sections. The non-destructive nature of the technique, together with the ability to directly probe biochemical changes without the addition of stains or contrast agents, enables a range of complementary analyses. This review focuses on the application of Fourier transform infrared (FTIR) microspectroscopy to analyse central nervous system tissues, with the aim of understanding the biochemical and structural changes associated with neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, transmissible spongiform encephalopathies, multiple sclerosis, as well as brain tumours. Modern biospectroscopic methods that combine FTIR microspectroscopy with bioinformatic analysis constitute a powerful new methodology that can discriminate pathology from normal healthy tissue in a rapid, unbiased fashion, with high sensitivity and specificity. Notably, the ability to detect protein secondary structural changes associated with Alzheimer's plaques, neurons in Parkinson's disease, and in some spectra from meningioma, as well as in the animal models of Alzheimer's disease, transmissible spongiform encephalopathies, and multiple sclerosis, illustrates the power of this technology. The capacity to offer insight into the biochemical and structural changes underpinning aetio-pathogenesis of diseases in tissues provides both a platform to investigate early pathologies occurring in a variety of experimentally induced and naturally occurring central nervous system diseases, and the potential to evaluate new therapeutic approaches.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , HumanosRESUMEN
Demyelination coincides with numerous changes of gene expression in the central nervous system (CNS). Cystatin F, which is a papain-like lysosomal cysteine proteinase inhibitor that is normally expressed by immune cells and not in the brain, is massively induced in the CNS during acute demyelination. We found that microglia, which are monocyte/macrophage-lineage cells in the CNS, express cystatin F only during demyelination. By using several demyelinating animal models and the spinal cord tissues from multiple sclerosis (MS) patients, we examined spatiotemporal expression pattern of cystatin F by in situ hybridization and immunohistochemistry. We found that the timing of cystatin F induction matches with ongoing demyelination, and the places with cystatin F expression overlapped with the remyelinating area. Most interestingly, cystatin F induction ceased in chronic demyelination, in which remyelinating ability was lost. These findings demonstrate that the expression of cystatin F indicates the occurrence of ongoing demyelination/remyelination and the absence of cystatin F expression indicates the cessation of remyelination in the demyelinating area.
Asunto(s)
Cistatinas/biosíntesis , Enfermedades Autoinmunes Desmielinizantes SNC/metabolismo , Microglía/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Animales , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/deficiencia , Biomarcadores de Tumor/metabolismo , Células Cultivadas , Enfermedad Crónica , Cistatinas/deficiencia , Cistatinas/metabolismo , Enfermedades Autoinmunes Desmielinizantes SNC/genética , Enfermedades Autoinmunes Desmielinizantes SNC/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Mutantes Neurológicos , Ratones Transgénicos , Microglía/patología , Fibras Nerviosas Mielínicas/patología , Regeneración Nerviosa/genética , Recuperación de la Función/genéticaRESUMEN
OBJECTIVE: Clinical studies indicate that anti-CD20 B-cell depletion may be an effective multiple sclerosis (MS) therapy. We investigated mechanisms of anti-CD20-mediated immune modulation using 2 paradigms of experimental autoimmune encephalomyelitis (EAE). METHODS: Murine EAE was induced by recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or MOG peptide (p)35-55, which does not require B cells. RESULTS: In EAE induced by rMOG, B cells became activated and, when serving as antigen-presenting cells (APCs), promoted differentiation of proinflammatory MOG-specific Th1 and Th17 cells. B-cell depletion prevented or reversed established rMOG-induced EAE, which was associated with less central nervous system (CNS) inflammation, elimination of meningeal B cells, and reduction of MOG-specific Th1 and Th17 cells. In contrast, in MOG p35-55-induced EAE, B cells did not become activated or efficiently polarize proinflammatory MOG-specific T cells, similar to naive B cells. In this setting, anti-CD20 treatment exacerbated EAE, and did not impede development of Th1 or Th17 cells. Irrespective of the EAE model used, B-cell depletion reduced the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), and increased the proinflammatory polarizing capacity of remaining myeloid APCs. INTERPRETATION: Our study highlights distinct roles for B cells in CNS autoimmunity. Clinical benefit from anti-CD20 treatment may relate to inhibition of proinflammatory B cell APC function. In certain clinical settings, however, elimination of unactivated B cells, which participate in regulation of T cells and other APC, may be undesirable. Differences in immune responses to MOG protein and peptide may be important considerations when choosing an EAE model for testing novel B cell-targeting agents for MS.
Asunto(s)
Anticuerpos/uso terapéutico , Antígenos CD20/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/fisiopatología , Activación de Linfocitos/inmunología , Animales , Antígenos CD20/genética , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Citometría de Flujo/métodos , Factores de Transcripción Forkhead/metabolismo , Glicoproteínas/efectos adversos , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos/efectos adversos , Estadísticas no Paramétricas , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Despite progress in understanding immunogenetic aspects of this disease, the mechanisms involved in lesion formation are unknown. To gain new insights into the neuropathology of MS, we used an innovative integration of Fourier transform infrared (FT-IR) microspectroscopy, bioinformatics, and a synchrotron light source to analyze macromolecular changes in the CNS during the course and prevention of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. We report that subtle chemical and structural changes not observed by conventional histology were detected before the onset of clinical signs of EAE. Moreover, trained artificial neural networks (ANNs) could discriminate, with excellent sensitivity and specificity, pathology from surrounding tissues and the early stage of the disease progression. Notably, we show that this novel measurement platform can detect characteristic differences in biochemical composition of lesion pathology in animals partially protected against EAE by vaccination with Nogo-A, an inhibitor of neural outgrowth, demonstrating the potential for automated screening and evaluation of new therapeutic agents.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/terapia , Animales , Automatización , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Femenino , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple , Proteínas de la Mielina/inmunología , Proteínas de la Mielina/uso terapéutico , Redes Neurales de la Computación , Proteínas Nogo , Péptidos/inmunología , Péptidos/uso terapéutico , Sensibilidad y Especificidad , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Factores de Tiempo , VacunaciónRESUMEN
Autoimmune diseases are incurable. We have hypothesized that these diseases can be cured by the transplantation of bone marrow (BM) stem cells that have been genetically engineered to express self-Ag. Here we have tested this hypothesis in experimental autoimmune encephalomyelitis (EAE) induced by the self-Ag myelin oligodendrocyte glycoprotein (MOG). We show that, in mice, transplantation of BM genetically modified to express MOG prevented the induction and progression of EAE, and combined with antecedent corticosteroid treatment, induced long-term remission of established disease. Mice remained resistant to EAE development upon subsequent rechallenge with MOG. Transfer of BM from these mice rendered recipients resistant to EAE. Splenocytes from these mice failed to proliferate or produce IL-17, IFN-gamma, and GM-CSF in response to MOG(35-55) peptide stimulation and they failed to produce MOG autoantibody. Mechanistically, we demonstrated in vivo reduction in development of CD4(+) MOG(35-55)-specific thymocytes, indicative of clonal deletion with no evidence for selection of Ag-specific regulatory T cells. These findings validate our hypothesis that transplantation of genetically modified BM expressing disease-causative self-Ag provides a curative approach by clonal deletion of disease-causative self-reactive T cells.
Asunto(s)
Autoantígenos/inmunología , Trasplante de Médula Ósea , Supresión Clonal/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Glicoproteínas/inmunología , Tolerancia Inmunológica/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T Reguladores/inmunología , Corticoesteroides/farmacología , Animales , Autoanticuerpos/genética , Autoanticuerpos/inmunología , Autoantígenos/genética , Supresión Clonal/efectos de los fármacos , Supresión Clonal/genética , Citocinas/genética , Citocinas/inmunología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/terapia , Femenino , Expresión Génica/genética , Expresión Génica/inmunología , Glicoproteínas/genética , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/genética , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Ratones , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos/genética , Timo/inmunología , Transducción GenéticaRESUMEN
Inhibitors associated with CNS myelin are thought to be important in the failure of axons to regenerate after spinal cord injury and in other neurodegenerative disorders. Here we show that targeting the CNS-specific inhibitor of neurite outgrowth Nogo A by active immunization blunts clinical signs, demyelination and axonal damage associated with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Mice vaccinated against Nogo A produce Nogo-specific antibodies that block the neurite outgrowth inhibitory activity associated with CNS myelin in vitro. Passive immunization with anti-Nogo IgGs also suppresses EAE. Our results identify Nogo A as an important determinant of the development of EAE and suggest that its blockade may help to maintain and/or to restore the neuronal integrity of the CNS after autoimmune insult in diseases such as MS. Our finding that Nogo A is involved in CNS autoimmune demyelination indicates that this molecule may have a far more complex role than has been previously anticipated.
Asunto(s)
Encefalomielitis Aguda Diseminada , Proteínas de la Mielina/fisiología , Neuritas/fisiología , Animales , Animales Recién Nacidos , Anticuerpos/uso terapéutico , Células Cultivadas , Citocinas/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Encefalomielitis Aguda Diseminada/metabolismo , Encefalomielitis Aguda Diseminada/fisiopatología , Encefalomielitis Aguda Diseminada/terapia , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple , Proteínas de la Mielina/genética , Proteínas de la Mielina/inmunología , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , Proteínas Nogo , Ratas , Ratas Sprague-Dawley , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/fisiología , Tinción con Nitrato de Plata/métodos , Médula Espinal/metabolismo , Médula Espinal/patología , Linfocitos T/inmunología , Linfocitos T/fisiología , Factores de Tiempo , Vacunación/efectos adversosRESUMEN
The diversity of autoimmune responses poses a formidable challenge to the development of antigen-specific tolerizing therapy. We developed 'myelin proteome' microarrays to profile the evolution of autoantibody responses in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS). Increased diversity of autoantibody responses in acute EAE predicted a more severe clinical course. Chronic EAE was associated with previously undescribed extensive intra- and intermolecular epitope spreading of autoreactive B-cell responses. Array analysis of autoantigens targeted in acute EAE was used to guide the choice of autoantigen cDNAs to be incorporated into expression plasmids so as to generate tolerizing vaccines. Tolerizing DNA vaccines encoding a greater number of array-determined myelin targets proved superior in treating established EAE and reduced epitope spreading of autoreactive B-cell responses. Proteomic monitoring of autoantibody responses provides a useful approach to monitor autoimmune disease and to develop and tailor disease- and patient-specific tolerizing DNA vaccines.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Inmunoensayo/métodos , Vaina de Mielina/inmunología , Análisis por Matrices de Proteínas/métodos , Vacunas de ADN/inmunología , Vacunas de ADN/uso terapéutico , Animales , Tolerancia a Medicamentos , Encefalomielitis Autoinmune Experimental/diagnóstico , Ratones , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Mapeo de Interacción de Proteínas/métodos , Resultado del TratamientoRESUMEN
OBJECTIVE: To investigate the role of very late antigen-4 (VLA-4) on regulatory B cells (Breg) in CNS autoimmune disease. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in mice selectively deficient for VLA-4 on B cells (CD19cre/α4(f/f)) by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p)35-55 or recombinant human (rh) MOG protein. B-cell and T-cell populations were examined by flow cytometry and immunohistochemistry. Breg were evaluated by intracellular IL-10 staining of B cells and, secondly, by coexpression of CD1d and CD5. RESULTS: As previously reported, EAE was less severe in B-cell VLA-4-deficient vs control CD19cre mice when induced by rhMOG, a model that is B-cell-dependent and leads to efficient B-cell activation and antibody production. Paradoxically, B-cell VLA-4-deficient mice developed more severe clinical disease than control mice when EAE was induced with MOG p35-55, a B-cell-independent encephalitogen that does not efficiently activate B cells. Peripheral T-cell and humoral immune responses were not altered in B-cell VLA-4-deficient mice. In MOG p35-55-induced EAE, B-cell VLA-4 deficiency reduced CNS accumulation of B but not T cells. Breg were detected in the CNS of control mice with MOG p35-55-induced EAE. However, more severe EAE in B-cell VLA-4-deficient mice was associated with virtual absence of CNS Breg. CONCLUSIONS: Our results demonstrate that CNS accumulation of Breg is VLA-4-dependent and suggest that Breg may contribute to regulation of CNS autoimmunity in situ. These observations underscore the need to choose the appropriate encephalitogen when studying how B cells contribute to pathogenesis or regulation of CNS autoimmunity.
RESUMEN
OBJECTIVE: To evaluate the influence of oral laquinimod, a candidate multiple sclerosis (MS) treatment, on induction of T follicular helper cells, development of meningeal B cell aggregates, and clinical disease in a spontaneous B cell-dependent MS model. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by immunization with recombinant myelin oligodendrocyte glycoprotein (rMOG) protein. Spontaneous EAE was evaluated in C57BL/6 MOG p35-55-specific T cell receptor transgenic (2D2) × MOG-specific immunoglobulin (Ig)H-chain knock-in (IgHMOG-ki [Th]) mice. Laquinimod was administered orally. T cell and B cell populations were examined by flow cytometry and immunohistochemistry. RESULTS: Oral laquinimod treatment (1) reduced CD11c+CD4+ dendritic cells, (2) inhibited expansion of PD-1+CXCR5+BCL6+ T follicular helper and interleukin (IL)-21-producing activated CD4+CD44+ T cells, (3) suppressed B cell CD40 expression, (4) diminished formation of Fas+GL7+ germinal center B cells, and (5) inhibited development of MOG-specific IgG. Laquinimod treatment not only prevented rMOG-induced EAE, but also inhibited development of spontaneous EAE and the formation of meningeal B cell aggregates. Disability progression was prevented when laquinimod treatment was initiated after mice developed paralysis. Treatment of spontaneous EAE with laquinimod was also associated with increases in CD4+CD25hiFoxp3+ and CD4+CD25+IL-10+ regulatory T cells. CONCLUSIONS: Our observations that laquinimod modulates myelin antigen-specific B cell immune responses and suppresses both development of meningeal B cell aggregates and disability progression in spontaneous EAE should provide insight regarding the potential application of laquinimod to MS treatment. Results of this investigation demonstrate how the 2D2 × Th spontaneous EAE model can be used successfully for preclinical evaluation of a candidate MS treatment.
RESUMEN
Studies in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), have shown that regulatory B cells modulate the course of the disease via the production of suppressive cytokines. While data indicate a role for transforming growth factor (TGF)-ß1 expression in regulatory B cell functions, this mechanism has not yet been tested in autoimmune neuroinflammation. Transgenic mice deficient for TGF-ß1 expression in B cells (B-TGF-ß1-/-) were tested in EAE induced by recombinant mouse myelin oligodendrocyte glycoprotein (rmMOG). In this model, B-TGF-ß1-/- mice showed an earlier onset of neurologic impairment compared to their littermate controls. Exacerbated EAE susceptibility in B-TGF-ß1-/- mice was associated with augmented CNS T helper (Th)1/17 responses. Moreover, selective B cell TGF-ß1-deficiency increased the frequencies and activation of myeloid dendritic cells, potent professional antigen-presenting cells (APCs), suggesting that B cell-derived TGF-ß1 can constrain Th1/17 responses through inhibition of APC activity. Collectively our data suggest that B cells can down-regulate the function of APCs, and in turn encephalitogenic Th1/17 responses, via TGF-ß1, findings that may be relevant to B cell-targeted therapies.
Asunto(s)
Linfocitos B Reguladores/inmunología , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/genética , Células TH1/inmunología , Células Th17/inmunología , Factor de Crecimiento Transformador beta1/genética , Animales , Linfocitos B Reguladores/patología , Comunicación Celular/inmunología , Células Dendríticas/patología , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Eliminación de Gen , Expresión Génica , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Células TH1/patología , Células Th17/patología , Factor de Crecimiento Transformador beta1/inmunologíaRESUMEN
Experimental autoimmune encephalomyelitis (EAE) models multiple sclerosis (MS) and is characterized by marked mononuclear cell influx in the brain. Several studies have demonstrated a role for chemokines during EAE. It remains to be determined whether these mediators modulate EAE primarily by mediating leukocyte influx into the CNS or by modifying lymphocyte activation and/or trafficking into lymphoid organs. After induction of EAE with MOG(35-55), leukocyte recruitment peaked on day 14 and correlated with symptom onset, TNF-alpha production and production of CCL2 and CCL5. Levels of CXCL-10 and CCL3 were not different from control animals. Using intravital microscopy, we demonstrated that leukocyte rolling and adhesion also peaked at day 14. Treatment with anti-CCL2 or anti-CCL5 antibodies just prior to the intravital microscopy prevented leukocyte adhesion, but not rolling. Our data suggest that induction of leukocyte adhesion to the brain microvasculature is an important mechanism by which CCL2 and CCL5 participate in the pathophysiology of EAE.