RESUMEN
After natalizumab (NAT) cessation, some multiple sclerosis (MS) patients experience a severe disease rebound. The rebound pathophysiology is still unclear; however, it has been linked to interleukin-17-producing T-helper (Th17) cells. We demonstrate that during NAT treatment, MCAM+CCR6+Th17 cells gradually acquire a pathogenic profile, including proinflammatory cytokine production, pathogenic transcriptional signatures, brain endothelial barrier impairment, and oligodendrocyte damage via induction of apoptotic pathways. This is accompanied by an increase in Th17 cell frequencies in the cerebrospinal fluid of NAT-treated patients. Notably, Th17 cells derived from NAT-treated patients, who later developed a disease rebound upon treatment cessation, displayed a distinct transcriptional pathogenicity profile associated with altered migratory properties. Accordingly, increased brain infiltration of patient Th17 cells was illustrated in a humanized mouse model and brain histology from a rebound patient. Therefore, peripheral blood-accumulated MCAM+CCR6+Th17 cells might be involved in rebound pathophysiology, and monitoring of changes in Th17 cell pathogenicity in patients before/during NAT treatment cessation might enable rebound risk assessment in the future.
Asunto(s)
Esclerosis Múltiple , Células Th17 , Animales , Ratones , Natalizumab/farmacología , Natalizumab/uso terapéutico , Virulencia , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/líquido cefalorraquídeo , EncéfaloRESUMEN
Aging affects all cell types in the CNS and plays an important role in CNS diseases. However, the underlying molecular mechanisms driving these age-associated changes and their contribution to diseases are only poorly understood. The white matter in the aging brain as well as in diseases, such as Multiple sclerosis is characterized by subtle abnormalities in myelin sheaths and paranodes, suggesting that oligodendrocytes, the myelin-maintaining cells of the CNS, lose the capacity to preserve a proper myelin structure and potentially function in age and certain diseases. Here, we made use of directly converted oligodendrocytes (dchiOL) from young, adult and old human donors to study age-associated changes. dchiOL from all three age groups differentiated in an comparable manner into O4 + immature oligodendrocytes, but the proportion of MBP + mature dchiOL decreased with increasing donor age. This was associated with an increased ROS production and upregulation of cellular senescence markers such as CDKN1A, CDKN2A in old dchiOL. Comparison of the transcriptomic profiles of dchiOL from adult and old donors revealed 1324 differentially regulated genes with limited overlap with transcriptomic profiles of the donors' fibroblasts or published data sets from directly converted human neurons or primary rodent oligodendroglial lineage cells. Methylome analyses of dchiOL and human white matter tissue samples demonstrate that chronological and epigenetic age correlate in CNS white matter as well as in dchiOL and resulted in the identification of an age-specific epigenetic signature. Furthermore, we observed an accelerated epigenetic aging of the myelinated, normal appearing white matter of multiple sclerosis (MS) patients compared to healthy individuals. Impaired differentiation and upregulation of cellular senescence markers could be induced in young dchiOL in vitro using supernatants from pro-inflammatory microglia. In summary, our data suggest that physiological aging as well as inflammation-induced cellular senescence contribute to oligodendroglial pathology in inflammatory demyelinating diseases such as MS.
Asunto(s)
Envejecimiento , Senescencia Celular , Esclerosis Múltiple , Oligodendroglía , Humanos , Oligodendroglía/patología , Oligodendroglía/metabolismo , Senescencia Celular/fisiología , Envejecimiento/patología , Esclerosis Múltiple/patología , Esclerosis Múltiple/metabolismo , Adulto , Anciano , Persona de Mediana Edad , Masculino , Femenino , Adulto Joven , Inflamación/patología , Inflamación/metabolismo , Sustancia Blanca/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la CiclinaRESUMEN
Multiple sclerosis (MS) is a focal inflammatory and demyelinating disease. The inflammatory infiltrates consist of macrophages/microglia, T and B cells. Remyelination (RM) is an endogenous repair process which frequently fails in MS patients. In earlier studies, T cells either promoted or impaired RM. Here, we used the combined cuprizone/MOG-EAE model to further dissect the functional role of T cells for RM. The combination of MOG immunization with cuprizone feeding targeted T cells to the corpus callosum and increased the extent of axonal injury. Global gene expression analyses demonstrated significant changes in the inflammatory environment; however, additional MOG immunization did not alter the course of RM. Our results suggest that the inflammatory environment in the combined model affects axons and oligodendrocytes differently and that oligodendroglial lineage cells might be less susceptible to T cell mediated injury.
Asunto(s)
Enfermedades Desmielinizantes , Esclerosis Múltiple , Remielinización , Animales , Ratones , Axones , Cuerpo Calloso/metabolismo , Cuprizona/toxicidad , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Esclerosis Múltiple/metabolismo , Vaina de Mielina/fisiología , Oligodendroglía/metabolismo , Remielinización/inmunología , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
Multiple sclerosis (MS) is the most frequent inflammatory and demyelinating disease of the CNS. The disease course in MS is highly variable and driven by a combination of relapse-driven disease activity and relapse-independent disease progression. The formation of new focal demyelinating lesions is associated with clinical relapses; however, the pathological mechanisms driving disease progression are less well understood. Current concepts suggest that ongoing focal and diffuse inflammation within the CNS in combination with an age-associated failure of compensatory and repair mechanisms contribute to disease progression. The aim of our study was to characterize the diffuse microglia activation in periplaque white matter (PPWM) of MS patients, to identify factors modulating its extent and to determine its potential correlation with loss or preservation of oligodendrocytes. We analyzed microglial and oligodendroglial numbers in PPWM in a cohort of 96 tissue blocks from 32 MS patients containing 100 lesions as well as a control cohort (n = 37). Microglia activation in PPWM was dependent on patient age, proximity to lesion, lesion type, and to a lesser degree on sex. Oligodendrocyte numbers were decreased in PPWM; however, increased microglia densities did not correlate with lower oligodendroglial cell counts, indicating that diffuse microglia activation is not sufficient to drive oligodendroglial loss in PPWM. In summary, our findings support the notion of the close relationship between focal and diffuse inflammation in MS and that age is an important modulator of MS pathology.
Asunto(s)
Esclerosis Múltiple , Sustancia Blanca , Humanos , Esclerosis Múltiple/patología , Sustancia Blanca/patología , Microglía/patología , Oligodendroglía/patología , Progresión de la Enfermedad , Inflamación/patología , RecurrenciaRESUMEN
A consensus is yet to be reached regarding the exact prevalence of epileptic seizures or epilepsy in multiple sclerosis (MS). In addition, the underlying pathophysiological basis of the reciprocal interaction among neuroinflammation, demyelination, and epilepsy remains unclear. Therefore, a better understanding of cellular and network mechanisms linking these pathologies is needed. Cuprizone-induced general demyelination in rodents is a valuable model for studying MS pathologies. Here, we studied the relationship among epileptic activity, loss of myelin, and pro-inflammatory cytokines by inducing acute, generalized demyelination in a genetic mouse model of human absence epilepsy, C3H/HeJ mice. Both cellular and network mechanisms were studied using in vivo and in vitro electrophysiological techniques. We found that acute, generalized demyelination in C3H/HeJ mice resulted in a lower number of spike-wave discharges, increased cortical theta oscillations, and reduction of slow rhythmic intrathalamic burst activity. In addition, generalized demyelination resulted in a significant reduction in the amplitude of the hyperpolarization-activated inward current (Ih) in thalamic relay cells, which was accompanied by lower surface expression of hyperpolarization-activated, cyclic nucleotide-gated channels, and the phosphorylated form of TRIP8b (pS237-TRIP8b). We suggest that demyelination-related changes in thalamic Ih may be one of the factors defining the prevalence of seizures in MS.
Asunto(s)
Enfermedades Desmielinizantes , Epilepsia Tipo Ausencia , Animales , Corteza Cerebral/fisiología , Cuprizona/metabolismo , Cuprizona/toxicidad , Citocinas/metabolismo , Enfermedades Desmielinizantes/inducido químicamente , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Ratones , Ratones Endogámicos C3H , Neuronas/fisiología , Nucleótidos Cíclicos/metabolismo , Convulsiones , Tálamo/fisiologíaRESUMEN
A close interaction between gut immune responses and distant organ-specific autoimmunity including the CNS in multiple sclerosis has been established in recent years. This so-called gut-CNS axis can be shaped by dietary factors, either directly or via indirect modulation of the gut microbiome and its metabolites. Here, we report that dietary supplementation with conjugated linoleic acid, a mixture of linoleic acid isomers, ameliorates CNS autoimmunity in a spontaneous mouse model of multiple sclerosis, accompanied by an attenuation of intestinal barrier dysfunction and inflammation as well as an increase in intestinal myeloid-derived suppressor-like cells. Protective effects of dietary supplementation with conjugated linoleic acid were not abrogated upon microbiota eradication, indicating that the microbiome is dispensable for these conjugated linoleic acid-mediated effects. Instead, we observed a range of direct anti-inflammatory effects of conjugated linoleic acid on murine myeloid cells including an enhanced IL10 production and the capacity to suppress T-cell proliferation. Finally, in a human pilot study in patients with multiple sclerosis (n = 15, under first-line disease-modifying treatment), dietary conjugated linoleic acid-supplementation for 6 months significantly enhanced the anti-inflammatory profiles as well as functional signatures of circulating myeloid cells. Together, our results identify conjugated linoleic acid as a potent modulator of the gut-CNS axis by targeting myeloid cells in the intestine, which in turn control encephalitogenic T-cell responses.
Asunto(s)
Suplementos Dietéticos , Enteritis/patología , Ácidos Linoleicos Conjugados/farmacología , Monocitos/inmunología , Esclerosis Múltiple Recurrente-Remitente/patología , Adulto , Animales , Autoinmunidad/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Enteritis/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Esclerosis Múltiple Recurrente-Remitente/inmunología , Proyectos Piloto , Prueba de Estudio ConceptualRESUMEN
Dimethyl fumarate, an approved treatment for relapsing-remitting multiple sclerosis, exerts pleiotropic effects on immune cells as well as CNS resident cells. Here, we show that dimethyl fumarate exerts a profound alteration of the metabolic profile of human CD4+ as well as CD8+ T cells and restricts their antioxidative capacities by decreasing intracellular levels of the reactive oxygen species scavenger glutathione. This causes an increase in mitochondrial reactive oxygen species levels accompanied by an enhanced mitochondrial stress response, ultimately leading to impaired mitochondrial function. Enhanced mitochondrial reactive oxygen species levels not only result in enhanced T-cell apoptosis in vitro as well as in dimethyl fumarate-treated patients, but are key for the well-known immunomodulatory effects of dimethyl fumarate both in vitro and in an animal model of multiple sclerosis, i.e. experimental autoimmune encephalomyelitis. Indeed, dimethyl fumarate immune-modulatory effects on T cells were completely abrogated by pharmacological interference of mitochondrial reactive oxygen species production. These data shed new light on dimethyl fumarate as bona fide immune-metabolic drug that targets the intracellular stress response in activated T cells, thereby restricting mitochondrial function and energetic capacity, providing novel insight into the role of oxidative stress in modulating cellular immune responses and T cell-mediated autoimmunity.
Asunto(s)
Antioxidantes/farmacología , Autoinmunidad/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Dimetilfumarato/farmacología , Inmunosupresores/farmacología , Adulto , Animales , Antioxidantes/uso terapéutico , Autoinmunidad/fisiología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Estudios de Cohortes , Dimetilfumarato/uso terapéutico , Femenino , Humanos , Inmunosupresores/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/inmunología , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Adulto JovenRESUMEN
Pericytes at the blood-brain barrier (BBB) are located between the tight endothelial cell layer of the blood vessels and astrocytic endfeet. They contribute to central nervous system (CNS) homeostasis by regulating BBB development and maintenance. Loss of pericytes results in increased numbers of infiltrating immune cells in the CNS in experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis (MS). However, little is known about their competence to modulate immune cell activation or function in CNS autoimmunity. To evaluate the capacity of pericytes to directly interact with T cells in an antigen-specific fashion and potentially (re)shape their function, we depleted major histocompatibility complex (MHC) class II from pericytes in a cell type-specific fashion and performed T cell-pericyte cocultures and EAE experiments. We found that pericytes present antigen in vitro to induce T cell activation and proliferation. In an adoptive transfer EAE experiment, pericyte-specific MHC II KO resulted in locally enhanced T cell infiltration in the CNS; even though, overall disease course of mice was not affected. Thus, pericytes may serve as non-professional antigen-presenting cells affecting states of T cell activation, thereby locally shaping lesion formation in CNS inflammation but without modulating disease severity.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Ratones , Animales , Encefalomielitis Autoinmune Experimental/patología , Pericitos/patología , Linfocitos T , Sistema Nervioso Central/patología , Barrera Hematoencefálica/patología , Antígenos , Antígenos de Histocompatibilidad Clase II , Ratones Endogámicos C57BLRESUMEN
Oligodendroglial progenitor cells (OPCs) are highly proliferative and migratory cells, which differentiate into complex myelin forming and axon ensheathing mature oligodendrocytes during myelination. Recent studies indicate that the oligodendroglial cell population is heterogeneous on transcriptional and functional level depending on the location in the central nervous system. Here, we compared intrinsic properties of OPC from spinal cord and brain on functional and transcriptional level. Spinal cord OPC demonstrated increased migration as well as differentiation capacity. Moreover, transcriptome analysis revealed differential expression of several genes between both OPC populations. In spinal cord OPC, we confirmed upregulation of SKAP2, a cytoplasmatic adaptor protein known for its implication in cytoskeletal remodeling and migration in other cell types. Recent findings suggest that actin dynamics determine not only oligodendroglial migration, but also differentiation: Whereas actin polymerization is important for process extension, actin destabilization and depolymerization is required for myelin sheath formation. Downregulation or complete lack of SKAP2 in OPC resulted in reduced migration and impaired morphological maturation in oligodendrocytes. In contrast, overexpression of SKAP2 as well as constitutively active SKAP2 increased OPC migration suggesting that SKAP2 function is dependent on activation by phosphorylation. Furthermore, lack of SKAP2 enhanced the positive effect on OPC migration after integrin activation suggesting that SKAP2 acts as modulator of integrin dependent migration. In summary, we demonstrate the presence of intrinsic differences between spinal cord and brain OPC and identified SKAP2 as a new regulator of oligodendroglial migration and sheath formation.
Asunto(s)
Vaina de Mielina , Oligodendroglía , Diferenciación Celular/fisiología , Sistema Nervioso Central , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Médula EspinalRESUMEN
Oligodendrocytes are extensively coupled to astrocytes, a phenomenon ensuring glial homeostasis and maintenance of central nervous system myelin. Molecular disruption of this communication occurs in demyelinating diseases such as multiple sclerosis. Less is known about the vulnerability and reconstruction of the panglial network during adult demyelination-remyelination. Here, we took advantage of lysolcithin-induced demyelination to investigate the expression dynamics of the oligodendrocyte specific connexin 47 (Cx47) and to some extent that of astrocyte Cx43, and whether this dynamic could be modulated by grafted induced pluripotent stem cell (iPSC)-neural progeny. Our data show that disruption of Cx43-Cx47 mediated hetero-cellular gap-junction intercellular communication following demyelination is larger in size than demyelination. Loss of Cx47 expression is timely rescued during remyelination and accelerated by the grafted neural precursors. Moreover, mouse and human iPSC-derived oligodendrocytes express Cx47, which co-labels with astrocyte Cx43, indicating their integration into the panglial network. These data suggest that in rodents, full lesion repair following transplantation occurs by panglial reconstruction in addition to remyelination. Targeting panglial elements by cell therapy or pharmacological compounds may help accelerating or stabilizing re/myelination in myelin disorders.
Asunto(s)
Conexinas , Células Madre Pluripotentes Inducidas , Esclerosis Múltiple , Remielinización , Animales , Astrocitos , Conexina 43/genética , Ratones , OligodendroglíaRESUMEN
Multiple sclerosis is a chronic inflammatory, demyelinating disease, although it has been suggested that in the progressive late phase, inflammatory lesion activity declines. We recently showed in the Netherlands Brain Bank multiple sclerosis-autopsy cohort considerable ongoing inflammatory lesion activity also at the end stage of the disease, based on microglia/macrophage activity. We have now studied the role of T cells in this ongoing inflammatory lesion activity in chronic multiple sclerosis autopsy cases. We quantified T cells and perivascular T-cell cuffing at a standardized location in the medulla oblongata in 146 multiple sclerosis, 20 neurodegenerative control and 20 non-neurological control brain donors. In addition, we quantified CD3+, CD4+, and CD8+ T cells in 140 subcortical white matter lesions. The location of CD8+ T cells in either the perivascular space or the brain parenchyma was determined using CD8/laminin staining and confocal imaging. Finally, we analysed CD8+ T cells, isolated from fresh autopsy tissues from subcortical multiple sclerosis white matter lesions (n = 8), multiple sclerosis normal-appearing white matter (n = 7), and control white matter (n = 10), by flow cytometry. In normal-appearing white matter, the number of T cells was increased compared to control white matter. In active and mixed active/inactive lesions, the number of T cells was further augmented compared to normal-appearing white matter. Active and mixed active/inactive lesions were enriched for both CD4+ and CD8+ T cells, the latter being more abundant in all lesion types. Perivascular clustering of T cells in the medulla oblongata was only found in cases with a progressive disease course and correlated with a higher percentage of mixed active/inactive lesions and a higher lesion load compared to cases without perivascular clusters in the medulla oblongata. In all white matter samples, CD8+ T cells were located mostly in the perivascular space, whereas in mixed active/inactive lesions, 16.3% of the CD8+ T cells were encountered in the brain parenchyma. CD8+ T cells from mixed active/inactive lesions showed a tissue-resident memory phenotype with expression of CD69, CD103, CD44, CD49a, and PD-1 and absence of S1P1. They upregulated markers for homing (CXCR6), reactivation (Ki-67), and cytotoxicity (GPR56), yet lacked the cytolytic enzyme granzyme B. These data show that in chronic progressive multiple sclerosis cases, inflammatory lesion activity and demyelinated lesion load is associated with an increased number of T cells clustering in the perivascular space. Inflammatory active multiple sclerosis lesions are populated by CD8+ tissue-resident memory T cells, which show signs of reactivation and infiltration of the brain parenchyma.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Esclerosis Múltiple/inmunología , Tejido Parenquimatoso/inmunología , Sustancia Blanca/inmunología , Adulto , Autopsia , Linfocitos T CD8-positivos/patología , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Humanos , Memoria Inmunológica/inmunología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patología , Esclerosis Múltiple Crónica Progresiva/inmunología , Esclerosis Múltiple Crónica Progresiva/patología , Enfermedades del Sistema Nervioso/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Sustancia Blanca/patologíaRESUMEN
T cells critically depend on reprogramming of metabolic signatures to meet the bioenergetic demands during activation and clonal expansion. Here we identify the transcription factor Nur77 as a cell-intrinsic modulator of T cell activation. Nur77-deficient T cells are highly proliferative, and lack of Nur77 is associated with enhanced T cell activation and increased susceptibility for T cell-mediated inflammatory diseases, such as CNS autoimmunity, allergic contact dermatitis and collagen-induced arthritis. Importantly, Nur77 serves as key regulator of energy metabolism in T cells, restricting mitochondrial respiration and glycolysis and controlling switching between different energy pathways. Transcriptional network analysis revealed that Nur77 modulates the expression of metabolic genes, most likely in close interaction with other transcription factors, especially estrogen-related receptor α. In summary, we identify Nur77 as a transcriptional regulator of T cell metabolism, which elevates the threshold for T cell activation and confers protection in different T cell-mediated inflammatory diseases.
Asunto(s)
Autoinmunidad , Activación de Linfocitos , Mitocondrias , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Consumo de Oxígeno/inmunología , Linfocitos T , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Perfilación de la Expresión Génica , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/inmunología , Mitocondrias/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/inmunología , Receptores de Estrógenos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Oligodendroglial pathology is central to de- and dysmyelinating, but also contributes to neurodegenerative and psychiatric diseases as well as brain injury. The understanding of oligodendroglial biology in health and disease has been significantly increased during recent years by experimental in vitro and in vivo preclinical studies as well as histological analyses of human tissue samples. However, for many of these diseases the underlying pathology is still not fully understood and treatment options are frequently lacking. This is at least partly caused by the limited access to human oligodendrocytes from patients to perform functional studies and drug screens. The induced pluripotent stem cell technology (iPSC) represents a possibility to circumvent this obstacle and paves new ways to study human disease and to develop new treatment options for so far incurable central nervous system (CNS) diseases. In this review, we summarize the differences between human and rodent oligodendrocytes, provide an overview of the different techniques to generate oligodendrocytes from human progenitor or stem cells and describe the results from studies using iPSC derived oligodendroglial lineage cells. Furthermore, we discuss future perspectives and challenges of the iPSC technology with respect to disease modeling, drug screen, and cell transplantation approaches.
Asunto(s)
Enfermedades Desmielinizantes/patología , Células Madre Pluripotentes Inducidas/patología , Leucoencefalopatías/patología , Oligodendroglía/patología , Diferenciación Celular/fisiología , HumanosRESUMEN
Multiple sclerosis (MS) is the most frequent demyelinating disease and a leading cause for disability in young adults. Despite significant advances in immunotherapies in recent years, disease progression still cannot be prevented. Remyelination, meaning the formation of new myelin sheaths after a demyelinating event, can fail in MS lesions. Impaired differentiation of progenitor cells into myelinating oligodendrocytes may contribute to remyelination failure and, therefore, the development of pharmacological approaches which promote oligodendroglial differentiation and by that remyelination, represents a promising new treatment approach. However, this generally accepted concept has been challenged recently. To further understand mechanisms contributing to remyelination failure in MS, we combined detailed histological analyses assessing oligodendroglial cell numbers, presence of remyelination as well as the inflammatory environment in different MS lesion types in white matter with in vitro experiments using induced-pluripotent stem cell (iPSC)-derived oligodendrocytes (hiOL) and supernatants from polarized human microglia. Our findings suggest that there are multiple reasons for remyelination failure in MS which are dependent on lesion stage. These include lack of myelin sheath formation despite the presence of mature oligodendrocytes in a subset of active lesions as well as oligodendroglial loss and a hostile tissue environment in mixed active/inactive lesions. Therefore, we conclude that better in vivo and in vitro models which mimic the pathological hallmarks of the different MS lesion types are required for the successful development of remyelination promoting drugs.
Asunto(s)
Diferenciación Celular/fisiología , Vaina de Mielina/patología , Oligodendroglía/metabolismo , Remielinización/fisiología , Adulto , Anciano , Enfermedades Desmielinizantes/patología , Humanos , Microglía/patología , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Vaina de Mielina/metabolismo , Células Madre/metabolismoRESUMEN
Multiple sclerosis (MS) is the most frequent demyelinating disease in young adults and despite significant advances in immunotherapy, disease progression still cannot be prevented. Promotion of remyelination, an endogenous repair mechanism resulting in the formation of new myelin sheaths around demyelinated axons, represents a promising new treatment approach. However, remyelination frequently fails in MS lesions, which can in part be attributed to impaired differentiation of oligodendroglial progenitor cells into mature, myelinating oligodendrocytes. The reasons for impaired oligodendroglial differentiation and defective remyelination in MS are currently unknown. To determine whether intrinsic oligodendroglial factors contribute to impaired remyelination in relapsing-remitting MS (RRMS), we compared induced pluripotent stem cell-derived oligodendrocytes (hiOL) from RRMS patients and controls, among them two monozygous twin pairs discordant for MS. We found that hiOL from RRMS patients and controls were virtually indistinguishable with respect to remyelination-associated functions and proteomic composition. However, while analyzing the effect of extrinsic factors we discovered that supernatants of activated peripheral blood mononuclear cells (PBMCs) significantly inhibit oligodendroglial differentiation. In particular, we identified CD4+ T cells as mediators of impaired oligodendroglial differentiation; at least partly due to interferon-gamma secretion. Additionally, we observed that blocked oligodendroglial differentiation induced by PBMC supernatants could not be restored by application of oligodendroglial differentiation promoting drugs, whereas treatment of PBMCs with the immunomodulatory drug teriflunomide prior to supernatant collection partly rescued oligodendroglial differentiation. In summary, these data indicate that the oligodendroglial differentiation block is not due to intrinsic oligodendroglial factors but rather caused by the inflammatory environment in RRMS lesions which underlines the need for drug screening approaches taking the inflammatory environment into account. Combined, these findings may contribute to the development of new remyelination promoting strategies.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Oligodendroglía/patología , Remielinización/inmunología , Diferenciación Celular/fisiología , Humanos , Células Madre Pluripotentes Inducidas , Interferón gamma/inmunología , Células Precursoras de Oligodendrocitos/patologíaRESUMEN
Rapid and efficient protocols to generate oligodendrocytes (OL) from human induced pluripotent stem cells (iPSC) are currently lacking, but may be a key technology to understand the biology of myelin diseases and to develop treatments for such disorders. Here, we demonstrate that the induction of three transcription factors (SOX10, OLIG2, NKX6.2) in iPSC-derived neural progenitor cells is sufficient to rapidly generate O4+ OL with an efficiency of up to 70% in 28 d and a global gene-expression profile comparable to primary human OL. We further demonstrate that iPSC-derived OL disperse and myelinate the CNS of Mbpshi/shiRag-/- mice during development and after demyelination, are suitable for in vitro myelination assays, disease modeling, and screening of pharmacological compounds potentially promoting oligodendroglial differentiation. Thus, the strategy presented here to generate OL from iPSC may facilitate the studying of human myelin diseases and the development of high-throughput screening platforms for drug discovery.
Asunto(s)
Diferenciación Celular/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Factores de Transcripción/genética , Animales , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Muerte Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Análisis por Conglomerados , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Expresión Génica Ectópica , Perfilación de la Expresión Génica , Humanos , Ratones , Mutación , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Estrés Oxidativo , Médula Espinal/metabolismo , Médula Espinal/patología , Médula Espinal/ultraestructura , Factores de Transcripción/metabolismo , Transcriptoma , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
The tightly controlled processes of myelination and remyelination require the participation of the cytoskeleton. The reorganization of the cytoskeleton is controlled by small GTPases of the RhoA family. Here, we report that Vav3, a Rho GTPase regulating guanine nucleotide exchange factor (GEF) is involved in oligodendrocyte maturation, myelination and remyelination. When Vav3 was eliminated by genetic recombination, oligodendrocyte precursor cell (OPC) differentiation toward mature oligodendrocytes was accelerated. In contrast, Vav3-deficient oligodendrocytes displayed a reduced capacity to myelinate synthetic microfibers in vitro. Furthermore, remyelination was impaired in Vav3 knockout cerebellar slice cultures that were demyelinated by the addition of lysolecithin. In agreement with these observations, remyelination was compromised when the cuprizone model of myelin lesion was performed in Vav3-deficient mice. When Vav3-deficient oligodendrocytes were examined with Förster resonance energy transfer (FRET)-based biosensors, an altered activation profile of RhoA GTPases was revealed on the cellular level, which could be responsible for an impaired remyelination. Taken together, this study highlights Vav3 as a novel regulator of oligodendrocyte maturation and remyelination, suggesting that manipulation of the Vav3-dependent signaling pathway could help to improve myelin repair.
Asunto(s)
Diferenciación Celular/genética , Leucoencefalopatías/patología , Células Precursoras de Oligodendrocitos/fisiología , Proteínas Proto-Oncogénicas c-vav/metabolismo , Remielinización/genética , Animales , Caspasa 3/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Quelantes/toxicidad , Cuprizona/toxicidad , Modelos Animales de Enfermedad , GTP Fosfohidrolasas/metabolismo , Antígeno Ki-67/metabolismo , Leucoencefalopatías/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Básica de Mielina/metabolismo , Fibras Nerviosas Mielínicas/patología , Células Precursoras de Oligodendrocitos/patología , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas c-vav/genética , Remielinización/efectos de los fármacos , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
In multiple sclerosis, demyelination occurs as a consequence of chronic autoimmunity in the central nervous system causing progressive neurological impairment in patients. After a demyelinating event, new myelin sheaths are formed by adult oligodendroglial progenitor cells; a process called remyelination. However, remyelination often fails in multiple sclerosis due to insufficient recruitment and differentiation of oligodendroglial precursor cells. A pivotal role for the two-pore-domain potassium (K2P ) channel, TASK1, has already been proven for an animal model of multiple sclerosis. However, the mechanisms underlying the TASK1-mediated effects are still elusive. Here, we tested the role of TASK1 channels in oligodendroglial differentiation and remyelination after cuprizone-induced demyelination in male mice. We found TASK1 channels to be functionally expressed on primary murine and human, pluripotent stem cell-derived oligodendrocytes. Lack of TASK1 channels resulted in an increase of mature oligodendrocytes in vitro as well as a higher number of mature oligodendrocytes and accelerated developmental myelination in vivo. Mechanistically, Task1-deficient cells revealed a higher amount of phosphorylated WNK1, a kinase known to be involved in the downstream signaling of the myelination regulator LINGO-1. Furthermore, we analyzed the effect of genetic TASK1 ablation or pharmacological TASK1 inhibition on disease-related remyelination. Neither channel inhibition nor lack of TASK1 channels promoted remyelination after pathological demyelination. In summary, we conclude that functional TASK1 channels participate in the modulation of differentiating oligodendroglial cells in a previously unknown manner. However, while being involved in developmental myelination our data suggest that TASK1 channels have no major effect on remyelination.
Asunto(s)
Diferenciación Celular/genética , Enfermedades Desmielinizantes/patología , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Anestésicos Locales/farmacología , Animales , Animales Recién Nacidos , Bupivacaína/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Células Cultivadas , Cuprizona/toxicidad , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidores de la Monoaminooxidasa/toxicidad , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Proteínas de la Mielina/ultraestructura , Proteínas del Tejido Nervioso/genética , Células Precursoras de Oligodendrocitos/efectos de los fármacos , Células Precursoras de Oligodendrocitos/fisiología , Células Precursoras de Oligodendrocitos/ultraestructura , Oligodendroglía/efectos de los fármacos , Oligodendroglía/fisiología , Oligodendroglía/ultraestructura , Canales de Potasio de Dominio Poro en Tándem/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacologíaRESUMEN
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood-brain barrier, CD56(bright) NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α4ß1 integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4(+) T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4(+) T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor's ligand CD155 on CD4(+) T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4(+) T cells and the cytolytic activity of NK cells.
Asunto(s)
Barrera Hematoencefálica/inmunología , Linfocitos T CD4-Positivos/inmunología , Movimiento Celular/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Esclerosis Múltiple/inmunología , Receptores de Interleucina-2/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Barrera Hematoencefálica/patología , Linfocitos T CD4-Positivos/patología , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Células K562 , Células Asesinas Naturales/patología , Masculino , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patología , Natalizumab/administración & dosificación , Receptores Virales/inmunologíaRESUMEN
Molecular mechanisms that determine lesion localization or phenotype variation in multiple sclerosis are mostly unidentified. Although transmigration of activated encephalitogenic T cells across the blood-brain barrier (BBB) is a crucial step in the disease pathogenesis of CNS autoimmunity, the consequences on brain endothelial barrier integrity upon interaction with such T cells and subsequent lesion formation and distribution are largely unknown. We made use of a transgenic spontaneous mouse model of CNS autoimmunity characterized by inflammatory demyelinating lesions confined to optic nerves and spinal cord (OSE mice). Genetic ablation of a single immune-regulatory molecule in this model [i.e., B7-homolog 1 (B7-H1, PD-L1)] not only significantly increased incidence of spontaneous CNS autoimmunity and aggravated disease course, especially in the later stages of disease, but also importantly resulted in encephalitogenic T-cell infiltration and lesion formation in normally unaffected brain regions, such as the cerebrum and cerebellum. Interestingly, B7-H1 ablation on myelin oligodendrocyte glycoprotein-specific CD4+ T cells, but not on antigen-presenting cells, amplified T-cell effector functions, such as IFN-γ and granzyme B production. Therefore, these T cells were rendered more capable of eliciting cell contact-dependent brain endothelial cell dysfunction and increased barrier permeability in an in vitro model of the BBB. Our findings suggest that a single immune-regulatory molecule on T cells can be ultimately responsible for localized BBB breakdown, and thus substantial changes in lesion topography in the context of CNS autoimmunity.