RESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with no effective cure. Astrocytes display a toxic phenotype in ALS and contribute to motoneuron (MN) degeneration. Modulating astrocytes' neurotoxicity can reduce MN death. Our previous studies showed the beneficial effect of mesenchymal stem cell (MSC) administration in SOD1G93A ALS mice, but the mechanisms are still unclear. We postulated that the effects could be mediated by extracellular vesicles (EVs) secreted by MSCs. We investigated, by immunohistochemical, molecular, and in vitro functional analyses, the activity of MSC-derived EVs on the pathological phenotype and neurotoxicity of astrocytes isolated from the spinal cord of symptomatic SOD1G93A mice and human astrocytes (iAstrocytes) differentiated from inducible neural progenitor cells (iNPCs) of ALS patients. In vitro EV exposure rescued mouse and human ALS astrocytes' neurotoxicity towards MNs. EVs significantly dampened the pathological phenotype and neuroinflammation in SOD1G93A astrocytes. In iAstrocytes, exposure to EVs increased the antioxidant factor Nrf2 and reduced reactive oxygen species. We previously found nine miRNAs upregulated in MSC-derived EVs. Here, the transfection of SOD1G93A astrocytes with single miRNA mimics reduced astrocytes' activation and the expression of neuroinflammatory factors. Moreover, miR-466q and miR-467f mimics downregulate Mapk11, while miR-466m-5p and miR-466i-3p mimics promote the nuclear translocation of Nrf2. In iAstrocytes, transfection with miR-29b-3p mimic upregulated NQO1 antioxidant activity and reduced neurotoxicity towards MNs. MSC-derived EVs modulate astrocytes' reactive phenotype and neurotoxicity through anti-inflammatory and antioxidant-shuttled miRNAs, thus representing a therapeutic strategy in ALS.
Assuntos
Esclerose Lateral Amiotrófica , Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , Doenças Neurodegenerativas , Humanos , Camundongos , Animais , Esclerose Lateral Amiotrófica/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Doenças Neurodegenerativas/metabolismo , Camundongos Transgênicos , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
Multiple sclerosis (MS) is a neurological disorder characterized by an autoimmune response, demyelinating plaques and axonal damage. Intense immunosuppression (II) followed by autologous hematopoietic stem cell transplantation has been proposed as a treatment in severe forms of MS. We have used murine relapsing-remitting (RR) experimental autoimmune encephalomyelitis (RR-EAE) to evaluate the transplantation of syngeneic bone marrow cells (BMC) after II, in combination with mesenchymal stem cells (MSCs) as a new therapeutic adjunct capable of improving immune reconstitution. In EAE-affected mice treated with BMC alone, we observed a drastic reduction in the clinical course only during the early RR phase of the disease. There was no difference in the RR-EAE clinical course between mice treated with BMC alone and co-transplanted mice. To analyze the immune reconstitution, we quantified the circulating immune cells in naïve and RR-EAE-affected mice after II, with BMC alone or in combination with MSC. Although II resulted in reduced numbers of circulating immune cells, reconstitution did not differ in co-transplanted mice. During the early phase of the disease, IL-4 was significantly elevated in co-transplanted mice, as compared to those treated with BMC alone. These data suggest that BMC transplantation after II transiently ameliorates the clinical symptoms of RR-EAE, but that co-transplantation with MSC has no synergistic effect.
RESUMO
Neuroinflammation is associated with synapse dysfunction and cognitive decline in patients and animal models. One candidate for translating the inflammatory stress into structural and functional changes in neural networks is the transcriptional repressor RE1-silencing transcription factor (REST) that regulates the expression of a wide cluster of neuron-specific genes during neurogenesis and in mature neurons. To study the cellular and molecular pathways activated under inflammatory conditions mimicking the experimental autoimmune encephalomyelitis (EAE) environment, we analyzed REST activity in neuroblastoma cells and mouse cortical neurons treated with activated T cell or microglia supernatant and distinct pro-inflammatory cytokines. We found that REST is activated by a variety of neuroinflammatory stimuli in both neuroblastoma cells and primary neurons, indicating that a vast transcriptional change is triggered during neuroinflammation. While a dual activation of REST and its dominant-negative splicing isoform REST4 was observed in N2a neuroblastoma cells, primary neurons responded with a pure full-length REST upregulation in the absence of changes in REST4 expression. In both cases, REST upregulation was associated with activation of Wnt signaling and increased nuclear translocation of ß-catenin, a well-known intracellular transduction pathway in neuroinflammation. Among single cytokines, IL-1ß caused a potent and prompt increase in REST transcription and translation in neurons, which promoted a delayed and strong synaptic downscaling specific for excitatory synapses, with decreased frequency and amplitude of spontaneous synaptic currents, decreased density of excitatory synaptic connections, and decreased frequency of action potential-evoked Ca2+ transients. Most important, the IL-1ß effects on excitatory transmission were strictly REST dependent, as conditional deletion of REST completely occluded the effects of IL-1ß activation on synaptic transmission and network excitability. Our results demonstrate that REST upregulation represents a new pathogenic mechanism for the synaptic dysfunctions observed under neuroinflammatory conditions and identify the REST pathway as therapeutic target for EAE and, potentially, for multiple sclerosis.
Assuntos
Córtex Cerebral/metabolismo , Inflamação/metabolismo , Interleucina-1beta/farmacologia , Proteínas Repressoras/metabolismo , Transmissão Sináptica , Animais , Córtex Cerebral/citologia , Técnicas de Cocultura , Meios de Cultivo Condicionados , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas Repressoras/biossíntese , Transmissão Sináptica/efeitos dos fármacos , Linfócitos T/metabolismo , Regulação para CimaRESUMO
Mesenchymal stromal/stem cells (MSCs) are characterized by neuroprotective, immunomodulatory, and neuroregenerative properties, which support their therapeutic potential for inflammatory/neurodegenerative diseases, including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). One mode of action through which MSCs exert their immunomodulatory effects is release of extracellular vesicles that carry proteins, mRNAs, and microRNAs (miRNAs), which, once transferred, modify the function of target cells. We identified nine miRNAs significantly dysregulated in IFN-γ-primed MSCs, but present at different levels in their derived small extracellular vesicles (s-EV). We show that miR-467f and miR-466q modulate the pro-inflammatory phenotype of activated N9 microglia cells and of primary microglia acutely isolated from late symptomatic SOD1G93A mice, a murine ALS model, by downregulating Tnf and Il1b expression. Further analysis of the mode of action of miR-467f and miR-466q indicated that they dampen the pro-inflammatory phenotype of microglia by modulating p38 MAPK signaling pathway via inhibition of expression of their target genes, Map3k8 and Mk2. Finally, we demonstrated that in vivo administration of s-EV leads to decreased expression of neuroinflammation markers in the spinal cord of EAE-affected mice, albeit without affecting disease course. Overall, our data suggest that MSC-derived exosomes could affect neuroinflammation possibly through specific immunomodulatory miRNAs acting on microglia.
Assuntos
Esclerose Lateral Amiotrófica/terapia , Encefalite/terapia , Vesículas Extracelulares/genética , Inflamação/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , MicroRNAs/administração & dosagem , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Encefalite/genética , Encefalite/metabolismo , Encefalite/patologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Microglia/metabolismo , Neuroproteção , Transdução de SinaisRESUMO
Transplanted mesenchymal stromal/stem cells (MSC) ameliorate the clinical course of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), reducing inflammation and demyelination. These effects are mediated by instructive cross-talk between MSC and immune and neural cells. Astroglial reaction to injury is a prominent feature of both EAE and MS. Astrocytes constitute a relevant target to control disease onset and progression and, based on their potential to acquire stem cell properties in situ, to foster recovery in the post-acute phase of pathology. We have assessed how MSC impact astrocytes in vitro and ex vivo in EAE. Expression of astroglial factors implicated in EAE pathogenesis was quantified by real-time PCR in astrocytes co-cultured with MSC or isolated from EAE cerebral cortex; astrocyte morphology and expression of activation markers were analyzed by confocal microscopy. The acquisition of neural stem cell properties by astrocytes was evaluated by neurosphere assay. Our study shows that MSC prevented astrogliosis, reduced mRNA expression of inflammatory cytokines that sustain immune cell infiltration in EAE, as well as protein expression of endothelin-1, an astrocyte-derived factor that inhibits remyelination and contributes to neurodegeneration and disease progression in MS. Moreover, our data reveal that MSC promoted the acquisition of progenitor traits by astrocytes. These data indicate that MSC attenuate detrimental features of reactive astroglia and, based on the reacquisition of stem cell properties, also suggest that astrocytes may be empowered in their protective and reparative actions by MSC.
Assuntos
Encefalomielite Autoimune Experimental , Células-Tronco Mesenquimais , Esclerose Múltipla , Animais , Astrócitos , Camundongos , Camundongos Endogâmicos C57BL , FenótipoRESUMO
Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination.
Assuntos
Astrócitos/fisiologia , Doenças Desmielinizantes/fisiopatologia , Vesículas Extracelulares/fisiologia , Microglia/fisiologia , Bainha de Mielina/fisiologia , Remielinização/fisiologia , Animais , Astrócitos/patologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Técnicas de Cocultura , Corpo Caloso/patologia , Corpo Caloso/fisiopatologia , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Vesículas Extracelulares/patologia , Inflamação/patologia , Inflamação/fisiopatologia , Lisofosfatidilcolinas , Masculino , Células-Tronco Mesenquimais/fisiologia , Camundongos Endogâmicos C57BL , Microglia/patologia , Bainha de Mielina/patologia , Neuroproteção/fisiologia , Células Precursoras de Oligodendrócitos/patologia , Células Precursoras de Oligodendrócitos/fisiologia , Ratos Sprague-DawleyRESUMO
During experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis associated with blood-brain barrier (BBB) disruption, oligodendrocyte precursor cells (OPCs) overexpress proteoglycan nerve/glial antigen 2 (NG2), proliferate, and make contacts with the microvessel wall. To explore whether OPCs may actually be recruited within the neurovascular unit (NVU), de facto intervening in its cellular and molecular composition, we quantified by immunoconfocal morphometry the presence of OPCs in contact with brain microvessels, during postnatal cerebral cortex vascularization at postnatal day 6, in wild-type (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naïve and EAE-affected WT and NG2KO mice. As observed in WT mice during postnatal development, a higher number of juxtavascular and perivascular OPCs was revealed in adult WT mice during EAE compared to adult naïve WT mice. In EAE-affected mice, OPCs were mostly associated with microvessels that showed altered claudin-5 and occludin tight junction (TJ) staining patterns and barrier leakage. In contrast, EAE-affected NG2KO mice, which did not show any significant increase in vessel-associated OPCs, seemed to retain better preserved TJs and BBB integrity. As expected, absence of NG2, in both OPCs and pericytes, led to a reduced content of vessel basal lamina molecules, laminin, collagen VI, and collagen IV. In addition, analysis of the major ligand/receptor systems known to promote OPC proliferation and migration indicated that vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor-AA (PDGF-AA), and the transforming growth factor-ß (TGF-ß) were the molecules most likely involved in proliferation and recruitment of vascular OPCs during EAE. These results were confirmed by real time-PCR that showed Fgf2, Pdgfa and Tgfb expression on isolated cerebral cortex microvessels and by dual RNAscope-immunohistochemistry/in situ hybridization (IHC/ISH), which detected Vegfa and Vegfr2 transcripts on cerebral cortex sections. Overall, this study suggests that vascular OPCs, in virtue of their developmental arrangement and response to neuroinflammation and growth factors, could be integrated among the classical NVU cell components. Moreover, the synchronized activation of vascular OPCs and pericytes during both BBB development and dysfunction, points to NG2 as a key regulator of vascular interactions.
Assuntos
Antígenos/biossíntese , Barreira Hematoencefálica/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Microvasos/metabolismo , Oligodendroglia/metabolismo , Proteoglicanas/biossíntese , Células-Tronco/metabolismo , Animais , Antígenos/genética , Barreira Hematoencefálica/patologia , Movimento Celular/genética , Proliferação de Células/genética , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Claudina-5/genética , Claudina-5/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Camundongos , Camundongos Knockout , Microvasos/patologia , Oligodendroglia/patologia , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteoglicanas/genética , Células-Tronco/patologia , Junções Íntimas/genética , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The degenerative effects of multiple sclerosis at the level of the vascular and neuronal networks in the central nervous system are currently the object of intensive investigation. Preclinical studies have demonstrated the efficacy of mesenchymal stem cell (MSC) therapy in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis, but the neuropathology of specific lesions in EAE and the effects of MSC treatment are under debate. Because conventional imaging techniques entail protocols that alter the tissues, limiting the reliability of the results, we have used non-invasive X-ray phase-contrast tomography to obtain an unprecedented direct 3D characterization of EAE lesions at micro-to-nano scales, with simultaneous imaging of the vascular and neuronal networks. We reveal EAE-mediated alterations down to the capillary network. Our findings shed light on how the disease and MSC treatment affect the tissues, and promote X-ray phase-contrast tomography as a powerful tool for studying neurovascular diseases and monitoring advanced therapies.
Assuntos
Capilares/diagnóstico por imagem , Capilares/patologia , Esclerose Múltipla/diagnóstico por imagem , Esclerose Múltipla/patologia , Neurônios/patologia , Tomografia por Raios X , Animais , Capilares/ultraestrutura , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/diagnóstico por imagem , Encefalomielite Autoimune Experimental/patologia , Feminino , Imageamento Tridimensional , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Nanopartículas/química , Nanopartículas/ultraestruturaRESUMO
Natalizumab (NTZ) is a monoclonal antibody targeting the α4ß1 integrin (CD49d/CD29), very late antigen-4 (VLA-4), which is approved for treatment of relapsing-remitting multiple sclerosis (RR-MS). A possible association between NTZ treatment and a higher risk of melanoma is under debate. Natural Killer (NK) cells, which express VLA-4, represent an innate barrier limiting spreading of melanoma under steady state conditions. Indeed, because of their expression of activating receptors, they are very efficient in recognizing and killing melanoma cells without the need of a previous priming. For this reason, we aimed at assessing whether NK-cell functions might be impaired by sustained exposure to NTZ. To investigate this possibility we isolated NK cells from healthy donors and tested their cytotoxic and migratory functions against primary melanoma cells derived from subcutaneous and lymph node metastases. Flow cytometry analysis demonstrated expression of CD49d on both freshly isolated NK cells and activated NK cells. Moreover, VLA-4 and its receptor, vascular cell adhesion protein-1 (VCAM-1) were similarly expressed on freshly isolated NK cells. However, upon a short exposure to NTZ, expression of VLA-4 on NK cells decreased. Analysis of NK receptor expression upon exposure of NK cells from three healthy donors to NTZ indicated that DNAM-1 and NKp46 are apparently decreased, while NKG2A is increased. The degranulation of NK cells towards melanoma cells, which express both VLA-4 and VCAM-1, was not affected when NTZ was added to the co-culture or when both NK cells and melanoma cells were each pre-exposed to NTZ for over 12h. In contrast, degranulation was significantly inhibited after 48h of pre-incubation indicating that NTZ can influence NK-cell degranulation towards melanoma cells only after a prolonged exposure. Using a migration chamber assay, we observed that the migration of NK cells towards melanoma cells was dependent upon the concentration of melanoma cells in the lower chamber, and that it was significantly reduced in presence of NTZ. Our results show that upon exposure to NTZ both cytolytic activity and migration toward melanoma cells were affected, suggesting that binding of NTZ to NK cells affects pathways involved in these NK-cell functions. We analyzed the expression of CD49d on NK cells from MS patients treated with NTZ and observed that it decreases with time of treatment. These data suggest that blockade of VLA-4 on NK-cell surface alters some key functions involved in the immune surveillance toward melanoma by NK cells and may provide a mechanistic explanation for the reported occurrence of melanoma in MS patients treated with NTZ.
Assuntos
Vigilância Imunológica/efeitos dos fármacos , Integrina alfa4beta1/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Melanoma/imunologia , Melanoma/metabolismo , Adulto , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Estudos Transversais , Citotoxicidade Imunológica/efeitos dos fármacos , Feminino , Expressão Gênica , Humanos , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Natalizumab/farmacologia , Receptores de Células Matadoras Naturais/metabolismo , Adulto JovemRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) display a therapeutic plasticity because of their ability to modulate immunity, foster tissue repair, and differentiate into mesodermal cells. IFN-γ has been described to differently affect human mesenchymal stem cell (hMSC) and mouse mesenchymal stem cell (mMSC) immunomodulation and differentiation, depending on the inflammatory milieu. OBJECTIVE: We aimed at dissecting the relevant intracellular pathways through which IFN-γ affects MSC plasticity and the consequence of their manipulation on MSC functions. METHODS: Modification of relevant IFN-γ-dependent pathways in mMSCs was carried out in vitro through gene silencing or chemical inhibition of key components. Functional outcomes were assessed by means of Western blotting, real-time PCR, differentiation, and proliferation assays on MSCs. The effect on T cells was addressed by T-cell proliferation assays; the effect of mammalian target of rapamycin (mTOR) manipulation in MSCs was studied in vivo in a mouse model of delayed-type hypersensitivity assay. To address whether similar mechanisms are involved also in hMSCs on IFN-γ stimulation, the effect of chemical inhibition on the same intracellular pathways was assessed by means of Western blotting, and the final outcome on immunomodulatory properties was evaluated based on real-time PCR and T-cell proliferation. RESULTS: We revealed that in mMSCs IFN-γ-induced immunoregulation is mediated by early phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3, which is significantly enhanced by an extracellular signal-regulated kinase 1/2-dependent mTOR inhibition, thereby promoting pSTAT1 nuclear translocation. Accordingly, after intracellular mTOR inhibition, MSCs augmented their ability to inhibit T-cell proliferation and control delayed-type hypersensitivity in vivo. Similarly, on mTOR blockade, hMSCs also enhanced their immunoregulatory features. A sustained exposure to IFN-γ led to inhibition of STAT3 activity, which in mMSCs resulted in an impaired proliferation and differentiation. CONCLUSION: These results provide new insights about MSC intracellular pathways affected by IFN-γ, demonstrating that pharmacologic or genetic manipulation of MSCs can enhance their immunomodulatory functions, which could be translated into novel therapeutic approaches.
Assuntos
Interferon gama/farmacologia , Células-Tronco Mesenquimais/imunologia , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/imunologia , Serina-Treonina Quinases TOR/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Hipersensibilidade Tardia/imunologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
In adult CNS, nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte progenitor cells (OPCs) and is an early marker of pericyte activation in pathological conditions. NG2 could, therefore, play a role in experimental autoimmune encephalomyelitis (EAE), a disease associated with increased blood-brain barrier (BBB) permeability, inflammatory infiltrates, and CNS damage. We induced EAE in NG2 knock-out (NG2KO) mice and used laser confocal microscopy immunofluorescence and morphometry to dissect the effect of NG2 KO on CNS pathology. NG2KO mice developed milder EAE than their wild-type (WT) counterparts, with less intense neuropathology associated with a significant improvement in BBB stability. In contrast to WT mice, OPC numbers did not change in NG2KO mice during EAE. Through FACS and confocal microscopy, we found that NG2 was also expressed by immune cells, including T cells, macrophages, and dendritic cells (DCs). Assessment of recall T cell responses to the encephalitogen by proliferation assays and ELISA showed that, while WT and NG2KO T cells proliferated equally to the encephalitogenic peptide MOG35-55, NG2KO T cells were skewed towards a Th2-type response. Because DCs could be responsible for this effect, we assessed their expression of IL-12 by PCR and intracellular FACS. IL-12-expressing CD11c+ cells were significantly decreased in MOG35-55-primed NG2KO lymph node cells. Importantly, in WT mice, the proportion of IL-12-expressing cells was significantly lower in CD11c+ NG2- cells than in CD11c+ NG2+ cells. To assess the relevance of NG2 at immune system and CNS levels, we induced EAE in bone-marrow chimeric mice, generated with WT recipients of NG2KO bone-marrow cells and vice versa. Regardless of their original phenotype, mice receiving NG2KO bone marrow developed milder EAE than those receiving WT bone marrow. Our data suggest that NG2 plays a role in EAE not only at CNS/BBB level, but also at immune response level, impacting on DC activation and thereby their stimulation of reactive T cells, through controlling IL-12 expression.
Assuntos
Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/patologia , Células da Medula Óssea/imunologia , Transplante de Medula Óssea , Células Dendríticas/patologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de Doença , Medula Espinal/imunologia , Medula Espinal/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Microglia cells, the resident innate immune cells in the brain, are highly active, extending and retracting highly motile processes through which they continuously survey their microenvironment for 'danger signals' and interact dynamically with surrounding cells. Upon sensing changes in their central nervous system microenvironment, microglia become activated, undergoing morphological and functional changes. Microglia activation is not an 'all-or-none' process, but rather a continuum depending on encountered stimuli, which is expressed through a spectrum of molecular and functional phenotypes ranging from so-called 'classically activated', with a highly pro-inflammatory profile, to 'alternatively activated' associated with a beneficial, less inflammatory, neuroprotective profile. Microglia activation has been demonstrated in most neurological diseases of diverse aetiology and has been implicated as a contributor to neurodegeneration. The possibility to promote microglia's neuroprotective phenotype has therefore become a therapeutic goal. We have focused our discussion on the role of microglia in multiple sclerosis, a prototype of inflammatory, demyelinating, neurodegenerative disease, and on the effect of currently approved or on-trial anti-inflammatory therapeutic strategies that might mediate neuroprotection at least in part through their effect on microglia by modifying their behaviour via a switch of their functional phenotype from a detrimental to a protective one. In addition to pharmaceutical approaches, such as treatment with glatiramer acetate, interferon-ß, fingolimod or dimethyl fumarate, we address the alternative therapeutic approach of treatment with mesenchymal stem cells and their potential role in neuroprotection through their 'calming' effect on microglia.
Assuntos
Encéfalo/imunologia , Microglia/imunologia , Esclerose Múltipla/imunologia , Neurônios/imunologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Comunicação Celular , Humanos , Transplante de Células-Tronco Mesenquimais , Microglia/metabolismo , Microglia/patologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Transdução de SinaisRESUMO
The diagnosis of a neurological disease of the central nervous system (CNS) is often associated with the anticipation of an irreversible and untreatable disability. This is the case also of multiple sclerosis (MS) where approved treatments effectively modulate the autoimmune attack to myelin antigens, but poorly affect neurodegeneration and do not promote tissue repair. Thus, stem cell-based therapies are increasingly being considered a possible strategy for diseases of the CNS. Mesenchymal stem cells (MSC), the safety of which has been demonstrated in the last 20 years through clinical trials and case studies, are of particular interest in view not only of their neuroprotective, but also of their immunomodulatory properties. Here, we review the therapeutic features of MSC that make them relevant in the treatment of CNS illnesses and discuss the pioneer clinical experience with MSC-based therapy in neurological diseases.
Assuntos
Doenças do Sistema Nervoso Central/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Animais , HumanosRESUMO
Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS) yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and "epitope spread", have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such "multi-epitope-targeting" approach in murine experimental autoimmune encephalomyelitis (EAE) associated with a single ("classical") or multiple ("complex") anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc) encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as "multi-epitope-targeting" agents. Y-MSPc was superior to peptide(s) in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells). Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of "classical" or "complex EAE" or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a "multi-epitope-targeting" strategy is required for effective immune-specific therapy of organ-specific autoimmune diseases associated with complex and dynamic pathogenic autoimmunity, such as MS; our data further demonstrate that the "multi-epitope-targeting" approach to therapy is optimized through specifically designed multi-epitope-proteins, rather than myelin peptide cocktails, as "multi-epitope-targeting" agents. Such artificial multi-epitope proteins can be tailored to other organ-specific autoimmune diseases.
Assuntos
Epitopos/imunologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Peptídeos/imunologia , Engenharia de Proteínas/métodos , Proteínas/imunologia , Animais , Autoimunidade , Linhagem Celular , Regulação para Baixo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/imunologia , Peptídeos/administração & dosagem , Peptídeos/uso terapêutico , Proteínas/uso terapêuticoRESUMO
This review is dedicated to John Newsom-Davis, who was an exceptional colleague and friend, always exchanging ideas with respect and consideration. We shall not forget his involvement and passion in search for the truth on the role of thymectomy in the management of Myasthenia Gravis (MG). In this short review, we shall summarize what we learnt from DNA microarrays applied to MG thymus. We shall focus on three main comparisons of the thymic transcriptomes: 1) highly hyperplastic MG patients versus non-MG adults; 2) corticosteroid-treated versus untreated seropositive MG patients; and 3) seronegative versus seropositive MG patients.