RESUMO
The cross-talk between T cells and astrocytes occurring under physiological and, even more, neuroinflammatory conditions may profoundly impact the generation of adaptive immune responses in the nervous tissue. In this study, we used a standardized in vitro co-culture assay to investigate the immunomodulatory properties of astrocytes differing for age, sex, and species. Mouse neonatal astrocytes enhanced T cell vitality but suppressed T lymphocyte proliferation in response to mitogenic stimuli or myelin antigens, regardless of the Th1, Th2 or Th17 T cell phenotype. Studies comparing glia cells from adult and neonatal animals showed that adult astrocytes were more efficient in inhibiting T lymphocyte activation than neonatal astrocytes, regardless of their sex. Differently from primary cultures, mouse and human astrocytes derived from reprogrammed fibroblasts did not interfere with T cell proliferation. Overall, we describe a standardized astrocyte-T cell interaction in vitro assay and demonstrate that primary astrocytes and iAstrocytes may differ in modulating T cell function.
Assuntos
Ativação Linfocitária , Células Th17 , Animais , Humanos , Camundongos , Astrócitos , Proliferação de Células , Neuroglia , Masculino , FemininoRESUMO
Differently from other myeloid cells, microglia derive exclusively from precursors originating within the yolk sac and migrate to the CNS under development, without any contribution from fetal liver or postnatal hematopoiesis. Consistent with their unique ontology, microglia may express specific physiological markers, which have been partly described in recent years. Here we wondered whether profiles distinguishing microglia from peripheral macrophages vary with age and under pathology. To this goal, we profiled transcriptomes of microglia throughout the lifespan and included a parallel comparison with peripheral macrophages under physiological and neuroinflammatory settings using age- and sex-matched wild-type and bone marrow chimera mouse models. This comprehensive approach demonstrated that the phenotypic differentiation between microglia and peripheral macrophages is age-dependent and that peripheral macrophages do express some of the most commonly described microglia-specific markers early during development, such as Fcrls, P2ry12, Tmem119, and Trem2. Further, during chronic neuroinflammation CNS-infiltrating macrophages and not peripheral myeloid cells acquire microglial markers, indicating that the CNS niche may instruct peripheral myeloid cells to gain the phenotype and, presumably, the function of the microglia cell. In conclusion, our data provide further evidence about the plasticity of the myeloid cell and suggest caution in the strict definition and application of microglia-specific markers.SIGNIFICANCE STATEMENT Understanding the respective role of microglia and infiltrating monocytes in neuroinflammatory conditions has recently seemed possible by the identification of a specific microglia signature. Here instead we provide evidence that peripheral macrophages may express some of the most commonly described microglia markers at some developmental stages or pathological conditions, in particular during chronic neuroinflammation. Further, our data support the hypothesis about phenotypic plasticity and convergence among distinct myeloid cells so that they may act as a functional unit rather than as different entities, boosting their mutual functions in different phases of disease. This holds relevant implications in the view of the growing use of myeloid cell therapies to treat brain disease in humans.
Assuntos
Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Macrófagos/metabolismo , Microglia/metabolismo , Transcriptoma , Animais , Encéfalo/citologia , Plasticidade Celular/fisiologia , Inflamação/metabolismo , Macrófagos/citologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microglia/citologia , Fenótipo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismoRESUMO
Myelin loss occurring in demyelinating diseases, including multiple sclerosis, is the leading cause of long-lasting neurological disability in adults. While endogenous remyelination, driven by resident oligodendrocyte precursor cells (OPCs), might partially compensate myelin loss in the early phases of demyelinating disorders, this spontaneous reparative potential fails at later stages. To investigate the cellular mechanisms sustaining endogenous remyelination in demyelinating disorders, we focused our attention on endogenous neural precursor cells (eNPCs) located within the subventricular zone (SVZ) since this latter area is considered one of the primary sources of new OPCs in the adult forebrain. First, we fate mapped SVZ-eNPCs in cuprizone-induced demyelination and found that SVZ endogenous neural stem/precursor cells are recruited during the remyelination phase to the corpus callosum (CC) and are capable of forming new oligodendrocytes. When we ablated SVZ-derived eNPCs during cuprizone-induced demyelination in female mice, the animals displayed reduced numbers of oligodendrocytes within the lesioned CC. Although this reduction in oligodendrocytes did not impact the ensuing remyelination, eNPC-ablated mice experienced increased axonal loss. Our results indicate that, in toxic models of demyelination, SVZ-derived eNPCs contribute to support axonal survival.SIGNIFICANCE STATEMENT One of the significant challenges in MS research is to understand the detrimental mechanisms leading to the failure of CNS tissue regeneration during disease progression. One possible explanation is the inability of recruited oligodendrocyte precursor cells (OPCs) to complete remyelination and to sustain axonal survival. The contribution of endogenous neural precursor cells (eNPCs) located in the subventricular zone (SVZ) to generate new OPCs in the lesion site has been debated. Using transgenic mice to fate map and to selectively kill SVZ-derived eNPCs in the cuprizone demyelination model, we observed migration of SVZ-eNPCs after injury and their contribution to oligodendrogenesis and axonal survival. We found that eNPCs are dispensable for remyelination but protect partially from increased axonal loss.
Assuntos
Corpo Caloso/metabolismo , Doenças Desmielinizantes/metabolismo , Ventrículos Laterais/citologia , Bainha de Mielina/metabolismo , Células-Tronco Neurais/citologia , Animais , Movimento Celular , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/patologia , Cuprizona/toxicidade , Doenças Desmielinizantes/etiologia , Doenças Desmielinizantes/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Oligodendroglia/citologia , Oligodendroglia/metabolismoRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Oligodendroglia/patologia , Remielinização/imunologia , Diferenciação Celular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas , Interferon gama/imunologia , Células Precursoras de Oligodendrócitos/patologiaRESUMO
Astrocytes greatly participate to inflammatory and neurotoxic reactions occurring in neurodegenerative diseases and are valuable pharmacological targets to support neuroprotection. Here we used human astrocytes generated from reprogrammed fibroblasts as a cellular model to study the effect of the compound Laquinimod and its active metabolite de-Laquinimod on astrocyte functions and the astrocyte-neuron interaction. We show that human iAstrocytes expressed the receptor for the inflammatory mediator IL1 and responded to it via nuclear translocation of NFκB, an event that did not occur if cells were treated with Laquinimod, indicating a direct anti-inflammatory activity of the drug on the human astrocyte. Similarly, while exposure to IL1 downregulated glial glutamate transporters GLAST and GLT1, treatment with Laquinimod supported maintenance of physiological levels of these proteins despite the inflammatory milieu. Laquinimod also induced nuclear translocation of the aryl hydrocarbon receptor (AHR), suggesting that drug action was mediated by activation of the AHR pathway. However, the drug was effective despite AHR inhibition via CH223191, indicating that AHR signaling in the astrocyte is dispensable for drug responses. Finally, in vitro experiments with rat spinal neurons showed that laquinimod did not exert neuroprotection directly on the neuron but dampened astrocyte-induced neurodegeneration. Our findings indicate that fibroblast-derived human astrocytes represent a suitable model to study astrocyte-neuron crosstalk and demonstrate indirect, partial neuroprotective efficacy for laquinimod.
Assuntos
Astrócitos/metabolismo , Inflamação/patologia , Neurotoxinas/toxicidade , Quinolonas/farmacologia , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Interleucina-1beta/metabolismo , NF-kappa B/metabolismo , Degeneração Neural/patologia , Quinolonas/química , Ratos Sprague-Dawley , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Myeloid cells, such as macrophages and microglia, play a crucial role in neuroinflammation and have been recently identified as a novel therapeutic target, especially for chronic forms. The general aim would be to change the phenotype of myeloid cells from pro- to anti-inflammatory, favoring their tissue-trophic and regenerative functions. Myeloid cells, however, display a number of functional phenotypes, not immediately identifiable as pro- or anti-inflammatory, and associated to ambiguous markers. METHODS: We employed in vitro assays to study macrophage polarization/differentiation in the presence of classical polarizing stimuli such as IFNγ (pro-inflammatory) and IL4 (anti-inflammatory). We induced neuroinflammation in mice by immunization with a myelin antigen and treated diseased mice with intracisternal delivery of an IL4-expressing lentiviral vector. We analyzed clinical, pathological, and immunological outcomes with a focus on myeloid cells. RESULTS: We found that IL6, usually considered a pro-inflammatory cytokine, was released in vitro by macrophages treated with the anti-inflammatory cytokine IL4. We show the existence of macrophages expressing IL6 along with classical anti-inflammatory markers such as CD206 and demonstrate that these cells are immunosuppressive in vitro. In neuroinflamed mice, we show that IL4 delivery in the central nervous system (CNS) is associated with clinical and pathological protection from disease, associated with increased IL6 expression in infiltrating macrophages. CONCLUSIONS: IL6 is known to mediate both pro- and anti-inflammatory effects, having two distinct ways to induce cell-signaling: either through the membrane bound receptor (anti-inflammatory) or through trans-signaling (pro-inflammatory). We show here that IL6-expressing macrophages are associated to protection from neuroinflammation, suggesting that IL6 anti-inflammatory properties prevail in the CNS, and calling for a general reconsideration of IL6 in macrophage polarization.
Assuntos
Mediadores da Inflamação/metabolismo , Interleucina-4/farmacologia , Interleucina-6/biossíntese , Macrófagos/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Feminino , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/imunologia , Interleucina-4/imunologia , Interleucina-6/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The role of central nervous system (CNS) glia in sustaining self-autonomous inflammation and driving clinical progression in multiple sclerosis (MS) is gaining scientific interest. We applied a single transcription factor (SOX10)-based protocol to accelerate oligodendrocyte differentiation from human induced pluripotent stem cell (hiPSC)-derived neural precursor cells, generating self-organizing forebrain organoids. These organoids include neurons, astrocytes, oligodendroglia, and hiPSC-derived microglia to achieve immunocompetence. Over 8 weeks, organoids reproducibly generated mature CNS cell types, exhibiting single-cell transcriptional profiles similar to the adult human brain. Exposed to inflamed cerebrospinal fluid (CSF) from patients with MS, organoids properly mimic macroglia-microglia neurodegenerative phenotypes and intercellular communication seen in chronic active MS. Oligodendrocyte vulnerability emerged by day 6 post-MS-CSF exposure, with nearly 50% reduction. Temporally resolved organoid data support and expand on the role of soluble CSF mediators in sustaining downstream events leading to oligodendrocyte death and inflammatory neurodegeneration. Such findings support the implementation of this organoid model for drug screening to halt inflammatory neurodegeneration.
Assuntos
Encéfalo , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Esclerose Múltipla , Neuroglia , Organoides , Fenótipo , Humanos , Esclerose Múltipla/patologia , Esclerose Múltipla/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Organoides/patologia , Organoides/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Encéfalo/patologia , Encéfalo/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Microglia/metabolismo , Microglia/patologiaRESUMO
OBJECTIVE: Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are emerging as new biomarkers of tissue damage. Our previous data indicate that reactive microglia/macrophages release MVs in vitro. The aim of the study was to evaluate whether MVs are released by microglia/macrophages in vivo and whether their number varies in brain inflammatory conditions, such as multiple sclerosis (MS). METHODS: Electron and fluorescence microscopy and flow cytometry were used to detect myeloid MVs in the cerebrospinal fluid (CSF) of healthy controls, MS patients, and rodents affected by experimental autoimmune encephalomyelitis (EAE), the animal model of MS. RESULTS: Myeloid MVs were detected in CSF of healthy controls. In relapsing and remitting EAE mice, the concentration of myeloid MVs in the CSF was significantly increased and closely associated with disease course. Analysis of MVs in the CSF of 28 relapsing patients and 28 patients with clinical isolated syndrome from 2 independent cohorts revealed higher levels of myeloid MVs than in 13 age-matched controls, indicating a clinical value of MVs as a companion tool to capture disease activity. Myeloid MVs were found to spread inflammatory signals both in vitro and in vivo at the site of administration; mice impaired in MV shedding were protected from EAE, suggesting a pathogenic role for MVs in the disease. Finally, FTY720, the first approved oral MS drug, significantly reduced the amount of MVs in the CSF of EAE-treated mice. INTERPRETATION: These findings identify myeloid MVs as a marker and therapeutic target of brain inflammation.
Assuntos
Biomarcadores/líquido cefalorraquidiano , Doenças do Sistema Nervoso Central/líquido cefalorraquidiano , Doenças do Sistema Nervoso Central/tratamento farmacológico , Inflamação/líquido cefalorraquidiano , Inflamação/tratamento farmacológico , Medula Espinal/metabolismo , Animais , Western Blotting , Sinalização do Cálcio/fisiologia , Comunicação Celular , Células Cultivadas , Encefalite/líquido cefalorraquidiano , Encefalite/patologia , Citometria de Fluxo , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Microscopia de Fluorescência , Esclerose Múltipla/patologia , Doença Autoimune do Sistema Nervoso Experimental/líquido cefalorraquidiano , Doença Autoimune do Sistema Nervoso Experimental/tratamento farmacológico , Neuroglia/metabolismo , Neuroglia/fisiologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/fisiologiaRESUMO
BACKGROUND: There are two generally accepted strategies for treating multiple sclerosis (MS), preventing central nervous system (CNS) damage indirectly through immunomodulatory interventions and/or repairing CNS damage by promoting remyelination. Both approaches also provide neuroprotection since they can prevent, indirectly or directly, axonal damage. OBJECTIVE: Recent experimental and clinical evidence indicates that the novel immunomodulatory drug laquinimod can exert a neuroprotective role in MS. Whether laquinimod-mediated neuroprotection is exerted directly on neuronal cells or indirectly via peripheral immunomodulation is still unclear. METHODS: C57Bl/6 experimental autoimmune encephalomyelitis (EAE) mice, immunised with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, were treated for 26 days with subcutaneous daily injections of laquinimod (from 1 to 25 mg/kg). Patch clamp electrophysiology was performed on acute brain striatal slices from EAE mice treated with daily (25 mg/kg) laquinimod and on acute brain striatal slices from control mice bathed with laquinimod (1-30 µM). RESULTS: Both preventive and therapeutic laquinimod treatment fully prevented the alterations of GABAergic synapses induced by EAE, the first limiting also glutamatergic synaptic alterations. This dual effect might, in turn, have limited glutamatergic excitotoxicity, a phenomenon previously observed early during EAE and possibly correlated with later axonal damage. Furthermore, laquinimod treatment also preserved cannabinoid CB1 receptor sensitivity, normally lost during EAE. Finally, laquinimod per se was able to regulate synaptic transmission by increasing inhibitory post-synaptic currents and, at the same time, reducing excitatory post-synaptic currents. CONCLUSIONS: Our data suggest a novel neuroprotective mechanism by which laquinimod might in vivo protect from neuronal damage occurring as a consequence of inflammatory immune-mediated demyelination.
Assuntos
Encéfalo/efeitos dos fármacos , Encefalomielite Autoimune Experimental/patologia , Inflamação/patologia , Fármacos Neuroprotetores/farmacologia , Quinolonas/farmacologia , Sinapses/efeitos dos fármacos , Animais , Encéfalo/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Sinapses/patologiaRESUMO
Innovative pro-regenerative treatment strategies for progressive multiple sclerosis (PMS), combining neuroprotection and immunomodulation, represent an unmet need. Neural precursor cells (NPCs) transplanted in animal models of multiple sclerosis have shown preclinical efficacy by promoting neuroprotection and remyelination by releasing molecules sustaining trophic support and neural plasticity. Here we present the results of STEMS, a prospective, therapeutic exploratory, non-randomized, open-label, single-dose-finding phase 1 clinical trial ( NCT03269071 , EudraCT 2016-002020-86), performed at San Raffaele Hospital in Milan, Italy, evaluating the feasibility, safety and tolerability of intrathecally transplanted human fetal NPCs (hfNPCs) in 12 patients with PMS (with evidence of disease progression, Expanded Disability Status Scale ≥6.5, age 18-55 years, disease duration 2-20 years, without any alternative approved therapy). The safety primary outcome was reached, with no severe adverse reactions related to hfNPCs at 2-year follow-up, clearly demonstrating that hfNPC therapy in PMS is feasible, safe and tolerable. Exploratory secondary analyses showed a lower rate of brain atrophy in patients receiving the highest dosage of hfNPCs and increased cerebrospinal fluid levels of anti-inflammatory and neuroprotective molecules. Although preliminary, these results support the rationale and value of future clinical studies with the highest dose of hfNPCs in a larger cohort of patients.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Esclerose Múltipla , Células-Tronco Neurais , Adolescente , Adulto , Humanos , Pessoa de Meia-Idade , Adulto Jovem , Esclerose Múltipla/terapia , Estudos Prospectivos , Transplante de Células-Tronco/métodosRESUMO
We investigated the potential of antibody-vectorialized superparamagnetic iron oxide (SPIO) particles as cellular specific magnetic resonance contrast agents to image lymphocyte populations within the central nervous system (CNS), with the final goal of obtaining a reliable tool for noninvasively detecting and tracking specific cellular populations in vivo. We used superparamagnetic particles bound to a monoclonal antibody. The particle is the contrast agent, by means of its T2* relaxation properties; the antibody is the targeting vector, responsible for homing the particle to target a surface antigen. To investigate the efficiency of particle vectorialization by these antibodies, we compared two types of antibody-vectorialized CD3-specific particles in vivo. We successfully employed vectorialized SPIO particles to image B220⺠cells in a murine model of B-cell lymphoma. Likewise, we were able to identify CD3⺠infiltrates in a murine model of multiple sclerosis. The specificity of the technique was confirmed by immunohistochemistry and electron microscopy of corresponding sections. Our findings suggest that indirect binding of the antibody to a streptavidinated particle allows for enhanced particle vectorialization compared to covalent binding of the antibody to the particle.
Assuntos
Anticorpos Monoclonais , Encéfalo/citologia , Dextranos , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita , Linfócitos T/citologia , Animais , Dextranos/administração & dosagem , Encefalomielite Autoimune Experimental/diagnóstico , Feminino , Humanos , Injeções , Linfoma de Células B/diagnóstico , Nanopartículas de Magnetita/administração & dosagem , Camundongos , Sensibilidade e EspecificidadeRESUMO
CD4(+)Foxp3(+) regulatory T cells (Tregs) have been considered crucial in controlling immune system homeostasis, and their derangement is often associated to autoimmunity. Tregs identification is, however, difficult because most markers, including CD25 and Foxp3, are shared by recently activated T cells. We show in this paper that CD4(+)Foxp3(+) T cells are generated in peripheral lymphoid organs on immunization and readily accumulate in the target organ of an autoimmune reaction, together with classical inflammatory cells, constituting up to 50% of infiltrating CD4(+) T cells. Most CD4(+)Foxp3(+) T cells are, however, CD25(-) and express proinflammatory cytokines such as IL-17 and IFN-γ, questioning their suppressive nature. Moreover, in vitro CD4(+) T lymphocytes from naive and autoimmune mice, stimulated to differentiate into Th1, Th2, Th17, and induced Tregs, display early mixed expression of lineage-specific markers. These results clearly point to an unprecedented plasticity of naive CD4(+) T cells, that integrating inflammatory signals may change their fate from the initial lineage commitment to a different functional phenotype.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Fatores de Transcrição Forkhead/imunologia , Interferon gama/imunologia , Interleucina-17/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Fatores de Transcrição Forkhead/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologiaRESUMO
The adult brain retains over life endogenous neural stem/precursor cells (eNPCs) within the subventricular zone (SVZ). Whether or not these cells exert physiological functions is still unclear. In the present work, we provide evidence that SVZ-eNPCs tune structural, electrophysiological, and behavioural aspects of striatal function via secretion of insulin-like growth factor binding protein-like 1 (IGFBPL1). In mice, selective ablation of SVZ-eNPCs or selective abrogation of IGFBPL1 determined an impairment of striatal medium spiny neuron morphology, a higher failure rate in GABAergic transmission mediated by fast-spiking interneurons, and striatum-related behavioural dysfunctions. We also found IGFBPL1 expression in the human SVZ, foetal and induced-pluripotent stem cell-derived NPCs. Finally, we found a significant correlation between SVZ damage, reduction of striatum volume, and impairment of information processing speed in neurological patients. Our results highlight the physiological role of adult SVZ-eNPCs in supporting cognitive functions by regulating striatal neuronal activity.
Assuntos
Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Ventrículos Laterais , Células-Tronco Neurais , Proteínas Supressoras de Tumor , Animais , Humanos , Camundongos , Eletrofisiologia Cardíaca , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Células-Tronco Neurais/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Ventrículos Laterais/fisiologiaRESUMO
Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS) with heterogeneous pathophysiology. In its progressive course oligodendrocyte and neuroaxonal damage is sustained by compartmentalized inflammation due to glial dysregulation. Siponimod (BAF312), a modulator of two sphingosine-1-phosphate (S1P) receptors (S1P1 and S1P5) is the first oral treatment specifically approved for active secondary progressive MS. To address potential direct effects of BAF312 on glial function and glia-neuron interaction, we set up a series of in vitro functional assays with astrocytes generated from human fibroblasts. These cells displayed the typical morphology and markers of astroglia, and were susceptible to the action of inflammatory mediators and BAF312, because expressing receptors for IL1, IL17, and S1P (namely S1P1 and S1P3). Targeting of S1P signaling by BAF312 inhibited NFκB translocation evoked by inflammatory cytokines, indicating a direct anti-inflammatory activity of the drug on the human astrocyte. Further, while glia cells exposed to IL1 or IL17 downregulated protein expression of glutamate transporters, BAF312-treated astrocytes maintained high levels of GLAST and GLT1 regardless of the presence of inflammatory mediators. Interestingly, despite potential glial susceptibility to S1P signaling via S1P3, which is not targeted by BAF312, NFκB translocation and downregulation of glutamate transporters in response to S1P were inhibited at similar levels by BAF312 and FTY720, another S1P signaling modulator targeting also S1P3. Accordingly, specific inhibition of S1P1 via NIBR-0213 blocked S1P-evoked NFκB translocation, demonstrating that modulation of S1P1 is sufficient to dampen signaling via other S1P receptors. Considering that NFκB-dependent responses are regulated by Nrf2, we measured activation of this critical transcription factor for anti-oxidant reactions, and observed that BAF312 rapidly induced nuclear translocation of Nrf2, but this effect was attenuated in the presence of an inflammatory milieu. Finally, in vitro experiments with spinal neurons exposed to astrocyte-conditioned media showed that modulation of S1P or cytokine signaling in astrocytes via BAF312 prevented neurons from astrocyte-induced degeneration. Overall, these experiments on human astrocytes suggest that during neuroinflammation targeting of S1P1 via BAF312 may modulate key astrocyte functions and thereby attain neuroprotection indirectly.
Assuntos
Astrócitos/fisiologia , Azetidinas/farmacologia , Compostos de Benzil/farmacologia , Fibroblastos/fisiologia , Esclerose Múltipla/imunologia , Fator 2 Relacionado a NF-E2/metabolismo , Doenças Neurodegenerativas/imunologia , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologia , Diferenciação Celular , Células Cultivadas , Resistência à Doença , Regulação para Baixo , Transportador 2 de Aminoácido Excitatório/metabolismo , Humanos , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (iß). However, the gene expression profile and secretory function of iß still need to be validated in comparison with native ß cells. METHODS: Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into iß and compared with donor pancreatic islets and EndoC-ßH1, an immortalized human ß cell line. RESULTS: Both clones of iPSCs differentiated into insulin+ cells with high efficiency (up to 20%). iß were negative for pluripotency markers (Oct4, Sox2, Ssea4) and positive for Pdx1, Nkx6.1, Chromogranin A, PC1/3, insulin, glucagon and somatostatin. iß basally secreted C-peptide, glucagon and ghrelin and released insulin in response either to increasing concentration of glucose or a depolarizing stimulus. The comparison revealed that iß are remarkably similar to donor derived islets in terms of gene and protein expression profile and similar level of heterogeneity. The ability of iß to respond to glucose instead was more related to that of EndoC-ßH1. DISCUSSION: We demonstrated that insulin-producing cells generated from iPSCs recapitulate fundamental gene expression profiles and secretory function of native human ß cells.
Assuntos
Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células Secretoras de Insulina/citologia , Transcriptoma , Células Cultivadas , Técnicas de Reprogramação Celular , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Vírus Sendai/genéticaRESUMO
Regulatory T cells (Treg) are necessary to maintain immunological tolerance and are key players in the control of autoimmune disease susceptibility. Expression of the transcription factor FOXP3 is essential for differentiation of Treg cells and indispensable for their suppressive function. However, there is still a lack of knowledge about the mechanisms underlying its regulation. Here, we demonstrate that pro-autophagy protein AMBRA1 is also a key modulator of T cells, regulating the complex network that leads to human Treg differentiation and maintenance. Indeed, through its ability to interact with the phosphatase PP2A, AMBRA1 promotes the stability of the transcriptional activator FOXO3, which, in turn, triggers FOXP3 transcription. Furthermore, we found that AMBRA1 plays a significant role in vivo by regulating Treg cell induction in mouse models of both tumor growth and multiple sclerosis, thus highlighting the role of AMBRA1 in the control of immune homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Diferenciação Celular , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Células HeLa , Homeostase , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/metabolismo , Proteína Fosfatase 2/metabolismo , Linfócitos T/citologiaRESUMO
IL-27 and IL-35 are heterodimeric cytokines, members of the IL-12 family and considered to have immunomodulatory properties. Their role during neuroinflammation had been investigated using mutant mice devoid of either one of their subunits or lacking components of their receptors, yielding conflicting results. We sought to understand the therapeutic potential of IL-27 and IL-35 delivered by gene therapy in neuroinflammation. We constructed lentiviral vectors expressing IL-27 and IL-35 from a single polypeptide chain, and we validated in vitro their biological activity. We injected IL-27 and IL-35-expressing lentiviral vectors into the cerebrospinal fluid (CSF) of mice affected by experimental neuroinflammation (EAE), and performed clinical, neuropathological and immunological analyses. Both cytokines interfere with neuroinflammation, but only IL-27 significantly modulates disease development, both clinically and neuropathologically. IL-27 protects from autoimmune inflammation by inhibiting granulocyte macrophages colony-stimulating factor (GM-CSF) expression in CD4+ T cells and by inducing program death-ligand 1 (PD-L1) expression in both CNS-resident and CNS-infiltrating myeloid cells. We demonstrate here that IL-27 holds therapeutic potential during neuroinflammation and that IL-27 inhibits GM-CSF and induces pd-l1 mRNA in vivo.
Assuntos
Sistema Nervoso Central/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Inflamação/metabolismo , Interleucina-27/metabolismo , Interleucinas/metabolismo , Leucócitos/metabolismo , Animais , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Líquido Cefalorraquidiano/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Terapia Genética , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
In the past decade, tissue microarray (TMA) technology has evolved as an innovative tool for high-throughput proteomics analysis and mainly for biomarker validation. Similarly, enormous amount of data can be obtained from the cell line macroarray (CLMA) technology, which developed from the TMA using formalin-fixed, paraffin-embedded cell pellets. Here, we applied CLMA technology in stem cell research and in particular to identify bona fide neogenerated human induced pluripotent stem cell (hiPSC) clones suitable for down the line differentiation. All hiPSC protocols generate tens of clones, which need to be tested to determine genetically stable cell lines suitable for differentiation. Screening methods generally rely on fluorescence-activated cell sorting isolation and coverslip cell growth followed by immunofluorescence; these techniques could be cumbersome. Here, we show the application of CLMA to identify neogenerated pluripotent cell colonies and neuronal differentiated cell products. We also propose the use of the automated image analyzer, TissueQuest, as a reliable tool to quickly select the best clones, based upon the level of expression of multiple pluripotent biomarkers.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Análise Serial de Tecidos/métodos , Diferenciação Celular , Linhagem Celular , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologiaRESUMO
Multiple sclerosis is an inflammatory disease of the CNS leading to the destruction of oligodendrocytes (OLs), myelin sheaths and axons. The mediators of tissue injury remain unknown. Glutamate, which can be released by activated immune cells or produced within the CNS, has been implicated as a potential mediator of tissue injury in multiple sclerosis. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) and kainate are highly toxic when added to rodent OL cultures. Using OLs derived from human adult surgical specimens, we investigated AMPA/kainate receptor expression and the effects of receptor stimulation on the viability of human OLs. We find that human adult OLs in vitro express low levels of ionotropic glutamate receptors and are resistant to excitotoxicity mediated by high and sustained doses of AMPA or kainate, even when receptor desensitization is blocked. In contrast, rat OLs show strong AMPA receptor expression and are susceptible to excitotoxicity, as previously demonstrated. Furthermore, we show in human brain sections that OLs do not express AMPA receptors in situ and that glial expression of AMPA receptors is limited to astrocytes. The apparent lack of glutamate receptor expression on human OLs and their resistance to AMPA/kainate toxicity should be considered when postulating mechanisms of tissue injury in multiple sclerosis.
Assuntos
Oligodendroglia/efeitos dos fármacos , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia , Adulto , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Astrócitos/metabolismo , Western Blotting , Encéfalo/metabolismo , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Feminino , Ácido Glutâmico/fisiologia , Humanos , Ácido Caínico/farmacologia , Masculino , Pessoa de Meia-Idade , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da EspécieRESUMO
Calcitonin Gene-Related Peptide (CGRP) inhibits microglia inflammatory activation in vitro. We here analyzed the involvement of CGRP and Receptor Component Protein (RCP) in experimental autoimmune encephalomyelitis (EAE). Alpha-CGRP deficiency increased EAE scores which followed the scale alpha-CGRP null>heterozygote>wild type. In wild type mice, CGRP delivery into the cerebrospinal fluid (CSF) 1) reduced chronic EAE (C-EAE) signs, 2) inhibited microglia activation (revealed by quantitative shape analysis), and 3) did not alter GFAP expression, cell density, lymphocyte infiltration, and peripheral lymphocyte production of IFN-gamma, TNF-alpha, IL-17, IL-2, and IL-4. RCP (probe for receptor involvement) was expressed in white matter microglia, astrocytes, oligodendrocytes, and vascular-endothelial cells: in EAE, also in infiltrating lymphocytes. In relapsing-remitting EAE (R-EAE) RCP increased during relapse, without correlation with lymphocyte density. RCP nuclear localization (stimulated by CGRP in vitro) was I) increased in microglia and decreased in astrocytes (R-EAE), and II) increased in microglia by CGRP CSF delivery (C-EAE). Calcitonin like receptor was rarely localized in nuclei of control and relapse mice. CGRP increased in motoneurons. In conclusion, CGRP can inhibit microglia activation in vivo in EAE. CGRP and its receptor may represent novel protective factors in EAE, apparently acting through the differential cell-specific intracellular translocation of RCP.