RESUMEN
The receptor for colony stimulating factor 1 (CSF-1R) is important for the survival and function of myeloid cells that mediate pathology during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). CSF-1 and IL-34, the ligands of CSF-1R, have similar bioactivities but distinct tissue and context-dependent expression patterns, suggesting that they have different roles. This could be the case in EAE, given that CSF-1 expression is up-regulated in the CNS, while IL-34 remains constitutively expressed. We found that targeting CSF-1 with neutralizing antibody halted ongoing EAE, with efficacy superior to CSF-1R inhibitor BLZ945, whereas IL-34 neutralization had no effect, suggesting that pathogenic myeloid cells were maintained by CSF-1. Both antiCSF-1 and BLZ945 treatment greatly reduced the number of monocyte-derived cells and microglia in the CNS. However, antiCSF-1 selectively depleted inflammatory microglia and monocytes in inflamed CNS areas, whereas BLZ945 depleted virtually all myeloid cells, including quiescent microglia, throughout the CNS. AntiCSF-1 treatment reduced the size of demyelinated lesions and microglial activation in the gray matter. Lastly, we found that bone marrowderived immune cells were the major mediators of CSF-1Rdependent pathology, while microglia played a lesser role. Our findings suggest that targeting CSF-1 could be effective in ameliorating MS pathology, while preserving the homeostatic functions of myeloid cells, thereby minimizing risks associated with ablation of CSF-1Rdependent cells.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Factor Estimulante de Colonias de Macrófagos , Esclerosis Múltiple , Animales , Benzotiazoles/farmacología , Benzotiazoles/uso terapéutico , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Ácidos Picolínicos/farmacología , Ácidos Picolínicos/uso terapéutico , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidoresRESUMEN
Extracellular vesicles (EVs), a broad term for the lipid microparticles known as microvesicles and exosomes, are discharged by cells into their surrounding space. Microvesicles are discharged upon outward plasma membrane budding, while exosomes are secreted after multivesicular body (MVB) fusion with the plasma membrane. The majority of information regarding EV biology comes from studies performed in non-polarized cells. Here we characterize EV release in polarized cells. We found a substantial asymmetry in the number and composition of EVs produced and released from the apical membrane of epithelial cells as compared to the basolateral membrane. We showed that the quantitative difference is related to the polarized distribution of two phosphoinositide species between the two cell surfaces and that the peculiar biochemical composition of resultant EVs reflects their site of origin. In particular, apical and basolateral exosomes may derive from distinct classes of MVBs originating from and fusing with the same plasma membrane. We identify VAMP8/Endobrevin as a regulator of the basolateral release of exosomes, whereas the mechanism responsible for apical EV release requires further study.
Asunto(s)
Micropartículas Derivadas de Células , Exosomas , Vesículas Extracelulares , Polaridad Celular , Cuerpos MultivesicularesRESUMEN
Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are neuroinflammatory conditions where inflammatory CD4+ T cells play a major role. Forkhead box P3 (Foxp3)+ regulatory T (Treg) cells suppress inflammation and an increase in their numbers and activity is beneficial for MS and EAE. However, studies have shown that Treg cells can transdifferentiate to pathogenic Th17 cells under inflammatory conditions. Drugs that stimulate Treg cell induction and their resistance to inflammatory stimuli are necessary to develop effective therapies to treat MS. Here, we show that primaquine (PQ), an anti-malarial drug, suppresses EAE through the stimulation of Foxp3+ Treg cells. PQ-elicited Treg cells are refractory to inflammatory stimuli and suppress EAE. Additionally, PQ-elicited Foxp3+ Treg cells were more efficient in suppressing the proliferation of responder cells compared to PBS-elicited Treg cells. Although PQ does not directly induce Foxp3+ Treg cell differentiation from naïve T cells, it modulated dendritic cells (DCs) to induce Foxp3+ Treg cells in an indoleamine 2,3 dioxygenase (IDO)-dependent manner. Together, our results show that PQ elicits Foxp3+ Treg cells with a superior suppressive activity to reduce EAE. PQ has the potential as a safe and effective treatment for MS and other CNS autoimmune inflammatory diseases.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Primaquina/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Antimaláricos/farmacología , Autoinmunidad , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental , Activación de Linfocitos/inmunología , Ratones , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/etiología , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th17/metabolismoRESUMEN
Extracellular vesicles (EVs) play a major role in cell-to-cell communication in physiological and pathological conditions, and their manipulation may represent a promising therapeutic strategy. Microglia, the parenchymal mononuclear phagocytes of the brain, modulate neighboring cells also through the release of EVs. The production of custom EVs filled with desired molecules, possibly targeted to make their uptake cell specific, and their administration in biological fluids may represent a valid approach for drug delivery. We engineered a murine microglia cell line, BV-2, to release EVs overexpressing the endogenous "eat me" signal Lactadherin (Mfg-e8) on the surface to target phagocytes and containing the anti-inflammatory cytokine IL-4. A single injection of 107 IL-4+Mfg-e8+ EVs into the cisterna magna modulated established neuroinflammation and significantly reduced clinical signs in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Injected IL-4+Mfg-e8+ EVs target mainly phagocytes (i.e., macrophages and microglia) surrounding liquoral spaces, and their cargo promote the upregulation of anti-inflammatory markers chitinase 3-like 3 (ym1) and arginase-1 (arg1), significantly reducing tissue damage. Engineered EVs may represent a biological drug delivery tool able to deliver multiple functional molecules simultaneously to treat neuroinflammatory diseases.
Asunto(s)
Vesículas Extracelulares/metabolismo , Interleucina-4/metabolismo , Esclerosis Múltiple/metabolismo , Animales , Antígeno CD11b/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/ultraestructura , Línea Celular , Células Cultivadas , Modelos Animales de Enfermedad , Vesículas Extracelulares/ultraestructura , Femenino , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Mediadores de Inflamación/metabolismo , Interleucina-4/farmacología , Interleucina-6/biosíntesis , Macrófagos/metabolismo , Animales , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Femenino , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/prevención & control , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/inmunología , Interleucina-4/inmunología , Interleucina-6/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
GM-CSF-producing T helper (Th) cells play a crucial role in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Recent studies have identified a distinct population of GM-CSF-producing Th cells, named ThGM cells, that also express cytokines TNF, IL-2, and IL-3, but lack expression of master transcription factors (TF) and signature cytokines of commonly recognized Th cell lineages. ThGM cells are highly encephalitogenic in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Similar to Th17 cells, in response to IL-12, ThGM cells upregulate expression of T-bet and IFN-γ and switch their phenotype to Th1. Here we show that in addition to T-bet, TF RUNX3 also contributes to the Th1 switch of ThGM cells. T-bet-deficient ThGM cells in the CNS of mice with EAE had low expression of RUNX3, and knockdown of RUNX3 expression in ThGM cells abrogated the Th1-inducing effect of IL-12. Comparison of ThGM and Th1 cell transcriptomes showed that ThGM cells expressed a set of TFs known to inhibit the development of other Th lineages. Lack of expression of lineage-specific cytokines and TFs by ThGM cells, together with expression of TFs that inhibit the development of other Th lineages, suggests that ThGM cells are a non-polarized subset of Th cells with lineage characteristics.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Citocinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-12/metabolismo , Ratones , Fenotipo , Células TH1 , Células Th17 , Factores de Transcripción/metabolismoRESUMEN
Astrocytes are highly heterogeneous in their phenotype and function, which contributes to CNS disease, repair, and aging; however, the molecular mechanism of their functional states remains largely unknown. Here, we show that activation of sirtuin 1 (SIRT1), a protein deacetylase, played an important role in the detrimental actions of reactive astrocytes, whereas its inactivation conferred these cells with antiinflammatory functions that inhibited the production of proinflammatory mediators by myeloid cells and microglia and promoted the differentiation of oligodendrocyte progenitor cells. Mice with astrocyte-specific Sirt1 knockout (Sirt1-/-) had suppressed progression of experimental autoimmune encephalomyelitis (EAE), an animal model of CNS inflammatory demyelinating disease. Ongoing EAE was also suppressed when Sirt1 expression in astrocytes was diminished by a CRISPR/Cas vector, resulting in reduced demyelination, decreased numbers of T cells, and an increased rate of IL-10-producing macrophages and microglia in the CNS, whereas the peripheral immune response remained unaffected. Mechanistically, Sirt1-/- astrocytes expressed a range of nuclear factor erythroid-derived 2-like 2 (Nfe2l2) target genes, and Nfe2l2 deficiency shifted the beneficial action of Sirt1-/- astrocytes to a detrimental one. These findings identify an approach for switching the functional state of reactive astrocytes that will facilitate the development of astrocyte-targeting therapies for inflammatory neurodegenerative diseases such as multiple sclerosis.
Asunto(s)
Astrocitos , Encefalomielitis Autoinmune Experimental , Sirtuina 1 , Animales , Ratones , Astrocitos/enzimología , Astrocitos/patología , Autoinmunidad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Ratones Endogámicos C57BL , Fenotipo , Sirtuina 1/genética , Sirtuina 1/metabolismo , Ratones NoqueadosRESUMEN
Targeted and effective drug delivery to central nervous system (CNS) lesions is a major challenge in the treatment of multiple sclerosis (MS). Extracellular vesicles (EVs) have great promise as a drug delivery nanosystem given their unique characteristics, including a strong cargo-loading capacity, low immunogenicity, high biocompatibility, inherent stability, high delivery efficiency, ease of manipulation, and blood-brain barrier (BBB) penetration. Clinical applications are, however, limited by their insufficient targeting capability and "dilution effects" upon systemic administration. Neural stem cells (NSCs) provide an abundant source of EVs because of their remarkable capacity for self-renewal. Here, we developed a novel therapeutic strategy for local delivery and treatment using EVPs, which are derived from NSCs with the expression of the CNS lesion targeting ligand-PDGFRα. Furthermore, we used EVPs as a targeting carrier for encapsulating Bryostatin-1 (Bryo-1), a natural compound with remarkable anti-inflammation ability. Our data showed that Bryo-1 delivered by EVPs was more stable and concentrated in the CNS than native Bryo-1. Systemic injection of a low dosage (1 × 108 particles) of EVPs + Bryo-1, versus only EVPs or Bryo-1 administration, significantly ameliorated clinical disease development, decreased the infiltration of pro-inflammatory cells, blocked myelin loss and astrogliosis, protected BBB integrity, and altered microglia pro-inflammatory phenotype in the CNS of EAE mice. Taken as a whole, our study showed that engineered EVs have a CNS targeting capacity, and it provides potentially powerful therapeutic effects for the treatment of various neuroinflammatory diseases.
Asunto(s)
Vesículas Extracelulares , Esclerosis Múltiple , Animales , Brioestatinas/farmacología , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/tratamiento farmacológico , Enfermedades NeuroinflamatoriasRESUMEN
Extracellular vesicles (EVs) are heterogeneous cell-derived membranous structures that arise from the endosome system or directly detach from the plasma membrane. In recent years, many advances have been made in the understanding of the clinical definition and pathogenesis of neurodegenerative diseases, but translation into effective treatments is hampered by several factors. Current research indicates that EVs are involved in the pathology of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). Besides, EVs are also involved in the process of myelin formation, and can also cross the blood-brain barrier to reach the sites of CNS injury. It is suggested that EVs have great potential as a novel therapy for the treatment of neurodegenerative diseases. Here, we reviewed the advances in understanding the role of EVs in neurodegenerative diseases and addressed the critical function of EVs in the CNS. We have also outlined the physiological mechanisms of EVs in myelin regeneration and highlighted the therapeutic potential of EVs in neurodegenerative diseases.
Asunto(s)
Vesículas Extracelulares , Enfermedades Neurodegenerativas , Enfermedad de Alzheimer , Barrera Hematoencefálica , Humanos , Enfermedades Neurodegenerativas/terapia , Enfermedad de ParkinsonRESUMEN
Inducible conditional knockout mice are important tools for studying gene function and disease therapy, but their generation is costly and time-consuming. We introduced clustered regularly interspaced short palindromic repeats (CRISPR) and Cre into an LSL-Cas9 transgene-carrying mouse line by using adeno-associated virus (AAV)-PHP.eB to rapidly knockout gene(s) specifically in central nervous system (CNS) cells of adult mice. NeuN in neurons and GFAP in astrocytes were knocked out 2 weeks after an intravenous injection of vector, with an efficiency comparable to that of inducible Cre-loxP conditional knockout. For functional testing, we generated astrocyte-specific Act1 knockout mice, which exhibited a phenotype similar to mice with Cre-loxP-mediated Act1 knockout, in an animal model of multiple sclerosis (MS), an autoimmune disorder of the CNS. With this novel technique, neural cell-specific knockout can be induced rapidly (few weeks) and cost-effectively. Our study provides a new approach to building inducible conditional knockout mice, which would greatly facilitate research on CNS biology and disease.
RESUMEN
IFN-ß has been the treatment for multiple sclerosis (MS) for almost three decades, but understanding the mechanisms underlying its beneficial effects remains incomplete. We have shown that MS patients have increased numbers of GM-CSF+ Th cells in circulation, and that IFN-ß therapy reduces their numbers. GM-CSF expression by myelin-specific Th cells is essential for the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. These findings suggested that IFN-ß therapy may function via suppression of GM-CSF production by Th cells. In the current study, we elucidated a feedback loop between monocytes and Th cells that amplifies autoimmune neuroinflammation, and found that IFN-ß therapy ameliorates central nervous system (CNS) autoimmunity by inhibiting this proinflammatory loop. IFN-ß suppressed GM-CSF production in Th cells indirectly by acting on monocytes, and IFN-ß signaling in monocytes was required for EAE suppression. IFN-ß increased IL-10 expression by monocytes, and IL-10 was required for the suppressive effects of IFN-ß. IFN-ß treatment suppressed IL-1ß expression by monocytes in the CNS of mice with EAE. GM-CSF from Th cells induced IL-1ß production by monocytes, and, in a positive feedback loop, IL-1ß augmented GM-CSF production by Th cells. In addition to GM-CSF, TNF and FASL expression by Th cells was also necessary for IL-1ß production by monocyte. IFN-ß inhibited GM-CSF, TNF, and FASL expression by Th cells to suppress IL-1ß secretion by monocytes. Overall, our study describes a positive feedback loop involving several Th cell- and monocyte-derived molecules, and IFN-ß actions on monocytes disrupting this proinflammatory loop.
Asunto(s)
Autoinmunidad , Comunicación Celular , Interferón beta/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Autoinmunidad/efectos de los fármacos , Comunicación Celular/genética , Comunicación Celular/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Interferón beta/farmacología , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/efectos de los fármacosRESUMEN
Since extracellular vesicles (EVs) were discovered in 1983 in sheep reticulocytes samples, they have gradually attracted scientific attention and become a topic of great interest in the life sciences field. EVs are small membrane particles, released by virtually every cell that carries a variety of functional molecules. Their main function is to deliver messages to the surrounding area in both physiological and pathological conditions. Initially, they were thought to be either cell debris, signs of cell death, or unspecific structures. However, accumulating evidence support a theory that EVs are a universal mechanism of communication. Thanks to their biological characteristics and functions, EVs are likely to represent a promising strategy for obtaining pathogen information, identifying therapeutic targets and selecting specific biomarkers for a variety of diseases, such as autoimmune diseases. In this review, we provide a brief overview of recent progress in the study of the biology and functions of EVs. We also discuss their roles in diagnosis and therapy, with particular emphasis on autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/fisiopatología , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Animales , Enfermedades Autoinmunes/metabolismo , HumanosRESUMEN
Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are neuroinflammatory diseases of the central nervous system (CNS), where leukocytes and CNS resident cells play important roles in disease development and pathogenesis. The antimalarial drug chloroquine (CQ) has been shown to suppress EAE by modulating dendritic cells (DCs) and Th17 cells. However, the mechanism of action by which CQ modulates EAE is far from being elucidated. Here, we comprehensively analyzed the CNS of CQ and PBS-treated EAE mice to identify and characterize the cells that are affected by CQ. Our results show that leukocytes are largely modulated by CQ and have a reduction in the expression of inflammatory markers. Intriguingly, CQ vastly modulated the CNS resident cells astrocytes, oligodendrocytes (OLs) and microglia (MG), with the latter producing IL-10 and IL-12p70. Overall, our results show a panoramic view of the cellular components that are affect by CQ and provide further evidence that drug repurposing of CQ will be beneficial to MS patients.
RESUMEN
Antigen (Ag)-specific tolerance induction by intravenous (i. v.) injection of high-dose auto-Ags has been explored for therapy of autoimmune diseases, including multiple sclerosis (MS). It is thought that the advantage of such Ag-specific therapy over non-specific immunomodulatory treatments would be selective suppression of a pathogenic immune response without impairing systemic immunity, thus avoiding adverse effects of immunosuppression. Auto-Ag i.v. tolerance induction has been extensively studied in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and limited clinical trials demonstrated that it is safe and beneficial to a subset of MS patients. Nonetheless, the mechanisms of i.v. tolerance induction are incompletely understood, hampering the development of better approaches and their clinical application. Here, we describe a pathway whereby auto-Ag i.v. injected into mice with ongoing clinical EAE induces interferon-gamma (IFN-γ) secretion by auto-Ag-specific CD4+ T cells, triggering interleukin (IL)-27 production by conventional dendritic cells type 1 (cDC1). IL-27 then, via signal transducer and activator of transcription 3 activation, induces programmed death ligand 1 (PD-L1) expression by monocyte-derived dendritic cells (moDCs) in the central nervous system of mice with EAE. PD-L1 interaction with programmed cell death protein 1 on pathogenic CD4+ T cells leads to their apoptosis/anergy, resulting in disease amelioration. These findings identify a key role of the IFN-γ/IL-27/PD-L1 axis, involving T cells/cDC1/moDCs in the induction of i.v. tolerance.
Asunto(s)
Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/inmunología , Sistema Nervioso Central/inmunología , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Monocitos/inmunología , Esclerosis Múltiple/inmunología , Animales , Autoinmunidad , Antígeno B7-H1/genética , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Tolerancia Inmunológica , Interferón gamma/metabolismo , Interleucina-27/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Elevation of granulocyte-macrophage colony-stimulating factor (GM-CSF)producing T helper (TH) cells has been associated with several autoimmune diseases, suggesting a potential role in the pathogenesis of autoimmunity. However, the identity of GM-CSFproducing TH cells has not been closely examined. Using single-cell RNA sequencing and high-dimensional single-cell mass cytometry, we identified eight populations of antigen-experienced CD45RA−CD4+ T cells in blood of healthy individuals including a population of GM-CSFproducing cells, known as THGM, that lacked expression of signature transcription factors and cytokines of established TH lineages. Using GM-CSF-reporter/fate reporter mice, we show that THGM cells are present in the periphery and central nervous system in a mouse model of experimental autoimmune encephalomyelitis. In addition to GM-CSF, human and mouse THGM cells also expressed IL-2, tumor necrosis factor (TNF), IL-3, and CCL20. THGM cells maintained their phenotype through several cycles of activation but up-regulated expression of T-bet and interferon-γ (IFN-γ) upon exposure to IL-12 in vitro and in the central nervous system of mice with autoimmune neuroinflammation. Although T-bet was not required for the development of THGM cells, it was essential for their encephalitogenicity. These findings demonstrate that THGM cells constitute a distinct population of TH cells with lineage characteristics that are poised to adopt a TH1 phenotype and promote neuroinflammation.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Esclerosis Múltiple/inmunología , Proteínas de Dominio T Box/metabolismo , Células TH1/inmunología , Animales , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Femenino , Regulación de la Expresión Génica/inmunología , Voluntarios Sanos , Humanos , Masculino , Ratones , Ratones Transgénicos , Esclerosis Múltiple/genética , Esclerosis Múltiple/patología , RNA-Seq , Análisis de la Célula Individual , Células TH1/metabolismoRESUMEN
Autoimmune diseases such as multiple sclerosis (MS) develop because of failed peripheral immune tolerance for a specific self-antigen (Ag). Numerous approaches for Ag-specific suppression of autoimmune neuroinflammation have been proven effective in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. One such approach is intravenous tolerance induction by injecting a myelin Ag used for triggering EAE. However, the translation of this and similar experimental strategies into therapy for MS has been hampered by uncertainty regarding relevant myelin Ags in MS patients. To address this issue, we developed a therapeutic strategy that relies on oligodendrocyte (Ol)-derived extracellular vesicles (Ol-EVs), which naturally contain multiple myelin Ags. Intravenous Ol-EV injection reduced disease pathophysiology in a myelin Ag-dependent manner, both prophylactically and therapeutically, in several EAE models. The treatment was safe and restored immune tolerance by inducing immunosuppressive monocytes and apoptosis of autoreactive CD4+ T cells. Furthermore, we showed that human Ols also released EVs containing most relevant myelin Ags, providing a basis for their use in MS therapy. These findings introduce an approach for suppressing central nervous system (CNS) autoimmunity in a myelin Ag-specific manner, without the need to identify the target Ag.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Vesículas Extracelulares , Esclerosis Múltiple , Animales , Encefalomielitis Autoinmune Experimental/terapia , Humanos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/tratamiento farmacológico , Glicoproteína Mielina-Oligodendrócito , OligodendroglíaRESUMEN
Oligodendrocyte progenitor cells (OPCs) are a subtype of glial cells responsible for myelin regeneration. Oligodendrocytes (OLGs) originate from OPCs and are the myelinating cells in the central nervous system (CNS). OLGs play an important role in the context of lesions in which myelin loss occurs. Even though many protocols for isolating OPCs have been published, their cellular yield remains a limit for clinical application. The protocol proposed here is novel and has practical value; in fact, OPCs can be generated from a source of autologous cells without gene manipulation. Our method represents a rapid, and high-efficiency differentiation protocol for generating mouse OLGs from bone marrow-derived cells using growth-factor defined media. With this protocol, it is possible to obtain mature OLGs in 7-8 weeks. Within 2-3 weeks from bone marrow (BM) isolation, after neurospheres formed, the cells differentiate into Nestin+ Sox2+ neural stem cells (NSCs), around 30 days. OPCs specific markers start to be expressed around day 38, followed by RIP+O4+ around day 42. CNPase+ mature OLGs are finally obtained around 7-8 weeks. Further, bone marrow-derived OPCs exhibited therapeutic effect in shiverer (Shi) mice, promoting myelin regeneration and reducing the tremor. Here, we propose a method by which OLGs can be generated starting from BM cells and have similar abilities to subventricular zone (SVZ)-derived cells. This protocol significantly decreases the timing and costs of the OLGs differentiation within 2 months of culture.
RESUMEN
Microvesicles (MVs) are membrane particles of 200-500 nm released by all cell types constitutively. MVs of myeloid origin are found increased in the cerebrospinal fluid (CSF) of patients suffering from neuroinflammatory disorders, although the factors triggering their production have never been defined. Here, we report that both pro- and anti-inflammatory cytokines, specifically interferon-γ and interleukin-4, are equally able to stimulate the production of MVs from microglia cells and monocytes. Additionally, we found this process to be independent from the best characterized molecular pathway so far described for membrane shedding, which is centered on the purinergic receptor P2X7, whose activation by high concentrations of extracellular ATP (exATP) results in membrane blebbing operated by the secreted enzyme acid sphingomyelinase (ASMase). Moreover, a potent inhibitor of ASMase, injected in a mouse model of multiple sclerosis, failed to reduce the number of MVs in their CSF. This suggests that cytokines, rather than exATP, may exert a long-term control of MV production in the context of chronic inflammation, where both pro- and anti-inflammatory factors play coordinated roles.
Asunto(s)
Micropartículas Derivadas de Células/inmunología , Citocinas/inmunología , Esclerosis Múltiple/inmunología , Células Mieloides/inmunología , Transducción de Señal/inmunología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/inmunología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Línea Celular , Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Imipramina/farmacología , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/metabolismo , Células Mieloides/metabolismo , Receptores Purinérgicos P2X7/inmunología , Receptores Purinérgicos P2X7/metabolismo , Retinoides/farmacología , Transducción de Señal/efectos de los fármacos , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/inmunología , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
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.
Asunto(s)
Sistema Nervioso Central/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Inflamación/metabolismo , Interleucina-27/metabolismo , Interleucinas/metabolismo , Leucocitos/metabolismo , Animales , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Líquido Cefalorraquídeo/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Terapia Genética , Lentivirus/genética , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Blood-borne monocyte derived cells play a pivotal, initially unrecognized, role in most central nervous system disorders, including diseases initially classified as purely neurodegenerative (i.e., Alzheimer's disease, Parkinson's disease, and ALS). Their trafficking to the brain and spinal cord has been extensively studied in classical neuroinflammatory disorders such as multiple sclerosis. Central nervous system resident myeloid cells, namely microglia and perivascular macrophages, also are in the spotlight of investigations on neurological disorders. Myeloid cells, such as infiltrating macrophages and microglia, have been described as having both protective and destructive features in neurological disorders, thus identification of their functional phenotype during disease evolution would be of paramount importance. Extracellular vesicles, namely exosomes and shed vesicles, are released by virtually any cell type and can be detected and identified in terms of cell origin in biological fluids. They therefore constitute an ideal tool to access information on cells residing in an inaccessible site such as the brain. We will review here available information on extracellular vesicles detection in neurological disorders with special emphasis on neurodegenerative diseases.