RESUMEN
Demyelination is a key pathogenic feature of multiple sclerosis (MS). Here, we evaluated the astrocyte contribution to myelin loss and focused on the neurotrophin receptor TrkB, whose up-regulation on the astrocyte finely demarcated chronic demyelinated areas in MS and was paralleled by neurotrophin loss. Mice lacking astrocyte TrkB were resistant to demyelination induced by autoimmune or toxic insults, demonstrating that TrkB signaling in astrocytes fostered oligodendrocyte damage. In vitro and ex vivo approaches highlighted that astrocyte TrkB supported scar formation and glia proliferation even in the absence of neurotrophin binding, indicating TrkB transactivation in response to inflammatory or toxic mediators. Notably, our neuropathological studies demonstrated copper dysregulation in MS and model lesions and TrkB-dependent expression of copper transporter (CTR1) on glia cells during neuroinflammation. In vitro experiments evidenced that TrkB was critical for the generation of glial intracellular calcium flux and CTR1 up-regulation induced by stimuli distinct from neurotrophins. These events led to copper uptake and release by the astrocyte, and in turn resulted in oligodendrocyte loss. Collectively, these data demonstrate a pathogenic demyelination mechanism via the astrocyte release of copper and open up the possibility of restoring copper homeostasis in the white matter as a therapeutic target in MS.
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Astrocitos/metabolismo , Astrocitos/patología , Cobre/metabolismo , Esclerosis Múltiple/metabolismo , Animales , Transporte Biológico , Enfermedad Crónica , Cicatriz/patología , Cuprizona , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental , Humanos , Inflamación/patología , Ligandos , Proteínas de Transporte de Membrana/metabolismo , Ratones Noqueados , Vaina de Mielina/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Receptor trkB/metabolismo , Regulación hacia Arriba , Sustancia Blanca/patologíaRESUMEN
Hepatocyte nuclear factor 4 α (HNF4α), a transcription factor (TF) essential for embryonic development, has been recently shown to regulate the expression of inflammatory genes. To characterize HNF4a function in immunity, we measured the effect of HNF4α antagonists on immune cell responses in vitro and in vivo. HNF4α blockade reduced immune activation in vitro and disease severity in the experimental model of multiple sclerosis (MS). Network biology studies of human immune transcriptomes unraveled HNF4α together with SP1 and c-myc as master TF regulating differential expression at all MS stages. TF expression was boosted by immune cell activation, regulated by environmental MS risk factors and higher in MS immune cells compared to controls. Administration of compounds targeting TF expression or function demonstrated non-synergic, interdependent transcriptional control of CNS autoimmunity in vitro and in vivo. Collectively, we identified a coregulatory transcriptional network sustaining neuroinflammation and representing an attractive therapeutic target for MS and other inflammatory disorders.
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Autoinmunidad , Factor Nuclear 4 del Hepatocito , Esclerosis Múltiple , Humanos , Autoinmunidad/genética , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Transcriptoma , Genes mycRESUMEN
Alteration in endogenous Interferon (IFN) system may profoundly impact immune cell function in autoimmune diseases. Here, we provide evidence that dysregulation in IFN-regulated genes and pathways are involved in B cell- and monocyte-driven pathogenic contribution to Multiple Sclerosis (MS) development and maintenance. In particular, by using an Interferome-based cell type-specific approach, we characterized an increased susceptibility to an IFN-linked caspase-3 dependent apoptotic cell death in both B cells and monocytes of MS patients that may arise from their chronic activation and persistent stimulation by activated T cells. Ongoing caspase-3 activation functionally impacts on MS monocyte properties influencing the STAT-3/IL-16 axis, thus, driving increased expression and massive release of the bio-active IL-16 triggering and perpetuating CD4+ T cell migration. Importantly, our analysis also identified a previously unknown multi-component defect in type I IFN-mediated signaling and response to virus pathways specific of MS B cells, impacting on induction of anti-viral responses and Epstein-barr virus infection control in patients. Taking advantage of cell type-specific transcriptomics and in-depth functional validation, this study revealed pathogenic contribution of endogenous IFN signaling and IFN-regulated cell processes to MS pathogenesis with implications on fate and functions of B cells and monocytes that may hold therapeutic potential.
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Linfocitos B/inmunología , Linfocitos B/metabolismo , Interferón Tipo I/genética , Monocitos/inmunología , Monocitos/metabolismo , Esclerosis Múltiple/etiología , Esclerosis Múltiple/metabolismo , Transcriptoma , Adulto , Apoptosis , Biomarcadores , Estudios de Casos y Controles , Susceptibilidad a Enfermedades , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Interferón Tipo I/metabolismo , Interleucina-16/genética , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patología , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Regiones Promotoras Genéticas , Transducción de SeñalRESUMEN
Erectile dysfunction is a common disease characterized by endothelial dysfunction. The aetiology of ED is often multifactorial but evidence is being accumulated in favor of the proper function of the vascular endothelium that is essential to achieving and maintaining penile erection. Uric acid itself causes endothelial dysfunction via decreased nitric oxide production. This study aims to evaluate the serum uric acid (SUA) levels in 180 ED patients, diagnosed with the International Index of Erectile Function-5 (IIEF-5) and 30 non-ED control. Serum uric acid was analyzed with a commercially available kit using ModularEVO (Roche, Monza, Italy). Within-assay and between-assay variations were 3.0% and 6.0%, respectively. Out of the ED patients, 85 were classified as arteriogenic (A-ED) and 95 as non-arteriogenic (NA-ED) with penile-echo-color-Doppler. Uric acid levels (median and range in mg/dL) in A-ED patients (5.8, 4.3-7.5) were significantly higher (p < .001) than in NA-ED patients (4.4, 2.6-5.9) and in control group (4.6, 3.1-7.2). There was a significant difference (p < .001) between uric acid levels in patients with mild A-ED (IIEF-5 16-20) and severe/complete A-ED (IIEF-5 ≤ 10) that were 5.4 (range 4.3-6.5) mg/dL and 6.8 (range 6.4-7.2) mg/dL, respectively. There was no difference between the levels of uric acid in patients with different degree of NA-ED. Our findings reveal that SUA is a marker of ED but only of ED of arteriogenic aetiology.
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Impotencia Vasculogénica/sangre , Ácido Úrico/sangre , Adulto , Estudios de Casos y Controles , Endotelio Vascular/fisiopatología , Humanos , Impotencia Vasculogénica/diagnóstico , Impotencia Vasculogénica/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Erección Peniana/fisiología , Encuestas y Cuestionarios , Ultrasonografía Doppler en ColorRESUMEN
OBJECTIVE: Although astrocytes participate in glial scar formation and tissue repair, dysregulation of the NFκB pathway and of nitric oxide (NO) production in these glia cells contributes to neuroinflammation and neurodegeneration. Here we investigated the role of the crosstalk between sphingosine-1-phosphate (S1P) and cytokine signaling cascades in astrocyte activation and inflammation-mediated neurodegeneration, and addressed the effects of fingolimod on astrocyte-neuron interaction and NO synthesis in vivo. METHODS: Immunohistochemistry, immunofluorescence, and confocal microscopy were used to detect S1P receptors, interleukin (IL) 1R, IL17RA, and nitrosative stress in multiple sclerosis (MS) plaques, experimental autoimmune encephalomyelitis (EAE) spinal cord, and the spinal cord of fingolimod-treated EAE mice. An in vitro model was established to study the effects of S1P, IL1, and IL17 stimulation on NFkB translocation and NO production in astrocytes, on spinal neuron survival, and on astrocyte-neuron interaction. Furthermore, fingolimod efficacy in blocking astrocyte-mediated neurodegeneration was evaluated. RESULTS: We found coordinated upregulation of IL1R, IL17RA, S1P1, and S1P3 together with nitrosative markers in astrocytes within MS and EAE lesions. In vitro studies revealed that S1P, IL17, and IL1 induced NFκB translocation and NO production in astrocytes, and astrocyte conditioned media triggered neuronal death. Importantly, fingolimod blocked the 2 activation events evoked in astrocytes by either S1P or inflammatory cytokines, resulting in inhibition of astrocyte-mediated neurodegeneration. Finally, therapeutic administration of fingolimod to EAE mice hampered astrocyte activation and NO production. INTERPRETATION: A neuroprotective effect of fingolimod in vivo may result from its inhibitory action on key astrocyte activation steps.
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Astrocitos/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inmunosupresores/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Óxido Nítrico/antagonistas & inhibidores , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Anciano , Animales , Astrocitos/metabolismo , Células Cultivadas , Cerebro/metabolismo , Cerebro/patología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Clorhidrato de Fingolimod , Humanos , Inmunosupresores/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Óxido Nítrico/biosíntesis , Glicoles de Propileno/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptores Tipo I de Interleucina-1/metabolismo , Receptores de Interleucina-17/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Esfingosina/administración & dosificación , Esfingosina/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
In this study we investigated the contribution of gender to global gene expression in peripheral blood mononuclear cells from multiple sclerosis (MS) patients and healthy controls. We observed that, in contrast to the conventional approach, gender-based case-control comparisons resulted in genelists with significantly reduced heterogeneity in human populations. In addition, MS was characterized by significant changes both in the quantity and in the quality of the sex-specific genes. Application of stringent statistics defined gender-based signatures which classified a second independent MS population with high precision. The global unsupervised cluster analyses for 60 subjects showed that 29/31 female and 27/29 male samples were properly identified. Notably, MS was associated in women and in men with distinct gene signatures which however shared several molecular functions, biological processes and interactors. Issues regarding epigenetic control of gene expression appeared as the main common theme for disease, with a central role for the functional modules related to histone deacetylase, NF-kappaB and androgen receptor signaling. Moreover, in silico analyses predicted that the differential expression in MS women and men were depending on the transcription factor SP1. Specific targeting of this pathway by the bis-anthracycline WP631 impaired T cell responses in vitro and in vivo, and reduced the incidence and the severity of experimental autoimmune encephalomyelitis, indicating that SP1 dependent gene transcription sustains neuroinflammation. Thus, the gender-based approach with its reduced heterogeneity and the systems biology tools with the identification of the molecular and functional networks successfully uncovered the differences but also the commonalities associated to multiple sclerosis in women and men. In conclusion, we propose gender-based systems biology as a novel tool to gain fundamental information on disease-associated functional processes.
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Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Factor de Transcripción Sp1/metabolismo , Transcriptoma , Adulto , Animales , Análisis por Conglomerados , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Epigénesis Genética , Femenino , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Reproducibilidad de los Resultados , Factores Sexuales , Transcripción GenéticaRESUMEN
Peripheral blood mononuclear cells (PBMCs) bear specific dysregulations in genes and pathways at distinct stages of multiple sclerosis (MS) that may help with classifying MS and non-MS subjects, specifying the early stage of disease, or discriminating among MS courses. Here we describe an unbiased machine learning workflow to build MS stage-specific classifiers based on PBMC transcriptomics profiles from more than 300 individuals, including healthy subjects and patients with clinically isolated syndromes, relapsing-remitting MS, primary or secondary progressive MS, or other neurological disorders. The pipeline, designed to optimize and compare the performance of distinct machine learning algorithms in the training cohort, generates predictive models not influenced by demographic features, such as age and gender, and displays high accuracy in the independent validation cohort. Proper application of machine learning to transcriptional profiles of circulating blood cells may allow identification of disease state and stage in MS.
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Perfilación de la Expresión Génica/métodos , Esclerosis Múltiple/clasificación , Esclerosis Múltiple/genética , Adulto , Biomarcadores/sangre , Femenino , Expresión Génica/genética , Humanos , Leucocitos Mononucleares/metabolismo , Aprendizaje Automático , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple Crónica Progresiva/sangre , Esclerosis Múltiple Recurrente-Remitente/sangre , Transcriptoma/genéticaRESUMEN
OBJECTIVE: To perform systematic transcriptomic analysis of multiple sclerosis (MS) risk genes in peripheral blood mononuclear cells (PBMCs) of subjects with distinct MS stages and describe the pathways characterized by dysregulated gene expressions. METHODS: We monitored gene expression levels in PBMCs from 3 independent cohorts for a total of 297 cases (including clinically isolated syndromes (CIS), relapsing-remitting MS, primary and secondary progressive MS) and 96 healthy controls by distinct microarray platforms and quantitative PCR. Differential expression and pathway analyses for distinct MS stages were defined and validated by literature mining. RESULTS: Genes located in the vicinity of MS risk variants displayed altered expression in peripheral blood at distinct stages of MS compared with the healthy population. The frequency of dysregulation was significantly higher than expected in CIS and progressive forms of MS. Pathway analysis for each MS stage-specific gene list showed that dysregulated genes contributed to pathogenic processes with scientific evidence in MS. CONCLUSIONS: Systematic gene expression analysis in PBMCs highlighted selective dysregulation of MS susceptibility genes playing a role in novel and well-known pathogenic pathways.
RESUMEN
OBJECTIVE: To track the effects of fingolimod, an approved drug for multiple sclerosis (MS), on the activation of myeloid cells from the periphery to the CNS. METHODS: In vitro and ex vivo immunologic studies coupled with flow cytometry were performed to evaluate the action of fingolimod on lipopolysaccharide (LPS)-induced expression of activation markers in human monocytes from healthy participants, participants with untreated MS, and participants with fingolimod-treated MS. In vivo administration of fingolimod during experimental autoimmune encephalomyelitis (EAE) was established to verify the activation state of splenic, CNS infiltrating, and CNS resident myeloid cells ex vivo at flow cytometer. RESULTS: We found that in vitro exposure of human monocytes to fingolimod inhibited LPS-induced CD25 and CD150 expression and tumor necrosis factor-α (TNF-α) secretion without altering immune cell survival. Further, EAE treatment with fingolimod led to reduced amounts of TNF-α produced by myeloid cells in vivo in the spleen and CNS. Finally, while displaying normal induction of CD25 and CD150 levels at high LPS concentration, monocytes from patients with fingolimod-treated MS showed significantly higher activation threshold at suboptimal LPS stimulation than controls. CONCLUSIONS: The inhibition of myeloid cell activation may be part of the immunosuppressive action of fingolimod and take place in the periphery and in the CNS.
RESUMEN
OBJECTIVE: To investigate the potential role of prokineticin 2 (PK2), a bioactive peptide involved in multiple biological functions including immune modulation, in CNS autoimmune demyelinating disease. METHODS: We investigated the expression of PK2 in mice with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), and in patients with relapsing-remitting MS. We evaluated the biological effects of PK2 on expression of EAE and on development of T-cell response against myelin by blocking PK2 in vivo with PK2 receptor antagonists. We treated with PK2 immune cells activated against myelin antigen to explore the immune-modulating effects of this peptide in vitro. RESULTS: Pk2 messenger RNA was upregulated in spinal cord and lymph node cells (LNCs) of mice with EAE. PK2 protein was expressed in EAE inflammatory infiltrates and was increased in sera during EAE. In patients with relapsing-remitting MS, transcripts for PK2 were significantly increased in peripheral blood mononuclear cells compared with healthy controls, and PK2 serum concentrations were significantly higher. A PK2 receptor antagonist prevented or attenuated established EAE in chronic and relapsing-remitting models, reduced CNS inflammation and demyelination, and decreased the production of interferon (IFN)-γ and interleukin (IL)-17A cytokines in LNCs while increasing IL-10. PK2 in vitro increased IFN-γ and IL-17A and reduced IL-10 in splenocytes activated against myelin antigen. CONCLUSION: These data suggest that PK2 is a critical immune regulator in CNS autoimmune demyelination and may represent a new target for therapy.
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Along with their established role in allergic reactions, histamine and its receptors have been implicated in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis. In this study we analyzed the gene expression of histamine receptor 1 (HRH1), HRH2 and HRH4 in peripheral blood mononuclear cells derived from patients with clinically isolated syndrome (CIS), relapsing-remitting (RR) MS, secondary-progressive (SP) MS, primary-progressive (PP) MS, and healthy controls (HC). We found that HRH1 transcript was significantly down-modulated in SP-MS compared with HC, and HRH4 was increased in this group compared to HC, CIS and RR-MS. No other differences in the expression of histamine receptors were observed between HC, CIS and other clinical forms of definite MS.