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An increasing body of evidence associates low vitamin D levels with increased risk of multiple sclerosis (MS), suggesting the possibility of a gene-environment interaction for this environmental factor in MS pathogenesis. Moreover, it has been shown that vitamin D downregulates major histocompatibility complex (MHC) class II expression in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We here report about the impact of a dietary vitamin D supplementation on EAE in the rat strains having functionally relevant allelic variations in the CIITA (Mhc2ta) gene, a master regulator of MHC class II expression. Full length myelin oligodendrocyte glycoprotein (MOG)-EAE was induced in DA.PVGav1-Vra4 congenic rats harboring the Vra4 locus from PVG strain in the EAE- susceptible DA background, and compared to the parental strains. The congenic rats fed with either vitamin D supplemented, deprived or regular diet developed an intermediate clinical EAE phenotype, in contrast to DA and PVG strains. Immunopathological studies revealed vitamin D dose-dependent effect on demyelination and inflammatory infiltration of the central nervous system (CNS), expression of MHC class II and CIITA, as well as downregulation of a range of pro-inflammatory genes. Taken together, our findings demonstrate an impact of vitamin D on the target tissue pathology and peripheral immune response during EAE in DA.PVGav1-Vra4 congenic strain. Thereby, our data provide evidence of a modulatory effect of vitamin D in context of genetic variances in the Vra4 locus/Mhc2ta gene in MS-like neuroinflammation, with potential relevance for the human demyelinating disease.
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Dimethyl fumarate (DMF) is a first-line-treatment for relapsing-remitting multiple sclerosis (RRMS). The redox master regulator Nrf2, essential for redox balance, is a target of DMF, but its precise therapeutic mechanisms of action remain elusive. Here we show impact of DMF on circulating monocytes and T cells in a prospective longitudinal RRMS patient cohort. DMF increases the level of oxidized isoprostanes in peripheral blood. Other observed changes, including methylome and transcriptome profiles, occur in monocytes prior to T cells. Importantly, monocyte counts and monocytic ROS increase following DMF and distinguish patients with beneficial treatment-response from non-responders. A single nucleotide polymorphism in the ROS-generating NOX3 gene is associated with beneficial DMF treatment-response. Our data implicate monocyte-derived oxidative processes in autoimmune diseases and their treatment, and identify NOX3 genetic variant, monocyte counts and redox state as parameters potentially useful to inform clinical decisions on DMF therapy of RRMS.
Asunto(s)
Dimetilfumarato/uso terapéutico , Inmunosupresores/uso terapéutico , Monocitos/inmunología , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , NADPH Oxidasas/genética , Adulto , Metilación de ADN/efectos de los fármacos , Dimetilfumarato/farmacología , Epigénesis Genética/efectos de los fármacos , Femenino , Humanos , Inmunosupresores/farmacología , Recuento de Leucocitos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Esclerosis Múltiple Recurrente-Remitente/sangre , Esclerosis Múltiple Recurrente-Remitente/inmunología , NADPH Oxidasas/metabolismo , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/metabolismo , Polimorfismo de Nucleótido Simple , Estudios Prospectivos , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Resultado del TratamientoRESUMEN
The human leukocyte antigen (HLA) haplotype DRB1*15:01 is the major risk factor for multiple sclerosis (MS). Here, we find that DRB1*15:01 is hypomethylated and predominantly expressed in monocytes among carriers of DRB1*15:01. A differentially methylated region (DMR) encompassing HLA-DRB1 exon 2 is particularly affected and displays methylation-sensitive regulatory properties in vitro. Causal inference and Mendelian randomization provide evidence that HLA variants mediate risk for MS via changes in the HLA-DRB1 DMR that modify HLA-DRB1 expression. Meta-analysis of 14,259 cases and 171,347 controls confirms that these variants confer risk from DRB1*15:01 and also identifies a protective variant (rs9267649, p < 3.32 × 10-8, odds ratio = 0.86) after conditioning for all MS-associated variants in the region. rs9267649 is associated with increased DNA methylation at the HLA-DRB1 DMR and reduced expression of HLA-DRB1, suggesting a modulation of the DRB1*15:01 effect. Our integrative approach provides insights into the molecular mechanisms of MS susceptibility and suggests putative therapeutic strategies targeting a methylation-mediated regulation of the major risk gene.
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Metilación de ADN , Predisposición Genética a la Enfermedad/genética , Cadenas HLA-DRB1/genética , Esclerosis Múltiple/genética , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Células Cultivadas , Estudios de Cohortes , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Metaanálisis como Asunto , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Factores de Riesgo , Adulto JovenRESUMEN
BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system caused by genetic and environmental factors. DNA methylation, an epigenetic mechanism that controls genome activity, may provide a link between genetic and environmental risk factors. OBJECTIVE: We sought to identify DNA methylation changes in CD4+ T cells in patients with relapsing-remitting (RR-MS) and secondary-progressive (SP-MS) disease and healthy controls (HC). METHODS: We performed DNA methylation analysis in CD4+ T cells from RR-MS, SP-MS, and HC and associated identified changes with the nearby risk allele, smoking, age, and gene expression. RESULTS: We observed significant methylation differences in the VMP1/MIR21 locus, with RR-MS displaying higher methylation compared to SP-MS and HC. VMP1/MIR21 methylation did not correlate with a known MS risk variant in VMP1 or smoking but displayed a significant negative correlation with age and the levels of mature miR-21 in CD4+ T cells. Accordingly, RR-MS displayed lower levels of miR-21 compared to SP-MS, which might reflect differences in age between the groups, and healthy individuals and a significant enrichment of up-regulated miR-21 target genes. CONCLUSION: Disease-related changes in epigenetic marking of MIR21 in RR-MS lead to differences in miR-21 expression with a consequence on miR-21 target genes.
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Linfocitos T CD4-Positivos/fisiología , Regulación de la Expresión Génica/fisiología , MicroARNs/genética , Esclerosis Múltiple Crónica Progresiva/genética , Esclerosis Múltiple Recurrente-Remitente/genética , Adulto , Metilación de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Crónica Progresiva/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Regulación hacia ArribaRESUMEN
The initiation, commitment, and terminal differentiation of the B cell lineage is stringently controlled by the coordinated action of various transcription factors. Among these, Arid3a has previously been implicated in regulating early B lymphopoiesis, humoral immune responses to phosphocholine, and furthermore to promote the B1 over the B2 cell lineage. We have now interrogated the function of Arid3a in the adult mouse using conditional mutagenesis. We demonstrate that loss of Arid3a does not affect early B cell development or lineage commitment but rather loss of this transcription factor results in a broad expansion of bone marrow B lymphopoiesis in a manner that reflects its developmental expression pattern. Furthermore, loss of Arid3a resulted in expanded splenic B cell numbers with the exception of the B1 lineage that was maintained at normal numbers. However, B1a lymphoyctes were reduced in the peritoneal cavity. In addition, antibody responses to phosphocholine were attenuated in the absence of Arid3a. Hence, functional Arid3a is required in mature B cells for specific immune responses and for generating normal numbers of B cells in a subset dependent manner.
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Besides its vital role in immunity, the complement system also contributes to the shaping of the synaptic circuitry of the brain. We recently described that soluble Complement Receptor 2 (sCR2) is part of the nerve injury response in rodents. We here study CR2 in context of multiple sclerosis (MS) and explore the molecular effects of CR2 on C3 activation. Significant increases in sCR2 levels were evident in cerebrospinal fluid (CSF) from both patients with relapsing-remitting MS (n=33; 6.2ng/mL) and secondary-progressive MS (n=9; 7.0ng/mL) as compared to controls (n=18; 4.1ng/mL). Furthermore, CSF sCR2 levels correlated significantly both with CSF C3 and C1q as well as to a disease severity measure. In vitro, sCR2 inhibited the cleavage and down regulation of C3b to iC3b, suggesting that it exerts a modulatory role in complement activation downstream of C3. These results propose a novel function for CR2/sCR2 in human neuroinflammatory conditions.
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Complemento C3/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Esclerosis Múltiple/inmunología , Receptores de Complemento 3d/inmunología , Adulto , Activación de Complemento/inmunología , Complemento C1q/líquido cefalorraquídeo , Complemento C1q/inmunología , Complemento C3/líquido cefalorraquídeo , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/patología , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/patología , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
OBJECTIVE: We aimed to examine the regulation of lipocalin-2 (LCN2) in multiple sclerosis (MS) and its potential functional relevance with regard to myelination and neurodegeneration. METHODS: We determined LCN2 levels in 3 different studies: (1) in CSF and plasma from a case-control study comparing patients with MS (n = 147) with controls (n = 50) and patients with relapsing-remitting MS (n = 75) with patients with progressive MS (n = 72); (2) in CSF and brain tissue microdialysates from a case series of 7 patients with progressive MS; and (3) in CSF at baseline and 60 weeks after natalizumab treatment in a cohort study of 17 patients with progressive MS. Correlation to neurofilament light, a marker of neuroaxonal injury, was tested. The effect of LCN2 on myelination and neurodegeneration was studied in a rat in vitro neuroglial cell coculture model. RESULTS: Intrathecal production of LCN2 was increased predominantly in patients with progressive MS (p < 0.005 vs relapsing-remitting MS) and displayed a positive correlation to neurofilament light (p = 0.005). Levels of LCN2 in brain microdialysates were severalfold higher than in the CSF, suggesting local production in progressive MS. Treatment with natalizumab in progressive MS reduced LCN2 levels an average of 13% (p < 0.0001). LCN2 was found to inhibit remyelination in a dose-dependent manner in vitro. CONCLUSIONS: LCN2 production is predominantly increased in progressive MS. Although this moderate increase does not support the use of LCN2 as a biomarker, the correlation to neurofilament light and the inhibitory effect on remyelination suggest that LCN2 might contribute to neurodegeneration through myelination-dependent pathways.
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Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system, with a high rate of neurocognitive symptoms for which the molecular background is still uncertain. There is accumulating evidence for dysregulation of the kynurenine pathway (KP) in different psychiatric and neurodegenerative conditions. We here report the first comprehensive analysis of cerebrospinal fluid (CSF) kynurenine metabolites in MS patients of different disease stages and in relation to neurocognitive symptoms. Levels of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QUIN) were determined with liquid chromatography mass spectrometry in cell-free CSF. At the group level MS patients (cohort 1; n=71) did not differ in absolute levels of TRP, KYN, KYNA or QUIN as compared to non-inflammatory neurological disease controls (n=20). Stratification of patients into different disease courses revealed that both absolute QUIN levels and the QUIN/KYN ratio were increased in relapsing-remitting MS (RRMS) patients in relapse. Interestingly, secondary progressive MS (SPMS) displayed a trend for lower TRP and KYNA, while primary progressive (PPMS) patients displayed increased levels of all metabolites, similar to a group of inflammatory neurological disease controls (n=13). In the second cohort (n=48), MS patients with active disease and short disease duration were prospectively evaluated for neuropsychiatric symptoms. In a supervised multivariate analysis using orthogonal projection to latent structures (OPLS-DA) depressed patients displayed higher KYNA/TRP and KYN/TRP ratios, mainly due to low TRP levels. Still, this model had low predictive value and could not completely separate the clinically depressed patients from the non-depressed MS patients. No correlation was evident for other neurocognitive measures. Taken together these results demonstrate that clinical disease activity and differences in disease courses are reflected by changes in KP metabolites. Increased QUIN levels of RRMS patients in relapse and generally decreased levels of TRP in SPMS may relate to neurotoxicity and failure of remyelination, respectively. In contrast, PPMS patients displayed a more divergent pattern more resembling inflammatory conditions such as systemic lupus erythematosus. The pattern of KP metabolites in RRMS patients could not predict neurocognitive symptoms.
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Progresión de la Enfermedad , Quinurenina/líquido cefalorraquídeo , Esclerosis Múltiple/líquido cefalorraquídeo , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/complicaciones , Triptófano/líquido cefalorraquídeoRESUMEN
BACKGROUND: Activation of the complement system has been implicated in both acute and chronic states of neurodegeneration. However, a detailed understanding of this complex network of interacting components is still lacking. METHODS: Large-scale global expression profiling in a rat F2(DAxPVG) intercross identified a strong cis-regulatory influence on the local expression of complement receptor 2 (Cr2) in the spinal cord after ventral root avulsion (VRA). Expression of Cr2 in the spinal cord was studied in a separate cohort of DA and PVG rats at different time-points after VRA, and also following sciatic nerve transection (SNT) in the same strains. Consequently, Cr2 (-/-) mice and Wt controls were used to further explore the role of Cr2 in the spinal cord following SNT. The in vivo experiments were complemented by astrocyte and microglia cell cultures. RESULTS: Expression of Cr2 in naïve spinal cord was low but strongly up regulated at 5-7 days after both VRA and SNT. Levels of Cr2 expression, as well as astrocyte activation, was higher in PVG rats than DA rats following both VRA and SNT. Subsequent in vitro studies proposed astrocytes as the main source of Cr2 expression. A functional role for Cr2 is suggested by the finding that transgenic mice lacking Cr2 displayed increased loss of synaptic nerve terminals following nerve injury. We also detected increased levels of soluble CR2 (sCR2) in the cerebrospinal fluid of rats following VRA. CONCLUSIONS: These results demonstrate that local expression of Cr2 in the central nervous system is part of the axotomy reaction and is suggested to modulate subsequent complement mediated effects.
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Receptores de Complemento 3d/metabolismo , Médula Espinal/metabolismo , Raíces Nerviosas Espinales/lesiones , Raíces Nerviosas Espinales/patología , Regulación hacia Arriba/fisiología , Análisis de Varianza , Animales , Antígenos CD/metabolismo , Astrocitos/metabolismo , Antígeno CD11b/metabolismo , Células Cultivadas , Lateralidad Funcional , Redes Reguladoras de Genes , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones Transgénicos , Análisis por Micromatrices , Microglía/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores de Complemento 3d/genética , Neuropatía Ciática/metabolismo , Neuropatía Ciática/patología , Sinaptofisina/metabolismoRESUMEN
Dysregulation of the complement system is evident in many CNS diseases but mechanisms regulating complement activation in the CNS remain unclear. In a recent large rat genome-wide expression profiling and linkage analysis we found co-regulation of complement C3 immediately downstream of butyrylcholinesterase (BuChE), an enzyme hydrolyzing acetylcholine (ACh), a classical neurotransmitter with immunoregulatory effects. We here determined levels of neurofilament-light (NFL), a marker for ongoing nerve injury, C3 and activity of the two main ACh hydrolyzing enzymes, acetylcholinesterase (AChE) and BuChE, in cerebrospinal fluid (CSF) from patients with MS (n = 48) and non-inflammatory controls (n = 18). C3 levels were elevated in MS patients compared to controls and correlated both to disability and NFL. C3 levels were not induced by relapses, but were increased in patients with ≥9 cerebral lesions on magnetic resonance imaging and in patients with progressive disease. BuChE activity did not differ at the group level, but was correlated to both C3 and NFL levels in individual samples. In conclusion, we show that CSF C3 correlates both to a marker for ongoing nerve injury and degree of disease disability. Moreover, our results also suggest a potential link between intrathecal cholinergic activity and complement activation. These results motivate further efforts directed at elucidating the regulation and effector functions of the complement system in MS, and its relation to cholinergic tone.
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Butirilcolinesterasa/líquido cefalorraquídeo , Complemento C3/líquido cefalorraquídeo , Traumatismos del Nervio Craneal/líquido cefalorraquídeo , Nervios Craneales/metabolismo , Esclerosis Múltiple/líquido cefalorraquídeo , Proteínas de Neurofilamentos/líquido cefalorraquídeo , Acetilcolinesterasa/líquido cefalorraquídeo , Adulto , Biomarcadores/líquido cefalorraquídeo , Estudios de Casos y Controles , Traumatismos del Nervio Craneal/tratamiento farmacológico , Traumatismos del Nervio Craneal/inmunología , Traumatismos del Nervio Craneal/patología , Nervios Craneales/efectos de los fármacos , Nervios Craneales/inmunología , Nervios Craneales/patología , Evaluación de la Discapacidad , Femenino , Proteínas Ligadas a GPI/líquido cefalorraquídeo , Humanos , Factores Inmunológicos/uso terapéutico , Imagen por Resonancia Magnética , Masculino , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Recurrencia , Inducción de Remisión , Índice de Severidad de la EnfermedadRESUMEN
The complement system is activated in a wide spectrum of CNS diseases and is suggested to play a role in degenerative phenomena such as elimination of synaptic terminals. Still, little is known of mechanisms regulating complement activation in the CNS. Loss of synaptic terminals in the spinal cord after an experimental nerve injury is increased in the inbred DA strain compared with the PVG strain and is associated with expression of the upstream complement components C1q and C3, in the absence of membrane attack complex activation and neutrophil infiltration. To further dissect pathways regulating complement expression, we performed genome-wide expression profiling and linkage analysis in a large F2(DA × PVG) intercross, which identified quantitative trait loci regulating expression of C1qa, C1qb, C3, and C9. Unlike C1qa, C1qb, and C9, which all displayed distinct coregulation with different cis-regulated C-type lectins, C3 was regulated in a coexpression network immediately downstream of butyrylcholinesterase. Butyrylcholinesterase hydrolyses acetylcholine, which exerts immunoregulatory effects partly through TNF-α pathways. Accordingly, increased C3, but not C1q, expression was demonstrated in rat and mouse glia following TNF-α stimulation, which was abrogated in a dose-dependent manner by acetylcholine. These findings demonstrate new pathways regulating CNS complement expression using unbiased mapping in an experimental in vivo system. A direct link between cholinergic activity and complement activation is supported by in vitro experiments. The identification of distinct pathways subjected to regulation by naturally occurring genetic variability is of relevance for the understanding of disease mechanisms in neurologic conditions characterized by neuronal injury and complement activation.
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Sistema Nervioso Central/metabolismo , Fibras Colinérgicas/fisiología , Activación de Complemento , Complemento C3/biosíntesis , Regulación de la Expresión Génica/inmunología , Redes Reguladoras de Genes , Acetilcolina/farmacología , Acetilcolina/fisiología , Animales , Animales Congénicos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Lesiones Encefálicas/inmunología , Lesiones Encefálicas/fisiopatología , Butirilcolinesterasa/fisiología , Células Cultivadas , Sistema Nervioso Central/química , Sistema Nervioso Central/patología , Complemento C1q/biosíntesis , Complemento C1q/genética , Complemento C3/genética , Desnervación , Factores de Transcripción Forkhead/metabolismo , Ligamiento Genético , Estudio de Asociación del Genoma Completo , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Sitios de Carácter Cuantitativo , Ratas , Rizotomía , Organismos Libres de Patógenos Específicos , Raíces Nerviosas Espinales/cirugía , Sinaptofisina/análisis , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer's disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer's disease and multiple sclerosis in particular.
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Enfermedad de Alzheimer/enzimología , Astrocitos/enzimología , Colina O-Acetiltransferasa/sangre , Colina O-Acetiltransferasa/líquido cefalorraquídeo , Esclerosis Múltiple/enzimología , Acetilcolina/metabolismo , Acetilcolinesterasa/sangre , Acetilcolinesterasa/líquido cefalorraquídeo , Enfermedad de Alzheimer/genética , Animales , Astrocitos/citología , Astrocitos/metabolismo , Butirilcolinesterasa/sangre , Butirilcolinesterasa/líquido cefalorraquídeo , Células Cultivadas , Colina O-Acetiltransferasa/genética , Regulación Enzimológica de la Expresión Génica , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Linfocitos/citología , Linfocitos/enzimología , Ratones , Esclerosis Múltiple/genéticaRESUMEN
Butyrylcholinesterase (BuChE) activity is associated with activated astrocytes in Alzheimer's disease brain. The BuChE-K variant exhibits 30%-60% reduced acetylcholine (ACh) hydrolyzing capacity. Considering the increasing evidence of an immune-regulatory role of ACh, we investigated if genetic heterogeneity in BuChE affects cerebrospinal fluid (CSF) biomarkers of inflammation and cholinoceptive glial function. Alzheimer's disease patients (n = 179) were BCHE-K-genotyped. Proteomic and enzymatic analyses were performed on CSF and/or plasma. BuChE genotype was linked with differential CSF levels of glial fibrillary acidic protein, S100B, interleukin-1ß, and tumor necrosis factor (TNF)-α. BCHE-K noncarriers displayed 100%-150% higher glial fibrillary acidic protein and 64%-110% higher S100B than BCHE-K carriers, who, in contrast, had 40%-80% higher interleukin-1ß and 21%-27% higher TNF-α compared with noncarriers. A high level of CSF BuChE enzymatic phenotype also significantly correlated with higher CSF levels of astroglial markers and several factors of the innate complement system, but lower levels of proinflammatory cytokines. These individuals also displayed beneficial paraclinical and clinical findings, such as high cerebral glucose utilization, low ß-amyloid load, and less severe progression of clinical symptoms. In vitro analysis on human astrocytes confirmed the involvement of a regulated BuChE status in the astroglial responses to TNF-α and ACh. Histochemical analysis in a rat model of nerve injury-induced neuroinflammation, showed focal assembly of astroglial cells in proximity of BuChE-immunolabeled sites. In conclusion, these results suggest that BuChE enzymatic activity plays an important role in regulating intrinsic inflammation and activity of cholinoceptive glial cells and that this might be of clinical relevance. The dissociation between astroglial markers and inflammatory cytokines indicates that a proper activation and maintenance of astroglial function is a beneficial response, rather than a disease-driving mechanism. Further studies are needed to explore the therapeutic potential of manipulating BuChE activity or astroglial functional status.
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Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/patología , Astrocitos/metabolismo , Butirilcolinesterasa/metabolismo , Citocinas/líquido cefalorraquídeo , Acetilcolina/farmacología , Acetilcolinesterasa/metabolismo , Anciano , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/diagnóstico por imagen , Compuestos de Anilina , Astrocitos/efectos de los fármacos , Butirilcolinesterasa/genética , Proteínas de Unión al Calcio , Células Cultivadas , Trastornos del Conocimiento/etiología , Proteínas del Sistema Complemento/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Fluorodesoxiglucosa F18 , Proteína Ácida Fibrilar de la Glía/líquido cefalorraquídeo , Humanos , Masculino , Escala del Estado Mental , Proteínas de Microfilamentos , Pruebas Neuropsicológicas , Polimorfismo de Nucleótido Simple , Cintigrafía , Subunidad beta de la Proteína de Unión al Calcio S100/líquido cefalorraquídeo , TiazolesRESUMEN
BACKGROUND: C-type lectin (CLEC) receptors are important for initiating and shaping immune responses; however, their role in inflammatory reactions in the central nervous system after traumatic injuries is not known. The antigen-presenting lectin-like receptor gene complex (Aplec) contains a few CLEC genes, which differ genetically among inbred rat strains. It was originally thought to be a region that regulates susceptibility to autoimmune arthritis, autoimmune neuroinflammation and infection. METHODS: The inbred rat strains DA and PVG differ substantially in degree of spinal cord motor neuron death following ventral root avulsion (VRA), which is a reproducible model of localized nerve root injury. A large F2 (DAxPVG) intercross was bred and genotyped after which global expressional profiling was performed on spinal cords from F2 rats subjected to VRA. A congenic strain, Aplec, created by transferring a small PVG segment containing only seven genes, all C-type lectins, ontoDA background, was used for further experiments together with the parental strains. RESULTS: Global expressional profiling of F2 (DAxPVG) spinal cords after VRA and genome-wide eQTL mapping identified a strong cis-regulated difference in the expression of Clec4a3 (Dcir3), a C-type lectin gene that is a part of the Aplec cluster. Second, we demonstrate significantly improved motor neuron survival and also increased T-cell infiltration into the spinal cord of congenic rats carrying Aplec from PVG on DA background compared to the parental DA strain. In vitro studies demonstrate that the Aplec genes are expressed on microglia and upregulated upon inflammatory stimuli. However, there were no differences in expression of general microglial activation markers between Aplec and parental DA rats, suggesting that the Aplec genes are involved in the signaling events rather than the primary activation of microglia occurring upon nerve root injury. CONCLUSIONS: In summary, we demonstrate that a genetic variation in Aplec occurring among inbred strains regulates both survival of axotomized motor neurons and the degree of lymphocyte infiltration. These results demonstrate a hitherto unknown role for CLECs for intercellular communication that occurs after damage to the nervous system, which is relevant for neuronal survival.
Asunto(s)
Lectinas Tipo C/genética , Neuronas Motoras/fisiología , Familia de Multigenes/genética , Radiculopatía/genética , Radiculopatía/patología , Linfocitos T/fisiología , Animales , Animales Congénicos , Presentación de Antígeno , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Astrocitos/metabolismo , Recuento de Células , Supervivencia Celular/fisiología , Células Cultivadas , Femenino , Citometría de Flujo , Inmunohistoquímica , Lectinas Tipo C/metabolismo , Análisis por Micromatrices , Microglía/metabolismo , Proteínas de la Mielina/metabolismo , Oligodendroglía/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Raíces Nerviosas Espinales/patologíaRESUMEN
AIM: Genetic factors are important for outcome after traumatic brain injury (TBI), although exact knowledge of relevant genes/pathways is still lacking. We here used an unbiased approach to define differentially activated pathways between the inbred DA and PVG rat strains. The results prompted us to study further if a naturally occurring genetic variation in glutathione-S-transferase alpha 4 (Gsta4) affects the outcome after TBI. RESULTS: Survival of neurons after experimental TBI is increased in PVG compared to the DA strain. Global expression profiling analysis shows the glutathione metabolism pathway to be the most regulated between the strains, with increased Gsta4 in PVG among top regulated transcripts. A congenic strain (R5) with a PVG genomic insert containing the Gsta4 gene on DA background displays a reversal of the strain pattern for Gsta4 expression and increased survival of neurons compared to DA. Gsta4 is known to effectively reduce 4-hydroxynonenal (4-HNE), a noxious by-product of lipid peroxidation. Immunostaining of 4-HNE was evident in both rat and human TBI. Intracerebral injection of 4-HNE resulted in neurodegeneration with increased levels of a marker for nerve injury in cerebrospinal fluid of DA compared to R5. INNOVATION: These findings provide strong support for the notion that the inherent capability of coping with increased 4-HNE after TBI affects outcome in terms of nerve cell loss. CONCLUSION: A naturally occurring variation in Gsta4 expression in rats affects neurodegeneration after TBI. Further studies are needed to explore if genetic variability in Gsta4 can be associated to outcome also in human TBI.
Asunto(s)
Lesiones Encefálicas/enzimología , Lesiones Encefálicas/patología , Glutatión Transferasa/genética , Isoenzimas/genética , Neuronas/enzimología , Neuronas/patología , Aldehídos/metabolismo , Aldehídos/toxicidad , Animales , Lesiones Encefálicas/genética , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Isoenzimas/metabolismo , Peroxidación de Lípido , Masculino , Ratones , Ratones TransgénicosRESUMEN
Increasing evidence suggests that genetic background affects outcome of traumatic brain injuries (TBI). Still, there is limited detailed knowledge on what pathways/processes are affected by genetic heterogeneity. The inbred rat strains DA and PVG differ in neuronal survival following TBI. We here carried out global expressional profiling to identify differentially regulated pathways governing the response to an experimental controlled brain contusion injury. One of the most differentially regulated molecular networks concerned immune cell trafficking. Subsequent characterization of the involved cells using flow cytometry demonstrated greater infiltration of neutrophils and monocytes, as well as a higher degree of microglia activation in DA compared to PVG rats. In addition, DA rats displayed a higher number of NK cells and a higher ratio of CD161bright compared to CD161dim NK cells. Local expression of complement pathway molecules such as C1 and C3 was higher in DA and both the key complement component C3 and membrane-attack complex (MAC) could be demonstrated on axons and nerve cells. A stronger activation of the complement system in DA was associated with higher cerebrospinal fluid levels of neurofilament-light, a biomarker for nerve/axonal injury. In summary, we demonstrate substantial differences between DA and PVG rats in activation of inflammatory pathways; in particular, immune cell influx and complement activation associated with neuronal/axonal injury after TBI. These findings suggest genetic influences acting on inflammatory activation to be of importance in TBI and motivate further efforts using experimental forward genetics to identify genes/pathways that affect outcome.
Asunto(s)
Lesiones Encefálicas , Activación de Complemento , Leucocitos , ARN Mensajero/análisis , Ratas Endogámicas , Animales , Lesiones Encefálicas/genética , Lesiones Encefálicas/inmunología , Movimiento Celular/genética , Activación de Complemento/genética , Activación de Complemento/inmunología , Complemento C1q/genética , Complemento C1q/inmunología , Complemento C3/genética , Complemento C3/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/inmunología , Citocinas/genética , Citocinas/inmunología , Perfilación de la Expresión Génica , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Leucocitos/citología , Leucocitos/inmunología , Microglía/citología , Microglía/inmunología , Monocitos/citología , Monocitos/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Neutrófilos/citología , Neutrófilos/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Endogámicas/genética , Ratas Endogámicas/inmunologíaRESUMEN
Genetic regulation of autoimmune neuroinflammation is a well known phenomenon, but genetic influences on inflammation following traumatic nerve injuries have received little attention. In this study we examined the inflammatory response in a rat traumatic brain injury (TBI) model, with a particular focus on major histocompatibility class II (MHC II) presentation, in two inbred rat strains that have been extensively characterized in experimental autoimmune encephalomyelitis (EAE); DA and PVG. In addition, MHC and Vra4 congenic strains on these backgrounds were studied to give information on MHC and non-MHC gene contribution. Thus, allelic differences in Vra4, harboring the Ciita gene, was found to regulate expression of the invariant chain at the mRNA level, with a much smaller effect exerted by the MHC locus itself. Notably, however, at the protein level the MHC congenic PVG-RT1(av1) strain displayed much stronger MHCII(+) presentation, as shown both by immunolabeling and flow cytometry, than the PVG strain, dwarfing the effect of Ciita. The PVG-RT1(av1) strain had significantly more T-cell influx than both DA and PVG, suggesting regulation both by MHC and non-MHC genes. Finally, in terms of outcome, the EAE susceptible DA strain displayed a significantly smaller resulting lesion volume than the resistant PVG-RT1(av1) strain. These results provide additional support for a role of adaptive immune response after neurotrauma and demonstrate that outcome is significantly affected by host genetic factors.