RESUMO
Parent-of-origin effects comprise a range of genetic and epigenetic mechanisms of inheritance. Recently, detection of such effects implicated epigenetic mechanisms in the etiology of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. We here sought to dissect the magnitude and the type of parent-of-origin effects in the pathogenesis of experimental neuroinflammation under controlled environmental conditions. We investigated inheritance of an MS-like disease in rat, experimental autoimmune encephalomyelitis (EAE), using a backcross strategy designed to identify the parental origin of disease-predisposing alleles. A striking 37-54% of all detected disease-predisposing loci depended on parental transmission. Additionally, the Y chromosome from the susceptible strain contributed to disease susceptibility. Accounting for parent-of-origin enabled more powerful and precise identification of novel risk factors and increased the disease variance explained by the identified factors by 2-4-fold. The majority of loci displayed an imprinting-like pattern whereby a gene expressed only from the maternal or paternal copy exerts an effect. In particular, a locus on chromosome 6 comprises a well-known cluster of imprinted genes including the paternally expressed Dlk1, an atypical Notch ligand. Disease-predisposing alleles at the locus conferred lower Dlk1 expression in rats and, together with data from transgenic overexpressing Dlk1 mice, demonstrate that reduced Dlk1 drives more severe disease and modulates adaptive immune reactions in EAE. Our findings suggest a significant epigenetic contribution to the etiology of EAE. Incorporating these effects enables more powerful and precise identification of novel risk factors with diagnostic and prognostic implications for complex disease.
Assuntos
Encefalomielite Autoimune Experimental/genética , Epigênese Genética , Impressão Genômica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Alelos , Animais , Proteínas de Ligação ao Cálcio , Cromossomos Humanos Par 6/genética , Encefalomielite Autoimune Experimental/patologia , Feminino , Predisposição Genética para Doença , Humanos , Camundongos , Camundongos Transgênicos , Ratos , Fatores de RiscoRESUMO
Genetic variation in the major histocompatibility complex (MHC) affects CD4â¶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4â¶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4â¶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of â¼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Complexo Principal de Histocompatibilidade/genética , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Alelos , Animais , Apresentação de Antígeno , Diferenciação Celular/genética , Linhagem da Célula , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Ratos , Recombinação Genética , Seleção GenéticaRESUMO
Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca(2+) ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca(2+) or completely rescued using alkaline Ca(2+) chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1-yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Pectinas/metabolismo , Mucilagem Vegetal/metabolismo , Sementes/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Cálcio/farmacologia , Parede Celular/genética , Parede Celular/metabolismo , Quelantes/farmacologia , Endossomos/enzimologia , Endossomos/genética , Endossomos/metabolismo , Esterificação , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Concentração de Íons de Hidrogênio , Epiderme Vegetal/genética , Epiderme Vegetal/metabolismo , Mucilagem Vegetal/genética , Sementes/efeitos dos fármacos , Sementes/genética , Ubiquitina-Proteína Ligases/genética , Água/metabolismoRESUMO
The experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system commonly used to study multiple sclerosis (MS). We combined clinical EAE phenotypes with genome-wide expression profiling in spleens from 150 backcross rats between susceptible DA and resistant PVG rat strains during the chronic EAE phase. This enabled correlation of transcripts with genotypes, other transcripts and clinical EAE phenotypes and implicated potential genetic causes and pathways in EAE. We detected 2285 expression quantitative trait loci (eQTLs). Sixty out of 599 cis-eQTLs overlapped well-known EAE QTLs and constitute positional candidate genes, including Ifit1 (Eae7), Atg7 (Eae20-22), Klrc3 (eEae22) and Mfsd4 (Eae17). A trans-eQTL that overlaps Eae23a regulated a large number of small RNAs and implicates a master regulator of transcription. We defined several disease-correlated networks enriched for pathways involved in cell-mediated immunity. They include C-type lectins, G protein coupled receptors, mitogen-activated protein kinases, transmembrane proteins, suppressors of transcription (Jundp2 and Nr1d1) and STAT transcription factors (Stat4) involved in interferon signaling. The most significant network was enriched for T cell functions, similar to genetic findings in MS, and revealed both established and novel gene interactions. Transcripts in the network have been associated with T cell proliferation and differentiation, the TCR signaling and regulation of regulatory T cells. A number of network genes and their family members have been associated with MS and/or other autoimmune diseases. Combining disease and genome-wide expression phenotypes provides a link between disease risk genes and distinct molecular pathways that are dysregulated during chronic autoimmune inflammation.
Assuntos
Autoimunidade/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Mapeamento Cromossômico , Análise por Conglomerados , Encefalomielite Autoimune Experimental/metabolismo , Epistasia Genética , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Interferons/metabolismo , Masculino , Fenótipo , Locos de Características Quantitativas , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
MicroRNAs (miRNAs) are known to regulate most biological processes and have been found dysregulated in a variety of diseases, including multiple sclerosis (MS). In this study, we characterized miRNAs that associate with susceptibility to develop experimental autoimmune encephalomyelitis (EAE) in rats, a well-established animal model of MS. Using Illumina next-generation sequencing, we detected 544 miRNAs in the lymph nodes of EAE-susceptible Dark Agouti and EAE-resistant Piebald Virol Glaxo rats during immune activation. Forty-three miRNAs were found differentially expressed between the two strains, with 81% (35 out of 43) showing higher expression in the susceptible strain. Only 33% of tested miRNAs displayed differential expression in naive lymph nodes, suggesting that a majority of regulated miRNAs are EAE dependent. Further investigation of a selected six miRNAs indicates differences in cellular source and kinetics of expression. Several of the miRNAs, including miR-146a, miR-21, miR-181a, miR-223, and let-7, have previously been implicated in immune system regulation. Moreover, 77% (33 out of 43) of the miRNAs were associated with MS and other autoimmune diseases. Target genes likely regulated by the miRNAs were identified using computational predictions combined with whole-genome expression data. Differentially expressed miRNAs and their targets involve functions important for MS and EAE, such as immune cell migration through targeting genes like Cxcr3 and cellular maintenance and signaling by regulation of Prkcd and Stat1. In addition, we demonstrated that these three genes are direct targets of miR-181a. Our study highlights the impact of multiple miRNAs, displaying diverse kinetics and cellular sources, on development of pathogenic autoimmune inflammation.
Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , MicroRNAs/genética , Análise de Sequência de RNA , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , MicroRNAs/biossíntese , Ratos , Ratos Endogâmicos , Análise de Sequência de RNA/métodos , Análise de Sequência de RNA/tendências , Especificidade da EspécieRESUMO
Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the central nervous system (CNS) in young adults. Chronic treatments with histone deacetylase inhibitors (HDACis) have been reported to ameliorate experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, by targeting immune responses. We have recently shown that the HDAC inhibition/knockdown in the presence of thyroid hormone (T3) can also promote oligodendrocyte (OL) differentiation and expression of myelin genes in neural stem cells (NSCs) and oligodendrocyte precursors (OPCs). In this study, we found that treatment with an HDACi, valproic acid (VPA), and T3, alone or in combination, directly affects encephalitogenic CD4+ T cells. VPA, but not T3, compromised their proliferation, while both molecules reduced the frequency of IL-17-producing cells. Transfer of T3, VPA and VPA/T3 treated encephalitogenic CD4+ T cells into naïve rats induced less severe EAE, indicating that the effects of these molecules are persistent and do not require their maintenance after the initial stimuli. Thus, we investigated the effect of acute treatment with VPA and l-thyroxine (T4), a precursor of T3, on myelin oligodendrocyte glycoprotein-induced EAE in Dark Agouti rats, a close mimic of MS. We found that a brief treatment after disease onset led to sustained amelioration of EAE and prevention of inflammatory demyelination in the CNS accompanied with a higher expression of myelin-related genes in the brain. Furthermore, the treatment modulated immune responses, reduced the number of CD4+ T cells and affected the Th1 differentiation program in the brain. Our data indicate that an acute treatment with VPA and T4 after the onset of EAE can produce persistent clinically relevant therapeutic effects by limiting the pathogenic immune reactions while promoting myelin gene expression.
Assuntos
Encéfalo/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Inibidores Enzimáticos/uso terapêutico , Tiroxina/uso terapêutico , Ácido Valproico/uso terapêutico , Análise de Variância , Animais , Encéfalo/patologia , Antígeno CD11b/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/etiologia , Citometria de Fluxo , Interleucina-17/metabolismo , Antígeno Ki-67/metabolismo , Proteína Básica da Mielina/imunologia , Proteína Básica da Mielina/toxicidade , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , RatosRESUMO
BACKGROUND: Recently, a polymorphism in the LIGHT gene was shown to increase the risk of multiple sclerosis (MS) in a genome-wide association study (GWAS). OBJECTIVE: Our aim was to investigate if serum levels of LIGHT were affected by this polymorphism and by the disease itself. METHODS: Serum levels of LIGHT were investigated in four cohorts; 1) MS (n = 159) and controls (n = 160) in relation to rs1077667 genotype; 2) MS at relapse (n = 30) vs. healthy controls (n = 26); 3) MS (n = 27) vs. other neurological disease (OND, n = 33); and 4) MS patients before and after one year of treatment with natalizumab (n = 30). RESULTS: Carriers of the GG genotype had the lowest serum levels of LIGHT (p=0.02). Serum levels of LIGHT were increased in MS at relapse in two separate cohorts: vs. healthy controls (p=0.00005) and vs. remission (p=0.00006), other neurological disease (OND) (p=0.002) and OND with signs of inflammation (iOND; p=0.00005). Furthermore, serum levels of LIGHT were decreased by natalizumab treatment (p=0.001). CONCLUSION: Soluble LIGHT is an inhibitor of T-cell activation and GG carriers of rs1077667, with the highest risk for MS, had the lowest serum levels. The increased levels of LIGHT at times of increased MS activity suggest that soluble LIGHT is protective and may act to limit inflammation.
Assuntos
Predisposição Genética para Doença/genética , Esclerose Múltipla/sangue , Esclerose Múltipla/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/sangue , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Dysregulation of TNF is an important pathophysiological phenotype for many diseases. Recently, certain genetically regulated loci have been identified to regulate several inflammatory diseases. We hypothesized that a region on rat chromosome 4 known to regulate experimental autoimmune encephalomyelitis, experimental arthritis and experimental autoimmune neuritis harbors a gene regulating central inflammatory molecules, such as TNF. We therefore mapped TNF production using linkage analysis in the 12th generation of an advanced intercross line between DA and PVG.AV1 rats, which differ in susceptibility to several inflammatory conditions. A single TNF-regulating quantitative trait locus with a logarithm of odds score of 6.2 was identified and its biological effect was confirmed in a congenic rat strain. The profound TNF regulation mapped in congenic strains to the macrophage population. Several TLR signaling cascades led to the same reduced proinflammatory phenotype in congenic macrophages, indicating control of a convergence point for innate inflammatory activity. The decreased TNF potential and reduced proinflammatory macrophage phenotype in congenic rats was also associated with reduced clinical severity in experimental autoimmune encephalomyelitis, pristane-induced arthritis and sepsis experimental models. Determination of genes and mechanisms involved in this genetically determined TNF regulation will be valuable in understanding disease pathogenesis and aid treatment development.
Assuntos
Artrite Experimental/genética , Artrite Experimental/imunologia , Mediadores da Inflamação/fisiologia , Macrófagos/imunologia , Sepse/genética , Sepse/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Animais , Animais Congênicos , Artrite Experimental/induzido quimicamente , Células Cultivadas , Cruzamentos Genéticos , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Ligação Genética , Imunofenotipagem/métodos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Neurite Autoimune Experimental/genética , Neurite Autoimune Experimental/imunologia , Neurite Autoimune Experimental/patologia , Mapeamento Físico do Cromossomo , Locos de Características Quantitativas/imunologia , Ratos , Sepse/patologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Terpenos/toxicidade , Receptores Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Definition of dysregulated immune components in multiple sclerosis may help in the identification of new therapeutic targets. Deviation of the interleukin 18 receptor 1 (IL18R1) is of particular interest since the receptor is critical for experimental neuroinflammation. The objective of this study was to determine whether expression of IL18R1 varies between multiple sclerosis patients and controls, and to test genetic association of IL18R1 with multiple sclerosis. We used quantitative real-time PCR to assess mRNA levels of IL18R1 in cerebrospinal fluid and peripheral blood mononuclear cells of 191 patients with multiple sclerosis, 61 patients with clinically isolated syndrome and 168 controls having other neurological disorders. Association was tested in 2153 patients with multiple sclerosis and 1733 controls using 13 tagging single nucleotide polymorphisms within the IL18R1 gene. We found that patients with multiple sclerosis had increased IL18R1 mRNA expression in both cerebrospinal fluid cells (p < 0.05) and peripheral blood mononuclear cells (p < 0.05) compared with controls. Patients with clinically isolated syndrome had elevated levels compared with controls in cerebrospinal fluid cells (p < 0.001) but not in peripheral blood mononuclear cells. The gene was not associated to multiple sclerosis. We conclude that the increased expression of IL18R1 may contribute pathogenically to disease and is therefore a potential therapeutic target. The absence of a genetic association in the IL18R1 gene itself suggests regulation from other parts of the genome, or as part of the inflammatory cascade in multiple sclerosis without a prime genetic cause.
Assuntos
Subunidade alfa de Receptor de Interleucina-18/genética , Esclerose Múltipla Crônica Progressiva/genética , Esclerose Múltipla Recidivante-Remitente/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Estudos de Associação Genética , Haplótipos , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Crônica Progressiva/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/sangue , RNA Mensageiro/líquido cefalorraquidiano , Suécia , Regulação para Cima , Adulto JovemRESUMO
INTRODUCTION: Previous studies suggest that multiple sclerosis (MS) patients have a greater stroke risk than the general population but there is limited evidence of stroke risk in patients receiving disease-modifying treatment. We assessed stroke risk in MS patients treated with subcutaneous interferon-ß1a (sc IFN-ß1a) using pooled data from clinical trials and post-marketing surveillance. METHODS: Seventeen phase II-IV Merck KGaA-sponsored trials of sc IFN-ß1a were assessed to estimate the stroke incidence rate (IR) and IR ratio (IRR) per 100 patient-years (PY), and associated 95% confidence intervals (CI). The association of treatment duration with stroke was assessed through a Cox model. IR, IRR, and hazard ratio (HR) were adjusted by age, sex, presence of any comorbidity, and MS duration. Individual case safety reports were retrieved from the Global Patient Safety Database. The reporting rates of stroke were calculated and classified as medically confirmed or non-medically confirmed according to the source of each report. RESULTS: In 17 clinical trials, 4412 patients were treated with sc IFN-ß1a for a total of 10,622 PY and 1055 patients with placebo for 2005 PY. The IR/100 PY (95% CI) of stroke was 0.025 (0.004, 0.150) in sc IFN-ß1a patients and 0.051 (0.008, 0.349) in placebo patients. The IRR for sc IFN-ß1a vs placebo was 0.486 (0.238, 0.995) and the HR was 0.496 (0.235, 1.043) for time to stroke-related event for sc IFN-ß1a treatment at any dose compared with placebo. Among sc IFN-ß1a patients, the IRR in those treated for < 2 years was 0.602 (0.159, 2.277) and for ≥ 2 years 0.469 (0.196, 1.124). Analysis of the safety database showed that the overall reporting rate for stroke was 13.286/10,000 PY. CONCLUSION: Safety data from both clinical trial and post-marketing settings indicate that treatment with sc IFN-ß1a does not increase stroke risk in patients with MS. FUNDING: Merck KGaA, Darmstadt, Germany.
Assuntos
Interferon beta-1a , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Vigilância de Produtos Comercializados , Medição de Risco/métodos , Acidente Vascular Cerebral/epidemiologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Adulto , Ensaios Clínicos como Assunto/estatística & dados numéricos , Bases de Dados Factuais/estatística & dados numéricos , Feminino , Alemanha , Humanos , Injeções Subcutâneas , Interferon beta-1a/administração & dosagem , Interferon beta-1a/efeitos adversos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/epidemiologia , Farmacovigilância , Vigilância de Produtos Comercializados/métodos , Vigilância de Produtos Comercializados/estatística & dados numéricosRESUMO
Defining genetic variants that predispose for diseases is an important initiative that can improve biological understanding and focus therapeutic development. Genetic mapping in humans and animal models has defined genomic regions controlling a variety of phenotypes known as quantitative trait loci (QTL). Causative disease determinants, including single nucleotide polymorphisms (SNPs), lie within these regions and can often be identified through effects on gene expression. We previously identified a QTL on rat chromosome 4 regulating macrophage phenotypes and immune-mediated diseases including experimental autoimmune encephalomyelitis (EAE). Gene analysis and a literature search identified lysine-specific demethylase 3A (Kdm3a) as a potential regulator of these phenotypes. Genomic sequencing determined only two synonymous SNPs in Kdm3a. The silent synonymous SNP in exon 15 of Kdm3a caused problems with quantitative PCR detection in the susceptible strain through reduced amplification efficiency due to altered secondary cDNA structure. Shape Probability Shift analysis predicted that the SNP often affects RNA folding; thus, it may impact protein translation. Despite these differences in rats, genetic knockout of Kdm3a in mice resulted in no dramatic effect on immune system development and activation or EAE susceptibility and severity. These results provide support for tools that analyze causative SNPs that impact nucleic acid structures.
Assuntos
DNA/química , Éxons/genética , Inativação Gênica , Histona Desmetilases com o Domínio Jumonji/deficiência , Histona Desmetilases com o Domínio Jumonji/genética , Polimorfismo de Nucleotídeo Único/genética , RNA/química , Animais , Sequência de Bases , DNA Complementar/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Feminino , Técnicas de Silenciamento de Genes , Camundongos , Fenótipo , RatosRESUMO
Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.
Assuntos
Ansiedade/genética , Mapeamento Cromossômico/métodos , Cardiopatias/genética , Esclerose Múltipla/genética , Análise de Sequência de DNA/métodos , Animais , Animais não Endogâmicos , Variação Genética/genética , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genética , RatosRESUMO
Macrophages are multifunctional immune cells that may either drive or modulate disease pathogenesis depending on their activation phenotype. Autoimmune type 1 diabetes (T1D) is a chronic proinflammatory condition characterized by unresolved destruction of pancreatic islets. Adoptive cell transfer of macrophages with immunosuppressive properties represents a novel immunotherapy for treatment of such chronic autoimmune diseases. We used a panel of cytokines and other stimuli to discern the most effective regimen for in vitro induction of immunosuppressive macrophages (M2r) and determined interleukin (IL)-4/IL-10/transforming growth factor-ß (TGF-ß) to be optimal. M2r cells expressed programmed cell death 1 ligand-2, fragment crystallizable region γ receptor IIb, IL-10, and TGF-ß, had a potent deactivating effect on proinflammatory lipopolysaccharide/interferon-γ-stimulated macrophages, and significantly suppressed T-cell proliferation. Clinical therapeutic efficacy was assessed after adoptive transfer in NOD T1D mice, and after a single transfer of M2r macrophages, >80% of treated NOD mice were protected against T1D for at least 3 months, even when transfer was conducted just prior to clinical onset. Fluorescent imaging analyses revealed that adoptively transferred M2r macrophages specifically homed to the inflamed pancreas, promoting ß-cell survival. We suggest that M2r macrophage therapy represents a novel intervention that stops ongoing autoimmune T1D and may have relevance in a clinical setting.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Imunoterapia Adotiva , Células Secretoras de Insulina/imunologia , Macrófagos/imunologia , Macrófagos/transplante , Animais , Proliferação de Células , Rastreamento de Células , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Feminino , Regulação da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Testes de Liberação de Interferon-gama , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Ativação de Macrófagos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pâncreas/imunologia , Pâncreas/metabolismo , Pâncreas/patologia , Estado Pré-Diabético/imunologia , Estado Pré-Diabético/metabolismo , Estado Pré-Diabético/patologia , Estado Pré-Diabético/terapia , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Multiple sclerosis (MS) is a polygenic disease characterized by inflammation and demyelination in the central nervous system (CNS), which can be modeled in experimental autoimmune encephalomyelitis (EAE). The Eae18b locus on rat chromosome 10 has previously been linked to regulation of beta-chemokine expression and severity of EAE. Moreover, the homologous chemokine cluster in humans showed evidence of association with susceptibility to MS. We here established a congenic rat strain with Eae18b locus containing a chemokine cluster (Ccl2, Ccl7, Ccl11, Ccl12 and Ccl1) from the EAE- resistant PVG rat strain on the susceptible DA background and utilized myelin oligodendrocyte glycoprotein (MOG)-induced EAE to characterize the mechanisms underlying the genetic regulation. Congenic rats developed a milder disease compared to the susceptible DA strain, and this was reflected in decreased demyelination and in reduced recruitment of inflammatory cells to the brain. The congenic strain also showed significantly increased Ccl11 mRNA expression in draining lymph nodes and spinal cord after EAE induction. In the lymph nodes, macrophages were the main producers of CCL11, whereas macrophages and lymphocytes expressed the main CCL11 receptor, namely CCR3. Accordingly, the congenic strain also showed significantly increased Ccr3 mRNA expression in lymph nodes. In the CNS, the main producers of CCL11 were neurons, whereas CCR3 was detected on neurons and CSF producing ependymal cells. This corresponded to increased levels of CCL11 protein in the cerebrospinal fluid of the congenic rats. Increased intrathecal production of CCL11 in congenic rats was accompanied by a tighter blood brain barrier, reflected by more occludin(+) blood vessels. In addition, the congenic strain showed a reduced antigen specific response and a predominant anti-inflammatory Th2 phenotype. These results indicate novel mechanisms in the genetic regulation of neuroinflammation.
Assuntos
Quimiocina CCL11/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Regulação da Expressão Gênica/imunologia , Animais , Barreira Hematoencefálica/metabolismo , Loci Gênicos/genética , Homeostase/genética , Homeostase/imunologia , Hibridização Genética , Inflamação/genética , Inflamação/imunologia , Linfonodos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Família Multigênica/genética , Ratos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
BACKGROUND: To elucidate mechanisms involved in multiple sclerosis (MS), we studied genetic regulation of experimental autoimmune encephalomyelitis (EAE) in rats, assuming a conservation of pathogenic pathways. In this study, we focused on Eae23, originally identified to regulate EAE in a (LEW.1AV1xPVG.1AV1)F2 cross. Our aim was to determine whether one or more genes within the 67 Mb region regulate EAE and to define candidate risk genes. METHODOLOGY/PRINCIPAL FINDINGS: We used high resolution quantitative trait loci (QTL) analysis in the 10th generation (G10) of an advanced intercross line (AIL) to resolve Eae23 into two QTLs that independently regulate EAE, namely Eae23a and Eae23b. We established a congenic strain to validate the effect of this region on disease. PVG alleles in Eae23 resulted in significant protection from EAE and attenuated CNS inflammation/demyelination. Disease amelioration was accompanied with increased levels of Foxp3(+) cells in the CNS of the congenic strain compared to DA. We then focused on candidate gene investigation in Eae23b, a 9 Mb region linked to all clinical phenotypes. Affymetrix exon arrays were used to study expression of the genes in Eae23b in the parental strains, where none showed differential expression. However, we found lower expression of exon 4 of ZEB1, which is specific for splice-variant Zfhep1. ZEB1 is an interleukin 2 (IL2) repressor involved in T cell development. The splice-specific variance prompted us to next analyze the expression of ZEB1 and its two splice variants, Zfhep1 and Zfhep2, in both lymph node and spleen. We demonstrated that ZEB1 splice-variants are differentially expressed; severity of EAE and higher IL2 levels were associated with down-regulation of Zfhep1 and up-regulation of Zfhep2. CONCLUSIONS/SIGNIFICANCE: We speculate that the balance between splice-variants of ZEB1 could influence the regulation of EAE. Further functional studies of ZEB1 and the splice-variants may unravel novel pathways contributing to MS pathogenesis and inflammation in general.
Assuntos
Encefalomielite Autoimune Experimental/genética , Proteínas de Homeodomínio/genética , Esclerose Múltipla/genética , Locos de Características Quantitativas , Fatores de Transcrição/genética , Animais , Mapeamento Cromossômico , Encefalomielite Autoimune Experimental/imunologia , Feminino , Regulação da Expressão Gênica , Proteínas de Homeodomínio/imunologia , Humanos , Masculino , Esclerose Múltipla/imunologia , Splicing de RNA , Ratos , Fatores de Transcrição/imunologia , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
The immunological mechanisms underlying autoimmunity are being elucidated through genetic and functional analyses in both humans and rodent models. However, acceptance of models as valid equivalents of human disease is variable, and the validation of defined human candidate molecules in experimental models is hitherto limited. We thus aimed to determine the kinetic expression of several Multiple Sclerosis (MS) candidate genes in the myelin oligodendrocyte glycoprotein (MOG)-induced rat experimental autoimmune encephalomyelitis (EAE) model using susceptible DA and resistant PVG inbred strains. Increased expression of MS candidate genes IL2RA and IL7RA associated with disease susceptibility. Higher expression of these candidate genes and IL18R1 in susceptible rats may lead to enhancement of the disease-driving T(H)1 and T(H)17 pathways. Susceptible DA rats had augmented marker molecules of these pathways and upon restimulation with autoantigen produced increased effector molecules including IFN-gamma, IL-17F and IL-22. The altered T helper cell differentiation pathways led to differences in a MOG-specific proliferative and autoantibody response, which ultimately results in infiltration in the central nervous system and EAE induction. Our results validate the MOG-induced EAE model as having similar mechanisms to human MS and determined the kinetics of several disease mechanisms in relevant tissues.
Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Regulação da Expressão Gênica/imunologia , Ativação Linfocitária/genética , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Animais , Antígenos de Superfície/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/fisiopatologia , Glicoproteínas/imunologia , Subunidade alfa de Receptor de Interleucina-18/genética , Subunidade alfa de Receptor de Interleucina-2/genética , Cinética , Linfonodos/citologia , Linfonodos/imunologia , Esclerose Múltipla/fisiopatologia , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Ratos , Ratos Endogâmicos , Receptores de Interleucina-7/genética , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
BACKGROUND: Multiple Sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate complex pathogenic mechanisms. Transcriptional control through isoform selection and mRNA levels determines pathway activation and ultimately susceptibility to disease. METHODOLOGY/PRINCIPAL FINDINGS: We have studied the role of alternative splicing and differential expression in lymph node cells from EAE-susceptible Dark Agouti (DA) and EAE-resistant Piebald Virol Glaxo.AV1 (PVG) inbred rat strains using Affymetrix Gene Chip Rat Exon 1.0 ST Arrays. Comparing the two strains, we identified 11 differentially spliced and 206 differentially expressed genes at day 7 post-immunization, as well as 9 differentially spliced and 144 differentially expressed genes upon autoantigen re-stimulation. Functional clustering and pathway analysis implicate genes for glycosylation, lymphocyte activation, potassium channel activity and cellular differentiation in EAE susceptibility. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that alternative splicing occurs during complex disease and may govern EAE susceptibility. Additionally, transcriptome analysis not only identified previously defined EAE pathways regulating the immune system, but also novel mechanisms. Furthermore, several identified genes overlap known quantitative trait loci, providing novel causative candidate targets governing EAE.