RESUMO
Systemic autoimmune diseases are characterized by the overexpression of type I IFN stimulated genes, and accumulating evidence indicate a role for type I IFNs in these diseases. However, the underlying mechanisms for this are still poorly understood. To explore the role of type I IFN regulated miRNAs in systemic autoimmune disease, we characterized cellular expression of miRNAs during both acute and chronic type I IFN responses. We identified a T cell-specific reduction of miR-31-5p levels, both after intramuscular injection of IFNß and in patients with Sjögren's syndrome (SjS). To interrogate the role of miR-31-51p in T cells we transfected human CD4+ T cells with a miR-31-5p inhibitor and performed metabolic measurements. This identified an increase in basal levels of glucose metabolism after inhibition of miR-31-5p. Furthermore, treatment with IFN-α also increased the basal levels of human CD4+ T-cell metabolism. In all, our results suggest that reduced levels of miR-31-5p in T cells of SjS patients support autoimmune T-cell responses during chronic type I IFN exposure.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Metabolismo Energético/imunologia , MicroRNAs/imunologia , Síndrome de Sjogren/imunologia , Linfócitos T CD4-Positivos/patologia , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Interferon-alfa/imunologia , Interferon-alfa/farmacologia , Interferon beta/imunologia , Interferon beta/farmacologia , Masculino , Síndrome de Sjogren/patologiaRESUMO
Vitamin D exerts multiple immunomodulatory functions and has been implicated in the etiology and treatment of several autoimmune diseases, including multiple sclerosis (MS). We have previously reported that in juvenile/adolescent rats, vitamin D supplementation protects from experimental autoimmune encephalomyelitis (EAE), a model of MS. Here we demonstrate that this protective effect associates with decreased proliferation of CD4+ T cells and lower frequency of pathogenic T helper (Th) 17 cells. Using transcriptome, methylome, and pathway analyses in CD4+ T cells, we show that vitamin D affects multiple signaling and metabolic pathways critical for T-cell activation and differentiation into Th1 and Th17 subsets in vivo. Namely, Jak/Stat, Erk/Mapk, and Pi3K/Akt/mTor signaling pathway genes were down-regulated upon vitamin D supplementation. The protective effect associated with epigenetic mechanisms, such as (i) changed levels of enzymes involved in establishment and maintenance of epigenetic marks, i.e., DNA methylation and histone modifications; (ii) genome-wide reduction of DNA methylation, and (iii) up-regulation of noncoding RNAs, including microRNAs, with concomitant down-regulation of their protein-coding target RNAs involved in T-cell activation and differentiation. We further demonstrate that treatment of myelin-specific T cells with vitamin D reduces frequency of Th1 and Th17 cells, down-regulates genes in key signaling pathways and epigenetic machinery, and impairs their ability to transfer EAE. Finally, orthologs of nearly 50% of candidate MS risk genes and 40% of signature genes of myelin-reactive T cells in MS changed their expression in vivo in EAE upon supplementation, supporting the hypothesis that vitamin D may modulate risk for developing MS.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Vitamina D/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Genômica/métodos , Ativação Linfocitária/efeitos dos fármacos , Esclerose Múltipla/tratamento farmacológico , Ratos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: To explore circulating microRNAs (miRNAs) in cell-free CSF as novel biomarkers for multiple sclerosis (MS). METHODS: Profiling of miRNAs in CSF of pooled patients with clinically isolated syndrome (CIS), patients with relapsing-remitting MS, and inflammatory and noninflammatory neurologic disease controls was performed using TaqMan miRNA arrays. Two independent patient cohorts (n = 142 and n = 430) were used for validation with real-time PCR. RESULTS: We reliably detected 88 CSF miRNAs in the exploratory cohort. Subsequent validation in 2 cohorts demonstrated significantly higher levels of miR-150 in patients with MS. Higher miR-150 levels were also observed in patients with CIS who converted to MS compared to nonconverters, and in patients initiating natalizumab treatment. Levels of miR-150 correlated with immunologic parameters including CSF cell count, immunoglobulin G index, and presence of oligoclonal bands, and with candidate protein biomarkers C-X-C motif chemokine 13, matrix metallopeptidase 9, and osteopontin. Correlation with neurofilament light chain (NFL) was observed only when NFL was adjusted for age using a method that requires further validation. Additionally, miR-150 discriminated MS from controls and CIS converters from nonconverters equally well as the most informative protein biomarkers. Following treatment with natalizumab, but not fingolimod, CSF levels of miR-150 decreased, while plasma levels increased with natalizumab and decreased with fingolimod, suggesting immune cells as a source of miR-150. CONCLUSIONS: Our findings demonstrate miR-150 as a putative novel biomarker of inflammatory active disease with the potential to be used for early diagnosis of MS. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that CSF miR-150 distinguishes patients with MS from patients with other neurologic conditions.
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
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
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
Herpes simplex encephalitis (HSE) is a fatal infection of the central nervous system (CNS) predominantly caused by Herpes simplex virus type 1. Factors regulating the susceptibility to HSE are still largely unknown. To identify host gene(s) regulating HSE susceptibility we performed a genome-wide linkage scan in an intercross between the susceptible DA and the resistant PVG rat. We found one major quantitative trait locus (QTL), Hse1, on rat chromosome 4 (confidence interval 24.3-31 Mb; LOD score 29.5) governing disease susceptibility. Fine mapping of Hse1 using recombinants, haplotype mapping and sequencing, as well as expression analysis of all genes in the interval identified the calcitonin receptor gene (Calcr) as the main candidate, which also is supported by functional studies. Thus, using unbiased genetic approach variability in Calcr was identified as potentially critical for infection and viral spread to the CNS and subsequent HSE development.
Assuntos
Encefalite por Herpes Simples/genética , Predisposição Genética para Doença/genética , Neurônios/virologia , Receptores da Calcitonina/genética , Animais , Mapeamento Cromossômico/métodos , Citometria de Fluxo , Estudo de Associação Genômica Ampla , Genótipo , Haplótipos , Herpesvirus Humano 1 , Locos de Características Quantitativas , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Calcitonina/metabolismo , TransfecçãoRESUMO
A homozygous mutation in the complex III chaperone BCS1L causes GRACILE syndrome (intrauterine growth restriction, aminoaciduria, cholestasis, hepatic iron overload, lactacidosis). In control and patient fibroblasts we localized BCS1L in inner mitochondrial membranes. In patient liver, kidney, and heart BCS1L and Rieske protein levels, as well as the amount and activity of complex III, were decreased. Major histopathology was found in kidney and liver with cirrhosis and iron deposition, but of iron-related proteins only ferritin levels were high. In placenta from a GRACILE fetus, the ferrooxidases ceruloplasmin and hephaestin were upregulated suggesting association between iron overload and placental dysfunction.