RESUMEN

OBJECTIVE: To identify single-cell transcriptional signatures of dendritic cells (DCs) that are associated with autoimmunity, and determine whether those DC signatures are correlated with the clinical heterogeneity of autoimmune disease. METHODS: Blood-derived DCs were single-cell sorted from the peripheral blood of patients with rheumatoid arthritis, systemic lupus erythematosus, or type 1 diabetes as well as healthy individuals. DCs were analyzed using single-cell gene expression assays, performed immediately after isolation or after in vitro stimulation of the cells. In addition, protein expression was measured using fluorescence-activated cell sorting. RESULTS: CD1c+ conventional DCs and plasmacytoid DCs from healthy individuals exhibited diverse transcriptional signatures, while the DC transcriptional signatures in patients with autoimmune disease were altered. In particular, distinct DC clusters, characterized by up-regulation of TAP1, IRF7, and IFNAR1, were abundant in patients with systemic autoimmune disease, whereas DCs from patients with type 1 diabetes had decreased expression of the regulatory genes PTPN6, TGFB, and TYROBP. The frequency of CD1c+ conventional DCs that expressed a systemic autoimmune profile directly correlated with the extent of disease activity in patients with rheumatoid arthritis (Spearman's r = 0.60, P = 0.03). CONCLUSION: DC transcriptional signatures are altered in patients with autoimmune disease and are associated with the level of disease activity, suggesting that immune cell transcriptional profiling could improve our ability to detect and understand the heterogeneity of these diseases, and could guide treatment choices in patients with a complex autoimmune disease.

Asunto(s)

Enfermedades Autoinmunes/genética , Células Dendríticas/metabolismo , Inflamación/genética , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Enfermedades Autoinmunes/inmunología , Estudios de Casos y Controles , Células Dendríticas/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Inflamación/inmunología , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/inmunología , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/inmunología , Índice de Severidad de la Enfermedad , Análisis de la Célula Individual , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Regulación hacia Arriba

RESUMEN

The nonobese diabetic (NOD) mouse strain is a predominant animal model of type 1 diabetes. However, this mouse strain is considered to be non-permissive for embryonic stem cell (ESC) derivation using conventional methods. We examined small molecule inhibition of glycogen synthase kinase 3 (GSK3) to block spontaneous cell differentiation and promote pluripotency persistence. Here we show a single pharmacological GSK3 inhibitor, 6-bromoindirubin-3'-oxime (BIO), in combination with leukemia inhibition factor (LIF), promoted generation of stable NOD ESC lines at >80% efficiency. Significantly, expansion of the established NOD ESC lines no longer required treatment with BIO. These NOD ESC lines contributed to chimeric mice and transmitted to germline progeny that spontaneously developed diabetes. By contrast, 5-aza-2'-deoxycytidine (AZA), a small molecule inhibitor of DNA methylation, and trichostatin A (TSA) and valproic acid (VPA), small molecule inhibitors of histone deacetylase, could not promote generation of NOD ESCs by epigenetic remodeling. These combined findings provide strategic insights for imposing pluripotency in cells isolated from a non-permissive strain.

Asunto(s)

Células Madre Embrionarias/metabolismo , Células Germinativas/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Animales , Diferenciación Celular , Línea Celular , Células Cultivadas , Ratones , Ratones Endogámicos NOD

RESUMEN

Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

RESUMEN

Viral infections are associated with autoimmunity in type 1 diabetes. Here, we asked whether this association could be explained by variations in host immune response to a putative type 1 etiological factor, namely coxsackie B viruses (CVB). Heterogeneous antibody responses were observed against CVB capsid proteins. Heterogeneity was largely defined by different binding to VP1 or VP2. Antibody responses that were anti-VP2 competent but anti-VP1 deficient were unable to neutralize CVB, and were characteristic of children who developed early insulin-targeting autoimmunity, suggesting an impaired ability to clear CVB in early childhood. In contrast, children who developed a GAD-targeting autoimmunity had robust VP1 and VP2 antibody responses to CVB. We further found that 20% of memory CD4(+) T cells responding to the GAD65247-266 peptide share identical T cell receptors to T cells responding to the CVB4 p2C30-51 peptide, thereby providing direct evidence for the potential of molecular mimicry as a mechanism for GAD autoimmunity. Here, we highlight functional immune response differences between children who develop insulin-targeting and GAD-targeting autoimmunity, and suggest that children who lose B cell tolerance to insulin within the first years of life have a paradoxical impaired ability to mount humoral immune responses to coxsackie viruses.

Asunto(s)

Autoinmunidad , Infecciones por Coxsackievirus/inmunología , Diabetes Mellitus Tipo 1/virología , Enterovirus Humano B/inmunología , Insulina/inmunología , Adolescente , Anticuerpos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proteínas de la Cápside/inmunología , Niño , Preescolar , Diabetes Mellitus Tipo 1/inmunología , Femenino , Glutamato Descarboxilasa/inmunología , Glutamato Descarboxilasa/metabolismo , Humanos , Masculino

RESUMEN

A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying "natural" alleles in the human population is to engineer "artificial" alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis.

Asunto(s)

Elementos Transponibles de ADN , Diabetes Mellitus Tipo 1/genética , Estudios de Asociación Genética , Vectores Genéticos/genética , Mutagénesis Insercional , Animales , Puntos de Rotura del Cromosoma , Modelos Animales de Enfermedad , Femenino , Dosificación de Gen , Expresión Génica , Genes Reporteros , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Mutación , Sitios de Carácter Cuantitativo

RESUMEN

Plasmacytoid dendritic cells (pDC) compose one of the many distinct dendritic cell subsets. The primary function of pDC is to potently produce type 1 IFNs upon stimulation, which is highly relevant in antiviral responses. Consequently, the ability to manipulate the size of the pDC compartment in vivo may increase the capacity to clear viral infections. In an attempt to identify genetic loci affecting the size of the pDC compartment, defined by both the proportion and absolute number of pDC, we undertook an unbiased genetic approach. Linkage analysis using inbred mouse strains identified a locus on chromosome 7 (Pdcc1) significantly linked to both the proportion and the absolute number of pDC in the spleen. Moreover, loci on either chromosome 11 (Pdcc2) or 9 (Pdcc3) modified the effect of Pdcc1 on chromosome 7 for the proportion and absolute number of pDC, respectively. Further analysis using mice congenic for chromosome 7 confirmed Pdcc1, demonstrating that variation within this genetic interval can regulate the size of the pDC compartment. Finally, mixed bone marrow chimera experiments showed that both the proportion and the absolute number of pDC are regulated by cell-intrinsic hematopoietic factors. Our findings highlight the multigenic regulation of the size of the pDC compartment and will facilitate the identification of genes linked to this trait.

Asunto(s)

Compartimento Celular/inmunología , Cromosomas de los Mamíferos/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Genes Dominantes/inmunología , Animales , Compartimento Celular/genética , Cromosomas de los Mamíferos/genética , Femenino , Genes Dominantes/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Endogámicos NZB , Ratones Noqueados , Quimera por Radiación , Bazo/citología , Bazo/inmunología

RESUMEN

OBJECTIVE: The NOD mouse strain has been widely used to investigate the pathology and genetic susceptibility for type 1 diabetes. Induced pluripotent stem cells (iPSCs) derived from this unique mouse strain would enable new strategies for investigating type 1 diabetes pathogenesis and potential therapeutic targets. The objective of this study was to determine whether somatic fibroblasts from NOD mice could be reprogrammed to become iPSCs, providing an alternative source of stem cells for the production of genetically modified NOD cells and mice. RESEARCH DESIGN AND METHODS: Adult tail-tip fibroblasts from male NOD mice were reprogrammed by retroviral transduction of the coding sequences of three transcription factors, OCT4, SOX2, and KLF4, in combination with a histone deacetylase inhibitor, valproic acid. RESULTS: Eighteen NOD iPSC lines were generated, and three of these cell lines were further characterized. All three cell lines exhibited silencing of the three reprogramming transgenes and reactivation of endogenous pluripotent markers (OCT4, SOX2, NANOG, REX1, and SSEA1). These NOD iPSCs readily differentiated in vitro to form embryoid bodies and in vivo by teratoma formation in immunodeficient mice. Moreover, NOD iPSCs were successfully transfected with a reporter transgene and were capable of contributing to the inner cell mass of C57BL/6 blastocysts, leading to the generation of a chimeric mouse. CONCLUSIONS: Adult tail-tip fibroblasts from NOD mice can be reprogrammed, without constitutive ectopic expression of transcription factors, to produce iPSCs that exhibit classic mouse embryonic stem cell (ESC) features. These NOD iPSCs can be maintained and propagated under normal ESC culture conditions to produce genetically altered cell lines, differentiated cells, and chimeric mice.

Asunto(s)

Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Cola (estructura animal)/citología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Citometría de Flujo , Genotipo , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Repeticiones de Microsatélite/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

More than 25 loci have been linked to type 1 diabetes (T1D) in the nonobese diabetic (NOD) mouse, but identification of the underlying genes remains challenging. We describe here the positional cloning of a T1D susceptibility locus, Idd11, located on mouse chromosome 4. Sequence analysis of a series of congenic NOD mouse strains over a critical 6.9-kb interval in these mice and in 25 inbred strains identified several haplotypes, including a unique NOD haplotype, associated with varying levels of T1D susceptibility. Haplotype diversity within this interval between congenic NOD mouse strains was due to a recombination hotspot that generated four crossover breakpoints, including one with a complex conversion tract. The Idd11 haplotype and recombination hotspot are located within a predicted gene of unknown function, which exhibits decreased expression in relevant tissues of NOD mice. Notably, it was the recombination hotspot that aided our mapping of Idd11 and confirms that recombination hotspots can create genetic variation affecting a common polygenic disease. This finding has implications for human genetic association studies, which may be affected by the approximately 33,000 estimated hotspots in the genome.

Asunto(s)

Intercambio Genético/genética , Diabetes Mellitus Tipo 1/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Biología Computacional , Haplotipos/genética , Ratones , Ratones Endogámicos NOD , Datos de Secuencia Molecular , Análisis de Secuencia de ADN