RESUMO
Scleroderma (SSc) is a complex disease that involves activation of the immune system, vascular complications, and tissue fibrosis. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) mediates trimethylation of lysine 27 of histone 3 (H3K27me3), which acts as a repressive epigenetic mark. Both EZH2 and H3K27me3 were elevated in SSc dermal fibroblasts and endothelial cells compared with healthy controls. EZH2 inhibitor DZNep halted fibrosis both in vitro and in vivo. In SSc fibroblasts, DZNep dose-dependently reduced the expression of profibrotic genes and inhibited migratory activity of SSc fibroblasts. We show that epigenetic dysregulation and overexpression of LRRC16A explains EZH2-mediated fibroblast migration in SSc. In endothelial cells, inhibition of EZH2 restored normal angiogenesis in SSc via activating the Notch pathway, specifically by up-regulating the Notch ligand DLL4. Our results demonstrate that overexpression of EZH2 in SSc fibroblasts and endothelial cells is profibrotic and antiangiogenic. Targeting EZH2 or EZH2-regulated genes might be of therapeutic potential in SSc.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , Fibrose/genética , Proteínas dos Microfilamentos/genética , Esclerodermia Difusa/genética , Animais , Bleomicina/toxicidade , Movimento Celular/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Repressão Epigenética/genética , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/induzido quimicamente , Fibrose/patologia , Regulação da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Metilação , Camundongos , Neovascularização Fisiológica , Receptores Notch/genética , Transdução de SinaisRESUMO
OBJECTIVE: We examined genome-wide DNA methylation changes in CD8+ T cells from patients with lupus and controls and investigated the functional relevance of some of these changes in lupus. METHODS: Genome-wide DNA methylation of lupus and age, sex and ethnicity-matched control CD8+ T cells was measured using the Infinium MethylationEPIC arrays. Measurement of relevant cell subsets was performed via flow cytometry. Gene expression was quantified by qPCR. Inhibiting STAT1 and CIITA was performed using fludarabine and CIITA siRNA, respectively. RESULTS: Lupus CD8+ T cells had 188 hypomethylated CpG sites compared with healthy matched controls. Among the most hypomethylated were sites associated with HLA-DRB1. Genes involved in the type-I interferon response, including STAT1, were also found to be hypomethylated. IFNα upregulated HLA-DRB1 expression on lupus but not control CD8+ T cells. Lupus and control CD8+ T cells significantly increased STAT1 mRNA levels after treatment with IFNα. The expression of CIITA, a key interferon/STAT1 dependent MHC-class II regulator, is induced by IFNα in lupus CD8+ T cells, but not healthy controls. CIITA knockdown and STAT1 inhibition experiments revealed that HLA-DRB1 expression in lupus CD8+ T cells is dependent on CIITA and STAT1 signalling. Coincubation of naïve CD4+ T cells with IFNα-treated CD8+ T cells led to CD4+ T cell activation, determined by increased expression of CD69 and cytokine production, in patients with lupus but not in healthy controls. This can be blocked by neutralising antibodies targeting HLA-DR. CONCLUSIONS: Lupus CD8+ T cells are epigenetically primed to respond to type-I interferon. We describe an HLA-DRB1+ CD8+ T cell subset that can be induced by IFNα in patients with lupus. A possible pathogenic role for CD8+ T cells in lupus that is dependent on a high type-I interferon environment and epigenetic priming warrants further characterisation.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Metilação de DNA , Cadeias HLA-DRB1/genética , Lúpus Eritematoso Sistêmico/genética , Fator de Transcrição STAT1/genética , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Estudos de Casos e Controles , Células Cultivadas , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica/imunologia , Estudo de Associação Genômica Ampla , Humanos , Interferon Tipo I/imunologia , Interferon-alfa/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , RNA Mensageiro/genética , Transativadores/genética , Regulação para Cima/imunologia , Adulto JovemRESUMO
OBJECTIVE: The goal of this study was to comprehensively characterize CD4+CD28+ T cells overexpressing CD11a and KIR genes, and examine the relationship between this T cell subset, genetic risk, and disease activity in lupus. METHODS: The size of the CD4+CD28+KIR+CD11ahi T cell subset was determined by flow cytometry, and total genetic risk for lupus was calculated in 105 female patients using 43 confirmed genetic susceptibility loci. Primary CD4+CD28+KIR+CD11ahi T cells were isolated from lupus patients or were induced from healthy individuals using 5-azacytidine. Genome-wide DNA methylation was analyzed using an array-based approach, and the transcriptome was assessed by RNA sequencing. Transcripts in the CDR3 region were used to assess the TCR repertoire. Chromatin accessibility was determined using ATAC-seq. RESULTS: A total of 31,019 differentially methylated sites were identified in induced KIR+CD11ahi T cells with >99% being hypomethylated. RNA sequencing revealed a clear pro-inflammatory transcriptional profile. TCR repertoire analysis suggests less clonotype diversity in KIR+CD11ahi compared to autologous KIR-CD11alow T cells. Similarly, primary KIR+CD11ahi T cells isolated from lupus patients were hypomethylated and characterized by a pro-inflammatory chromatin structure. We show that the genetic risk for lupus was significantly higher in African-American compared to European-American lupus patients. The demethylated CD4+CD28+KIR+CD11ahi T cell subset size was a better predictor of disease activity in young (age ≤ 40) European-American patients independent of genetic risk. CONCLUSION: CD4+CD28+KIR+CD11ahi T cells are demethylated and characterized by pro-inflammatory epigenetic and transcriptional profiles in lupus. Eliminating these cells or blocking their pro-inflammatory characteristics might present a novel therapeutic approach for lupus.
Assuntos
Negro ou Afro-Americano , Inflamação/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Antígeno CD11a/metabolismo , Antígenos CD28/metabolismo , Antígenos CD4/metabolismo , Células Cultivadas , Metilação de DNA , Progressão da Doença , Epigênese Genética , Feminino , Perfil Genético , Humanos , Imunofenotipagem , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/epidemiologia , Receptores KIR/metabolismo , Risco , Análise de Sequência de RNA , Estados Unidos/epidemiologiaRESUMO
Loss of heterozygosity (LOH) at tumor suppressor loci is a major contributor to cancer initiation and progression. Both deletions and mitotic recombination can lead to LOH. Certain chromosomal loci known as common fragile sites are susceptible to DNA lesions under replication stress, and replication stress is prevalent in early stage tumor cells. There is extensive evidence for deletions stimulated by common fragile sites in tumors, but the role of fragile sites in stimulating mitotic recombination that causes LOH is unknown. Here, we have used the yeast model system to study the relationship between fragile site instability and mitotic recombination that results in LOH. A naturally occurring fragile site, FS2, exists on the right arm of yeast chromosome III, and we have analyzed LOH on this chromosome. We report that the frequency of spontaneous mitotic BIR events resulting in LOH on the right arm of yeast chromosome III is higher than expected, and that replication stress by low levels of polymerase alpha increases mitotic recombination 12-fold. Using single-nucleotide polymorphisms between the two chromosome III homologs, we mapped the locations of recombination events and determined that FS2 is a strong hotspot for both mitotic reciprocal crossovers and break-induced replication events under conditions of replication stress.