Matlock Jeffries

Genome-wide DNA methylation patterns in CD4+ T cells from patients with systemic lupus erythematosus

Systemic lupus erythematosus is a chronic-relapsing autoimmune disease of incompletely understood etiology. Recent evidence strongly supports an epigenetic contribution to the pathogenesis of lupus. To understand the extent and nature of dysregulated DNA methylation in lupus T cells, we performed a genome-wide DNA methylation study in CD4+ T cells in lupus patients compared to normal healthy controls. Cytosine methylation was quantified in 27,578 CG sites located within the promoter regions of 14,495 genes. We identified 236 hypomethylated and 105 hypermethylated CG sites in lupus CD4+ T cells compared to normal controls, consistent with widespread DNA methylation changes in lupus T cells. Of interest, hypomethylated genes in lupus T cells include CD9, which is known to provide potent T-cell co-stimulation signals. Other genes with known involvement in autoimmunity such as MMP9 and PDGFRA were also hypomethylated. The BST2 gene, an interferon-inducible membrane-bound protein that helps restrict the release of retroviral particles was also hypomethylated in lupus patients. Genes involved in folate biosynthesis, which plays a role in DNA methylation, were overrepresented among hypermethylated genes. In addition, the transcription factor RUNX3 was hypermethylated in patients, suggesting an impact on T-cell maturation. Protein-protein interaction maps identified a transcription factor, HNF4a, as a regulatory hub affecting a number of differentially methylated genes. Apoptosis was also an overrepresented ontology in these interaction maps. Further, our data suggest that the methylation status of RAB22A, STX1B2, LGALS3BP, DNASE1L1 and PREX1 correlates with disease activity in lupus patients.

Defective T-cell ERK signaling induces interferon-regulated gene expression and overexpression of methylation-sensitive genes similar to lupus patients

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against a host of nuclear antigens. The pathogenesis of lupus is incompletely understood. Environmental factors may play a role via altering DNA methylation, a mechanism regulating gene expression. In lupus, genes including CD11a and CD70 are overexpressed in T cells as a result of promoter hypomethylation. T-cell DNA methyltransferase expression is regulated in part by the extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we investigate the effects of decreased ERK pathway signaling in T cells using transgenic animals. We generated a transgenic mouse that inducibly expresses a dominant-negative MEK in T cells in the presence of doxycycline. We show that decreased ERK pathway signaling in T cells results in decreased expression of DNA methyltransferase 1 and overexpression of the methylation-sensitive genes CD11a and CD70, similar to T cells in human lupus. Our transgenic animal model also develops anti-dsDNA antibodies. Interestingly, microarray expression assays revealed overexpression of several interferon-regulated genes in the spleen similar to peripheral blood cells of lupus patients. This model supports the contention that ERK pathway signaling defects in T cells contribute to the development of autoimmunity.

Genome-Wide DNA Methylation Study Identifies Significant Epigenomic Changes in Osteoarthritic Cartilage

To perform a genome‐wide DNA methylation study to identify DNA methylation changes in osteoarthritic (OA) cartilage tissue.

Variants within MECP2, a key transcription regulator, are associated with increased susceptibility to lupus and differential gene expression in patients with systemic lupus erythematosus

OBJECTIVE Both genetic and epigenetic factors play an important role in the pathogenesis of lupus. The aim of this study was to examine methyl-CpG-binding protein 2 gene (MECP2) polymorphisms in a large cohort of patients with lupus and control subjects, and to determine the functional consequences of the lupus-associated MECP2 haplotype. METHODS We genotyped 18 single-nucleotide polymorphisms within MECP2, located on chromosome Xq28, in a large cohort of patients with lupus and control subjects of European descent. We studied the functional effects of the lupus-associated MECP2 haplotype by determining gene expression profiles in B cell lines in female lupus patients with and those without the lupus-associated MECP2 risk haplotype. RESULTS We confirmed, replicated, and extended the genetic association between lupus and genetic markers within MECP2 in a large independent cohort of lupus patients and control subjects of European descent (odds ratio 1.35, P = 6.65 x 10(-11)). MECP2 is a dichotomous transcription regulator that either activates or represses gene expression. We identified 128 genes that are differentially expressed in lupus patients with the disease-associated MECP2 haplotype; most ( approximately 81%) were up-regulated. Genes that were up-regulated had significantly more CpG islands in their promoter regions compared with genes that were down-regulated. Gene ontology analysis using the differentially expressed genes revealed significant association with epigenetic regulatory mechanisms, suggesting that these genes are targets for MECP2 regulation in B cells. Furthermore, at least 13 of the 104 up-regulated genes are regulated by interferon. The disease-risk MECP2 haplotype was associated with increased expression of the MECP2 transcription coactivator CREB1 and decreased expression of the corepressor histone deacetylase 1. CONCLUSION Polymorphism in the MECP2 locus is associated with lupus and, at least in part, contributes to the interferon signature observed in lupus patients.

Defective DNA methylation and CD70 overexpression in CD4+ T cells in MRL/lpr lupus-prone mice

We have determined that abnormal DNA methylation in T cells coincides with the development of autoimmunity, using a mouse model that exhibits an age‐dependent lupus‐like disease (MRL/lpr mice). Splenic CD4+ T cells were isolated from these mice at 5 and 16 wk of age (before and after autoimmunity is established) and the expression of DNA methyltransferase 1 (Dnmt1) and the methylation‐sensitive gene Tnfsf7 (CD70) was measured. Bisulfite DNA sequencing was used to monitor the methylation status of the Tnfsf7 gene. We found that Dnmt1 steady‐state mRNA levels were significantly lower in 16‐wk‐old MRL/lpr mice, which had established autoimmunity, compared to the 5‐wk‐old MRL/lpr mice. Furthermore, the expression of CD70 was higher in MRL/lpr mice at 16 wk. CD70 was overexpressed in MRL/lpr mice compared to age‐ and sex‐matched MRL+/+ controls. Bisulfite DNA sequencing of the Tnfsf7 gene in MRL/lpr mice revealed that at 16 wk, CG pairs were hypomethylated compared to 5‐wk‐old mice, and that Tnfsf7 from MRL/lpr mice was hypomethylated at 16 wk relative to age‐matched MRL+/+ controls. Our data indicate that decreased expression of Dnmt1 and the corresponding T cell DNA hypomethylation correlate with the development of age‐dependent autoimmunity in MRL/lpr mice.

Uric Acid Directly Promotes Human T-Cell Activation

Background:Abnormally high serum uric acid levels have been associated with several disease conditions including gout and kidney stone disease. More recently, it was shown that uric acid crystals stimulate dendritic cell maturation, activate the NALP3 inflammasome, and enhance antigen-specific immune responses. We hypothesize that uric acid can also stimulate T cells directly and in the absence of antigen presentation. Methods:Purified primary human T cells were incubated with and without uric acid at concentrations of 50, 100, 150, and 200 &mgr;g/mL. The expression of T-cell activation markers CD25 and CD70 was assessed by flow cytometry. In other experiments, Jurkat T cells were used and the expression of the costimulatory molecule CD70 was determined at the mRNA level. Results:Uric acid directly activates primary human T cells in the absence of antigen presentation. Furthermore, primary human T cells and Jurkat T cells treated with uric acid overexpress the costimulatory molecule CD70, which plays an important role in T cell-B cell interaction and antibody production. Conclusions:The finding that uric acid directly promotes T-cell activation in an antigen-independent system is novel and might play a mechanistic role in the inflammatory response observed in gouty arthritis and other immune-mediated diseases.

Functional characterization of the MECP2/IRAK1 lupus risk haplotype in human T cells and a human MECP2 transgenic mouse

Genetic polymorphism in MECP2/IRAK1 on chromosome Xq28 is a confirmed and replicated susceptibility locus for lupus. High linkage disequilibrium in this locus suggests that both MECP2 and IRAK1 are candidate genes for the disease. DNA methylation changes in lupus T cells play a central role in the pathogenesis of lupus, and MeCp-2 (encoded by MECP2) is a master regulator of gene expression and is also known to recruit DNA methyltransferase 1 (DNMT1) during DNA synthesis. Using human T cells from normal individuals with either the lupus risk or the lupus protective haplotype in MECP2/IRAK1, we demonstrate that polymorphism in this locus increases MECP2 isoform 2 mRNA expression in stimulated but not unstimulated T cells. By assessing DNA methylation levels across over 485,000 methylation sites across the entire genome, we further demonstrate that the lupus risk variant in this locus is associated with significant DNA methylation changes, including in the HLA-DR and HLA-DQ loci, as well as interferon-related genes such as IFI6, IRF6, and BST2. Further, using a human MECP2 transgenic mouse, we show that overexpression of MECP2 alters gene expression in stimulated T cells. This includes overexpression of Eif2c2 that regulates the expression of multiple microRNAs (such as miR-21), and the histone demethylase Jhdm1d. In addition, we show that MECP2 transgenic mice develop antinuclear antibodies. Our data suggest that the lupus-associated variant in the MECP2/IRAK1 locus has the potential to affect all 3 epigenetic mechanisms: DNA methylation, microRNA expression, and histone modification. Importantly, these data support the notion that variants within the MECP2 gene can alter DNA methylation in other genetic loci including the HLA and interferon-regulated genes, thereby providing evidence for genetic-epigenetic interaction in lupus.

DNA methylation patterns in naïve CD4+ T cells identify epigenetic susceptibility loci for malar rash and discoid rash in systemic lupus erythematosus.

Objective Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterised by heterogeneous clinical manifestations, autoantibody production and epigenetic dysregulation in T cells. We sought to investigate the epigenetic contribution to the development of cutaneous manifestations in SLE. Methods We performed genome-wide DNA methylation analyses in patients with SLE stratified by a history of malar rash, discoid rash or neither cutaneous manifestation, and age, sex and ethnicity matched healthy controls. We characterised differentially methylated regions (DMRs) in naïve CD4+ T cells unique to each disease subset, and assessed functional relationships between DMRs using bioinformatic approaches. Results We identified 36 and 37 unique DMRs that contribute to the epigenetic susceptibility to malar rash and discoid rash, respectively. These DMRs were primarily localised to genes mediating cell proliferation and apoptosis. Hypomethylation of MIR886 and TRIM69, and hypermethylation of RNF39 were specific to patients with SLE with a history of malar rash. Hypomethylation of the cytoskeleton-related gene RHOJ was specific to patients with SLE with a history of discoid rash. In addition, discoid rash-specific hypomethylated DMRs were found in genes involved in antigen-processing and presentation such as TAP1 and PSMB8. Network analyses showed that DMRs in patients with SLE with but not without a history of cutaneous manifestations are associated with TAP-dependent processing and major histocompatibility-class I antigen cross-presentation (p=3.66×10−18 in malar rash, and 3.67×10−13 in discoid rash). Conclusions We characterised DNA methylation changes in naïve CD4+ T cells specific to malar rash and discoid rash in patients with SLE. These data suggest unique epigenetic susceptibility loci that predispose to or are associated with the development of cutaneous manifestations in SLE.

Autoimmune disease in the epigenetic era: how has epigenetics changed our understanding of disease and how can we expect the field to evolve?

Autoimmune diseases are complex and enigmatic, and have presented particular challenges to researchers seeking to define their etiology and explain progression. Previous studies have implicated epigenetic influences in the development of autoimmunity. Epigenetics describes changes in gene expression related to environmental influences without alterations in the underlying genomic sequence, generally classified into three main groups: cytosine genomic DNA methylation, modification of various sidechain positions of histone proteins and noncoding RNAs feedback. The purpose of this article is to review the most relevant literature describing alterations of epigenetic marks in the development and progression of four common autoimmune diseases: systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis and Sjögren’s syndrome. The contribution of DNA methylation, histone modification and noncoding RNA for each of these disorders is discussed, including examples both of candidate gene studies and larger epigenomics surveys, and in various tissue types important for the pathogenesis of each. The future of the field is speculated briefly, as is the possibility of therapeutic interventions targeting the epigenome.

Haemolytic anaemia in a multi-ethnic cohort of lupus patients: a clinical and serological perspective

Systemic lupus erythematosus is a chronic autoimmune disease that can be associated with a variety of haematological manifestations. We identified 76 patients with haemolytic anaemia in a cohort of 1251 unrelated female lupus patients enrolled in our studies. The presence of the various American College of Rheumatology clinical criteria for lupus and serological specificities were determined in lupus patients with haemolytic anaemia and compared with a group of race-matched control lupus patients without haemolytic anaemia. Clinical data were obtained from medical records, and serological specificities were determined in our clinical immunology laboratory at OMRF. The presence of haemolytic anaemia in lupus patients was associated with a higher frequency of proteinuria (OR = 2.70, P = 0.000031), urinary cellular casts (OR = 2.83, P = 0.000062), seizures (OR = 2.96, P = 0.00024), pericarditis (OR = 2.21, P = 0.0019), pleuritis (OR = 1.72, P = 0.028) and lymphopenia (OR = 1.79, P = 0.015). These findings were independent of the presence of thrombocytopenia, which was approximately five times more common in lupus patients with haemolytic anaemia. Lupus patients with haemolytic anaemia were about 8 years younger than lupus patients without haemolytic anaemia at the time of disease onset (P = 0.000001). In the absence of thrombocytopenia, lupus patients with haemolytic anaemia were approximately two times more likely to have anti-dsDNA antibodies (P = 0.024). The presence of haemolytic anaemia is associated with a subset of lupus characterized by a younger age of disease onset, and a more severe disease with a higher likelihood of renal involvement, seizures, serositis and other cytopenias.