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1.  CD4+ T Cells Epigenetically Modified by Oxidative Stress Cause Lupus-like Autoimmunity in Mice 
Journal of autoimmunity  2015;62:75-80.
Lupus develops when genetically predisposed people encounter environmental agents such as UV light, silica, infections and cigarette smoke that cause oxidative stress, but how oxidative damage modifies the immune system to cause lupus flares is unknown. We previously showed that oxidizing agents decreased ERK pathway signaling in human T cells, decreased DNA methyltransferase 1 and caused demethylation and overexpression of genes similar to those from patients with active lupus. The current study tested whether oxidant-treated T cells can induce lupus in mice. We adoptively transferred CD4+ T cells treated in vitro with oxidants hydrogen peroxide or nitric oxide or the demethylating agent 5-azacytidine into syngeneic mice and studied the development and severity of lupus in the recipients. Disease severity was assessed by measuring anti-dsDNA antibodies, proteinuria, hematuria and by histopathology of kidney tissues. The effect of the oxidants on expression of CD40L, CD70, KirL1 and DNMT1 genes and CD40L protein in the treated CD4+ T cells was assessed by Q-RT-PCR and flow cytometry. H2O2 and ONOO− decreased Dnmt1 expression in CD4+ T cells and caused the upregulation of genes known to be suppressed by DNA methylation in patients with lupus and animal models of SLE. Adoptive transfer of oxidant-treated CD4+ T cells into syngeneic recipients resulted in the induction of anti-dsDNA antibody and glomerulonephritis. The results show that oxidative stress may contribute to lupus disease by inhibiting ERK pathway signaling in T cells leading to DNA demethylation, upregulation of immune genes and autoreactivity.
doi:10.1016/j.jaut.2015.06.004
PMCID: PMC4529773  PMID: 26165613
Systemic Lupus Erythematosus (SLE); CD70; CD40L; KirL1
2.  T CELL PKCδ KINASE INACTIVATION INDUCES LUPUS-LIKE AUTOIMMUNITY IN MICE 
Clinical immunology (Orlando, Fla.)  2015;158(2):193-203.
Genetic and environmental factors contribute to the onset and progression of lupus. CD4+ T cells from patients with active lupus show a decreased ERK signaling pathway, which causes changes in gene expression. The defect points to its upstream regulator, PKCδ, which exhibits a deficient activity due to oxidative stress. Our aim was to investigate the effect of a defective PKCδ in the development of lupus.
We generated a double transgenic C57BL6 × SJL mouse that expresses a doxycycline-induced dominant negative PKCδ (dnPKCδ) in T cells. The transgenic mice displayed decreased T cell ERK signaling, decreased DNMT1 expression and overexpression of methylation sensitive genes involved in the exaggerated immune response in the pathogenesis of lupus. The mice developed anti-dsDNA autoantibodies and glomerulonephritis with IgG deposition.
The study indicates common pathogenic mechanisms with human lupus, suggesting that environmentally-mediated T cell PKCδ inactivation plays a causative role in lupus.
doi:10.1016/j.clim.2015.03.017
PMCID: PMC4464903  PMID: 25829232
Lupus; T cells; PKCδ; transgenic mouse model; extracellular signal-regulated kinase (ERK); autoimmunity
3.  Characterisation of an epigenetically altered CD4+ CD28+ Kir+ T cell subset in autoimmune rheumatic diseases by multiparameter flow cytometry 
Lupus Science & Medicine  2016;3(1):e000147.
Objectives
Antigen-specific CD4+ T cells epigenetically modified with DNA methylation inhibitors overexpress genes normally suppressed by this mechanism, including CD11a, CD70, CD40L and the KIR gene family. The altered cells become autoreactive, losing restriction for nominal antigen and responding to self-class II major histocompatibility complex (MHC) molecules without added antigen, and are sufficient to cause a lupus-like disease in syngeneic mice. T cells overexpressing the same genes are found in patients with active lupus. Whether these genes are co-overexpressed on the same or different cells is unknown. The goal of this study was to determine whether these genes are overexpressed on the same or different T cells and whether this subset of CD4+ T cells is also present in patients with lupus and other rheumatic diseases.
Methods
Multicolour flow cytometry was used to compare CD11a, CD70, CD40L and KIR expression on CD3+CD4+CD28+ T cells to their expression on experimentally demethylated CD3+CD4+CD28+ T cells and CD3+CD4+CD28+ T cells from patients with active lupus and other autoimmune diseases.
Results
Experimentally demethylated CD4+ T cells and T cells from patients with active lupus have a CD3+CD4+CD28+CD11ahiCD70+CD40LhiKIR+ subset, and the subset size is proportional to lupus flare severity. A similar subset is found in patients with other rheumatic diseases including rheumatoid arthritis, systemic sclerosis and Sjögren's syndrome but not retroperitoneal fibrosis.
Conclusions
Patients with active autoimmune rheumatic diseases have a previously undescribed CD3+CD4+CD28+CD11ahiCD70+CD40LhiKIR+ T cell subset. This subset may play an important role in flares of lupus and related autoimmune rheumatic diseases, provide a biomarker for disease activity and serve as a novel therapeutic target for the treatment of lupus flares.
doi:10.1136/lupus-2016-000147
PMCID: PMC4823547  PMID: 27099767
Systemic Lupus Erythematosus; T Cells; Kir; Autoimmune Diseases; DNA Methylation
4.  Environmental Exposures, Epigenetic Changes and the Risk of Lupus 
Lupus  2014;23(6):568-576.
A dose-dependent combination of environmental exposures, estrogenic hormones and genetic predisposition is thought to be required for lupus to develop and flare, but how the environment modifies the immune system in genetically predisposed people is unclear. Current evidence indicates that environmental agents that inhibit DNA methylation can convert normal antigen-specific CD4+ T lymphocytes into autoreactive, cytotoxic, pro-inflammatory cells that are sufficient to cause lupus-like autoimmunity in animal models, and that the same changes in DNA methylation characterize CD4+ T cells from patients with active lupus. Environmental agents implicated in inhibiting T cell DNA methylation include the lupus-inducing drugs procainamide and hydralazine, as well as diet, and agents causing oxidative stress, such as smoking, UV light exposure, and infections, which have been associated with lupus onset or disease activity. Other studies demonstrate that demethylated T cells cause only anti-DNA antibodies in mice lacking a genetic predisposition to lupus, but are sufficient to cause lupus-like autoimmunity in genetically predisposed mice and likely people, and that estrogens augment the disease. Collectively, these studies suggest that environmental agents that inhibit DNA methylation, together with lupus genes and estrogens or endocrine disruptors, combine in a dose-dependent fashion to cause lupus flares.
doi:10.1177/0961203313499419
PMCID: PMC4000546  PMID: 24763540
Lupus; epigenetics; environment; oxidative stress; mercury; endocrine disruptors; genetics
5.  Association of the interferon signature metric with serological disease manifestations but not global activity scores in multiple cohorts of patients with SLE 
Lupus Science & Medicine  2015;2(1):e000080.
Objectives
The interferon (IFN) signature (IS) in patients with systemic lupus erythematosus (SLE) includes over 100 genes induced by type I IFN pathway activation. We developed a method to quantify the IS using three genes—the IS metric (ISM)—and characterised the clinical characteristics of patients with SLE with different ISM status from multiple clinical trials.
Methods
Blood microarray expression data from a training cohort of patients with SLE confirmed the presence of the IS and identified surrogate genes. We assayed these genes in a quantitative PCR (qPCR) assay, yielding an ISM from the IS. The association of ISM status with clinical disease characteristics was assessed in patients with extrarenal lupus and lupus nephritis from four clinical trials.
Results
Three genes, HERC5, EPSTI and CMPK2, correlated well with the IS (p>0.96), and composed the ISM qPCR assay. Using the 95th centile for healthy control data, patients with SLE from different studies were classified into two ISM subsets—ISM-Low and ISM-High—that are longitudinally stable over 36 weeks. Significant associations were identified between ISM-High status and higher titres of anti-dsDNA antibodies, presence of anti extractable nuclear antigen autoantibodies, elevated serum B cell activating factor of the tumour necrosis factor family (BAFF) levels, and hypocomplementaemia. However, measures of overall clinical disease activity were similar for ISM-High and ISM-Low groups.
Conclusions
The ISM is an IS biomarker that divides patients with SLE into two subpopulations—ISM-High and ISM-Low—with differing serological manifestations. The ISM does not distinguish between high and low disease activity, but may have utility in identifying patients more likely to respond to treatment(s) targeting IFN-α.
Clinicaltrials.gov registration number
NCT00962832.
doi:10.1136/lupus-2014-000080
PMCID: PMC4379884  PMID: 25861459
Interferon; serological manifestations; SLE
6.  Epigenetics in 2013: DNA methylation and miRNA—key roles in systemic autoimmunity 
Nature reviews. Rheumatology  2014;10(2):72-74.
Several advances in 2013 have improved our understanding of how epigenetic mechanisms affect autoimmune disorders. Many new insights were made into the regulation of gene expression by DNA methylation in systemic lupus erythematosus. For rheumatoid arthritis, complex interrelationships between DNA methylation and microRNAs in regulating gene expression were described.
doi:10.1038/nrrheum.2013.211
PMCID: PMC4332710  PMID: 24418763
7.  Oxidative Stress, T Cell DNA Methylation, and Lupus 
Objective
Lupus develops when genetically predisposed people encounter environmental agents, such as ultraviolet light, silica, infections, and cigarette smoke, that cause oxidative stress, but how oxidative damage modifies the immune system to cause lupus flares is unknown. We previously showed that inhibiting DNA methylation in CD4+ T cells by blocking ERK pathway signaling is sufficient to alter gene expression, and that the modified cells cause lupus-like autoimmunity in mice. We also reported that T cells from patients with active lupus have decreased ERK pathway signaling, have decreased DNA methylation, and overexpress genes normally suppressed by DNA methylation. This study was undertaken to test whether oxidizing agents decrease ERK pathway signaling in T cells, decrease DNA methyltransferase levels, and cause demethylation and overexpression of T cell genes similar to that found in T cells from patients with active lupus.
Methods
CD4+ T cells were treated with the oxidizers H2O2 or ONOO−. Effects on ERK pathway signaling were measured by immunoblotting, DNA methyltransferase 1 (DNMT-1) levels were measured by reverse transcriptase–polymerase chain reaction (RT-PCR), and the methylation and expression of T cell genes were measured using flow cytometry, RT-PCR, and bisulfite sequencing.
Results
H2O2 and ONOO− inhibited ERK pathway signaling in T cells by inhibiting the upstream regulator protein kinase Cδ, decreased DNMT-1 levels, and caused demethylation and overexpression of genes previously shown to be suppressed by DNA methylation in T cells from patients with active lupus.
Conclusion
Our findings indicate that oxidative stress may contribute to human lupus flares by inhibiting ERK pathway signaling in T cells to decrease DNMT-1 and cause DNA demethylation.
doi:10.1002/art.38427
PMCID: PMC4141415  PMID: 24577881
8.  Oxidative Stress, T Cell DNA Methylation and Lupus 
Objective
Lupus develops when genetically predisposed people encounter environmental agents such as UV light, silica, infections and cigarette smoke that cause oxidative stress, but how oxidative damage modifies the immune system to cause lupus flares is unknown. We reported that inhibiting DNA methylation in CD4+ T cells by blocking ERK pathway signaling is sufficient to alter gene expression, and that the modified cells cause lupus-like autoimmunity in mice. We also reported that T cells from patients with active lupus have decreased ERK pathway signaling, decreased DNA methylation, and overexpress genes normally suppressed by DNA methylation. We therefore tested whether oxidizing agents decrease T cell ERK pathway signaling, decrease DNA methyltransferase levels, and cause demethylation and overexpression of T cell genes similar to that found in T cells from patients with active lupus.
Methods
CD4+ T cells were treated with the oxidizers H2O2 or ONOO−. Effects on ERK pathway signaling were measured by immunoblotting, Dnmt1 levels by RT-PCR, and the methylation and expression of T cell genes were measured using flow cytometry, RT-PCR and bisulfite sequencing.
Results
H2O2 and ONOO− inhibited T cell ERK pathway signaling by inhibiting the upstream regulator PKCδ, decreased Dnmt1 levels, and caused demethylation and overexpression genes previously shown to suppressed by DNA methylation in T cells from patients with active lupus.
Conclusions
Oxidative stress may contribute to human lupus flares by inhibiting T cell ERK pathway signaling to decrease Dnmt1 and cause DNA demethylation.
doi:10.1002/art.38427
PMCID: PMC4141415  PMID: 24577881
9.  Diet Influences Expression of Autoimmune Associated Genes and Disease Severity By Epigenetic Mechanisms in a Transgenic Lupus Model 
Arthritis and rheumatism  2013;65(7):1872-1881.
Objective
Lupus flares when genetically predisposed people encounter appropriate environmental agents. Current evidence indicates that the environment contributes by inhibiting T cell DNA methylation, causing overexpression of normally silenced genes. DNA methylation depends on both dietary transmethylation micronutrients and Erk-regulated DNA methyltransferase 1 (Dnmt1) levels. We used transgenic mice to study interactions between diet, Dnmt1 levels and genetic predisposition on the development and severity of lupus.
Methods
A doxycycline-inducible Erk defect was bred into lupus-resistant (C57BL/6) or lupus-susceptible (C57BL/6xSJL) mouse strains. Doxycycline treated mice were fed a standard commercial diet for eighteen weeks then switched to diets supplemented(MS) or restricted(MR) intransmethylation micronutrients. Disease severity was assessed by anti-dsDNA antibodies, proteinuria, hematuria and histopathology of kidney tissues. Pyrosequencing was used to determine micronutrient effects on DNA methylation.
Results
Doxycycline induced modest levels of anti-dsDNA antibodies in C57BL/6 mice and higher levels in C57BL/6xSJL mice. Doxycycline-treated C57BL/6xSJL mice developed hematuria and glomerulonephritis on the MR and standard but not the MS diet. In contrast C57BL/6 mice developed kidney disease only on the MR diet. Decreasing Erk signaling and methyl donors also caused demethylation and overexpression of the CD40lg gene in female mice, consistent with demethylation of the second X chromosome. Both the dietary methyl donor content and duration of treatment influenced methylation and expression of the CD40lg gene.
Conclusions
Dietary micronutrients that affect DNA methylation can exacerbate or ameliorate SLE disease in this transgenic murine lupus model, and contribute to lupus susceptibility and severity through genetic/epigenetic interactions.
doi:10.1002/art.37967
PMCID: PMC3735138  PMID: 23576011
Extracellular Receptor Kinase (Erk; Systemic Lupus Erythematosus (SLE; CD70; micronutrients; CD40L; KirL1
10.  Association of Endogenous Anti–Interferon-α Autoantibodies With Decreased Interferon-Pathway and Disease Activity in Patients With Systemic Lupus Erythematosus 
Arthritis and rheumatism  2011;63(8):2407-2415.
Objective
Numerous observations implicate interferon-α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti-IFNα autoantibodies (AIAAs) on IFN-pathway and disease activity is unclear. The aim of this study was to characterize IFN-pathway activity and the serologic and clinical profiles of AIAA-positive patients with SLE.
Methods
Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA-positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN-regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN-inducible chemokines, and other autoantibodies were measured using immunoassays.
Results
AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFNlow and IFNhigh, that differed in the levels of serum type I IFN bioactivity, IFN-regulated gene expression, BAFF, anti-ribosomal P, and anti-chromatin autoantibodies, and in AIAA status. The majority of AIAA-positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN-pathway activity, and lower disease activity compared with the IFNhigh patients. AIAA-positive sera were able to effectively neutralize type I IFN activity in vitro.
Conclusion
Patients with SLE commonly harbor AIAAs. AIAA-positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN-pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.
doi:10.1002/art.30399
PMCID: PMC4028124  PMID: 21506093
11.  MURINE MODELS OF LUPUS INDUCED BY HYPOMETHYLATED T CELLS 
Methods in molecular medicine  2004;102:285-294.
CD4+ T cell DNA hypomethylation may contribute to the development of drug induced and idiopathic human lupus. Inhibiting DNA methylation in mature CD4+ T cells causes MHC-specific autoreactivity in vitro. The lupus-inducing drugs hydralazine and procainamide also inhibit T cell DNA methylation and induce autoreactivity, and T cells from patients with active lupus have hypomethylated DNA and a similarly autoreactive T cell subset. Further, T cells treated with DNA methylation inhibitors demethylate the same sequences that demethylate in T cells from patients with active lupus. The pathologic significance of the autoreactivity induced by inhibiting T cell DNA methylation has been tested by treating murine T cells in vitro with drugs which modify DNA methylation, then injecting the cells into syngeneic female mice. Mice receiving CD4+ T cells demethylated by a variety of agents including procainamide and hydralazine develop a lupus-like disease. Further, transgenic mice with an inducible T cell DNA methylation defect also develop lupus-like autoimmunity. This chapter describes the protocols for inducing autoreactivity in murine T cells in vitro, and inducing autoimmunity in vivo using an adoptive transfer approach or transgenic animal models.
doi:10.1385/1-59259-805-6:285
PMCID: PMC4023250  PMID: 15286391
Lupus; drug-induced lupus; DNA methylation; animal models; autoimmunity
12.  Bariatric Endocrinology: Principles of Medical Practice 
Obesity, is a chronic, biological, preventable, and treatable disease. The accumulation of fat mass causes physical changes (adiposity), metabolic and hormonal changes due to adipose tissue dysfunction (adiposopathy), and psychological changes. Bariatric endocrinology was conceived from the need to address the neuro-endocrinological derangements that are associated with adiposopathy, and from the need to broaden the scope of the management of its complications. In addition to the well-established metabolic complications of overweight and obesity, adiposopathy leads to hyperinsulinemia, hyperleptinemia, hypoadiponectinemia, dysregulation of gut peptides including GLP-1 and ghrelin, the development of an inflammatory milieu, and the strong risk of vascular disease. Therapy for adiposopathy hinges on effectively lowering the ratio of orexigenic to anorexigenic signals reaching the the hypothalamus and other relevant brain regions, favoring a lower caloric intake. Adiposopathy, overweight and obesity should be treated indefinitely with the specific aims to reduce fat mass for the adiposity complications, and to normalize adipose tissue function for the adiposopathic complications. This paper defines the principles of medical practice in bariatric endocrinology—the treatment of overweight and obesity as means to treat adiposopathy and its accompanying metabolic and hormonal derangements.
doi:10.1155/2014/917813
PMCID: PMC4036612  PMID: 24899894
13.  Overexpression of X-Linked Genes in T Cells From Women With Lupus 
Journal of autoimmunity  2013;41:60-71.
Women develop lupus more frequently than men and the reason remains incompletely understood. Evidence that men with Klinefelter’s Syndrome (XXY) develop lupus at approximately the same rate as women suggests that a second X chromosome contributes. However, since the second X is normally inactivated, how it predisposes to lupus is unclear. DNA methylation contributes to the silencing of one X chromosome in women, and CD4+ T cell DNA demethylation contributes to the development of lupus-like autoimmunity. This suggests that demethylation of genes on the inactive X may predispose women to lupus, and this hypothesis is supported by a report that CD40LG, an immune gene encoded on the X chromosome, demethylates and is overexpressed in T cells from women but not men with lupus. Overexpression of other immune genes on the inactive X may also predispose women to this disease. We therefore compared mRNA and miRNA expression profiles in experimentally demethylated T cells from women and men as well as in T cells from women and men with lupus. T cells from healthy men and women were treated with the DNA methyltransferase inhibitor 5-azacytidine, then X-linked mRNAs were surveyed with oligonucleotide arrays, and X-linked miRNA’s surveyed with PCR arrays. CD40LG, CXCR3, OGT, miR-98, let-7f-2*, miR 188-3p, miR-421 and miR-503 were among the genes overexpressed in women relative to men. MiRNA target prediction analyses identified CBL, which downregulates T cell receptor signaling and is decreased in lupus T cells, as a gene targeted by miR-188-3p and miR-98. Transfection with miR-98 and miR-188-3p suppressed CBL expression. The same mRNA and miRNA transcripts were also demethylated and overexpressed in CD4+ T cells from women relative to men with active lupus. Together these results further support a role for X chromosome demethylation in the female predisposition to lupus.
doi:10.1016/j.jaut.2012.12.006
PMCID: PMC3622754  PMID: 23434382
Lupus; epigenetics; women’s health; X chromosome; gene expression; microRNA
14.  Epigenetic mechanisms in lupus 
Current opinion in rheumatology  2010;22(5):10.1097/BOR.0b013e32833ae915.
Purpose of review
Epigenetic mechanisms regulate gene expression, and epigenetic gene dysregulation is implicated in the pathogenesis of a growing number of disorders. Of the autoimmune diseases, epigenetic mechanisms are most clearly involved in human systemic lupus erythematosus (SLE). Herein, we summarize earlier work on epigenetic mechanisms contributing to human SLE. We first focus on the roles of DNA demethylation and DNA methyltransferase enzyme dysregulation, and we then review recent and important advances in this field.
Recent findings
Many advances in the past year have been made. The importance of DNA demethylation in SLE was confirmed through twin studies. New T lymphocyte immune genes that are activated by DNA demethylation, and that may participate in autoreactivity, were identified. Finally, novel mechanisms contributing to DNA demethylation in SLE were discovered.
Summary
A comprehensive understanding of the epigenetic mechanisms contributing to SLE will likely enable development of new therapeutic agents and strategies that target the dysregulated genes or correct the aberrant epigenetic modifications. Although specific agents have not yet been tested in SLE, the studies reviewed hold promise that these approaches will be useful in the treatment of human lupus.
doi:10.1097/BOR.0b013e32833ae915
PMCID: PMC3819175  PMID: 20445453
chromatin; DNA methylation; epigenetics; histone; lupus
15.  PKCδ OXIDATION CONTRIBUTES TO ERK INACTIVATION IN LUPUS T CELLS1 
Arthritis and rheumatism  2012;64(9):2964-2974.
Objective
CD4+ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T505 in the PKCδ activation loop. However, the mechanisms preventing PKCδ T505 phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We hypothesized that nitration inactivates PKCδ, contributing to impaired ERK pathway signaling in lupus T cells.
Methods
CD4+ T cells were purified from lupus patients and controls then stimulated with PMA. Signaling protein levels, nitration and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation.
Results
Treating CD4+ T cells with peroxynitrite nitrated PKCδ, preventing PKCδ T505 phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKCδ levels than controls which correlated directly with disease activity, and anti-nitrotyrosine immunoprecipitations demonstrated that nitrated PKCδ, but not unmodified PKCδ, was refractory to PMA stimulated T505 phosphorylation, similar to PKCδ in peroxynitrite treated cells.
Conclusions
Oxidative stress causes PKCδ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKCδ as a link between oxidative stress and the T cell epigenetic modifications in lupus.
doi:10.1002/art.34503
PMCID: PMC3414679  PMID: 22549474
Systemic lupus erythematosus; PKCδ; T cells; Signal transduction; Oxidative stress
16.  Dissecting complex epigenetic alterations in human lupus 
Systemic lupus erythematosus is a chronic relapsing autoimmune disease that primarily afflicts women, and both a genetic predisposition and appropriate environmental exposures are required for lupus to develop and flare. The genetic requirement is evidenced by an increased concordance in identical twins and by the validation of at least 35 single-nucleotide polymorphisms predisposing patients to lupus. Genes alone, though, are not enough. The concordance of lupus in identical twins is often incomplete, and when concordant, the age of onset is usually different. Lupus is also not present at birth, but once the disease develops, it typically follows a chronic relapsing course. Thus, genes alone are insufficient to cause human lupus, and additional factors encountered in the environment and over time are required to initiate the disease and subsequent flares. The nature of the environmental contribution, though, and the mechanisms by which environmental agents modify the immune response to cause lupus onset and flares in genetically predisposed people have been controversial. Reports that the lupus-inducing drugs procainamide and hydralazine are epigenetic modifiers, that epigenetically modified T cells are sufficient to cause lupus-like autoimmunity in animal models, and that patients with active lupus have epigenetic changes similar to those caused by procainamide and hydralazine have prompted a growing interest in how epigenetic alterations contribute to this disease. Understanding how epigenetic mechanisms modify T cells to contribute to lupus requires an understanding of how epigenetic mechanisms regulate gene expression. The roles of DNA methylation, histone modifications, and microRNAs in lupus pathogenesis will be reviewed here.
doi:10.1186/ar4125
PMCID: PMC3746410  PMID: 23374884
17.  Environmental Exposure, Estrogen and Two X Chromosomes are Required for Disease Development in an Epigenetic Model of Lupus 
Journal of Autoimmunity  2011;38(2-3):J135-J143.
Systemic lupus erythematosus (SLE) is an autoimmune disease primarily afflicting women. The reason for the gender bias is unclear, but genetic susceptibility, estrogen and environmental agents appear to play significant roles in SLE pathogenesis. Environmental agents can contribute to lupus susceptibility through epigenetic mechanisms. We used (C57BL/6 × SJL)F1 mice transgenic for a dominant-negative MEK (dnMEK) that was previously shown to be inducibly and selectively expressed in T cells. In this model, induction of the dnMEK by doxycycline treatment suppresses T cell ERK signaling, decreasing DNA methyltransferase expression and resulting in DNA demethylation, overexpression of immune genes Itgal (CD11a) and Tnfsf7 (CD70), and anti-dsDNA antibody. To examine the role of gender and estrogen in this model, male and female transgenic mice were neutered and implanted with time-release pellets delivering placebo or estrogen. Doxycycline induced IgG anti-dsDNA antibodies in intact and neutered, placebo-treated control female but not male transgenic mice. Glomerular IgG deposits were also found in the kidneys of female but not male transgenic mice, and not in the absence of doxycycline. Estrogen enhanced anti-dsDNA IgG antibodies only in transgenic, ERK-impaired female mice. Decreased ERK activation also resulted in overexpression and demethylation of the X-linked methylation-sensitive gene CD40lg in female but not male mice, consistent with demethylation of the second X chromosome in the females. The results show that both estrogen and female gender contribute to the female predisposition in lupus susceptibility through hormonal and epigenetic X chromosome effects and through suppression of ERK signaling by environmental agents.
doi:10.1016/j.jaut.2011.11.001
PMCID: PMC3312994  PMID: 22142890
Extracellular Receptor Kinase (ERK); Systemic Lupus erythematosus (SLE); Mouse
18.  Sex-specific differences in the relationship between genetic susceptibility, T cell DNA demethylation and lupus flare severity 
Journal of Autoimmunity  2012;38(2-3):J216-J222.
Lupus is less common in men than women, and the reason is incompletely understood. Current evidence indicates that lupus flares when genetically predisposed individuals encounter environmental agents that trigger the disease, and that the environmental contribution is mediated at least in part by T cell DNA demethylation. We hypothesized that lupus disease activity is directly related to total genetic risk and inversely related to T cell DNA methylation levels in each patient. Since women are predisposed to lupus in part because of their second X chromosome, we also hypothesized that men would require a greater genetic risk, a greater degree of autosomal T cell DNA demethylation, or both, to achieve a lupus flare equal in severity to women. Genetic risk was determined by genotyping men and women with lupus across 32 confirmed lupus susceptibility loci. The methylation status of two T cell autosomal genes known to demethylate in proportion to disease activity, KIR2DL4 (KIR) and PRF1, was measured by bisulfite sequencing. Lupus disease activity was determined by the SLEDAI. Interactions between genetic score, T cell DNA demethylation, and the SLEDAI score were compared between the men and women by regression analysis. Combining the degree of DNA demethylation with the genetic risk score for each patient demonstrated that the (genetic risk)/(DNA methylation) ratio increased directly with disease activity in both men and women with lupus. Importantly, men required a greater (genetic risk)/(DNA methylation) ratio to achieve a SLEDAI score equivalent to women (p=0.010 for KIR and p=0.0054 for PRF1). This difference was not explained by a difference in the genetic risk or T cell DNA demethylation alone, suggesting a genetic-epigenetic interaction. These results suggest that genetic risk and T cell DNA demethylation interact in lupus patients to influence the severity of lupus flares, and that men require a higher genetic risk and/or greater degree of T cell DNA demethylation to achieve a lupus flare equal in severity to women.
doi:10.1016/j.jaut.2011.11.008
PMCID: PMC3313010  PMID: 22305513
Genetic risk; epigenetics; DNA methylation; lupus; genetic-epigenetic interaction; sex-disparity
19.  Analysis of autosomal genes reveals gene–sex interactions and higher total genetic risk in men with systemic lupus erythematosus 
Annals of the Rheumatic Diseases  2011;71(5):694-699.
Objectives
Systemic lupus erythematosus (SLE) is a sexually dimorphic autoimmune disease which is more common in women, but affected men often experience a more severe disease. The genetic basis of sexual dimorphism in SLE is not clearly defined. A study was undertaken to examine sex-specific genetic effects among SLE susceptibility loci.
Methods
A total of 18 autosomal genetic susceptibility loci for SLE were genotyped in a large set of patients with SLE and controls of European descent, consisting of 5932 female and 1495 male samples. Sex-specific genetic association analyses were performed. The sex–gene interaction was further validated using parametric and nonparametric methods. Aggregate differences in sex-specific genetic risk were examined by calculating a cumulative genetic risk score for SLE in each individual and comparing the average genetic risk between male and female patients.
Results
A significantly higher cumulative genetic risk for SLE was observed in men than in women. (P = 4.52×10−8) A significant sex–gene interaction was seen primarily in the human leucocyte antigen (HLA) region but also in IRF5, whereby men with SLE possess a significantly higher frequency of risk alleles than women. The genetic effect observed in KIAA1542 is specific to women with SLE and does not seem to have a role in men.
Conclusions
The data indicate that men require a higher cumulative genetic load than women to develop SLE. These observations suggest that sex bias in autoimmunity could be influenced by autosomal genetic susceptibility loci.
doi:10.1136/annrheumdis-2011-200385
PMCID: PMC3324666  PMID: 22110124
20.  Evidence for gene-gene epistatic interactions among susceptibility loci for systemic lupus erythematosus 
Arthritis and Rheumatism  2012;64(2):485-492.
Objective
Several confirmed genetic susceptibility loci for lupus have been described. To date, no clear evidence for genetic epistasis is established in lupus. We test for gene-gene interactions in a number of known lupus susceptibility loci.
Methods
Eighteen SNPs tagging independent and confirmed lupus susceptibility loci were genotyped in a set of 4,248 lupus patients and 3,818 normal healthy controls of European descent. Epistasis was tested using a 2-step approach utilizing both parametric and non-parametric methods. The false discovery rate (FDR) method was used to correct for multiple testing.
Results
We detected and confirmed gene-gene interactions between the HLA region and CTLA4, IRF5, and ITGAM, and between PDCD1 and IL21 in lupus patients. The most significant interaction detected by parametric analysis was between rs3131379 in the HLA region and rs231775 in CTLA4 (Interaction odds ratio=1.19, z-score= 3.95, P= 7.8×10−5 (FDR≤0.05), PMDR= 5.9×10−45). Importantly, our data suggest that in lupus patients the presence of the HLA lupus-risk alleles in rs1270942 and rs3131379 increases the odds of also carrying the lupus-risk allele in IRF5 (rs2070197) by 17% and 16%, respectively (P= 0.0028 and 0.0047).
Conclusion
We provide evidence for gene-gene epistasis in systemic lupus erythematosus. These findings support a role for genetic interaction contributing to the complexity of lupus heritability.
doi:10.1002/art.33354
PMCID: PMC3268866  PMID: 21952918
21.  Identification of novel genetic susceptibility loci in African-American lupus patients using a candidate gene association study 
Arthritis and rheumatism  2011;63(11):3493-3501.
Objective
Candidate gene and genome-wide association studies have identified several disease susceptibility loci in lupus patients. These studies have been largely performed in European-derived and Asian lupus patients. In this study, we examine if some of these same susceptibility loci increase lupus risk in African-American individuals.
Methods
Single nucleotide polymorphisms tagging 15 independent lupus susceptibility loci were genotyped in a set of 1,724 lupus patients and 2,024 normal healthy controls of African-American descent. The loci examined included: PTPN22, FCGR2A, TNFSF4, STAT4, CTLA4, PDCD1, PXK, BANK1, MSH5 (HLA region), CFB (HLA region), C8orf13-BLK region, MBL2, KIAA1542, ITGAM, and MECP2/IRAK1.
Results
We provide the first evidence for genetic association between lupus and five susceptibility loci in African-American patients (C8orf13-BLK, BANK1, TNFSF4, KIAA1542 andCTLA4; P values= 8.0 × 10−6, 1.9 × 10−5, 5.7 × 10−5, 0.00099, 0.0045, respectively). Further, we confirm the genetic association between lupus and five additional lupus susceptibility loci (ITGAM, MSH5, CFB, STAT4, and FCGR2A; P values= 7.5 × 10−11, 5.2 × 10−8, 8.7 × 10−7, 0.0058, and 0.0070, respectively), and provide evidence for a genome-wide significance for the association between ITGAM and MSH5 (HLA region) for the first time in African-American lupus patients.
Conclusion
These findings provide evidence for novel genetic susceptibility loci for lupus in African-Americans and demonstrate that the majority of lupus susceptibility loci examined confer lupus risk across multiple ethnicities.
doi:10.1002/art.30563
PMCID: PMC3205224  PMID: 21792837
22.  Variation in the ICAM1–ICAM4–ICAM5 locus is associated with systemic lupus erythematosus susceptibility in multiple ancestries 
Annals of the rheumatic diseases  2012;71(11):1809-1814.
Objective
Systemic lupus erythematosus (SLE; OMIM 152700) is a chronic autoimmune disease for which the aetiology includes genetic and environmental factors. ITGAM, integrin αΜ (complement component 3 receptor 3 subunit) encoding a ligand for intracellular adhesion molecule (ICAM) proteins, is an established SLE susceptibility locus. This study aimed to evaluate the independent and joint effects of genetic variations in the genes that encode ITGAM and ICAM.
Methods
The authors examined several markers in the ICAM1–ICAM4–ICAM5 locus on chromosome 19p13 and the single ITGAM polymorphism (rs1143679) using a large-scale case–control study of 17 481 unrelated participants from four ancestry populations. The single marker association and gene–gene interaction were analysed for each ancestry, and a meta-analysis across the four ancestries was performed.
Results
The A-allele of ICAM1–ICAM4–ICAM5 rs3093030, associated with elevated plasma levels of soluble ICAM1, and the A-allele of ITGAM rs1143679 showed the strongest association with increased SLE susceptibility in each of the ancestry populations and the trans-ancestry meta-analysis (ORmeta=1.16, 95% CI 1.11 to 1.22; p=4.88×10−10 and ORmeta=1.67, 95% CI 1.55 to 1.79; p=3.32×10−46, respectively). The effect of the ICAM single-nucleotide polymorphisms (SNPs) was independent of the effect of the ITGAM SNP rs1143679, and carriers of both ICAM rs3093030-AA and ITGAM rs1143679-AA had an OR of 4.08 compared with those with no risk allele in either SNP (95% CI 2.09 to 7.98; p=3.91×10−5).
Conclusion
These findings are the first to suggest that an ICAM–integrin-mediated pathway contributes to susceptibility to SLE.
doi:10.1136/annrheumdis-2011-201110
PMCID: PMC3466387  PMID: 22523428
23.  Phenotypic associations of genetic susceptibility loci in systemic lupus erythematosus 
Annals of the rheumatic diseases  2011;70(10):1752-1757.
Objective
Systemic lupus erythematosus is a clinically heterogeneous autoimmune disease. A number of genetic loci that increase lupus susceptibility have been established. This study examines if these genetic loci also contribute to the clinical heterogeneity in lupus.
Materials and methods
4001 European-derived, 1547 Hispanic, 1590 African-American and 1191 Asian lupus patients were genotyped for 16 confirmed lupus susceptibility loci. Ancestry informative markers were genotyped to calculate and adjust for admixture. The association between the risk allele in each locus was determined and compared in patients with and without the various clinical manifestations included in the ACR criteria.
Results
Renal disorder was significantly correlated with the lupus risk allele in ITGAM (p=5.0×10−6, OR 1.25, 95% CI 1.12 to 1.35) and in TNFSF4 (p=0.0013, OR 1.14, 95% CI 1.07 to 1.25). Other significant findings include the association between risk alleles in FCGR2A and malar rash (p=0.0031, OR 1.11, 95% CI 1.17 to 1.33), ITGAM and discoid rash (p=0.0020, OR 1.20, 95% CI 1.06 to 1.33), STAT4 and protection from oral ulcers (p=0.0027, OR 0.89, 95% CI 0.83 to 0.96) and IL21 and haematological disorder (p=0.0027, OR 1.13, 95% CI 1.04 to 1.22). All these associations are significant with a false discovery rate of <0.05 and pass the significance threshold using Bonferroni correction for multiple testing.
Conclusion
Significant associations were found between lupus clinical manifestations and the FCGR2A, ITGAM, STAT4, TNSF4 and IL21 genes. The findings suggest that genetic profiling might be a useful tool to predict disease manifestations in lupus patients in the future.
doi:10.1136/ard.2011.154104
PMCID: PMC3232181  PMID: 21719445
24.  Early disease onset is predicted by a higher genetic risk for lupus and is associated with a more severe phenotype in lupus patients 
Annals of the rheumatic diseases  2010;70(1):151-156.
Background
Systemic lupus erythematosus (SLE) is a chronic, multiorgan, autoimmune disease that affects people of all ages and ethnicities.
Objectives
To explore the relationship between age at disease onset and many of the diverse manifestations of SLE. Additionally, to determine the relationship between age of disease onset and genetic risk in patients with SLE.
Methods
The relationship between the age at disease onset and SLE manifestations were explored in a multiracial cohort of 1317 patients. Patients with SLE were genotyped across 19 confirmed genetic susceptibility loci for SLE. Logistic regression was used to determine the relationships between the number of risk alleles present and age of disease onset.
Results
Childhood-onset SLE had higher odds of proteinuria, malar rash, anti-dsDNA antibody, haemolytic anaemia, arthritis and leucopenia (OR=3.03, 2.13, 2.08, 2.50, 1.89, 1.53, respectively; p values <0.0001, 0.0004, 0.0005, 0.0024, 0.0114, 0.045, respectively). In female subjects, the odds of having cellular casts were 2.18 times higher in childhood-onset than in adult-onset SLE (p=0.0027). With age of onset ≥50, the odds of having proteinuria, cellular casts, anti-nRNP antibody, anti-Sm antibody, anti-dsDNA antibody and seizures were reduced. However, late adult-onset patients with SLE have higher odds of developing photosensitivity than early adult-onset patients. Each SLE-susceptibility risk allele carried within the genome of patients with SLE increased the odds of having a childhood-onset disease in a race-specific manner: by an average of 48% in Gullah and 25% in African-Americans, but this was not significant in Hispanic and European-American lupus patients.
Conclusions
The genetic contribution towards predicting early-onset disease in patients with SLE is quantified for the first time. A more severe SLE phenotype is found in patients with early-onset disease in a large multi-racial cohort, independent of gender, race and disease duration.
doi:10.1136/ard.2010.141697
PMCID: PMC3034281  PMID: 20881011
25.  Age-dependent decreases in DNA methyltransferase levels and low transmethylation micronutrient levels synergize to promote overexpression of genes implicated in autoimmunity and acute coronary syndromes 
Experimental gerontology  2009;45(4):312-322.
T cell DNA methylation levels decline with age, activating genes such as KIR and TNFSF7 (CD70), implicated in lupus-like autoimmunity and acute coronary syndromes. The mechanisms causing age-dependent DNA demethylation are unclear. Maintenance of DNA methylation depends on DNA methyltransferase 1 (Dnmt1) and intracellular S-adenosylmethionine levels, and is inhibited by S-adenosylhomocysteine (SAH). SAM levels depend on dietary micronutrients including folate and methionine. SAH levels depend on serum homocysteine concentrations. T cell Dnmt1 levels also decline with age. We hypothesized that age-dependent Dnmt1 decreases synergize with low folate, low methionine or high homocysteine levels to demethylate and activate methylation-sensitive genes. T cells from healthy adults ages 22-81, stimulated and cultured with low folate, low methionine, or high homocysteine concentrations showed demethylation and overexpression of KIR and CD70 beginning at age ~50 and increased further with age. The effects were reproduced by Dnmt1 knockdowns in T cells from young subjects. These results indicate that maintenance of T cell DNA methylation patterns is more sensitive to low folate and methionine levels in older than younger individuals, due to low Dnmt1 levels, and that homocysteine further increases aberrant gene expression. Thus, attention to proper nutrition may be particularly important in the elderly.
doi:10.1016/j.exger.2009.12.008
PMCID: PMC2838973  PMID: 20035856
Aging; Epigenetics; DNA methylation; Senescence

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