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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  Key role of ERK pathway signaling in lupus 
Autoimmunity  2010;43(1):17.
Systemic lupus erythematosus is a poorly understood autoimmune disease, characterized by autoantibodies to nuclear antigens and immune complex deposition in organs like the kidney. Current evidence indicates that a pathologic CD4+T cell subset, characterized by impaired extracellular signal-regulated kinase (ERK) pathway signaling, DNA hypomethylation, and consequent aberrant gene expression contributes to disease pathogenesis. Hydralazine is a lupus-inducing drug that also decreases T cell DNA methylation by inhibiting the ERK signaling pathway, replicating the defect found in lupus T cells. These observations suggest that defective ERK pathway signaling alters gene expression in T cells by inhibiting DNA methylation, contributing to lupus pathogenesis. The signaling defect in hydralazine-treated and lupus T cells has now been mapped to protein kinase C δ. Understanding the mechanism causing decreased ERK pathway signaling in lupus may shed light on mechanisms contributing to disease development in genetically predisposed people.
doi:10.3109/08916930903374832
PMCID: PMC2819407  PMID: 19961364
Lupus T cells; epigenetics; DNA methylation; extracellular signal-regulated kinase pathway signaling; Protein Kinase δ
15.  T cell CD40LG gene expression and the production of IgG by autologous B cells in systemic lupus erythematosus 
Clinical immunology (Orlando, Fla.)  2009;132(3):362-370.
CD40 ligand (CD40LG), encoded on the X chromosome, has been reported to be overexpressed on lupus Tcells. Herein, we investigated the effect of DNA demethylation on Tcell CD40LG expression and the production of IgG by autologous B cells in lupus. We found normal human T cells transfected with CD40LG induced autologous B cell activation and plasma cell differentiation. Both female lupus CD4+ T cells and demethylating agents treated CD4+ T cells overexpressed CD40LG mRNA. Further, lupus T cells from both genders or demethylated CD4+ T cells from healthy women overstimulated autologous B cells, and this could be reversed with anti-CD40LG Ab in only females. We demonstrated that female lupus CD4+ T cells and demethylated CD4+ T cells express high level of CD40LG and overstimulate B cells to produce IgG. This is due to DNA demethylation and thereby reactivation of the inactive X chromosome in female.
doi:10.1016/j.clim.2009.05.011
PMCID: PMC2810511  PMID: 19520616
CD40 ligand; DNA methylation; Immunoglobulin G; Systemic lupus; erythematosus
16.  Stimulatory and Inhibitory Killer Immunoglobulin-Like Receptor Molecules are Expressed and Functional on Lupus T Cells1 
T cells from lupus patients have hypomethylated DNA and overexpress genes normally suppressed by DNA methylation that contribute to disease pathogenesis. We found that stimulatory and inhibitory killer cell immunoglobulin–like receptor (KIR3) genes are aberrantly overexpressed on experimentally demethylated T cells. We therefore asked if lupus T cells also overexpress KIR, and if the proteins are functional. T cells from lupus patients were found to overexpress KIR genes, and expression was proportional to disease activity. Antibodies to the stimulatory molecule KIR2DL4 triggered IFN-γ release by lupus T cells, and production was proportional to disease activity. Similarly, crosslinking the inhibitory molecule KIR3DL1 prevented the autoreactive macrophage killing that characterizes lupus T cells. These results indicate that aberrant T cell KIR expression may contribute to IFN overproduction and macrophage killing in human lupus, and suggest that antibodies to inhibitory KIR may be a treatment for this disease.
doi:10.4049/jimmunol.0900034
PMCID: PMC2730421  PMID: 19675166
Lupus; KIR genes; T cells; DNA methylation; epigenetics
17.  DECREASED DNA METHYLTRANSFERASE LEVELS CONTRIBUTE TO ABNORMAL GENE EXPRESSION IN “SENESCENT” CD4+CD28− T CELLS 
Clinical immunology (Orlando, Fla.)  2009;132(2):257-265.
A senescent CD4+CD28− T cell subset develops with aging and in chronic inflammatory diseases like rheumatoid arthritis, and is implicated in plaque rupture and myocardial infarctions. This subset is pro-inflammatory, cytotoxic for endothelial cells, and aberrantly expresses genes like CD70, perforin and killer-cell immunoglobulin-like receptor (KIR) genes. Why CD4+CD28− cells overexpress these genes is unclear. We found that the CD70, perforin and KIR2DL4 promoters are demethylated in CD4+CD28− T cells, and that DNA methyltransferase 1 (Dnmt1) and Dnmt3a levels are decreased in this subset. siRNA “knockdown” of Dnmt1, but not Dnmt3a, in CD4+CD28+ T cells caused similar demethylation and overexpression of KIR2DL4, perforin and CD70, while simultaneous knockdown of Dnmt1 and Dnmt3a caused greater demethylation and overexpression of these genes than Dnmt1 alone. We conclude that decreased Dnmt1 and Dnmt3a causes demethylation and overexpression of these and perhaps other genes in CD4+CD28− cells, potentially contributing to pathologic functions by this subset.
doi:10.1016/j.clim.2009.03.529
PMCID: PMC2710420  PMID: 19394279
T cell; DNA methylation; aging; rheumatoid arthritis; senescence
18.  The genetics and epigenetics of autoimmune diseases 
Journal of autoimmunity  2009;33(1):3.
Self tolerance loss is fundamental to autoimmunity. While understanding of immune regulation is expanding rapidly, the mechanisms causing loss of tolerance in most autoimmune diseases remain elusive. Autoimmunity is believed to develop when genetically predisposed individuals encounter environmental agents that trigger the disease. Recent advances in the genetic and environmental contributions to autoimmunity suggest that interactions between genetic elements and epigenetic changes caused by environmental agents may be responsible for inducing autoimmune disease. Genetic loci predisposing to autoimmunity are being identified through multi-center consortiums, and the number of validated genes is growing rapidly. Recent reports also indicate that the environment can contribute to autoimmunity by modifying gene expression through epigenetic mechanisms. This article will review current understanding of the genetics and epigenetics of lupus, rheumatoid arthritis, multiple sclerosis and type 1 diabetes, using systemic lupus erythematosus as the primary example. Other autoimmune diseases may have a similar foundation.
doi:10.1016/j.jaut.2009.03.007
PMCID: PMC2819418  PMID: 19349147
Epigenetics; Genetics; Lupus; Multiple Sclerosis; Rheumatoid Arthritis
19.  Epigenetics in human autoimmunity 
Autoimmunity  2008;41(4):278.
Epigenetic mechanisms are essential for normal development and function of the immune system. Similarly, a failure to maintain epigenetic homeostasis in the immune response due to factors including environmental influences, leads to aberrant gene expression, contributing to immune dysfunction and in some cases the development of autoimmunity in genetically predisposed individuals. This is exemplified by systemic lupus erythematosus, where environmentally induced epigenetic changes contribute to disease pathogenesis in those genetically predisposed. Similar interactions between genetically determined susceptibility and environmental factors are implicated in other systemic autoimmune diseases such as rheumatoid arthritis and scleroderma, as well as in organ specific autoimmunity. The skin is exposed to a wide variety of environmental agents, including UV radiation, and is prone to the development of autoimmune conditions such as atopic dermatitis, psoriasis and some forms of vitiligo, depending on environmental and genetic influences. Herein we review how disruption of epigenetic mechanisms can alter immune function using lupus as an example, and summarize how similar mechanisms may contribute to other human autoimmune rheumatic and skin diseases.
doi:10.1080/08916930802024616
PMCID: PMC2819669  PMID: 18432408
Epigenetics; DNA methylation; lupus; psoriasis; atopic dermatitis; vitiligo
20.  DNA METHYLATION INHIBITION INCREASES T CELL KIR EXPRESSION THROUGH EFFECTS ON BOTH PROMOTER METHYLATION AND TRANSCRIPTION FACTORS 
Clinical immunology (Orlando, Fla.)  2008;130(2):213-224.
Killer-cell immunoglobulin-like receptor (KIR) genes are a polymorphic family expressed on NK cells, and “senescent” CD28- T cells implicated in cardiovascular disease. KIR promoters are highly homologous, and NK expression is regulated by DNA methylation. T cell KIR regulation is poorly understood. We asked if epigenetic mechanisms and/or transcription factor alterations determine T cell KIR expression. DNA methylation inhibition activated multiple KIR genes in normal T cells. KIR2DL2 and KIR2DL4 were selected for further study. Expression of both was associated with promoter demethylation, and methylation of the promoters in reporter constructs suppressed expression. KIR reporter construct expression also increased in demethylated T cells and required Ets1, Sp1 and AML sites, implying effects on transcription factors. This was confirmed for Sp1. These results indicate that KIR genes are suppressed by DNA methylation in most T cells, and DNA demethylation promotes their expression through effects on both chromatin structure and transcription factors.
doi:10.1016/j.clim.2008.08.009
PMCID: PMC2655730  PMID: 18945643
KIR genes; T cells; DNA methylation; epigenetics
21.  Stronger inflammatory/cytotoxic T cell response in women identified by microarray analysis 
Genes and immunity  2009;10(5):509-516.
Women develop chronic inflammatory autoimmune diseases more often than men. The mechanisms causing the increased susceptibility are incompletely understood. Chronic immune stimulation characterizes many autoimmune disorders. We hypothesized that repeated stimulation may cause a different T cell response in women than men. Microarrays were used to compare gene expression in T cells from healthy men and women with and without repeated stimulation. Four days following a single stimulation only 25% of differentially expressed, gender-biased genes were expressed at higher levels in women. In contrast, following restimulation 72% were more highly expressed in women. Immune response genes were significantly over-represented among the genes upregulated in women and among the immune response genes, the inflammatory/cytotoxic effector genes interferon gamma (IFNG), lymphotoxin beta (LTB), granzyme A (GZMA), interleukin-12 receptor beta2 (IL12RB2), and granulysin (GNLY) were among those overexpressed to the greatest degree. In contrast, IL17A was the only effector gene more highly expressed in men. Estrogen response elements were identified in the promoters of half the overexpressed immune genes in women, and in <10% of the male biased genes. The differential expression of inflammatory/cytotoxic effector molecules in restimulated female T cells may contribute to the differences in autoimmune diseases between women and men.
doi:10.1038/gene.2009.12
PMCID: PMC2735332  PMID: 19279650
Interferon; granzyme A; granulysin; lymphotoxin B; autoimmunity; gender
22.  Aberrant T cell ERK pathway signaling and chromatin structure in lupus 
Autoimmunity reviews  2008;8(3):196-198.
Human systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibodies to nuclear components with subsequent immune complex formation and deposition in multiple organs. A combination of genetic and environmental factors is required for disease development, but how the environment interacts with the immune system in genetically predisposed hosts to cause lupus is unclear. Recent evidence suggests that environmental agents may alter T cell chromatin structure and gene expression through effects on DNA methylation, a repressive epigenetic mechanism promoting chromatin inactivation, to cause lupus in people with the appropriate genetic background. DNA methylation is regulated by ERK pathway signaling, and abnormalities in ERK pathway signaling may contribute to immune dysfunction in lupus through epigenetic effects on gene expression. This article reviews current evidence for epigenetic abnormalities, and in particular DNA demethylation, in the pathogenesis of idiopathic and some forms of drug induced lupus, and how impaired ERK pathway signaling may contribute to the development of human lupus through effects on T cell DNA methylation.
doi:10.1016/j.autrev.2008.07.043
PMCID: PMC2642928  PMID: 18723128
Lupus T cells; Epigenetics; DNA methylation; ERK pathway signaling; PKCδ
23.  Recent Advances and Opportunities in Research on Lupus: Environmental Influences and Mechanisms of Disease 
Environmental Health Perspectives  2008;116(6):695-702.
Objectives
In this review we summarize research on mechanisms through which environmental agents may affect the pathogenesis of lupus, discuss three exposures that have been the focus of research in this area, and propose recommendations for new research initiatives.
Data sources and synthesis
We examined studies pertaining to key mechanistic events and specific exposures. Apoptosis leading to increased production or decreased clearance of immunogenic intracellular self-antigens and defective apoptosis of autoreactive immune cells both have been implicated in the loss of self-tolerance. The adjuvant or bystander effect is also needed to produce a sustained autoimmune response. Activation of toll-like receptors is one mechanism through which these effects may occur. Abnormal DNA methylation may also contribute to the pathogenesis of lupus. Each of the specific exposures we examined—Epstein-Barr virus, silica, and trichloroethylene—has been shown, in humans or in mice, to act upon one or more of these pathogenic steps. Specific recommendations for the continued advancement of our understanding of environmental influences on lupus and other autoimmune diseases include the development and use of mouse models with varying degrees of penetrance and manifestations of disease, identification of molecular or physiologic targets of specific exposures, development and use of improved exposure assessment methodologies, and multisite collaborations designed to examine understudied environmental exposures in humans.
Conclusions
The advances made in the past decade concerning our understanding of mechanisms involved in the development of lupus and the influence of environmental agents on this process provide a strong foundation for further developments in this field.
doi:10.1289/ehp.11092
PMCID: PMC2430222  PMID: 18560522
adjuvant effect; apoptosis; autoimmune diseases; bystander effect; demethylation; epigenetics; Epstein-Barr virus; silica; systemic lupus erythematosus; trichloroethylene
24.  Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte Gene Expression 
Chromatin structure, determined in part by DNA methylation, is established during differentiation and prevents expression of genes unnecessary for the function of a given cell type. We reported that DNA methylation and chromatin structure contributes to lymphoid-specific ITGAL (CD11a) and PRF1 (perforin) expression. We used bisulfite sequencing to compare methylation patterns in the ITGAL promoter and 5' flanking region of T cells and fibroblasts, and in the PRF1 promoter and upstream enhancer of CD4+ and CD8+ T cells with fibroblasts. The effects of methylation on promoter function were tested using regional methylation of reporter constructs, and confirmed by DNA methyltransferase inhibition. The relationship between DNA methylation and chromatin structure was analyzed by DNaseI hypersensitivity. Herein we described the methods and results in greater detail.
doi:10.1251/bpo89
PMCID: PMC517978  PMID: 15448721
DNA methylation; T-lymphocytes; Gene expression regulation
25.  Benzodiazepine-induced superoxide signalsB cell apoptosis: mechanistic insight and potential therapeutic utility 
The Journal of Clinical Investigation  2002;110(8):1123-1132.
The properties of a proapoptotic 1,4-benzodiazepine, Bz-423, identified through combinatorial chemistry and phenotype screening are described. Bz-423 rapidly generated superoxide (O2–) in transformed Ramos B cells. This O2– response originated from mitochondria prior to mitochondrial transmembrane gradient collapse and opening of the permeability transition pore. Bz-423–induced O2– functioned as an upstream signal that initiated an apoptotic program characterized by cytochrome c release, mitochondrial depolarization, and caspase activation. Pretreatment of cells with agents that either block the formation of Bz-423–induced O2– or scavenge free radicals attenuated the death cascade, which demonstrated that cell killing by Bz-423 depends on O2–. Parallels between Ramos cells and germinal center B cells prompted experiments to determine whether Bz-423 had therapeutic activity in vivo. This possibility was tested using the (NZB × NZW)F1 murine model of lupus, in which the pathologically enhanced survival and expansion of germinal center B cells mediate disease. Administration of Bz-423 for 12 weeks specifically controlled germinal center hyperplasia and reduced the histological evidence of glomerulonephritis. Collectively, these studies define a new structure-function relationship for benzodiazepines and point to a new target and mechanism that could be of value for developing improved drugs to manage systemic lupus erythematosus and related disorders.
doi:10.1172/JCI16029
PMCID: PMC150800  PMID: 12393848

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