Abstract Genetic polymorphism in MECP2/IRAK1 is a confirmed susceptibility locus for lupus. DNA methylation changes in T cells play a central role in the pathogenesis of lupus, and MeCp-2 is a master regulator of gene expression and is known to recruit DNA methyltransferase 1 (DNMT1) during DNA synthesis. Using human T cells from normal individuals, we demonstrate that the lupus risk variant in MECP2/IRAK1 increases MECP2 mRNA expression in stimulated T cells. By assessing DNA methylation levels at ~ 485,000 methylation sites across the genome, we further show 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. We also 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. Importantly, we demonstrate 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.

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.

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/6xSJL)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.

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 multi-racial 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.

DNA methylation is an epigenetic mark that is critical in determining chromatin accessibility and regulating gene expression. This epigenetic mechanism has an important role in T-cell function. We used genome-wide methylation profiling to characterize the DNA methylome in primary human CD4+ T cells. We found that only 5% of CpG islands are methylated in CD4+ T cells, and that DNA methylation peak density is increased in subtelomeric chromosomal regions. We also found an inverse relationship between methylation peak density and chromosomal length. Our data indicate that DNA methylation in gene promoter regions is not always a repressive epigenetic mark. Indeed, about 27% of methylated genes are actively expressed in CD4+ T cells. We demonstrate that repressive methylation peaks are located closer to the transcription start site (TSS) compared with functionally non-repressive peaks (−893±110 bp versus −1342±218 bp (mean±s.e.m.), P-value <0.05). We also show that both a larger number and an increased CpG island density in promoter sequences predict transcriptional permissiveness of DNA methylation. TSS in the majority of genes with permissive DNA methylation peaks is in DNase I hypersensitive sites, indicating a failure of DNA methylation to induce chromatin inaccessibility in these loci.

OBJECTIVE:To assess whether genetically determined Amerindian ancestry predicts increased presence of risk alleles of known susceptibility genes for systemic lupus erythematosus (SLE). METHODS:Single-nucleotide polymorphisms (SNPs) within 16 confirmed genetic susceptibility loci for SLE were genotyped in a set of 804 Mestizo lupus patients and 667 Mestizo healthy controls. In addition, 347 admixture informative markers were genotyped. Individual ancestry proportions were determined using STRUCTURE. Association analysis was performed using PLINK, and correlation between ancestry and the presence of risk alleles was analyzed using linear regression. RESULTS:A meta-analysis of the genetic association of the 16 SNPs across populations showed that TNFSF4, STAT4, ITGAM, and IRF5 were associated with lupus in a Hispanic Mestizo cohort enriched for European and Amerindian ancestry. In addition, 2 SNPs within the major histocompatibility complex region, previously shown to be associated in a genome-wide association study in Europeans, were also associated in Mestizos. Using linear regression, we predicted an average increase of 2.34 risk alleles when comparing an SLE patient with 100% Amerindian ancestry versus an SLE patient with 0% Amerindian ancestry (P < 0.0001). SLE patients with 43% more Amerindian ancestry were predicted to carry 1 additional risk allele. CONCLUSION:Our results demonstrate that Amerindian ancestry is associated with an increased number of risk alleles for SLE.

Behçet's disease is a chronic systemic inflammatory disease that remains incompletely understood. Herein, we perform the first genome-wide association study in Behçet's disease.

OBJECTIVE:Interleukin-21 (IL-21) is a member of the type I cytokine superfamily that has a variety of effects on the immune system, including B cell activation, plasma cell differentiation, and immunoglobulin production. The expression of IL-21 receptor (IL-21R) is reduced in the B cells of patients with systemic lupus erythematosus (SLE), while serum IL-21 levels are increased both in lupus patients and in some murine lupus models. We recently reported that polymorphisms within the IL21 gene are associated with increased susceptibility to SLE. The aim of this study was to examine the genetic association between single-nucleotide polymorphisms (SNPs) within IL21R and SLE. METHODS:We genotyped 17 SNPs in the IL21R gene in 2 large cohorts of lupus patients (a European-derived cohort and a Hispanic cohort) and in ethnically matched healthy controls. RESULTS:We identified and confirmed the association between rs3093301 within the IL21R gene and SLE in the 2 cohorts (meta-analysis odds ratio 1.16 [95% confidence interval 1.08-1.25], P=1.0x10(-4)). CONCLUSION:Our findings indicate that IL21R is a novel susceptibility gene for SLE.

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.

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 inflamasome, 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 mu 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. Result: Uric acid directly activates primary human T cells in tire 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 ail antigen-independent system is novel and might play a mechanistic role in the inflammatory response observed in gouty arthritis and other immune-mediated diseases.

Interleukin (IL) 21 is a member of the type I cytokine superfamily that exerts a variety of effects on the immune system including B cell activation, plasma cell differentiation, and immunoglobulin production. The IL21 receptor (IL21R) is expressed on B cells, T cells, NK cells, and monocyte-derived dendritic cells. The expression of IL21R is reduced in B cells from lupus patients, while IL21 serum levels are increased in both lupus patients and some lupus-murine models. We recently reported that polymorphisms within the IL21 gene are associated with increased susceptibility to lupus. Herein, we genotyped SNPs in the IL21R gene located on chromosome 16p11. We identify and confirm the association between rs3093301 and lupus in two independent European-derived and Hispanic cohorts (p=7.2X10-5). In addition, the presence of the homozygous risk genotype in rs3093301 (A/A) was associated with the development of malar rash in the European-derived female lupus patients (OR=2.83, 95% CI=1.56-5.13, p=0.00045).

Systemic lupus erythematosus (SLE) is a predominantly female autoimmune disease that affects multiple organ systems. Herein, we report on an X-chromosome gene association with SLE. Methyl-CpG-binding protein 2 (MECP2) is located on chromosome Xq28 and encodes for a protein that plays a critical role in epigenetic transcriptional regulation of methylation-sensitive genes. Utilizing a candidate gene association approach, we genotyped 21 SNPs within and around MECP2 in SLE patients and controls. We identify and replicate association between SLE and the genomic element containing MECP2 in two independent SLE cohorts from two ethnically divergent populations. These findings are potentially related to the overexpression of methylation-sensitive genes in SLE.

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.

Abstract Purpose: 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 response. 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 μg/ml. This range of uric acid concentrations covers the normal physiologic serum uric acid concentration as well as a spectrum of hyperuricemic values in human. The expression of T cell activation markers CD25 and CD70 was assessed by flow cytometry. Purified human T cell proliferation response to the various uric acid concentrations was quantified using a colorimetric proliferation assay. Results: Uric acid treatment resulted in antigen-independent T cell activation as determined by increased expression of CD25 in both CD4+ as well as CD8+ T cells. CD4+ T cells also overexpressed CD70, a co-stimulation molecule important in T cell-dependent B cell activation. In addition, we demonstrate a dose response relationship between T cell proliferation and uric acid concentration in vitro. Conclusion: The finding that uric acid directly promotes T cell proliferation and activation in an antigen-independent system is novel and might play a mechanistic role in the inflammatory response observed in gout and other uric acid associated diseases. This work is supported by NIH Grant Number P20-RR015577 and the University of Oklahoma Health Sciences Center.

Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus. Beacon administration into the lateral ventricle of P. obesus stimulated food intake and body weight gain. In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon. CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon. Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon. Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism.

The global decline of amphibians and reptiles has resulted in an increase of studies focused on population. The Pierce Cedar Creek Institute in Hastings, MI is committed to the conservation of species, but no studies at the site have focused on population and distributions of colubrid species. The aim of this study is to assess the population, diversity, and microhabitat selection of the snake species on the Pierce Cedar Creek site. A system using drift fences, funnel traps, and an array of artificial cover areas was used to capture different snake species. PIT tags were used in order to identify snakes already captured. A total of 53 captures were recorded during the study, encompassing seven different species. The majority of captures and recaptures were Coluber constrictor foxi. An ANOVA analysis was used to determine habitat preferences, and habitat distribution of the species tagged. The data analysis showed that there was no significant habitat selection among the species, except for the Coluber constrictor foxi which preferred restored prairies with development.