Reprint of "The interaction between environmental triggers and epigenetics in autoimmunity".

Systemic lupus erythematosus flares when genetically predisposed people encounter environmental agents that cause oxidative stress, such as infections and sunlight. How these modify the immune system to initiate flares is unclear. Drug induced lupus models demonstrate that CD4+ T cells epigenetically altered with DNA methylation inhibitors cause lupus in animal models, and similar T cells are found in patients with active lupus. How infections and sun exposure inhibit T cell DNA methylation is unclear. DNA methylation patterns are replicated each time a cell divides in a process that requires DNA methyltransferase one (Dnmt1), which is upregulated as cells enter mitosis, as well as the methyl donor S-adenosylmethionine, created from dietary sources. Reactive oxygen species that inhibit Dnmt1 upregulation, and a diet poor in methyl donors, combine to cause lupus in animal models. Similar changes are found in patients with active lupus, indicating a mechanism contributing to lupus flares.

The interaction between environmental triggers and epigenetics in autoimmunity.

Systemic lupus erythematosus flares when genetically predisposed people encounter environmental agents that cause oxidative stress, such as infections and sunlight. How these modify the immune system to initiate flares is unclear. Drug induced lupus models demonstrate that CD4+ T cells epigenetically altered with DNA methylation inhibitors cause lupus in animal models, and similar T cells are found in patients with active lupus. How infections and sun exposure inhibit T cell DNA methylation is unclear. DNA methylation patterns are replicated each time a cell divides in a process that requires DNA methyltransferase one (Dnmt1), which is upregulated as cells enter mitosis, as well as the methyl donor S-adenosylmethionine, created from dietary sources. Reactive oxygen species that inhibit Dnmt1 upregulation, and a diet poor in methyl donors, combine to cause lupus in animal models. Similar changes are found in patients with active lupus, indicating a mechanism contributing to lupus flares.

Oxidative stress and dietary micronutrient deficiencies contribute to overexpression of epigenetically regulated genes by lupus T cells.

Patients with active lupus have altered T cells characterized by low DNA methyltransferase levels. We hypothesized that low DNA methyltransferase levels synergize with low methionine levels to cause greater overexpression of genes normally suppressed by DNA methylation. CD4+ T cells from lupus patients and controls were stimulated with PHA then cultured in custom media with normal or low methionine levels. Oxidative stress was induced by treating the normal CD4+ T cells with peroxynitrite prior to culture. Methylation sensitive gene expression was measured by flow cytometry. Results showed low methionine levels caused greater overexpression of methylation sensitive genes in peroxynitrite treated T cells relative to untreated T cells, and in T cells from lupus patients relative to T cells from healthy controls. In conclusion, low dietary transmethylation micronutrient levels and low DNA methyltransferase levels caused either by oxidative stress or lupus, have additive effects on methylation sensitive T cell gene expression.

CD4+CD28+KIR+CD11ahi T cells correlate with disease activity and are characterized by a pro-inflammatory epigenetic and transcriptional profile in lupus patients.

OBJECTIVE: The goal of this study was to comprehensively characterize CD4+CD28+ T cells overexpressing CD11a and KIR genes, and examine the relationship between this T cell subset, genetic risk, and disease activity in lupus. METHODS: The size of the CD4+CD28+KIR+CD11ahi T cell subset was determined by flow cytometry, and total genetic risk for lupus was calculated in 105 female patients using 43 confirmed genetic susceptibility loci. Primary CD4+CD28+KIR+CD11ahi T cells were isolated from lupus patients or were induced from healthy individuals using 5-azacytidine. Genome-wide DNA methylation was analyzed using an array-based approach, and the transcriptome was assessed by RNA sequencing. Transcripts in the CDR3 region were used to assess the TCR repertoire. Chromatin accessibility was determined using ATAC-seq. RESULTS: A total of 31,019 differentially methylated sites were identified in induced KIR+CD11ahi T cells with >99% being hypomethylated. RNA sequencing revealed a clear pro-inflammatory transcriptional profile. TCR repertoire analysis suggests less clonotype diversity in KIR+CD11ahi compared to autologous KIR-CD11alow T cells. Similarly, primary KIR+CD11ahi T cells isolated from lupus patients were hypomethylated and characterized by a pro-inflammatory chromatin structure. We show that the genetic risk for lupus was significantly higher in African-American compared to European-American lupus patients. The demethylated CD4+CD28+KIR+CD11ahi T cell subset size was a better predictor of disease activity in young (age ≤ 40) European-American patients independent of genetic risk. CONCLUSION: CD4+CD28+KIR+CD11ahi T cells are demethylated and characterized by pro-inflammatory epigenetic and transcriptional profiles in lupus. Eliminating these cells or blocking their pro-inflammatory characteristics might present a novel therapeutic approach for lupus.

T cell PKCδ kinase inactivation induces lupus-like autoimmunity in mice.

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.

CD4(+) T cells epigenetically modified by oxidative stress cause lupus-like autoimmunity in mice.

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.

DNA methylation and mRNA and microRNA expression of SLE CD4+ T cells correlate with disease phenotype.

Systemic lupus erythematosus (SLE) is an autoimmune disease well known for its clinical heterogeneity, and its etiology secondary to a cross-talk involving genetic predisposition and environmental stimuli. Although genome-wide analysis has contributed greatly to our understanding of the genetic basis of SLE, there is increasing evidence for a role of epigenetics. Indeed, recent data have demonstrated that in patients with SLE, there are striking alterations of DNA methylation, histone modifications, and deregulated microRNA expression, the sum of which contribute to over-expression of select autoimmune-related genes and loss of tolerance. To address this issue at the level of clinical phenotype, we performed DNA methylation, mRNA and microRNA expression screening using high-throughput sequencing of purified CD4+ T cells from patients with SLE, compared to age and sex matched controls. In particular, we studied 42 patients with SLE and divided this group into three clinical phenotypes: a) the presence of skin lesions without signs of systemic pathology; b) skin lesions but also chronic renal pathology; and c) skin lesions, chronic renal pathology and polyarticular disease. Interestingly, and as expected, sequencing data revealed changes in DNA methylation in SLE compared to controls. However, and more importantly, although there were common methylation changes found in all groups of SLE compared to controls, there was specific DNA methylation changes that correlated with clinical phenotype. These included changes in the novel key target genes NLRP2, CD300LB and S1PR3, as well as changes in the critical pathways, including the adherens junction and leukocyte transendothelial migration. We also noted that a significant proportion of genes undergoing DNA methylation changes were inversely correlated with gene expression and that miRNA screening revealed the existence of subsets with changes in expression. Integrated analysis of this data highlights specific sets of miRNAs controlled by DNA methylation, and genes that are altered by methylation and targeted by miRNAs. In conclusion, our findings suggest select epigenetic mechanisms that contribute to clinical phenotypes and further shed light on a new venue for basic SLE research.

Diet influences expression of autoimmune-associated genes and disease severity by epigenetic mechanisms in a transgenic mouse model of lupus.

OBJECTIVE: Lupus flares occur when genetically predisposed individuals 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 (DNMT-1) levels. We used transgenic mice to study the effect of interactions between diet, DNMT-1 levels, and genetic predisposition on the development and severity of lupus. METHODS: A doxycycline-inducible ERK defect was bred into lupus-resistant (C57BL/6) and lupus-susceptible (C57BL/6 × SJL) mouse strains. Doxycycline-treated mice were fed a standard commercial diet for 18 weeks and then switched to a transmethylation micronutrient-supplemented (MS) or -restricted (MR) diet. Disease severity was assessed by examining anti-double-stranded DNA (anti-dsDNA) antibody levels, the presence of proteinuria and hematuria, and by histopathologic analysis 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/6 × SJL mice. Doxycycline-treated C57BL/6 × SJL mice developed hematuria and glomerulonephritis on the MR and standard diets 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 the duration of treatment influenced methylation and expression of the CD40lg gene. CONCLUSION: Dietary micronutrients that affect DNA methylation can exacerbate or ameliorate disease in this transgenic murine lupus model, and contribute to lupus susceptibility and severity through genetic-epigenetic interactions.

Impaired DNA methylation and its mechanisms in CD4(+)T cells of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens. Although the exact cause of SLE is still unknown, the influence of environment, which is largely reflected by the epigenetic mechanisms, with DNA methylation changes in particular, are generally considered as key players in the pathogenesis of SLE. As an important post-translational modification, DNA methylation mainly suppresses the expression of relevant genes. Accumulating evidence has indicated that abnormal DNA hypomethylation in T cells is an important epigenetic hallmark in SLE. Apart from those classic methylation-sensitive autoimmunity-related genes in lupus, such as CD11a (ITGAL), Perforin (PRF1), CD70 (TNFSF7), CD40 ligand (TNFSF5) and PP2Acα, the genome-wide methylation pattern has also been explored recently, providing us a more and more full-scale picture of the abnormal status of DNA methylation in SLE. On the other hand, certain miRNAs, RFX1, defective ERK pathway signaling, Gadd45α and DNA hydroxymethylation have been proposed as potential mechanisms leading to DNA hypomethylation in lupus. In this review, we summarize current understanding of T cell DNA methylation changes and the consequently altered gene expressions in lupus, and how they contribute to the development of SLE. Possible mechanisms underlying these aberrancies are also discussed based on the reported literature and our own findings.

Overexpression of X-linked genes in T cells from women with lupus.

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.

Prostaglandin E2 increases fibroblast gene-specific and global DNA methylation via increased DNA methyltransferase expression.

Although alterations in DNA methylation patterns have been associated with specific diseases and environmental exposures, the mediators and signaling pathways that direct these changes remain understudied. The bioactive lipid mediator prostaglandin E(2) (PGE(2)) has been shown to exert a myriad of effects on cell survival, proliferation, and differentiation. Here, we report that PGE(2) also signals to increase global DNA methylation and DNA methylation machinery in fibroblasts. HumanMethylation27 BeadChip array analysis of primary fetal (IMR-90) and adult lung fibroblasts identified multiple genes that were hypermethylated in response to PGE(2). PGE(2), compared with nontreated controls, increased expression and activity (EC(50)∼10(7) M) of one specific isoform of DNA methyltransferase, DNMT3a. Silencing of DNMT3a negated the ability of PGE(2) to increase DNMT activity. The increase in DNMT3a expression was mediated by PGE(2) signaling via its E prostanoid 2 receptor and the second messenger cAMP. PGE(2), compared with the untreated control, increased the expression and activity of Sp1 and Sp3 (EC(50)∼3×10(7) M), transcription factors known to increase DNMT3a expression, and inhibition of these transcription factors abrogated the PGE(2) increase of DNMT3a expression. These findings were specific to fibroblasts, as PGE(2) decreased DNMT1 and DNMT3a expression in RAW macrophages. Taken together, these findings establish that DNA methylation is regulated by a ubiquitous bioactive endogenous mediator. Given that PGE(2) biosynthesis is modulated by environmental toxins, various disease states, and commonly used pharmacological agents, these findings uncover a novel mechanism by which alterations in DNA methylation patterns may arise in association with disease and certain environmental exposures.

Protein kinase Cδ oxidation contributes to ERK inactivation in lupus T cells.

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 T(505) in the protein kinase Cδ (PKCδ) activation loop. However, the mechanisms that prevent PKCδ T(505) 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 undertook this study to test our hypothesis 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 and then stimulated with phorbol myristate acetate (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 T(505) 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 did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKCδ, but not unmodified PKCδ, was refractory to PMA-stimulated T(505) phosphorylation, similar to PKCδ in peroxynitrite-treated cells. CONCLUSION: 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.

Evidence for gene-gene epistatic interactions among susceptibility loci for systemic lupus erythematosus.

OBJECTIVE: Several confirmed genetic susceptibility loci for lupus have been described. To date, no clear evidence for genetic epistasis in lupus has been established. The aim of this study was to test for gene-gene interactions in a number of known lupus susceptibility loci. METHODS: Eighteen single-nucleotide polymorphisms tagging independent and confirmed lupus susceptibility loci were genotyped in a set of 4,248 patients with lupus and 3,818 normal healthy control subjects of European descent. Epistasis was tested by a 2-step approach using both parametric and nonparametric 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 patients with lupus. 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 = 3.95, P = 7.8 × 10(-5) [FDR ≤0.05], P for multifactor dimensionality reduction = 5.9 × 10(-45)). Importantly, our data suggest that in patients with lupus, 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 P = 0.0047, respectively). 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.

Analysis of autosomal genes reveals gene-sex interactions and higher total genetic risk in men with systemic lupus erythematosus.

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 non-parametric 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=4.52x10-8) 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.

Variation in the ICAM1-ICAM4-ICAM5 locus is associated with systemic lupus erythematosus susceptibility in multiple ancestries.

OBJECTIVE: Systemic lupus erythematosus (SLE; OMIM 152700) is a chronic autoimmune disease for which the aetiology includes genetic and environmental factors. ITGAM, integrin α(M) (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 (OR(meta)=1.16, 95% CI 1.11 to 1.22; p=4.88×10(-10) and OR(meta)=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.

Sex-specific differences in the relationship between genetic susceptibility, T cell DNA demethylation and lupus flare severity.

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 autosomal genes known to demethylate in T cells 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.

Environmental exposure, estrogen and two X chromosomes are required for disease development in an epigenetic model of 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.

Association of endogenous anti-interferon-α autoantibodies with decreased interferon-pathway and disease activity in patients with systemic lupus erythematosus.

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, IFN(low) and IFN(high) , 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 IFN(high) 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.

Identification of novel genetic susceptibility loci in African American lupus patients in a candidate gene association study.

OBJECTIVE: Candidate gene and genome-wide association studies have identified several disease susceptibility loci in lupus patients. These studies have largely been performed in lupus patients who are Asian or of European ancestry. This study was undertaken to examine whether 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 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 found the first evidence of genetic association between lupus in African American patients and 5 susceptibility loci (C8orf13-BLK, BANK1, TNFSF4, KIAA1542, and CTLA4; P = 8.0 × 10⁻6, P = 1.9 × 10⁻5, P = 5.7 × 10⁻5, P = 0.00099, and P = 0.0045, respectively). Further, we confirmed the genetic association between lupus and 5 additional lupus susceptibility loci (ITGAM, MSH5, CFB, STAT4, and FCGR2A; P = 7.5 × 10⁻¹¹, P = 5.2 × 10⁻8, P = 8.7 × 10⁻7 , P = 0.0058, and P = 0.0070, respectively), and provided evidence, for the first time, of genome-wide significance for the association between lupus in African American patients and ITGAM and MSH5 (HLA region). CONCLUSION: These findings provide evidence of 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.

Early disease onset is predicted by a higher genetic risk for lupus and is associated with a more severe phenotype in lupus patients.

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.