Epigenetically Altered T Cells Contribute to Lupus Flares.

Lupus flares when genetically predisposed people encounter exogenous agents such as infections and sun exposure and drugs such as procainamide and hydralazine, but the mechanisms by which these agents trigger the flares has been unclear. Current evidence indicates that procainamide and hydralazine, as well as inflammation caused by the environmental agents, can cause overexpression of genes normally silenced by DNA methylation in CD4⁺ T cells, converting them into autoreactive, proinflammatory cytotoxic cells that are sufficient to cause lupus in mice, and similar cells are found in patients with active lupus. More recent studies demonstrate that these cells comprise a distinct CD4⁺ T cell subset, making it a therapeutic target for the treatment of lupus flares. Transcriptional analyses of this subset reveal proteins uniquely expressed by this subset, which may serve as therapeutic to deplete these cells, treating lupus flares.

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.

Contamination and risk implications of endocrine disrupting chemicals along the coastline of China: A systematic study using mussels and semipermeable membrane devices.

A systematic study has been carried out to assess the contamination of endocrine disrupting chemicals (EDCs) in five highly urbanized coastal cities spanning from temperate to subtropical environments along the coastline of China. In each of these cities, species of native mussels (Mytilus galloprovincialis, M. coruscus or Perna viridis) were deployed alongside with semipermeable membrane devices (SPMDs) for one month at a reference site and a polluted site. The level of 4-nonylphenol (4-NP), bisphenol A (BPA), 17ß-estradiol (E2) and 17α-ethynylestradiol (EE2) in SPMDs and transplanted mussels were determined and compared. The concentration of EDCs in mussels from polluted sites of Qingdao and Shenzhen ranged from 99.4±9.40 to 326.1±3.16ng/g dry wt. for 4-NP, Dalian and Shanghai from 170.3±4.00 to 437.2±36.8ng/g dry wt. for BPA, Dalian and Shenzhen from 82.9±3.03 to 315.6±6.50ng/g dry wt. for E2, and Shenzhen and Shanghai from 124.5±3.25 to 204.5±9.26ng/g dry wt. for EE2, respectively. These results demonstrate that concentrations of EDCs in mussels along the coastline of China are substantially higher than levels reported in mussels and seafood elsewhere. Despite high levels of EDCs and per capita seafood consumption in China, analysis indicated that 4-NP and BPA intake from mussels at polluted sites per se are still below the Tolerable Daily Intake (TDI). In contrast, the daily intake of E2 and EE2 (6.5 and 5.5µg/person/day, respectively) from mussel consumption exceeded the Acceptable Daily Intake (ADI) established by the WHO, USA and Australia by large margins, suggesting significant public health risks. A strong correlation was found between EDC concentrations in SPMDs and transplanted mussels, and the advantages of using mussels and SPMDs for monitoring EDCs in the aquatic environment are discussed.

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.

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.

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.

A novel approach for estimating the removal efficiencies of endocrine disrupting chemicals and heavy metals in wastewater treatment processes.

The wide occurrence of endocrine disrupting chemicals (EDCs) and heavy metals in coastal waters has drawn global concern, and thus their removal efficiencies in sewage treatment processes should be estimated. However, low concentrations coupled with high temporal fluctuations of these pollutants present a monitoring challenge. Using semi-permeable membrane devices (SPMDs) and Artificial Mussels (AMs), this study investigates a novel approach to evaluating the removal efficiency of five EDCs and six heavy metals in primary treatment, secondary treatment and chemically enhanced primary treatment (CEPT) processes. In general, the small difference between maximum and minimum values of individual EDCs and heavy metals measured from influents/effluents of the same sewage treatment plant suggests that passive sampling devices can smooth and integrate temporal fluctuations, and therefore have the potential to serve as cost-effective monitoring devices for the estimation of the removal efficiencies of EDCs and heavy metals in sewage treatment works.

Characterisation of an epigenetically altered CD4(+) CD28(+) Kir(+) T cell subset in autoimmune rheumatic diseases by multiparameter flow cytometry.

OBJECTIVES: Antigen-specific CD4(+) T cells epigenetically modified with DNA methylation inhibitors overexpress genes normally suppressed by this mechanism, including CD11a, CD70, CD40L and the KIR gene family. The altered cells become autoreactive, losing restriction for nominal antigen and responding to self-class II major histocompatibility complex (MHC) molecules without added antigen, and are sufficient to cause a lupus-like disease in syngeneic mice. T cells overexpressing the same genes are found in patients with active lupus. Whether these genes are co-overexpressed on the same or different cells is unknown. The goal of this study was to determine whether these genes are overexpressed on the same or different T cells and whether this subset of CD4(+) T cells is also present in patients with lupus and other rheumatic diseases. METHODS: Multicolour flow cytometry was used to compare CD11a, CD70, CD40L and KIR expression on CD3(+)CD4(+)CD28(+) T cells to their expression on experimentally demethylated CD3(+)CD4(+)CD28(+) T cells and CD3(+)CD4(+)CD28(+) T cells from patients with active lupus and other autoimmune diseases. RESULTS: Experimentally demethylated CD4(+) T cells and T cells from patients with active lupus have a CD3(+)CD4(+)CD28(+)CD11a(hi)CD70(+)CD40L(hi)KIR(+) subset, and the subset size is proportional to lupus flare severity. A similar subset is found in patients with other rheumatic diseases including rheumatoid arthritis, systemic sclerosis and Sjögren's syndrome but not retroperitoneal fibrosis. CONCLUSIONS: Patients with active autoimmune rheumatic diseases have a previously undescribed CD3(+)CD4(+)CD28(+)CD11a(hi)CD70(+)CD40L(hi)KIR(+) T cell subset. This subset may play an important role in flares of lupus and related autoimmune rheumatic diseases, provide a biomarker for disease activity and serve as a novel therapeutic target for the treatment of lupus flares.

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.

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.

Association of the interferon signature metric with serological disease manifestations but not global activity scores in multiple cohorts of patients with SLE.

OBJECTIVES: The interferon (IFN) signature (IS) in patients with systemic lupus erythematosus (SLE) includes over 100 genes induced by type I IFN pathway activation. We developed a method to quantify the IS using three genes-the IS metric (ISM)-and characterised the clinical characteristics of patients with SLE with different ISM status from multiple clinical trials. METHODS: Blood microarray expression data from a training cohort of patients with SLE confirmed the presence of the IS and identified surrogate genes. We assayed these genes in a quantitative PCR (qPCR) assay, yielding an ISM from the IS. The association of ISM status with clinical disease characteristics was assessed in patients with extrarenal lupus and lupus nephritis from four clinical trials. RESULTS: Three genes, HERC5, EPSTI and CMPK2, correlated well with the IS (p>0.96), and composed the ISM qPCR assay. Using the 95th centile for healthy control data, patients with SLE from different studies were classified into two ISM subsets-ISM-Low and ISM-High-that are longitudinally stable over 36 weeks. Significant associations were identified between ISM-High status and higher titres of anti-dsDNA antibodies, presence of anti extractable nuclear antigen autoantibodies, elevated serum B cell activating factor of the tumour necrosis factor family (BAFF) levels, and hypocomplementaemia. However, measures of overall clinical disease activity were similar for ISM-High and ISM-Low groups. CONCLUSIONS: The ISM is an IS biomarker that divides patients with SLE into two subpopulations-ISM-High and ISM-Low-with differing serological manifestations. The ISM does not distinguish between high and low disease activity, but may have utility in identifying patients more likely to respond to treatment(s) targeting IFN-α. CLINICALTRIALSGOV REGISTRATION NUMBER: NCT00962832.