Donna Ray

Demethylation of CD40LG on the Inactive X in T Cells from Women with Lupus

Why systemic lupus erythematosus primarily affects women is unknown. Recent evidence indicates that human lupus is an epigenetic disease characterized by impaired T cell DNA methylation. Women have two X chromosomes; one is inactivated by mechanisms including DNA methylation. We hypothesized that demethylation of sequences on the inactive X may cause gene overexpression uniquely in women, predisposing them to lupus. We therefore compared expression and methylation of CD40LG, a B cell costimulatory molecule encoded on the X chromosome, in experimentally demethylated T cells from men and women and in men and women with lupus. Controls included TNFSF7, a methylation-sensitive autosomal B cell costimulatory molecule known to be demethylated and overexpressed in lupus. Bisulfite sequencing revealed that CD40LG is unmethylated in men, while women have one methylated and one unmethylated gene. 5-Azacytidine, a DNA methyltransferase inhibitor, demethylated CD40LG and doubled its expression on CD4+ T cells from women but not men, while increasing TNFSF7 expression equally between sexes. Similar studies demonstrated that CD40LG demethylates in CD4+ T cells from women with lupus, and that women but not men with lupus overexpress CD40LG on CD4+ T cells, while both overexpress TNFSF7. These studies demonstrate that regulatory sequences on the inactive X chromosome demethylate in T cells from women with lupus, contributing to CD40LG overexpression uniquely in women. Demethylation of CD40LG and perhaps other genes on the inactive X may contribute to the striking female predilection of this disease.

Decreased ras-mitogen-activated protein kinase signaling may cause DNA hypomethylation in T lymphocytes from lupus patients

OBJECTIVE Previous studies have shown that inhibiting T cell DNA methylation causes a lupus-like disease by modifying gene expression. T cells from patients with lupus exhibit diminished levels of DNA methyltransferase (MTase) enzyme activity, hypomethylated DNA, and changes in gene expression similar to those exhibited by T cells treated with methylation inhibitors, suggesting that DNA hypomethylation may contribute to human lupus. Since it is known that DNA MTase levels are regulated by the ras-mitogen-activated protein kinase (MAPK) pathway, this study sought to determine whether decreased ras-MAPK signaling could account for the DNA hypomethylation in lupus T cells. METHODS DNA MTase messenger RNA (mRNA) from lupus patients and from healthy controls was quantitated by Northern analysis, and ras-MAPK signaling was determined by immunoblotting with antibodies to the activated forms of extracellular receptor-associated kinase (ERK). Results were compared with those in T cells in which ras-MAPK signaling was inhibited with a soluble inhibitor of MAPK ERK I (MEK1). RESULTS T cells from patients with active lupus had diminished DNA MTase mRNA levels and decreased signaling through the ras-MAPK pathway. Inhibiting signaling through the ras-MAPK pathway with the MEK1 inhibitor decreased DNA MTase mRNA and enzyme activity to the levels seen in lupus T cells, and resulted in DNA hypomethylation resembling that seen in lupus T cells. CONCLUSION These results suggest that a decrease in signaling through the ras-MAPK pathway may be responsible for the decreased MTase activity and DNA hypomethylation in patients with lupus.

DNA Methylation and Chromatin Structure Regulate T Cell Perforin Gene Expression

Perforin is a cytotoxic effector molecule expressed in NK cells and a subset of T cells. The mechanisms regulating its expression are incompletely understood. We observed that DNA methylation inhibition could increase perforin expression in T cells, so we examined the methylation pattern and chromatin structure of the human perforin promoter and upstream enhancer in primary CD4+ and CD8+ T cells as well as in an NK cell line that expresses perforin, compared with fibroblasts, which do not express perforin. The entire region was nearly completely unmethylated in the NK cell line and largely methylated in fibroblasts. In contrast, only the core promoter was constitutively unmethylated in primary CD4+ and CD8+ cells, and expression was associated with hypomethylation of an area residing between the upstream enhancer at −1 kb and the distal promoter at −0.3 kb. Treating T cells with the DNA methyltransferase inhibitor 5-azacytidine selectively demethylated this area and increased perforin expression. Selective methylation of this region suppressed promoter function in transfection assays. Finally, perforin expression and hypomethylation were associated with localized sensitivity of the 5′ flank to DNase I digestion, indicating an accessible configuration. These results indicate that DNA methylation and chromatin structure participate in the regulation of perforin expression in T cells.

TRAIL (Apo2 Ligand) and TWEAK (Apo3 Ligand) Mediate CD4+ T Cell Killing of Antigen-Presenting Macrophages

The human marrow produces ∼1010 monocytes daily, and this production must be balanced by a similar rate of destruction. Monocytes/macrophages can undergo apoptosis after activating CD4+ T cells, suggesting one mechanism that may contribute to macrophage homeostasis. Previous reports indicate that Fas-Fas ligand interactions are the principle molecules mediating this response. However, D10, an Iak-restricted cloned Th2 line, will similarly induce apoptosis in Ag-presenting macrophages, and D10 cells lack Fas ligand. To confirm that D10 cells kill macrophages through Fas-independent pathways, D10 cells were shown to kill MRL lpr/lpr (Iak) macrophages in an Ag-dependent fashion, indicating additional mechanisms. Recent reports demonstrate that TNF-related apoptosis-inducing ligand (TRAIL), interacting with Apo2, and TNF-like weak inducer of apoptosis (TWEAK), interacting with Apo3, will induce apoptosis in some cells. Using Abs to TRAIL and an Apo3-IgG Fc fusion protein, we demonstrated that D10 cells express both TRAIL and TWEAK. The Apo3 fusion protein, but not human IgG, inhibited D10-induced macrophage apoptosis, as did anti-TRAIL. Further studies demonstrated that AE7, a cloned Th1 line, and splenic T cells express TWEAK, TRAIL, and Fas ligand, and inhibiting these molecules also inhibited macrophage killing. These results indicate that D10 cells induce macrophage apoptosis through TRAIL- and TWEAK-dependent pathways. Because normal T cells also express these molecules, these results support the concept that T cells have multiple pathways by which to induce macrophage apoptosis. These pathways may be important in immune processes such as macrophage homeostasis as well as in down-regulation of immune responses and elimination of macrophages infected with intracellular organisms.

Defective T-cell ERK signaling induces interferon-regulated gene expression and overexpression of methylation-sensitive genes similar to lupus patients

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.

The role of epigenetics in aging and autoimmunity.

The decline in immunocompetence with age is accompanied by the increase in the incidence of autoimmune diseases. Aging of the immune system, or immunosenescence, is characterized by a decline of both T and B cell function, and paradoxically the presence of low-grade chronic inflammation. There is growing evidence that epigenetics, the study of inherited changes in gene expression that are not encoded by the DNA sequence itself, changes with aging. Interestingly, emerging evidence suggests a key role for epigenetics in human pathologies, including inflammatory and neoplastic disorders. Here, we will review the potential mechanisms that contribute to the increase in autoimmune responses in aging. In particular, we will discuss how epigenetic alterations, especially DNA methylation and histone acetylation, are accumulated during aging and how these events contribute to autoimmunity risk.

Autoreactive murine Th1 and Th2 cells kill syngeneic macrophages and induce autoantibodies

D10 cells, a cloned Th2 line, become autoreactive following treatment with DNA methylation inhibitors like 5-azacytidine (5-azaC), and induce anti-DNA antibodies if injected into unirradiated syngenic mice. The mechanism by which the autoreactive cells break tolerance is unknown. To further define effector functions required, we asked if 5-azaC-treated Th1 cells could also induce autoimmunity. AE7 cells, a cloned Th1 line, were treated with 5-azaC and shown to become autoreactive and induce anti-DNA antibodies in vivo. Comparison of effector mechanisms demonstrated that the two cell lines secreted a distinct repertoire of cytokines, and that only killing of syngeneic Mù was common to both AE7 and D10 cells. This suggests that Mù killing may be an early step in the induction of anti-DNA antibodies, providing antigenic nucleosomes and decreasing clearance of apoptotic material. Secretion of cytokines promoting B cell differentiation may play a role, but no one cytokine is required.

Murine Models of Lupus Induced by Hypomethylated T Cells

CD4+ T-cell DNA hypomethylation may contribute to the development of drug-induced and idiopathic human lupus. Inhibiting DNA methylation in mature CD4+ T cells causes autoreactivity specific to the major histocompatibility complex in vitro. The lupus-inducing drugs hydralazine and procainamide also inhibit T-cell DNA methylation and induce autoreactivity, and T cells from patients with active lupus have hypomethylated DNA and a similarly autoreactive T-cell subset. Further, T cells treated with DNA methylation inhibitors demethylate the same sequences that demethylate in T cells from patients with active lupus. The pathological significance of the autoreactivity induced by inhibiting T-cell DNA methylation has been tested by treating murine T cells in vitro with drugs that modify DNA methylation, then injecting the cells into syngeneic female mice. Mice receiving CD4+ T cells demethylated by a variety of agents, including procainamide and hydralazine, develop a lupuslike disease. This chapter describes the protocols for inducing autoreactivity in murine T cells in vitro and using the cells to induce autoimmunity in vivo.

Murine Models of Lupus Induced by Hypomethylated T Cells (DNA Hypomethylation and Lupus

CD4+ T cell DNA hypomethylation may contribute to the development of drug induced and idiopathic human lupus. Inhibiting DNA methylation in mature CD4+ T cells causes MHC-specific autoreactivity in vitro. The lupus-inducing drugs hydralazine and procainamide also inhibit T cell DNA methylation and induce autoreactivity, and T cells from patients with active lupus have hypomethylated DNA and a similarly autoreactive T cell subset. Further, T cells treated with DNA methylation inhibitors demethylate the same sequences that demethylate in T cells from patients with active lupus. The pathologic significance of the autoreactivity induced by inhibiting T cell DNA methylation has been tested by treating murine T cells in vitro with drugs which modify DNA methylation, then injecting the cells into syngeneic female mice. Mice receiving CD4+ T cells demethylated by a variety of agents including procainamide and hydralazine develop a lupus-like disease. Further, transgenic mice with an inducible T cell DNA methylation defect also develop lupus-like autoimmunity. This chapter describes the protocols for inducing autoreactivity in murine T cells in vitro and for inducing autoimmunity in vivo using an adoptive transfer approach or transgenic animal models.

T Cell Integrin Overexpression as a Model of Murine Autoimmunity.

Integrin adhesion molecules have important adhesion and signaling functions. They also play a central role in the pathogenesis of many autoimmune diseases. Over the past few years we have described a T cell adoptive transfer model to investigate the role of T cell integrin adhesion molecules in the development of autoimmunity. This report summarizes the methods we used in establishing this murine model. By treating murine CD4+ T cells with DNA hypomethylating agents and by transfection we were able to test thein vitro effects of integrin overexpression on T cell autoreactive proliferation, cytotoxicity, adhesion and trafficking. Furthermore, we showed that the ability to inducein vivo autoimmunity may be unique to the integrin lymphocyte function associated antigen-1 (LFA-1).