EPIGENETICS IN HUMAN AUTOIMMUNITY
DNA methylation and T-cell autoreactivity—Early studies used the DNA methyltransferase (Dnmt) inhibitor 5-azacytidine (5-azaC) to probe for functional changes caused by DNA methylation inhibition in mature T cells. One observation was that CD4 + T cells become autoreactive following 5-azaC treatment. CD4 + T cells normally respond to peptides presented in the antigen binding cleft of “self ” class II major histocompatibility antigen (MHC) molecules on antigen presenting cells. Following 5-azaC treatment, antigen specific CD4 + T cells lose the requirement for specific antigen and respond to antigen presenting cells without added antigen. The response is specific for self class II MHC molecules and is reversible, in that antigen responsiveness recovers 1–2 weeks after the Dnmt inhibitor is removed. The autoreactivity has been demonstrated with cloned and polyclonal human and murine CD4 + T cells. CD8 + T cells do not become autoreactive; the reason is unknown.
Mechanistic studies revealed that development of autoreactivity correlates with increased expression of the adhesion molecule LFA-1 (CD11a/CD18), caused by increased levels of CD11a (ITGAL) transcripts. The increase in ITGAL expression is due to demethylation of alu elements 5′to the ITGAL promoter. LFA-1 overexpression caused by transfection results in identical autoreactivity in human and murine T cells. LFA-1 surrounds the T-cell antigen receptor (TCR) to form the “immunologic synapse” providing both stability to the TCR–MHC interaction as well as co-stimulatory signals that activate T cells. Overstabilizing the lower affinity interaction between the TCR and “self” class II MHC molecules bearing inappropriate peptide fragments, together with increased costimulatory signaling, may be responsible for initiating the T-cell response to self MHC molecules without the appropriate antigen.
T-cell DNA demethylation and autoimmunity—The response of demethylated CD4 + cells to self class II MHC molecules demonstrates that normal, antigen reactive T cells can be modified by exogenous agents to become autoreactive, potentially contributing to an autoimmune disease. In animal models, CD4 + T cells responding directly to class II MHC molecules cause chronic graft-vs.-host disease, with many features of human lupus, including anti-nuclear antibodies (ANAs) and an immune complex kidney disease. This suggests that 5-azaC modified, class II responsive cells may also cause a disease resembling SLE. Pathogenicity of the autoreactive cells was demonstrated by injecting cloned or polyclonal 5-azaC treated CD4 + T cells intravenously into syngeneic mice. The recipients developed anti-DNA antibodies and an immune complex glomerulonephritis as well as other histologic features of autoimmunity, depending on the T cells treated and/or the strain LFA-1 transfected CD4 + T cells caused a similar lupus-like disease in the same system, indicating that LFA-1 overexpression contributes to the autoimmunity induced by demethylated T cells.
Another consequence of increased adhesion between T cells and antigen presenting cells is that demethylated or LFA-1-transfected CD4 + T cells kill autologous or syngeneic macrophages (Mø) without specific antigen, also in an MHC restricted fashio . The killing utilizes conventional mechanisms of apoptosis involving the death receptor Fas, TRAIL and TWEAK, but demethylated CD4 + T cells also acquire perforin expression through demethylation of PRF1, and perforin inhibitors prevent the killing. The autoreactive killing of Mø and perhaps other cells by hypomethylated T cells could stimulate ANAs by increasing antigenic apoptotic material. Injection of apoptotic cells into mice causes anti-DNA antibodies, as does impairment of molecules involved in clearing apoptotic debris. Mø killing would result in the release of apoptotic material. Further, since Mø remove apoptotic debris, the material will not be cleared effectively, amplifying the effect. A role for Mø apoptosis in lupus-like autoimmunity is supported by studies demonstrating that causing Mø apoptosis in vivo with clodronate-filled vesicles, selectively phagocytosed by Mø to cause their death, induces anti-DNA and antinucleosome antibodies in normal mice and accelerates autoimmunity in lupus-prone mice.
Anti-DNA antibody production may be augmented by B-cell overstimulation, due to overexpression of cytokines and cell surface costimulatory molecules by demethylated T cells. Co-culture of demethylated T cells with autologous B cells results in IgG hypersecretion, due in part to increased expression of Th1 and Th2 cytokines including IFN-γ, IL-4 and IL-6 and in part to overexpression of B-cell costimulatory molecules including CD70 and CD40L. IL- 4 and IL-6 are suppressed by methylation in Th1 cells, and IFN-γin Th2 cells, and expression is induced by 5-azaC. Similarly, T cells treated with DNA methylation inhibitors also overexpress the B-cell costimulatory molecule CD70 (TNFSF7), and overexpression is due to demethylation of a region flanking the TNFSF7 promoter. Coculture of B cells with autologous CD4 + T cells treated with DNA methylation inhibitors results in IgG overproduction that is reversed with anti-CD70 antibodies. CD40L is another methylation sensitive B-cell costimulatory molecule expressed on CD4 + T cells.
However, in contrast to CD70, CD40L (CD40LG) is encoded on the X chromosome, so men have one unmethylated gene in CD4 + T cells, while women have one methylated and one unmethylated gene. 5-azaC doubles CD40L expression on CD4 + T cells from women, due to demethylation of the gene on the inactive X, but has only minimal affects on CD40L in CD4 + T cells from men.
Together, these studies suggest that demethylated, autoreactive T cells could interact with Mø in vivo to cause apoptosis and release of antigenic nucleosomes, resulting in anti-DNA antibodies, and overstimulate B cells, increasing autoantibody production. Such cells might be more potent inducers of autoimmunity than cells made autoreactive by LFA-1 transfection alone. This was tested by injecting mice with LFA-1 overexpressing T cells caused by transfection, or T cells demethylated with a Dnmt inhibitor. Demethylated T cells were more potent in inducing autoimmunity than transfected cells.
DNA methylation and drug induced lupus—As noted above, procainamide and hydralazine are lupus-inducing drugs. Both cause ANAs in a majority of people and a lupuslike disease in a subset. Development of the systemic manifestations presumably requires the presence of lupus susceptibility genes. Interestingly, both are DNA methylation inhibitors. Procainamide is a competitive inhibitor of Dnmt 1 enzymatic activity, reducing the affinity of the enzyme for its substrates, hemimethylated DNA and S-adenosylmethionine. It has no effect on intracellular S-adenosylmethionine or S-adenosylhomocysteine pools. In contrast, hydralazine selectively inhibits T- and B-cell extracelluar signal-regulated kinase (ERK) pathway signaling, preventing upregulation of T-cell Dnmt 1 and 3a during mitosis, resulting in hypomethylation of the daughter cells, as well as receptor editing in B cells. Pathogenicity of decreased ERK pathway signaling was demonstrated by treating CD4 + T cells with U0126, a selective mitogen-activated protein kinase (MAPK) inhibitor that decreases ERK pathway signaling, and injecting the cells into syngeneic mice. The treated T cells overexpressed LFA-1 and became autoreactive, and mice receiving the treated cells developed anti-DNA antibodies, similar to procainamide treated cells. More recent studies mapped the hydralazine-induced ERK pathway defect to protein kinase C-delta (PCK-δ), and confirmed that transfection of T cells with a dominant negative PKC-δdemethylates T-cell DNA and causes CD70 overexpression. Interestingly, the PKC-δknockout mouse develops lupus.
DNA methylation and idiopathic lupus—Similar mechanisms contribute to idiopathic human lupus. T cells from patients with active lupus have decreased total deoxymethylcytosine content and decreased Dnmt 1 transcripts in lupus T cells relative to patients with inactive lupus and normal controls. Since lupus T cells have multiple signaling abnormalities, and Dnmt 1 expression is regulated by the ERK and Jun N-terminal kinase (JNK) pathways, T-cell signaling was examined in human lupus. Patients with active but not inactive lupus had decreased ERK phosphorylation in response to stimulation that was identical to hydralazine treated cells, while signaling through the JNK and p38 pathways was intact. Interestingly, the lupus ERK pathway defect maps to PKC-δ, also inhibited by hydralazine.
Other studies revealed additional functional and epigenetic similarities between lupus and experimentally demethylated T cells. Functional studies demonstrated that lupus T cells overstimulate autologous B-cell antibody production, similar to 5-azaC treated T cells, and that the overstimulation is inhibited with anti-CD70 or anti-CD40L. A subset of lupus T cells overexpresses LFA-1, and this subset spontaneously kills autologous Mø in an MHC restricted, autoreactive fashion identical to experimentally demethylated cells. Further, patients with active lupus have circulating, apoptotic monocytes in their peripheral blood, suggesting that a similar killing occurs in vivo.
Epigenetic studies demonstrated demethylation of the same ITGAL promoter sequences in CD4 + lupus T cells as in 5-azaC treated T cells. The degree of demethylation was proportional to disease activity. Similarly, CD4 + T cells from patients with active lupus demethylated the same PRF1 sequences and aberrantly expressed perforin. Concanamycin, a perforin antagonist, prevented the autoreactive Mø killing by lupus T cells, implicating perforin in this phenomenon.
Also similar to the in vitro model, bisulfite sequencing revealed that the core TNFSF7 promoter is normally demethylated in CD4 + T cells, and that 5-azaC extends the demethylated region upstream by ~300 bp. Cassette methylation confirmed transcriptional suppression when the region is methylated. Importantly, other DNA methylation inhibitors including procainamide, hydralazine, and the MEK inhibitor U0126 all demethylated the same sequence and increased CD70 expression as 5-azaC. CD4 + T cells from lupus patients overexpressed CD70 and demethylated the same region. Further, CD4 + T cells from women with active lupus also demethylated the CD40LG gene on the inactive X and overexpressed CD40L, while men with the same degree of lupus activity did not overexpress CD40L. This suggests that demethylation of the inactive X may contribute to increased incidence of lupus in women. Thus, for at least four genes (ITGAL, TNFSF7, PRF1 and CD40LG), identical changes in methylation, expression and function are found in experimentally demethylated and lupus T cells.
Histone modifications in lupus—The role of histone modifications in lupus is less well understood. Treating lupus T cells with histone deacetylase inhibitors including trichostatin A and suberoylanilide hydroxamic acid restores aberrant expression of some genes. However, these drugs also modify acetylation of transcription factors, nuclear transport proteins, and cytoskeleton proteins, and confirmatory studies at the chromatin level still need to be performed in lupus T cells. Summary—Identical epigenetic effects occur in experimentally demethylated and lupus T cells at the DNA, mRNA, protein, and functional levels for at least four genes contributing to T-cell autoreactivity, B-cell overstimulation and macrophage killing (ITGAL, TNFSF7, CD40LG and PRF1, respectively). Experimentally demethylated T cells cause a lupus-like disease in animal models. At least two lupus-inducing drugs are DNA methylation inhibitors with effects on T cells identical to those caused by 5-azaC and found in idiopathic lupus. In idiopathic and hydralazine-induced lupus, DNA demethylation appears to be caused by a failure to upregulate Dnmt 1 during mitosis, due to a defect in ERK pathway signaling that maps to PCK-δ, and the PKC-δknockout mouse develops lupus. It seems reasonable to propose that defective T-cell DNA methylation may contribute to the pathogenesis of lupus in genetically predisposed individuals.