CD4+ T cells epigenetically modified by oxidative stress cause lupus-like autoimmunity in mice
Introduction
Systemic lupus erythematosus (SLE) is a chronic relapsing autoimmune disease that primarily affects women. SLE develops and flares when genetically predisposed people encounter certain environmental agents. The genes predisposing to lupus are being identified, and epidemiologic evidence indicates that environmental agents which cause oxidative stress, such as infections, sun exposure, silica and smoking, are associated with lupus onset and flares [1]. However, how environmentally induced reactive oxygen species interact with the immune system to trigger lupus flares remains unclear.
Our group reported that CD4+ T cells epigenetically altered with DNA methylation inhibitors like 5-azacytidine (5-azaC), procainamide (Pca) or hydralazine (Hyd) cause lupus-like autoimmunity in animal models [2], and that similar epigenetically modified T cells are found in lupus patients during disease flares [1]. We traced the cause of the epigenetic defect to PKCδ inactivation, which prevents upregulation of DNA methyltransferase 1 (Dnmt1) during mitosis to copy methylation patterns [3]. Others reported that serum proteins are nitrated in patients with active lupus, caused by superoxide (O2-) combining with nitric oxide (NO) to form peroxynitrite (ONOO−), a highly reactive molecule that nitrates proteins and other molecules [4]. Our group subsequently found that PKCδ is similarly nitrated in T cells from patients with active lupus, and that nitrated PKCδ is unable to transmit signals that upregulate Dnmt1 to copy DNA methylation patterns in dividing T cells, causing demethylation and overexpression of genes normally suppressed by DNA methylation [5]. We extended these observations by demonstrating that treating human CD4+ T cells with the oxidizing agents H2O2 or ONOO− inhibits PKCδ activation, thereby decreasing ERK pathway signaling, decreasing Dnmt1 levels and causing demethylation and overexpression of CD70 and Kir genes similar to T cells from patients with active lupus [6]. However, whether the epigenetically modified T cells are sufficient to cause lupus-like autoimmunity was unknown.
We have now tested if female murine CD4+ T cells treated with H2O2 or ONOO− also overexpress methylation sensitive genes, and if the treated cells cause lupus-like autoimmunity in mice. The results demonstrate that the oxidized T cells overexpress the X-linked gene CD40L, one copy of which is silenced by DNA methylation in female human and mouse T cells [7], [8], and Kir genes, normally expressed by NK cells but silenced by DNA methylation in human and mouse T cells [9]. The results also demonstrate that adoptive transfer of the treated cells into syngeneic recipients causes anti-DNA antibodies and an immune complex glomerulonephritis, similar to T cells treated 5-azaC, Pca or Hyd. Together these studies support the contention that environmentally-induced oxidative stress may trigger lupus flares by epigenetically altering T cells through effects on T cell signaling and Dnmt1 expression.
Section snippets
Drugs and reagents
5-azaC and H2O2 were purchased from Sigma–Aldrich (St. Louis, MO), and ONOO− from Calbiochem (San Diego, CA).
Mice
Female SJL mice were purchased from Jackson Laboratories, housed in filter-protected cages, and provided with standard irradiated rodent diet 5053 (Lab Diet; PMI Nutrition International) and water ad libitum. Urinary protein and hemoglobin were measured using Chemstrip 7 dipsticks (Roche, Madison, WI). The mice were maintained in a specific pathogen–free facility by the Unit for
Murine CD4+ T cells treated with oxidizing agents overexpress methylation sensitive genes
We previously reported that inhibiting the replication of human CD4+ T cell DNA methylation patterns with the Dnmt inhibitor 5-azaC causes demethylation and overexpression of genes including CD40L on the female inactive X chromosome in humans and mice [8], [12] as well as the human KIR gene family, normally expressed by NK cells but not T cells [9], [13]. Initial studies compared the effects of H2O2 and ONOO− to 5-azaC on murine CD4+ T cell CD40L and Kir expression. We recently reported that
Discussion
A relatively extensive body of literature implicates CD4+ T cell DNA demethylation in lupus onset and flares. Early work demonstrated that inhibiting DNA methylation in CD4+ T cells with 5-azaC alters gene expression and converts polyclonal as well as cloned antigen-specific CD4+ T cells in autoreactive cells that respond to self class II MHC determinants without the addition of specific antigen [16], [17]. The autoreactivity was traced to overexpression of the adhesion molecule LFA-1
Acknowledgments
We thank Erin Grant for her excellent technical assistance and Ms. Stacy Fry for her excellent secretarial assistance. We also thank Dr. Gabriela Gorelik for helpful discussions and advice on oxidative stress and PKC-δ. This work was supported by PHS grants AR42525 and ES017885, a Merit grant from the Department of Veterans Affairs and the Lupus Insight Prize from the Lupus Foundation of America, the Alliance for Lupus Research and the Lupus Research Institute.
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