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

Abstract

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.

Keywords: CD40L; CD70; KirL1; Systemic lupus erythematosus (SLE).

Figure 1. 5-azaC, H₂O₂ and ONOO⁻ decrease Dnmt1 gene expression and increase the expression of methylation sensitive genes CD40L, CD70 and KirL1 mRNA in CD4⁺ T cells

The spleen, thymus and axillary lymph nodes were removed from female SJL mice, then mononuclear cells were isolated and stimulated with Con-A in vitro. 18-24 hours later CD4⁺ T cells were purified and treated with 5 μM 5-azaC, 20 μM H₂O₂ or 20 μM ONOO⁻ using previously described protocols [6]. 72 hours later the T cells were treated with PMA and ionomycin for their final 6 h culture then lysed and Dnmt1 (A), CD70 (B), CD40L (C) and KirL1 (D) transcripts measured relative to β-actin by RT-PCR. Results are expressed as percent of the untreated control (100%) and represent the mean +/− SEM of 3 experiments combined (*P≤0.05; **P<0.01; *** P≤ 0.001 by Student's t-Test.)

Figure 2. 5-azaC, H₂O₂ and ONOO⁻ increase CD40L protein expression in CD4⁺ T cells

Splenocytes from female SJL mice were stimulated with Con-A then treated with H₂O₂ or ONOO⁻ as in Figure 1. 72 hours later the cells were washed, stained with fluorochrome conjugated antibodies to CD4 and CD40L then analyzed by flow cytometry. * P=0.049, **P=0.004 by paired t-Test). Results are expressed as the mean±SEM (n=5 mice) and are from one of two independent experiments.

Figure 3. CD4⁺ T cells treated with 5-azaC, H₂O₂ or ONOO⁻ cause anti-DNA antibodies in syngeneic mice

CD4⁺ T cells treated as described for Fig. 1 were injected (5×10⁶ cells/recipient) into the tail veins of female SJL mice every 2 weeks for a total of 7 injections. Controls included mice receiving untreated T cells and HBSS alone. Blood was obtained 4, 8 and 12 weeks after the first injection and serum anti-dsDNA antibody levels measured by ELISA. Results are presented as the mean ± SEM of groups of 5-8 mice. The linear mixed model of ANOVA was used to assess the statistical significance of the effects of different treatments on antibody production relative to HBSS injections without cells over time. P values shown are versus mice treated with HBSS (no cells).

Figure 4. CD4⁺ T cells treated with 5-azaC, H₂O₂ or ONOO⁻ cause an immune complex glomerulonephritis in syngeneic mice

14 weeks after the first injection the mice were sacrificed and kidneys removed for histologic analysis. Hematoxylin and eosin stained kidney sections from mice receiving untreated (A), 5-azaC treated (C), H₂O₂ treated (E) or ONOO⁻ treated (G) CD4⁺ T cells. Kidney sections from the same mice tested for IgG deposition using immunoperoxidase staining. Glomeruli from mice receiving 5-azaC (D). H₂O₂ (F) and ONOO⁻ (H) treated CD4+ T cells all show IgG deposition, identified by the red deposits while mice receiving untreated T cells (B) do not. Sections were counterstained with hematoxylin.