Oxidative stress, T cell DNA methylation, and lupus

Abstract

Objective: Lupus develops when genetically predisposed people encounter environmental agents, such as ultraviolet 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 inhibiting DNA methylation in CD4+ T cells by blocking ERK pathway signaling is sufficient to alter gene expression, and that the modified cells cause lupus-like autoimmunity in mice. We also reported that T cells from patients with active lupus have decreased ERK pathway signaling, have decreased DNA methylation, and overexpress genes normally suppressed by DNA methylation. This study was undertaken to test whether oxidizing agents decrease ERK pathway signaling in T cells, decrease DNA methyltransferase levels, and cause demethylation and overexpression of T cell genes similar to that found in T cells from patients with active lupus.

Methods: CD4+ T cells were treated with the oxidizers H2 O2 or ONOO(-) . Effects on ERK pathway signaling were measured by immunoblotting, DNA methyltransferase 1 (DNMT-1) levels were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), and the methylation and expression of T cell genes were measured using flow cytometry, RT-PCR, and bisulfite sequencing.

Results: H2 O2 and ONOO(-) inhibited ERK pathway signaling in T cells by inhibiting the upstream regulator protein kinase Cδ, decreased DNMT-1 levels, and caused demethylation and overexpression of genes previously shown to be suppressed by DNA methylation in T cells from patients with active lupus.

Conclusion: Our findings indicate that oxidative stress may contribute to human lupus flares by inhibiting ERK pathway signaling in T cells to decrease DNMT-1 and cause DNA demethylation.

Figure 1

Effects of oxidative stress on killer cell immunoglobulin-like receptor (KIR) and CD70 gene expression in T cells. A–D, Peripheral blood mononuclear cells (PBMCs) from 3 healthy young donors were stimulated with phytohemagglutinin (PHA), treated with ONOO⁻ (A and B) or H₂O₂ (C and D), and then stained with fluorochrome-conjugated antibodies to CD4, CD8, and KIR proteins (A and C) or fluorochrome-conjugated anti-CD70 (B and D) and analyzed by flow cytometry. Values are the mean ± SEM of 3 determinations. P values were determined by analysis of variance. E and F, PBMCs from 4 healthy young donors were stimulated with PHA and then treated with the indicated concentrations of hydralazine (Hyd), H₂O₂, or ONOO⁻ as in A–D. CD4+ cells were then purified and KIR-2DL4 (E) or CD70 (F) transcripts were measured by reverse transcriptase–polymerase chain reaction. Results are presented relative to untreated T cells. Bars show the mean ± SEM. Values above the bars are P versus control, by t-test. G–J, PBMCs from healthy young donors were stimulated and then cultured alone (control) or with 1 mM N-acetylcysteine (NAC), 50 μM H₂O₂, 50 μM ONOO⁻, 1 mM NAC plus 50 μM H₂O₂, or 1 mM NAC plus 50 μM ONOO⁻. The cells were then stained for CD4 and KIR (G and I) or CD4 and CD70 (H and J) and analyzed as above. Bars show the mean ± SEM from 3 independent experiments in G and I and from 5 independent experiments in H and J. P values are versus H₂O₂ in G and H and versus ONOO⁻ in I and J.

Figure 2

H₂O₂ and ONOO⁻ inhibit T cell DNA methylation. Peripheral blood mononuclear cells from healthy young donors were stimulated with phytohemagglutinin and then washed and cultured alone or with 50 μM H₂O₂ or 50 μM ONOO⁻ added on days 1, 2, and 3. CD4+ cells were then isolated and DNA purified, and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4) (A) and TNFSF7 (CD70) (B) promoter methylation was measured by bisulfite sequencing. Results are presented as the average methylation of dC bases in CG pairs numbered relative to the transcription start site. Bars show the mean ± SEM of 4 experiments for KIR2DL4 and 3 experiments for TNFSF7. P values are versus control for each sequence. ANOVA = analysis of variance.

Figure 3

DNMT-1 mRNA levels in CD4+ T cells treated with H₂O₂ or ONOO⁻. Peripheral blood mononuclear cells from 4 young donors were stimulated and cultured alone or with 10 μM hydralazine (Hyd), 50 μM H₂O₂, or 50 μM ONOO⁻ as described in the Figure 1 legend. CD4+ cells were isolated 24 hours later, and RNA was isolated and DNMT-1 transcripts were measured relative to β-actin. Bars show the mean ± SEM. P values are versus control.

Figure 4

Decreased ERK pathway signaling in CD4+ T cells treated with H₂O₂ or ONOO⁻. CD4+ T cells from 6 healthy young donors were treated with 10 μM hydralazine (Hyd), 50 μM H₂O₂, or 50 μM ONOO⁻ for 60 minutes and then stimulated with phorbol myristate acetate (PMA) for 15 minutes. The cells were then lysed and Raf, MEK, and ERK phosphorylation was measured by immunoblotting. A, Representative immunoblots of Raf, MEK, and ERK phosphorylation in untreated cells and cells treated with hydralazine, H₂O₂, or ONOO⁻. B–D, Phosphorylation of Raf (B), MEK (C), and ERK (D) in cells treated with hydralazine, H₂O₂, or ONOO⁻. Bars show the mean ± SEM. Values above the bars are P versus control.

Figure 5

Protein kinase Cδ (PKCδ) inactivation in CD4+ T cells treated with hydralazine (Hyd), H₂O₂, or ONOO⁻. A, CD4+ T cells from a healthy young donor were cultured with 10 μM hydralazine for 60 minutes, stimulated with phorbol myristate acetate (PMA) for 15 minutes, and then lysed, and PKCδ phosphorylation was measured by immunoblotting. β-actin was used as a loading control. B, CD4+ T cells from a healthy young donor were cultured with 50 μM H₂O₂ and stimulated with PMA, and PKCδ phosphorylation was measured by immunoblotting. C, CD4+ T cells from a healthy young donor were cultured with 50 μM ONOO⁻ for 60 minutes and stimulated with PMA for 15 minutes, and PKCδ phosphorylation was measured by immunoblotting. D, PKCδ phosphorylation was quantified in CD4+ T cells treated with hydrazaline, H₂O₂, or ONOO⁻. Bars show the mean ± SEM from 6 similar experiments for each treatment. P values are versus control.