Hypomethylation of IL10 and IL13 promoters in CD4+ T cells of patients with systemic lupus erythematosus

Abstract

Interleukin- (IL-)10 and IL-13 play important roles in Th2 cell differentiation and production of autoantibodies in patients with (SLE). However, the mechanisms leading to IL10 and IL13 overexpression in SLE patients are not well understood. In this study, we confirm that the levels of both IL10 and IL13 mRNA in CD4+ T cells and of serum IL10 and IL13 proteins are increased in SLE patients. We show that the DNA methylation levels within IL10 and IL13 gene regulatory domains are reduced in SLE CD4+ T cells relative to healthy controls and negatively correlate with IL10 and IL13 mRNA expression. Moreover, treating healthy CD4+ T cells with the demethylating agent 5-azacytidine (5-azaC) increased IL10 and IL13 mRNA transcription. Together, our results show that promoter methylation is a determinant of IL10 and IL13 expression in CD4+ T cells, and we propose that DNA hypomethylation leads to IL10 and IL13 overexpression in SLE patients.

Figure 1

Serum IL-10 and IL-13 protein levels in SLE patients (n = 15) and healthy controls (n = 15). The mean concentration of IL-10 (a) and IL-13 (b) were detected by ELISA (P = .005; P = .007).

Figure 2

IL10 and IL13 mRNA expression in CD4⁺ T cells from SLE patients (n = 15) and healthy controls (n = 15) and the relationship between expression and disease activity. (a) and (b) IL10 and IL13 mRNA levels in CD4⁺ T cells were measured by quantitative real-time PCR and normalized to β-actin (SLE versus healthy control, P = .007 and P = .006, respectively). (c) In SLE patients, IL10 mRNA expression correlated (r = 0.746) with SLEDAI score (P = .001). (d) IL13 mRNA expression also correlated with disease activity (r = 0.628, P = .012).

Figure 3

IL10 enhancer methylation patterns in CD4⁺ T cells from SLE patients and healthy controls. (a) The mean methylation status for each of the 8 CG pairs within intron 4 of the IL10 locus in CD4⁺T cells from healthy controls (n = 10; upper panel) and SLE patients (n = 10; lower panel). (b) The combined average methylation status of 7 CG pairs (positions +3144, +3162, +3170, +3200, +3229, +3261, and +3265) in IL10 intron 4 in SLE CD4⁺ T cells was lower than healthy controls (P = .036). (c) Average IL10 promoter DNA methylation status correlated (r = −0.432) with relative mRNA level in SLE patients (P = .040).

Figure 4

IL13 promoter methylation patterns in CD4⁺ T cells from SLE patients and healthy controls. (a) The mean methylation status for each of the 20 CG pairs within the distal promoter of the IL13 locus in CD4⁺T cells from healthy controls (n = 10; upper panel) and SLE patients (n = 10; lower). The boxed area indicates the region (positions −2100, −2098, −2090, −2085, −2081, and −2076) with reduced methylation in SLE patients. (b) The combined average methylation status of the 6 hypomethylated CG pairs in the distal promoter of IL13 in SLE CD4⁺ T cells was reduced compared with the combined average methylation status of these positions in healthy controls (P = .020). (c) Average IL13 promoter (position −2100 to −2076) DNA methylation status correlated (r = −0.598) with relative mRNA level in SLE patients (P = .006).

Figure 5

Treatment of healthy CD4⁺ T cells with 5-azaC promotes IL10 and IL13 mRNA transcription. mRNA expression levels of IL10 (a) and IL13 (b) were measured by quantitative real-time PCR and normalized to β-actin. Treatment with 5-azaC results in a 2.6-fold increase of IL10 and a 5.7-fold increase of IL13, relative to untreated controls (P = .017 and P = .025, resp.).