Environmental exposure, estrogen and two X chromosomes are required for disease development in an epigenetic model of lupus

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease primarily afflicting women. The reason for the gender bias is unclear, but genetic susceptibility, estrogen and environmental agents appear to play significant roles in SLE pathogenesis. Environmental agents can contribute to lupus susceptibility through epigenetic mechanisms. We used (C57BL/6xSJL)F1 mice transgenic for a dominant-negative MEK (dnMEK) that was previously shown to be inducibly and selectively expressed in T cells. In this model, induction of the dnMEK by doxycycline treatment suppresses T cell ERK signaling, decreasing DNA-methyltransferase expression and resulting in DNA demethylation, overexpression of immune genes Itgal (CD11a) and Tnfsf7 (CD70), and anti-dsDNA antibody. To examine the role of gender and estrogen in this model, male and female transgenic mice were neutered and implanted with time-release pellets delivering placebo or estrogen. Doxycycline induced IgG anti-dsDNA antibodies in intact and neutered, placebo-treated control female but not male transgenic mice. Glomerular IgG deposits were also found in the kidneys of female but not male transgenic mice, and not in the absence of doxycycline. Estrogen enhanced anti-dsDNA IgG antibodies only in transgenic, ERK-impaired female mice. Decreased ERK activation also resulted in overexpression and demethylation of the X-linked methylation-sensitive gene CD40lg in female but not male mice, consistent with demethylation of the second X chromosome in the females. The results show that both estrogen and female gender contribute to the female predisposition in lupus susceptibility through hormonal and epigenetic X-chromosome effects and through suppression of ERK signaling by environmental agents.

Fig 1

Effects of decreased ERK signaling and gender on anti-dsDNA antibody. A. IgG anti-dsDNA antibody was measured in male (M) and female (F) (C57BL/6 × SJL)F1 mice bearing the dnMEK and CD2rtTA transgenes (+/+) and given 4 mg/ml DOX/5% glucose (DOX+) or 5% glucose alone (DOX−) in their drinking water for 4–24 weeks. B. Comparison between groups after 24 weeks of treatment. (*) p=0.01 Female (+/+) DOX+ vs. DOX−; (**) p<0.005 DOX-treated Female vs. Male (+/+). Mean ± S.E.M. 3–5 mice per group. Data are representative of 2 independent experiments.

Fig. 2

Glomerular IgG immune complex deposition in transgenic mice. Kidneys from female and male (C57BL/6 × SJL)F1 mice from Fig. 1 were bisected and half of the tissue snap frozen, the remaining half fixed in 10% formalin and paraffin embedded. Five micron sections of frozen tissue were stained with biotin-goat anti-mouse IgG (Fc specific) and FITC-streptavidin. Sections from formalin-fixed, paraffin embedded tissue were stained with H&E and examined by light microscopy. (A), Immunofluorescence (B) light microscopy of glomeruli from DOX-transgenic female mice. (C) 10× magnification of a section from a representative kidney of DOX+ female mice, (D) 40× view of area in box from (C), (E) light micrograph showing histopathology of a glomerulus from a DOX+ female mouse. Arrows indicate areas of abnormal histopathology. Sections shown are representatives of 5 mice per group.

Fig. 3

Effect of reduced ERK signaling on gene expression in male and female CD4⁺ T cells. Male and female transgenic mice were treated for 1 week with DOX or vehicle. Their spleen cells were isolated, treated for 24hr with Con-A, CD4⁺ T cells isolated then (A) dnMEK, (B) DNMT1, (C) CD70, and (D) CD11a, were measured by quantitative RT-PCR relative to β-actin. Mean ± S.E.M. from 5 mice per group.

Fig. 4

Estrogen and two X chromosomes are required to make anti-dsDNA antibody in response to impaired ERK pathway signaling. Neutered and estrogen or placebo supplemented dnMEK⁺CD2rtTA⁺ transgenic mice were given DOX/5% glucose (DOX+) or 5% glucose alone (DOX−) in their drinking water for 20 wks. P<0.001 female, estrogen treated DOX+ vs. DOX−. Mean ± S.D. of 3–5 mice per group.

Fig. 5

X chromosome gene CD40L expression and demethylation in CD4⁺ T cells. (A) CD4⁺ T cells from dnMEK⁺CD2rtTA⁺ male and female DOX-treated (DOX+) or control (DOX−) mice were stained with FITC-CD40L antibody then mean fluorescence intensity was analyzed by FACS and normalized to the males (p=0.02 female vs male in the DOX-treated cells). (B) The promoter region of the CD40L gene from female DOX+ transgenic mice. (C) spleen cells from female or male (D) mice were stimulated with Con-A, treated with 5Azacytadine, and 4 days later CD4⁺ T cells isolated and their DNA pyrosequenced to confirm demethylation of the CD40lg promoter region. Data shown are from a representative of 3 experiments of 3–5 mice per experiment. Values are Mean ± S.E.M. of 3–5 mice per group. CG pairs are numbered relative to the transcription start site described by Tsitsikov et al. [38]. Murine C57BL/6 CD40lg accession number VERSION NT_039706.7 GI:149271867 from the NCBI GenBank http://www.ncbi.nlm.nih.gov/nucleotide/149271867.