DNA methylation modulates HRES1/p28 expression in B cells from patients with Lupus

Abstract

Systemic lupus erythematosus (SLE) disease is an autoimmune disease of unknown aetiology that affects predominantly women of child bearing age. Since previous studies, including ours, have demonstrated that CD4+ T cells and B cells from SLE patients are defective in their ability to methylate their DNA upon antigen stimulation, the aim of this study was to investigate whether DNA demethylation affects the transcription of HRES-1 in B cells. HRES-1 is the prototype of Human Endogenous Retrovirus (HERV) overexpressed in SLE. We have observed that SLE B cells were characterized by their incapacity to methylate the HRES-1 promoter, both in unstimulated and in anti-IgM stimulated B cells. In turn, HRES-1/p28 expression was increased in SLE B cells after B cell receptor engagement, but not in controls. In SLE B cells the Erk/DNMT1 pathway was defective. In addition, blocking the autocrine-loop of IL-6 in SLE B cells with an anti-IL-6 receptor monoclonal antibody restores DNA methylation and control of HRES-1/p28 expression became effective. As a consequence, a better understanding of HERV dysregulation in SLE reinforces our comprehension of the disease and opens new therapeutic perspectives.

Keywords: B cells; DNA methylation; Erk; HRES-1; IL-6; systemic lupus erythematosus.

Figure 1

The HRES-1 promoter region is demethylated in B cells from SLE patients and DNA demethylation modifies HRES-1/p28 expression. A: Schematic representation of the organization of HRES-1 on chromosome 1q42 region (GenBank X16660). Circles identify the 9 CpG motifs within the 5′ U3-R-U5 LTR region of HRES-1. Of note, the 3′ LTR region of HRES-1 is lacking. The HpaII/MspI motif present in the 5′U3-R-U5 LTR region is represented (star) as well as HRES-1/p28 and HRES-1/Rab4 transcript positions, and primers used in the MS-PCR and quantitative RT-PCR (RT-qPCR). B: Analysis of HRES-1 promoter methylation by amplification of genomic DNA digested with the methylation sensitive HpaII, or with the methylation insensitive MspI enzyme. The 502 bp HRES-1 amplicon contains one HpaII/MspI site. C: Histograms representing HRES-1 promoter methylation status in 6 SLE patients and 6 healthy controls, the percentage of methylation was quantified by calculating the ratio of HpaII-digested to undigested bands. D: Quantitative RT-PCR results presenting as histograms revealing that a 24-h stimulation of B cells with an anti-IgM increases HRES-1/p28 transcription in B cells from SLE patients, but not from healthy controls. The symbol * represented p<0.05.

Figure 2

The Erk/DNMT pathway is defective in SLE B cells. A–B: Cytoplasmic staining of pErk (A) and DNMT1 (B) in permeabilized B cells stimulated with anti-IgM or not for 10 min (pErk) and 24 h (DNMT1). Representative flow cytometric profiles are represented. C: HRES-1 promoter analysis by MS-PCR reveals that the Erk/DNMT1 pathway was involved in HRES-1 promoter methylation when control B cells were BCR stimulated with an anti-IgM Ab. Inhibition of pErk and DNMTs was respectively achieved by incubating the cells 24 h with 50 μM of the ras signal blocker PD98059, and with 20 μM of 5-azacytidine (5-aza). D: Quantitative PCR measurement of HRES-1/P28 mRNA levels. The symbol * represented p<0.05.

Figure 3

The effect of IL-6 on HRES-1/P28 expression and HRES-1 promoter methylation status. A/C: HRES-1 promoter analysis by MS-PCR in SLE B cells (A) and healthy control B cells (C) after BCR engagement using an anti-IgM Ab. Based on our previous observation that anti-IgM stimulated SLE B cells, but not the controls, express high amounts of IL-6 [26], the influence of the autocrine loop of IL-6 was tested by adding 40 ng/ml of a blocking anti-IL-6 receptor Ab in SLE B, or by adding 100 ng/ml of rIL-6 in healthy control B cells. B/D: Quantitative PCR measurement of HRES-1/P28.