Patients with GSK-3 assay pSS and controls did not differ in methylation patterns of

the 8 CpG dinucleotides analysed in the promoter region (mean methylation level 52.7% ± 4.8% and 52.6% ± 6.9%, respectively, P = 0.87) (Fig. 3A). In the downstream enhancer region, the mean methylation levels for patients and controls were 53.2% ± 3.4% and 49.4% ± 4%, respectively (P = 0.09) (Fig. 3B). P values are adjusted for age. As expected, these results suggested that about half of the fragments were unmethylated in both patients and controls. We aimed to confirm these results by using the demethylating agent 5-AzaC. Treating CD4+ T cells with the demethylating agent 5-AzaC significantly demethylated the CpG sequences (Fig. 4).

The addition of 5-AzaC increased the protein level of CD40L by a mean of 60% and 72% in patients with pSS and controls, respectively, and the mRNA level of CD40L was approximately doubled for both patients and controls, with no difference between patients and controls in protein or mRNA level (P = 0.549 and P = 0.96, respectively) (Fig. 5A,B). Autoimmune diseases are more frequent in women, without a clear explanation. One explanation could be a more frequent X-inactivation escape in women with than without the diseases. CD40L, located on the long arm of the X chromosome (Xq26.3-q27.1), is a good candidate to assess this hypothesis. In fact, CD40L inactivation escape was first reported in SLE as leading to an overexpression of CD40L in this Maraviroc mouse autoimmune disease. The expression of membrane-bound CD40L and epigenetic regulation of CD40L expression have never been analysed in pSS. This study demonstrates that membrane-bound next CD40L is overexpressed in ex vivo activated CD4+ T cells from female patients with pSS through regulatory mechanisms that do not involve demethylated profiles of key regulatory regions of CD40L in contrast to what has been

reported for SLE [2]. CD40L is a type II membrane glycoprotein of the TNF family. Like other members of the family, CD40L forms trimeric structures that bind the CD40 receptor. CD40L–CD40 interaction can also lead to CD40L proteolysis and release of the soluble form of CD40L (sCD40L). CD40L is mainly expressed on activated T lymphocytes and platelets. It is expressed in a wide range of cell types, including B lymphocytes. CD40L–CD40 interaction leads to B-cell activation (immunoglobulin class switching, germinal centre formation and cytokines production) and dendritic cell maturation. Recently, a single common single nucleotide polymorphism at the CD40 locus (rs4810485) was found to be associated with rheumatoid arthritis [10]. The corresponding at-risk allele was associated with increased expression of CD40 on the surface of B lymphocytes. CD40–CD40L interaction has an important role in autoimmune diseases.