Christelle Le Dantec

Epigenetic dysregulation in salivary glands from patients with primary Sjögren's syndrome may be ascribed to infiltrating B cells.

Sjögrens syndrome (SS) is an autoimmune exocrinopathy characterized by an epithelium injury with dense lymphocytic infiltrates, mainly composed of activated T and B cells. Present at the interface of genetic and environmental risk factors, DNA methylation is suspected to play a key role in SS. To clarify this point, global DNA methylation was tested within salivary gland epithelial cells (SGEC), peripheral T cells and B cells from SS patients. Global DNA methylation was reduced in SGEC from SS patients, while no difference was observed in T and B cells. SGEC demethylation in SS patients was associated with a 7-fold decrease in DNA methyl transferase (DNMT) 1 and a 2-fold increase in Gadd45-alpha expression. The other DNA methylation/demethylation partners, tested by real time PCR (DNMT3a/b, PCNA, UHRF1, MBD2, and MBD4), were not different. Interestingly, SGEC demethylation may be attributed in part to the infiltrating B cells as suspected in patients treated with anti-CD20 antibodies to deplete B cells. Such hypothesis was confirmed using co-culture experiments with human salivary gland cells and B cells. Furthermore, B cell-mediated DNA demethylation could be ascribed to an alteration of the PKC delta/ERK/DNMT1 pathway. As a consequence, part of the SGEC dysfunction in SS may be linked to epigenetic modifications, thus opening new therapeutic perspectives in SS.

The contribution of epigenetics in Sjögren’s Syndrome

Sjögren’s syndrome (SS) is a chronic autoimmune epithelitis that combines exocrine gland dysfunctions and lymphocytic infiltrations. While the pathogenesis of SS remains unclear, its etiology is multifunctional and includes a combination of genetic predispositions, environmental factors, and epigenetic factors. Recently, interest has grown in the involvement of epigenetics in autoimmune diseases. Epigenetics is defined as changes in gene expression, that are inheritable and that do not entail changes in the DNA sequence. In SS, several epigenetic mechanisms are defective including DNA demethylation that predominates in epithelial cells, an abnormal expression of microRNAs, and abnormal chromatin positioning-associated with autoantibody production. Last but not least, epigenetic modifications are reversible as observed in minor salivary glands from SS patients after B cell depletion using rituximab. Thus epigenetic findings in SS open new perspectives for therapeutic approaches as well as the possible identification of new biomarkers.

Human Endogenous Retrovirus Group E and Its Involvement in Diseases

Human endogenous retrovirus group E (HERV-E) elements are stably integrated into the human genome, transmitted vertically in a Mendelian manner, and are endowed with transcriptional activity as alternative promoters or enhancers. Such effects are under the control of the proviral long terminal repeats (LTR) that are organized into three HERV-E phylogenetic subgroups, namely LTR2, LTR2B, and LTR2C. Moreover, HERV-E expression is tissue-specific, and silenced by epigenetic constraints that may be disrupted in cancer, autoimmunity, and human placentation. Interest in HERV-E with regard to these conditions has been stimulated further by concerns regarding the capacity of HERV-E elements to modify the expression of neighboring genes and/or to produce retroviral proteins, including immunosuppressive env peptides, which in turn may induce (auto)-antibody (Ab) production. Finally, better understanding of HERV-E elements may have clinical applications for prevention, diagnosis, prognosis, and therapy.

An in silico Approach Reveals Associations between Genetic and Epigenetic Factors within Regulatory Elements in B Cells from Primary Sjögren's Syndrome Patients.

Recent advances in genetics have highlighted several regions and candidate genes associated with primary Sjögren’s syndrome (SS), a systemic autoimmune epithelitis that combines exocrine gland dysfunctions, and focal lymphocytic infiltrations. In addition to genetic factors, it is now clear that epigenetic deregulations are present during SS and restricted to specific cell type subsets, such as lymphocytes and salivary gland epithelial cells. In this study, 72 single nucleotide polymorphisms (SNPs) associated with 43 SS gene risk factors were selected from publicly available and peer reviewed literature for further in silico analysis. SS risk variant location was tested revealing a broad distribution in coding sequences (5.6%), intronic sequences (55.6%), upstream/downstream genic regions (30.5%), and intergenic regions (8.3%). Moreover, a significant enrichment of regulatory motifs (promoter, enhancer, insulator, DNAse peak, and expression quantitative trait loci) characterizes SS risk variants (94.4%). Next, screening SNPs in high linkage disequilibrium (r2 ≥ 0.8 in Caucasians) revealed 645 new variants including 5 SNPs with missense mutations, and indicated an enrichment of transcriptionally active motifs according to the cell type (B cells > monocytes > T cells ≫ A549). Finally, we looked at SS risk variants for histone markers in B cells (GM12878), monocytes (CD14+) and epithelial cells (A548). Active histone markers were associated with SS risk variants at both promoters and enhancers in B cells, and within enhancers in monocytes. In conclusion and based on the obtained in silico results that need further confirmation, associations were observed between SS genetic risk factors and epigenetic factors and these associations predominate in B cells, such as those observed at the FAM167A–BLK locus.

How the Environment Influences Epigenetics, DNA Methylation, and Autoimmune Diseases

The list of environmental factors implicated in autoimmune diseases is growing and includes exogenous and endogenous infectious agents; drugs; diet; and agents that cause oxidative stress, such as smoking and UV light exposure. However, mechanisms for the role of environmental agents in inducing autoimmune diseases are poorly understood. For some of these factors, clues are emerging to support an effect on the epigenetic machinery, thus offering the possibility of preventing or treating autoimmune diseases in patients in novel ways.

Primary Sjögren’s Syndrome and Epigenetics

Abstract Primary Sjogrens syndrome (pSS) is a systemic autoimmune epithelitis characterized by exocrine gland dysfunction (with lymphocytic infiltrations predominantly in the salivary and lacrimal glands) and B-cell hyperactivation. The etiology of pSS is multifactorial, and there is increasing evidence to suggest that epigenetic factors may also be determinant in the physiopathology of pSS. Epigenetics is defined as stable heritable changes in gene expression not attributable to genomic DNA. Peripheral blood T cells, peripheral B cells, and minor salivary gland (MSG) tissue from pSS patients show abnormal DNA methylation. This process has been characterized and the consequences for MSGs include: (1) the overexpression of transposons; (2) an epigenetic change in genes controlling autoantigens, antigen presentation, interferon signaling, and calcium signaling; (3) defective control of microRNAs; and (4) altered chromatin positioning associated with autoantibody production. Cell-specific cross talk between epigenetic and genetic factors is also suspected to promote autoreactivity. Epigenetic modifications in MSG are reversible as observed following B-cell depletion. Thus the epigenetic findings in pSS will provide new perspectives for therapeutic approaches, as well as the possibility to identify new biomarkers.

A7.8 DNA Demethylation in Salivary Gland Epithelial Cells from Patients with Primary Sjögren’s Syndrome may be Ascribed to Infiltrating B Cells

Background and Objectives Sjögren’s syndrome (SS) is an autoimmune exocrinopathy characterised by an epithelium injury surrounded by dense lymphocytic infiltrates composed of activated T and B cells. Present at the interface of genetic and environmental risk factors, epigenetic modifications are suspected to play a key role in SS. Accordingly, we decided to further characterise DNA methylation in SS. Materials and Methods We tested, using a 5 methyl cytosine (5MeCyt) ELISA, global DNA methylation in long-term cultured salivary gland epithelial cells (SGEC), peripheral T cells and B cells from eight SS patients. DNA methylation/demethylation partners were assessed by real time quantitative PCR (DNA methyl transferase (DNMT)1, DNMT3a/b, PCNA, UHRF1, MBD2, MBD4, and Gadd45-alpha). Immunofluorescence was conducted on labial salivary gland biopsy. Co-culture experiments were performed associating the human salivary gland cell line (HSG) and B cells. Results Global DNA methylation was reduced in SGEC from SS patients (5MeCyt: 36.3 ± 3.2% in SS versus 43.1 ± 3.3% in controls, P = 0.01), while no difference was observed in T and B cells. SGEC demethylation in SS patients was associated with a 7-fold decrease of DNMT1 and a 1.8-fold increase of Gadd45-alpha expression. The other DNA methylation/demethylation partners tested were not differently expressed when compared to controls. Interestingly, SGEC demethylation may be attributed to the B cell infiltrate as DNA methylation increased in salivary gland biopsy after rituximab (anti-CD20 antibody) treatment. Such hypothesis was confirmed using co-culture experiments (HSG cells and B cells) revealing an alteration of the PKC-delta/ERK/DNMT1 pathway. Finally, DNA methylation was associated with the overexpression of several SGEC genes such as ICAM-1 and human endogenous retrovirus (HERV). Conclusions SGEC dysfunction in SS may be linked to epigenetic modifications and this tissue specific defect may be ascribed in part to infiltrating B cells. This observation opens new therapeutic perspectives in SS.