Epigenetic dysregulation in salivary glands from patients with primary Sjögren's syndrome may be ascribed to infiltrating B cells
Highlights
► In the etiology of Sjogren's syndrome (SS), epigenetic modifications are suspected. ► DNMT1 expression and global DNA methylation are altered in SS. ► Epigenetic defects are restricted to salivary gland epithelial cells. ► In exocrine glands, DNA methylation is controlled by infiltrating B cells.
Introduction
Sjögren's syndrome (SS) is a chronic autoimmune disease affecting exocrine glands, mainly the salivary and lacrimal glands, leading to its designation as an autoimmune epithelitis [1]. Systemic features may be present [2], autoantibodies are detected, and patients with SS have a 20–40 fold increased risk of developing B cell lymphoma [3]. Activated mononuclear cells infiltrate exocrine glands, mainly T and B cells, and such infiltration evolves with the lesion severity and disease manifestations. According to Christodoulou et al., B cells predominate in severe inflammatory lesions, and a B/T cell positive ratio correlated with the infiltration grade and the biopsy focus score [4].
The etiology of SS is multifactorial and arises from an interplay of genetic predispositions, immunological dysregulation, environmental factors and epigenetic modifications [5]. Experimental models support a role for DNA methylation in SS and lupus. Indeed, hydralazine and procainamide, two DNA methylation inhibitors, induce SS with immunological features of a systemic lupus erythematosus (SLE)-like disease [6], [7]. DNA methylation at the 5-carbon position of cytosines (5MeCyt) in CpG dinucleotides is tightly regulated during cell development and the cell cycle. The DNA methyl transferases DNMT3a/b and DNMT1 are responsible for the establishment and maintenance of CpG methylation patterns, respectively. To be effective, DNMTs need to be associated with DNA binding proteins such as the proliferation cell nuclear antigen (PCNA) and the DNA repair/transcription factor ubiquitin-like PHD and RING finger domains 1 (UHRF1) [8]. In contrast, DNA demethylation is related to the methyl-CpG-binding domain (MBD) protein-2 and -4 when associated with the stress protein Gadd-45α that can initiate DNA demethylation by replacing a 5MeCyt with an unmethylated cytosine [9].
Most of our knowledge regarding DNA demethylation in autoimmune diseases came from the analysis of CD4+ T cells and B cells in patients with SLE [10], [11], [12]. Several mechanisms have been described to explain DNA demethylation in SLE patients. The first one, described by Gorelik et al., is related to a defective PKC delta (PKCδ)/ERK pathway observed both in idiopathic and drug-induced SLE patients [13]. Interestingly, the PKCδ−/− mice reproduce the SS disease with reduced salivary gland function, B cell infiltration and autoantibody production [14]. The second mechanism is related to the expression of miRNAs that down regulate DNMT1 [15], [16], and the third to the overexpression of DNA demethylating factors such as Gadd-45α [17], and MBD2/4 [18].
Because DNA demethylation is commonly observed in autoimmune diseases [19], and suspected to be associated with autoantibody production [20], we decided to characterize DNA methylation in SS. The results indicate that salivary gland epithelial cells (SGEC) are demethylated in SS and that this defect may be attributed in part to the presence of infiltrating B cells.
Section snippets
Patients and controls
Eight patients fulfilling the American–European consensus group criteria for primary SS that displayed salivary gland (SG) focus score of at least 3 were enrolled in the study [21]. Peripheral blood was collected by phlebotomy using lithium heparin tubes, and minor labial SGs were obtained by biopsy. SGs obtained before and after two infusions of anti-CD20 at 4 months (Rituximab, Roche) were available for two patients [22], [23]. Peripheral blood controls were obtained from 8 healthy subjects
DNA global demethylation characterizes salivary glands epithelial cells from SS patients
To determine whether global DNA methylation may be altered in SS, 5MeCyt content was evaluated salivary gland (SG) epithelial cells, in peripheral T cells, and in peripheral B cells obtained from 8 patients with primary SS. As reported in Fig. 1, global DNA methylation was reduced in minor SG biopsy sections and in long term cultured SG epithelial cells (SGEC) from SS patients in contrast with the control group (5MeCyt: 36.3 ± 3.2% in SS versus 43.1 ± 3.3% in controls, P = 0.01). Surprisingly,
Discussion
Our study reveals that DNA demethylation characterizes SG from SS patients and that this defect persists in long term SGEC culture. In this context, it is not surprising that mice treated long term with demethylating drugs such as procainamide or 5-azacytidine, in addition to developing an SLE-like disease, present clinical features of SS (reviewed in Ref. [5]). Yin et al., have recently reported a decrease in TNFSF7 promoter methylation status in SS CD4+ T cells [26]. Testing global DNA
Acknowledgments
Thanks are due to Dr. Wesley H. Brooks for editorial assistance and to Geneviève Michel and Simone Forest for their help with typing of the paper. We are also grateful to the “Association Française du Gougerot Sjögren et des Syndromes Secs” for their support. Yosra Thabet is funded by the Islamic development bank merit scholarship program.
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