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Up-regulated expression of Ro52/tripartite motif-containing protein 21 ( TRIM21), Ro60/TROVE domain family, member 2 ( TROVE2) and lupus LA protein/Sjögren's syndrome antigen B ( La/ SSB) autoantigens has been described in the salivary gland epithelial cells ( SGEC) of patients with Sjögren's syndrome ( SS). SGECs, the key regulators of autoimmune SS responses, express high levels of surface functional Toll-like receptor ( TLR)-3, whereas Ro52/ TRIM21 negatively regulates TLR-3-mediated inflammation. Herein, we investigated the effect of TLR-3-signalling on the expression of Ro52/ TRIM21, as well as Ro60/ TROVE2 and La/ SSB autoantigens, by SGECs. The effect of TLR-3 or TLR-4 stimulation on autoantigen expression was evaluated by poly I: C or lipopolysaccharide ( LPS) treatment, respectively, of SGEC lines (10 from SS patients, 12 from non- SS controls) or HeLa cells, followed by analysis of mRNA and protein expression. Poly I: C, but not LPS, resulted in a two-step induction of Ro52/ TRIM21 mRNA expression by SGECs, a 12-fold increment at 6 h followed by a 2·5-fold increment at 24-48 h, whereas it induced a late two-fold up-regulation of Ro60/ TROVE2 and La/ SSB mRNAs at 48 h. Although protein expression levels were not affected significantly, the late up-regulation of Ro52/ TRIM21 mRNA was accompanied by protein redistribution, from nucleolar-like pattern to multiple coarse dots spanning throughout the nucleus. These late phenomena were mediated significantly by interferon ( IFN)-β production, as attested by cognate secretion and specific inhibition experiments and associated with IFN regulatory factor ( IRF)3 degradation. TLR-3-signalling had similar effects on SGECs obtained from SS patients and controls, whereas it did not affect the expression of these autoantigens in HeLa cells. TLR-3 signalling regulates the expression of autoantigens by SGECs, implicating innate immunity pathways in their over-expression in inflamed tissues and possibly in their exposure to the immune system. [ABSTRACT FROM AUTHOR]

Sjögren's syndrome (SS) is a chronic autoimmune epithelitis, and several lines of experiments indicate that multifactorial factors contribute to salivary gland epithelial cells (SGEC) dysfunctions including a combination of environmental factors, lymphocytic infiltrations, genetic predispositions as well as epigenetic defects. Such statement is reinforced by the observation that global DNA methylation (5MeCyt) is altered in minor salivary glands from pSS patients and that such defect is associated cytokeratin 19 (KRT19) overexpression. An epigenetic deregulation of the KRT19 gene was further tested by treating the human salivary gland (HSG) cell line with the DNA demethylating agent 5-azacytidin, and with the histone acetylase inhibitor trichostatin A. Blocking DNA methylation, but not histone acetylation, with 5-azacytidin was associated with KRT19 overexpression at both transcriptional and protein level. Next, analysis of the CpG genome-wide methylome array in the KTR19 locus from long term cultured SGEC obtained from 8 pSS patients revealed a more reduced DNA methylation level in those patients with defective global DNA methylation. Altogether, our data, therefore, suggest that alteration of DNA methylation in SGEC may contribute to pSS pathophysiology in part by controlling the expression of KRT19.

The pathogenesis of primary Sjögren's syndrome (pSS) is complex, in part due to DNA methylation abnormalities. This study was undertaken to evaluate the importance of global DNA methylation ((5m)C) as determined in minor salivary glands (MSG) from well characterized pSS patients. Twenty-two pSS patients and ten controls were selected, and MSG were stained with anti-(5m)C, anti-(5m)C/anti-cytokeratin (KRT)19, or with anti-SSB/La antibodies (Ab). The DNA methylation status at the SSB gene promoter P1 and P1' was evaluated by methylation-sensitive restriction enzymes (MSRE) coupled with PCR. The effect of the DNA demethylating drug 5 azacytidine (5-Aza) was tested in the human salivary gland (HSG) cell line. In pSS, the reduction of global DNA methylation ((5m)C) was associated with lymphocyte infiltration, the emergence of (5m)C(low) and KRT19(high) acini, and the detection of circulating anti-SSB/La Ab, but not with disease activity (ESSDAI). Next, treating HSG cells with 5-Aza was effective in inducing SSB expression. Finally in pSS patients positive for anti-SSB/La Ab, we further observed DNA demethylation at the SSB gene promoter P1 with consequent SSB overexpression at both the transcriptional and protein levels in salivary gland epithelial cells. In conclusion, our results highlight the importance of DNA methylation in the pathophysiology of pSS and to the emergence of anti-SSB/La Ab., (Copyright © 2015 Elsevier Ltd. All rights reserved.)