SummaryUp-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 polyI: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. PolyI: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.
1 These authors contributed equally to the study. SummaryThe elevated tissue expression of Ro/SSA and La/SSB autoantigens appears to be crucial for the generation and perpetuation of autoimmune humoral responses against these autoantigens in Sj€ ogren's syndrome (SS). The mechanisms that govern their expression are not known. miRNAs, the posttranscriptional regulators of gene expression, might be implicated. We have identified previously the miRNAs let7b, miR16, miR181a, miR200b-3p, miR200b-5p, miR223 and miR483-5p that are predicted to target Ro/SSA [Ro52/tripartite motif-containing protein 21 (TRIM21), Ro60/TROVE domain family, member 2 (TROVE2)] and La/SSB mRNAs. To study possible associations with autoantigen mRNA expression and disease features, their expression was investigated in minor salivary gland (MSG) tissues, peripheral blood mononuclear cells (PBMC) and long-term cultured non-neoplastic salivary gland epithelial cells (SGEC) from 29 SS patients (20 of 29 positive for autoantibodies to Ro/SSA and La/SSB) and 24 sicca-complaining controls. The levels of miR16 were up-regulated in MSGs, miR200b-3p in SGECs and miR223 and miR483-5p in PBMCs of SS patients compared to siccacomplaining controls. The MSG levels of let7b, miR16, miR181a, miR223 and miR483-5p were correlated positively with Ro52/TRIM21-mRNA. miR181a and miR200b-3p were correlated negatively with Ro52/TRIM21 and Ro60/ TROVE2 mRNAs in SGECs, respectively, whereas let7b, miR200b-5p and miR223 associated with La/SSB-mRNA. In PBMCs, let7b, miR16, miR181a and miR483-5p were correlated with Ro52/TRIM21, whereas let7b, miR16 and miR181a were also associated with La/SSB-mRNA expression. Significantly lower miR200b-5p levels were expressed in SS patients with mucosa-associated lymphoid tissue (MALT) lymphoma compared to those without. Our findings indicate that miR16, miR200b-3p, miR223 and miR483-5p are deregulated in SS, but the exact role of this deregulation in disease pathogenesis and autoantigen expression needs to be elucidated.
Background Ro52/TRIM21 is a major target of autoantigenic responses in Sjögren’s syndrome (SS). Up-regulated epithelial Ro52 expression has been reported at the salivary gland inflammatory lesions of SS patients; however, the factors that drive this effect have not been identified. Ro52 is an E3-ubiquitin ligase that negatively regulates TLR3-mediated inflammation by ubiquitinating and promoting proteasomal degradation of several downstream IRFs, whereas both type-I and type-II interferons (IFN) are considered to be potent inducers of Ro52. Long-term cultured non-neoplastic SGECs express constitutively high levels of surface TLR3. Moreover, TLR3 stimulation in SGECs has been shown to strongly induce several immune-modulatory molecules, cytokines including interferons and apoptosis Objectives To investigate whether TLR3 stimulation has a reciprocal regulatory effect in Ro52 expression by SGECs. Methods SGEC lines from SS patients (n=6) and non-SS controls (n=12) were treated with the TLR3 ligand analogue, polyinosinic:cytidylic acid (polyI:C; 5 µg/ml), and the TLR4-ligand, lipopolysaccharide (LPS, 1 µg/ml, control TLR treatment), for various time-points. The expression of Ro52/TRIM21, Ro60/TROVE2 and La/SSB mRNAs and proteins at 0, 6, 12, 24, 48 and 72 hrs of treatment was analyzed by real-time PCR, confocal microscopy and immunoblotting, respectively. mRNA expression was normalized by HPRT1 gene and calculated by the ΔΔCT method using HeLa as calibrator. Mann-Whitney test was employed to analyze statistical significances. Results SGECs obtained from SS patients and controls were found to respond similarly to TLR3 signaling. PolyI:C treatment strongly induced Ro52 mRNA expression by SGECs. This induction was evident at 6-hrs (mean fold induction of basal expression±SE: 13.12±4.5, p<0.0001) and remained stable until 12-hrs of treatment, whereas a further increment was observed at 48-hrs (mean fold induction 34.3±9.1 compared to basal levels, p<0.0001). Although, Ro52 protein expression was not significantly affected by polyI:C treatment, its nuclear localization was found to change at 48-72 hrs and to alter from nucleolar to nuclear dots. PolyI:C treatment was found to significantly affect the mRNA expression of Ro60 and La/SSB autoantigens at 48-hrs, however the fold induction was low, compared to that of Ro52 (mean fold induction±SE: 3.29±1.75, p<0.0001 and 0.86±0.18, p=0.002, respectively). Treatment with LPS was not found to significantly affect the mRNA or protein expression of the molecules studied (Ro52, Ro60 and La/SSB). Inhibition experiments using specific antibodies against IFNα, IFNβ, IFNγ and type I IFN receptor indicated that the second polyI:C-induced increment of Ro52 mRNA expression is mediated by IFNβ. Conclusions Our findings indicate a reciprocal regulation of TLR3 signaling in the expression of Ro52 autoantigen that is partially mediated by IFNβ production. However, further investigation is needed to clarify the exact mechanisms involved in this regulation. Disclosure of Interest Non...
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