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 None Declared