Background/Purpose: Previous studies have shown that the mRNA expression levels of the Ro52/TRIM21, Ro60/TROVE2 and La/SSB intracellular autoantigens are elevated within the immunopathologic lesions of SS. High surface constitutive expression of TLR-3 has been described in long-term cultured SGECs, whereas stimulation of TLR-3 via synthetic analogues or viral RNA has been shown to be potent inducer of several immune-modulatory molecules, interferons and other cytokines, as well as apoptosis, in SGEC. Given that TLR3-mediated inflammatory responses have been shown to be negatively regulated by Ro52/TRIM21 autoantigen we sought to investigate whether TLR3 stimulation has a reciprocal effect in Ro52/TRIM21, as well as Ro60/TROVE2 and La/SSB mRNA, expression by SGECs.

Methods: SGEC lines from SS patients (n=5) and non-SS controls (n=10), were treated with the analogue of the TLR3 ligand 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 was analyzed by real-time PCR at 0, 6, 12, 24, 48 and 72 hrs of treatment. The results were normalized by the expression of the HPRT1 gene and calculated by the ΔΔCT method using HeLa as calibrator. The expression of Ro52, Ro60 and La/SSB proteins was analyzed by immunoblotting, using specific antibodies. 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. The basal (constitutive) La/SSB and Ro60/TROVE2 mRNA levels were significantly upregulated following treatment of SGECs with polyI:C for 48-hrs (mean fold induction±SE: 0.86±0.18, p=0.003 and 3.29±1.75, respectively; p<0.0001 each). PolyI:C was found to be a strong inducer of Ro52/TRIM21 mRNA expression by SGECs at 6-hrs of treatment (mean fold induction of basal expression±SE: 13.12±4.5, p<0.0001). The polyI:C-induced Ro52/TRIM21 mRNA expression remained stable until 12-hrs of treatment, whereas a further increment was observed at 48-hrs of treatment (mean fold induction: 34.3±9.1 compared to basal levels, p<0.0001). The effect of polyI:C treatment in Ro52, Ro60 and La/SSB expression by SGECs  was further confirmed at the protein level by immunoblotting. Treatment with LPS was not found to affect the mRNA expression of the molecules studied (Ro52/TRIM21, Ro60/TROVE2 and La/SSB). PolyI:C or LPS treatment of HeLa cells did not show any effect on the expression levels of Ro52/TRIM21, Ro60/TROVE2 and La/SSB mRNAs.

Conclusion: Our findings indicate a reciprocal regulation of TLR3 signaling in SS-related intracellular autoantigens, and particularly Ro52 expression, by SGECs. Further investigation is needed to clarify the mechanisms involved in this regulation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tlr3-signaling-induces-the-expression-of-rossa-and-lassb-autoantigens-in-salivary-gland-epithelial-cells-sgecs/