OP0098 INFLAMMATORY GENE ENHANCERS ARE SHARED IN SYNOVIAL FIBROBLASTS AND MINOR SALIVARY GLAND FIBROBLASTS

Background

Cell type- and stimulus-specific enhancers are key regulatory elements in chronic inflammatory rheumatic diseases, such as rheumatoid arthritis (RA) and Sjögren`s syndrome (SjS). Recent transcriptome profiling of synovial fibroblasts (SF) from patients with RA and minor salivary gland-derived fibroblast (SGF) from patients with SjS point to similarities of fibroblast populations derived from these two chronic inflammatory diseases. Enhancer RNAs (eRNAs) are short-lived non-coding RNAs transcribed from cell type-specific enhancers that facilitate the transcription of their linked coding genes. The activation and interference with transcribed enhancers in SF and SGF has not been studied yet.

Objectives

To characterise the activation and regulation of transcribed inflammatory gene enhancers in RA SF and SGF.

Methods

SF were isolated from synovial tissues of patients with RA undergoing joint replacement surgery. SGF were isolated from minor salivary gland biopsies of patients with a suspected SjS, including samples of patients with SjS (n=3) and controls (n=3) that do not fulfil the classification criteria. The expression of eRNAs in SF (n=9) stimulated with TNF (24h; 10 ng/µl) or left untreated, was detected by cap analysis of gene expression followed by sequencing (CAGEseq). RA SF (n=7) and SGF (n=6) were stimulated with TNF (1, 3, 6, 24h; 10 ng/µl), IL1 (1, 24h; 1 ng/ml), or the Toll-like receptor agonists pIC (1, 24h; 10 μg/ml) and LPS (1, 24h; 100 ng/µl). The expression of selected eRNAs and their corresponding coding genes was analysed by real-time PCR. Samples containing the untranscribed RNA were measured in parallel. To study eRNA regulation, SF were treated with the bromodomain inhibitor I-BET (1 µM; 24h), prior to stimulation with TNF.

Results

We have identified and selected a set of TNF-induced eRNAs for the pro-inflammatory genes CCL2, IL8, IL6, CXCL1 and CCL20 for a more detailed analysis. These eRNAs were located upstream (eCCL2#1, eCCL2#2, eCXCL1#2, eIL6#1, eIL8#1-3), downstream (eCCL2#3, eCCL2#4, eCXCL1#1) and intronic (eCCL20) at distances between 300 bp to 35.6 kb relative to the transcription start sites of the corresponding coding genes. CAGEseq revealed a patient-dependent variability in eRNA expression, with none of the selected eRNAs being present in SF of all nine patients, and the existence of several TNF-induced eRNAs for individual coding genes. By performing TNF time course experiments in SF, we have detected different patterns of eRNAs: (a) eRNAs, that peaked at 1h (eCCL20, eIL8#2, eCCL2#1), (b) at 6h (eCXCL1#1), (c) or at 24h (eIL8#1, eIL8#3, eIL8#4, eCXCL1#2) after stimulation, and (d) eRNAs that were stably expressed over the time points (eCCL2#2, 3, 4). The same set of eRNAs and corresponding coding genes was induced in SGF, indicating that inflammatory gene enhancers are shared in fibroblasts from different localisation and diseases. All inflammatory stimuli induced the expression of eRNAs in SF and SGF. However, we detected some differences regarding the peak of eRNA expression (eCCL2#2, eCCL2#3, eCXCL1#2) and the potency of different stimuli to induce individual eRNAs. Treatment of SF with I-BET suppressed the expression of all eRNAs tested.

Conclusion

SGF, similar to SF, respond to different pro-inflammatory stimuli by inducing the expression of transcribed eRNAs and their corresponding coding genes. The expression of some eRNAs is maintained for up to 24h, contradicting previous reports that eRNAs are short-lived. Bromodomain inhibitors are sufficient to prevent the activation of eRNAs.

REFERENCES:

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Acknowledgements:

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Disclosure of Interests

Larissa Moser: None declared, Matthias Brunner: None declared, Miranda Houtman: None declared, Marco Sprecher: None declared, Muriel Elhai Consultant of: BMS, Grant/research support from: Support for travel (Janssen), Oliver Distler Consultant of: Abbvie, Britta Maurer Speakers bureau: Boehringer-Ingelheim, GSK, Novartis, Consultant of: Novartis, Boehringer Ingelheim, Janssen-Cilag, GSK, Grant/research support from: AbbVie, Protagen, Novartis Biomedical; congress support from Medtalk, Pfizer, Roche, Actelion, Mepha, and MSD, Caroline Ospelt: None declared, Kerstin Klein: None declared.