Mir-125a: A novel regulator of IL-6 and TLR driven pathways in RA pathogenesis

Rainey, A.-A., Gilchrist, D. S., Tange, C. E., Frleta, M., Crawford, L., Baxter, D., McInnes, I. B. and Kurowska-Stolarska, M. (2013) Mir-125a: A novel regulator of IL-6 and TLR driven pathways in RA pathogenesis. In: 77th Annual Meeting of the American College of Rheumatology / 48th Annual Meeting of the Association of Rheumatology Health Professionals, San Diego, CA, 26-30 Oct 2013., S404.

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Background/Purpose: Molecular mechanisms driving disease initiation and chronicity in RA are incompletely understood. There is increasing interest in the role played therein by microRNAs–small RNA species that mediate post-transcriptional regulation of integrated pathways in mammalian cells. We recently profiled miRs in RA synovial fluid (SF) CD14+ macrophages in comparison to peripheral blood (PB) monocytes and identified dysregulation of miR-125a, which has not previously been associated with RA pathophysiology. Herein we aimed to characterise its expression and functional significance. Methods: Matched PB and SF CD14+ cells were isolated from RA patients (n=10). Additional PB samples were obtained from RA DMARD good responders (n=17), recurrent non-responders (n=11) and matched healthy controls (n=17). Primary human monocytes and THP-1 cells were stimulated with TLR ligands (LPS, PAM3, PolyIC, CLO97) and differentiated using M-CSF or GM-CSF for up to 7 days. Copy number of miR-125a was evaluated using qPCR. Targeted pathways were identified using prediction algorithms (e.g. TargetScan) and transcriptional profiling of SF CD14+ cells. Direct molecular interactions were confirmed using luciferase reporters. miR-125a or control mimic were transfected into THP-1 cells and IL-6R expression was evaluated by flow cytometry. Results: miR-125a was up-regulated in RA SF CD14+ macrophages compared to PB controls (p=0.002). Moreover, miR-125a was up-regulated in RA PB CD14+ monocytes compared with healthy controls, regardless of DMARD response status (p <0.02). Copy number of miR-125a in resting control PB monocytes was low. Extensive activation profiling revealed M-CSF, GM-CSF and 10% RA SF all induced miR125a expression at discrete time-points between 24h and 7ds. Of TLR ligands tested, only TLR4 agonism increased miR125a. Prediction algorithms identified members of the IL-6 signalling pathway (IL-6R, gp130) as potential targets of miR-125a. Luciferase reporter assays thereafter confirmed functional target interactions. Transfection of THP-1 cells with miR-125a but not control mimic reduced IL-6R membrane expression measured by FACS. miR-125a also targeted the negative regulators of TLR signalling, TNFAIP3 and IRF4, assessed by TargetScan and luciferase reporter. Commensurate with this IRF4 was down-regulated in RA PB monocytes (both DMARD good and recurrent non-responders) compared to healthy donors. Conclusion: Inflammatory cytokines, maturation factors and articular DAMPs drive elevated miR-125a expression in monocyte/macrophage lineages. miR-125a, in turn represents a novel molecular pathway that cross regulates IL-6R and TLR pathway activation in RA macrophages. We conclude that miR-125a represents an intriguing molecular marker with therapeutic and biomarker potential.

Item Type:Conference Proceedings
Additional Information:<br>Special Issue: 2013 Annual Meeting Abstract Supplement</br> Arthritis and Rheumatism, 65(10):S404
Glasgow Author(s) Enlighten ID:McInnes, Professor Iain and Gilchrist, Dr Derek and Frleta-Gilchrist, Dr Marina and Kurowska-Stolarska, Professor Mariola and Tange, Miss Clare
Authors: Rainey, A.-A., Gilchrist, D. S., Tange, C. E., Frleta, M., Crawford, L., Baxter, D., McInnes, I. B., and Kurowska-Stolarska, M.
College/School:College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Journal Name:Arthritis and Rheumatism
ISSN (Online):1529-0131

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