Mir-34a in Rheumatoid Arthritis: characterisation of elevated synovial expression and association with treatment resistance

Tange, C. E., Alivernini, S., Gilchrist, D. S., Crawford, L., Rainey, A.-A., Baxter, D., McInnes, I. B. and Kurowska-Stolarska, M. (2013) Mir-34a in Rheumatoid Arthritis: characterisation of elevated synovial expression and association with treatment resistance. 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., S403.

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Abstract

Background/Purpose: Cells of the monocyte/macrophage lineage are critical to RA pathogenesis: unravelling mechanisms underlying macrophage inflammatory gene expression should elucidate novel disease-associated pathways and thereby biomarkers and therapeutic targets. MicroRNA (miR) comprise small, non-coding RNA species that are key regulators of mammalian gene expression. We sought to define the expression and regulation of novel miR species that regulate RA macrophage biology.

Methods: Blood (PB; healthy controls and RA patients with defined treatment characteristics) or synovial fluid (SF; RA) CD14+ cells were purified using histopaque centrifugation and autoMACS bead separation. CD14+ cells were matured with M-CSF or GM-CSF for up to 7 days, or stimulated with various TLR ligands. For T cell -macrophage interaction, CD4+ T cells were cultured with TNF, IL-2, IL-6, prior to 24hr co-culture with M-CSF-matured CD14+ cells. miR expression and copy number was variously quantified in cells or tissues via TLDA, validatory qPCR and in situ hybridisation with specific primers and DIG or FITC labelled probes, respectively.

Results: Prior TLDA elicited several dysregulated miRs in RA SF compared to PB CD14+ cells, including miR-34a. Fold change and copy number PCR assays confirmed elevated miR-34a in RA SF cells (n=10; p <0.01). Crucially miR-34a was also elevated in PB CD14+ cells from biologic-resistant RA patients compared to cDMARD good responders or matched healthy controls (n=19–30), associating miR-34a expression with chronic, treatment-resistance. miR-34a expression correlated with disease activity assessed by swollen joint count. In situ hybridisation demonstrated elevated miR-34a expression in RA compared with noninflammatory and inflammatory OA synovial tissues (n=6); double fluorescent staining confirmed expression in lining and sub-lining layer CD68+ macrophages, plus adjacent cells of FLS morphology. miR-34a was rapidly upregulated during monocyte maturation induced by adherence, M-CSF or GM-CSF, but was not further enhanced by addition of TLR ligands (LPS, CLO97, PAM3, PolyIC, CpG). Similarly co-culture with RA derived SF (10%) enhanced miR-34a expression in monocytes. Finally, cognate interactions between CD4+ T cells and macrophages further enhanced miR-34a expression in the latter cells.

Conclusion: miR-34a expression is elevated in RA SF macrophages and in PB monocytes of treatment resistant RA patients where it correlates with clinical disease activity measures. miR-34a is upregulated by the maturation rich synovial microenvironment and by interactions with a activated T cells. Putative miR-34a molecular targets include NOTCH1 rendering it a plausible novel immune regulator in synovitis.

Item Type:Conference Proceedings
Additional Information:
Special Issue: 2013 Annual Meeting Abstract Supplement
Arthritis and Rheumatism 65(10):403
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:McInnes, Professor Iain and Gilchrist, Dr Derek and Kurowska-Stolarska, Dr Mariola and Tange, Miss Clare and Alivernini, Mr Stefano
Authors: Tange, C. E., Alivernini, S., Gilchrist, D. S., Crawford, L., Rainey, A.-A., Baxter, D., McInnes, I. B., and Kurowska-Stolarska, M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Arthritis and Rheumatism
Publisher:Wiley-Blackwell
ISSN:0004-3591
ISSN (Online):1529-0131

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