Background/Purpose: Recently, it was shown that extracellular vesicles (EV) convey microRNAs (miR) from platelets to endothelial cells¹and regulate recipient cell gene expression. Interaction of synovial monocyte (MC)/macrophages with synovial fibroblasts (SF) is a key step to the inflammatory process in rheumatoid arthritis (RA). We hypothesised that MC/macrophage regulate SF behavior by delivering specific microRNA via EVs.

Methods: Characterization of EVs from human CD14⁺MCs and THP-1 cells was performed using scanning electron microscopy (SEM, n=3), nanosight trafficking analysis (NTA, n=3-7); and flow cytometry (n>4) with predefined sized beads for gating. smallRNA sequencing was performed on macrophages (n=4) and RASFs (n=4) as well as on macrophage-derived EVs (n=4). Macrophages were differentiated by culturing peripheral blood (PB) CD14⁺MCs from RA patients with M-CSF and either stimulated with LPS or not. In addition, matched RA PB and synovial fluid CD14⁺ (n=8), PB CD14⁺ from RA DMARD responders (n=16), DMARD non-responders (n=22), Biologic resistant (n=41), and age-matched healthy controls (n=21) were isolated and used for validation. (Pre-)miR-223 expression and copy number was quantified in cells or tissues via TLDA, qPCR & in situ hybridisation with specific primers and probes. Targeted pathways were identified using prediction algorithms (TargetScanHuman6.2). Transfer of miR-223 from MCs to SFs was determined by fluorescence microscopy, direct cell co-culture and FACS, as well as transwell co-culture. RASF Proliferation assays and IL-6 ELISAs served as read-out.

Results: SEM & NTA revealed a mean size of EVs from MCs & THP-1 cells of about 250nm (range 50-800nm). EV production was induced in myeloid cells by LPS (p<0.05). SmallRNA sequencing revealed high levels of miR-223 in macrophages as opposed to a lack of its expression in RASF. miR-223 levels were significantly higher in PB CD14⁺ cells of DMARD responders compared to DMARD non-responders and Biologic resistant RA patients (p<0.05 and p<0.01). miR-223 levels in CD14⁺ cells showed significant positive correlations to CDAI and SDAI. miR-223 was downregulated in synovial fluid MCs compared to matched PB controls (p< 0.01) and its expression was down-regulated in MCs/macrophages upon LPS stimulation. In contrast, EVs derived from LPS stimulated THP-1 cells contained higher levels of miR-223 suggesting stimulation driven packing of miR-223 into EVs. If co-cultured with MCs for 3d, RASF acquire miR-223 expression, but not pre-miR-223 to a significant extent (n=6, C_tvalues <30) confirming transfer of mature miR-223 between cells. Prediction algorithms identified FBXW7, a ubiquitin ligase targeting cyclin E, as a candidate target for miR-223 in RASF. Co-culture of RASF with THP-1-derived EV increased RASF proliferation (n=3, p<0.05). miR-223 transfection (25nM & 50nM) itself led to an increase of RASF proliferation after 72 and 96h (n=6-8, p<0.01 for all).

Conclusion: miR-223 transfer from MCs/macrophages to SFs by EVs during initial or chronic phases of synovial inflammation could promote SF proliferation and represent a novel intercellular mechanism underlying the pathogenesis of rheumatoid arthritis.

¹Laffont, B. et al. Blood (2013)

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mirna-223-delivery-to-synovial-fibroblasts-via-monocyte-derived-extracellular-vesicles-promotes-their-proliferation/