Background Microparticles (MPs) have been described to function as extracellular vehicles that shuttle microRNAs (miR) from platelets to endothelial cells1 and regulate recipient cell gene expression. Crosstalk of synovial monocytes (MC) with synovial fibroblasts (SF) is a key step to the inflammatory process in rheumatoid arthritis (RA).
Objectives Herein, we investigated whether delivery of specific miRs by MC-derived MPs affects RASF behaviour.
Methods Blood samples of healthy volunteers and RA patients were collected. MPs were obtained by differential centrifugation. CD14+ cells were isolated from PBMCs by using positive selection and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 6 days. Scanning electron microscopy, nanosight trafficking analysis and flow cytometry (FACS) have been applied to characterize MPs from human CD14+MCs and THP-1 cells. Total and smallRNA sequencing were performed on macrophages (n=4) and RASFs (n=4) as well as on macrophage-derived MPs (n=4). (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 with FGF-2 as positive control, as well as THP-1 derived MPs, miR-223 mimic, miR-223 inhibitor, control mimic and inhibitor were carried out.
Results MPs from MCs & THP-1 cells are about 250nm in size (range 50–800nm). SmallRNA sequencing revealed high levels of miR-223 in macrophages as opposed to a lack of its expression in RASF. If co-cultured with MCs for 3d, RASF acquire miR-223 expression, but not pre-miR-223 to a significant extent (n=6, Ct values <30) confirming transfer of mature miR-223 between cells. Prediction algorithms identified FBXW7 as a candidate target for miR-223 in RASF. FBXW7 acts as an ubiquitin ligase targeting cyclin E. Co-culture of RASF with THP-1 derived MPs increased RASF proliferation to a similar extent as FGF-2 (n=8, p<0.01). Transfection of RASF with miR-223 inhibitor prior to stimulation with THP-1 MPs abrogates their proliferative effect (n=5, 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).
Conclusions miR-223 transport from MCs to SFs by MPs represents a novel intercellular mechanism that could stimulate SF proliferation during initiation or chronicity of synovial inflammation in RA.
Laffont, B. et al. Blood (2013)
Acknowledgement We wish to thank Derek Baxter, Diane Vaughan, Margaret Mullin, Pawel Herzyk, Julie Galbraith, Russka Shumnalieva for their support and technical assistance.
Disclosure of Interest None declared