Search

ObjectivesIncreasing evidence suggests that inflammatory mechanisms play a key role in chronic tendon disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence disease perpetuation.MethodsT cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged tendon or interleukin-1β was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes.ResultsSignificant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte–T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio.ConclusionsInteraction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human tendon disorders.

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 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. References 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