Speaker
Description
"Introduction
Tendon injuries occur commonly in human and equine athletes. Post-injury, the healing response is inadequate leading to increased deposition of scar-tissue and high re-injury rates. This has motivated the development of novel treatments which promote superior tissue regeneration. Particular interest has surrounded the use of bone-marrow mesenchymal stromal cells (BM-MSCs), with clinical investigations showing promising results1. Nevertheless, how BM-MSCs encourage tendon healing is unclear. Tendon injuries invoke an inflammatory response, and although moderate levels of inflammation are required to initiate tendon repair, evidence suggests inadequate resolution of inflammation contributes to fibrotic healing2. Previously, IL-1β was evidenced to exhibit negative effects on equine tenocytes, yet these consequences could be rescued by exogenous IL-1 receptor antagonist protein (IL1Ra)3. In contrast, embryonic stem cell (ESC) derived tenocytes appeared to be protected from the adverse effects of IL-1β, making them the ideal model to investigate tendinopathy further.
Methodology
Three biological replicates of equine adult tenocytes and ESC-tenocytes were cultured with IFN-γ (100 ng/ml), TNFα (10 ng/ml) and IL-1β (1 nM) and/or IL1Ra (100 ng/ml) prior to gene expression analysis and immunocytochemistry to determine inflammatory pathway activation. A 3-D culture model was used to determine the effects of IFN-γ, TNFα and IL-1β on collagen gel contraction by adult or ESC-tenocytes over 14 days. Adult tenocytes were also stimulated with IFN-γ, TNFα and IL-1β alone or in co-culture with BM-MSCs or BM-MSC conditioned media and the effects on signal induction, gene expression and 3-D collagen gel contraction measured.
Results
Stimulation of adult tenocytes with IFN-γ, TNFα and IL-1β resulted in significant changes in tendon-associated gene expression. Furthermore, these cytokines significantly inhibited 3-D collagen gel contraction by adult tenocytes. Immunocytochemistry demonstrated this combination of cytokines activated NF-κB, but not STAT1, JNK or P38 MAPK in adult tenocytes. These adverse effects could not be rescued by IL1Ra or factors produced by BM-MSCs. Conversely, ESC-tenocytes appear to be protected from IL-1β, TNFα and IFN-γ; generating tendon-like constructs indistinguishable from controls and demonstrating little to no changes to gene expression. Additionally, inflammatory stimulation failed to activate key inflammatory signalling pathways in ESC-tenocytes.
Conclusion
We demonstrate IL-1β, TNFα and IFN-γ work synergistically to induce greater detrimental consequences for adult tendon function than when used individually. Moreover, these adverse effects cannot be rescued by direct suppression of IL-1β. However, ESC-tenocytes appear to be protected from inflammatory stimulation, exhibiting minimal effects on gene expression and no activation of NF-κB, suggesting an association between undesired cellular activities in tenocytes and NF-κB signalling. Our results suggest BM-MSCs are unable to protect adult tenocytes from the adverse effects of inflammation. Understanding the mechanisms by which NF-κB signalling is blocked in ESC-tenocytes, and how BM-MSCs facilitate healing, may enable us to identify novel interventions for tendon injuries which minimise scar-tissue formation, resulting in diminished re-injury rates and improved quality of life.
References
- 1. Smith, R. K. et al., PLoS ONE. 8(9), 1-14 (2013). 2. Abraham, A. C. et al., Tech Shoulder Elb Surg. 18(3), 84-90 (2017). 3. McClellan, A. et al., Sci. Rep. 9, 2755 (2019).
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