Exogenous interleukin-1 beta stimulation regulates equine tenocyte function and gene expression in three-dimensional culture which can be rescued by pharmacological inhibition of interleukin 1 receptor, but not nuclear factor kappa B, signaling

We investigated how Interleukin 1 beta (IL-1 beta) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-kappa B (NF-B-K) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1 beta for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF-B-K inhibitors on gel contraction and IL6 secretion were measured in 3D culture, with NF-B-K-P65 nuclear translocation by immunofluorescence and gene expression by qPCR measured in two-dimensional (2D) monolayer culture. In addition, daily 3D gel contraction and transcriptomic analysis was performed on interleukin 1 receptor antagonist-treated 3D gels at day 14. IL-1 beta increased NF-B-K-P65 nuclear translocation in 2D culture and IL6 secretion in 3D culture, but reduced daily tenocyte 3D gel contraction and impacted > 2500 genes at day 14, with enrichment for NF-B-K signaling. Administering direct pharmacological inhibitors of NF-B-K did reduce NF-B-K-P65 nuclear translocation, but had no effect on 3D gel contraction or IL6 secretion in the presence of IL-1 beta. However, IL1Ra restored 3D gel contraction and partially rescued global gene expression. Tenocyte 3D gel contraction and gene expression is adversely impacted by IL-1 beta which can only be rescued by blockade of interleukin 1 receptor, but not NF-B-K, signalling.