Speaker
Description
Introduction
Tendon injuries are common in equine athletes. Tissue healing occurs via biomechanically inferior scar tissue deposition, often resulting in re-injury. This is mediated, in part, by excessive proinflammatory cytokine release in the acute stages following injury. Stimulating equine tenocytes with interleukin 1 beta (IL-1β) alters tendon-associated and extracellular matrix remodelling gene expression in 2D culture and impairs 3D collagen gel contraction1. Similar findings were reported in humans tenocytes2, with activation of the NF-κB pathway suggested to facilitate these effects1,2. However, it is unknown how IL-1β modulates global gene expression in equine tenocytes in 3D culture and whether pharmacological NF-κB inhibition can attenuate the deleterious effects of IL-1β stimulation.
Methodology
Five biological replicates of adult tenocytes isolated from the equine superficial digital flexor tendon were cultured in a 3D collagen gel for 14 days under control or IL-1β-stimulated (1 nM) conditions, with media changes every 3-4 days. Daily gel contraction was examined before cells were harvested for RNA at day 14. Transcriptomic analysis was performed on a NovaSeq6000 platform, utilising Salmon-based alignment3 and differential expression analysis with DESeq2 (padj<0.05 and LogFC>1)4. Validation of several up-and-down regulated genes was undertaken with in-house qPCR. Gene Ontology and pathway analysis was performed with Panther Slim5 and Enrichr6, respectively. The interleukin 1 receptor antagonist (IL1Ra) protein1 and the NF-κB inhibitors JSH-237, IMD03548, and PF-066508339 were administered to tenocytes with-or-without IL-1β (1 nM); we are currently determining the rate of rescue for 3D collagen gel contraction, NF-κB P65 protein cytosol-nuclear shuttling after 60 min stimulation in 2D, gene expression after 72 hr in 2D, and IL-6 protein secretion with ELISA after 72 hr in 2D and during 14 days in 3D culture.
Results
Of the 18435 mapped genes, 2517 were differentially expressed following IL-1β exposure; 954 genes were upregulated and 1563 were downregulated. Gene ontology and pathway enrichment revealed that IL-1β enhanced NF-κB - but not JNK, P38, ERK, or STAT – signalling. Furthermore, IL-1β inhibited extracellular matrix organisation. IL1Ra fully rescued collagen gel contraction when co-administered with IL-1β, but only partially rescued tendon-associated and matrix remodelling gene expression at day 14. JSH-23 attenuated P65 nuclear translocation, reduced IL-6 secretion in 2D, and rescued matrix remodelling - but not tendon-associated - gene expression in 2D. Conversely, collagen gel contraction was not impacted by JSH-23 in the presence of IL-1β. Additional NF-κB inhibitor work is ongoing.
Conclusion
Exogenous IL-1β promotes NF-κB transcriptional signalling in equine tenocytes in 3D culture. These findings were verified with enhanced IL-6 secretion, a known target of P65. JSH-23 rescued IL-6 secretion and matrix remodelling gene expression after 72 hr in 2D, but had no influence on collagen gel contraction during 14 days in 3D culture. As IL1Ra fully rescued collagen gel contraction, this suggests a more global attenuation of NF-κB signalling is required to restore tenocyte function as other NF-κB proteins (i.e., c-REL, P50) may target tendon contraction genes. This will be addressed with the ongoing IMD0354 and PF-06650833 experiments.
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