# Inhibition of interleukin-1 receptor-associated kinase (IRAK)-4 provides partial rescue of interleukin-1 beta induced functional and gene expression changes in equine tenocytes

## TL;DR

This study shows that inhibiting IRAK-4 partially reverses some effects of IL-1β on horse tendon cells in 3D culture, but full recovery requires broader intervention.

## Contribution

The study identifies IRAK-4 inhibition as a partial rescue mechanism for IL-1β effects in equine tenocytes, revealing limitations of targeting single signaling components.

## Key findings

- IRAK-4 inhibitor PF-06650833 partially rescued collagen contraction and IL-6 production in equine tenocytes exposed to IL-1β.
- RNA sequencing showed partial gene expression rescue by PF-06650833, but not as complete as with IL1Ra.
- ENPP2 expression was increased by IL-1β and rescued by both IL1Ra and PF-06650833, but direct ENPP2 inhibition reduced collagen contraction.

## Abstract

Interleukin 1 beta (IL-1β) is upregulated following a tendon injury and in vitro studies have shown that it leads to numerous negative effects on tendon cell function and gene expression. IL-1β activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and we hypothesised that inhibiting NF-κB activation would mediate the negative effects of IL-1β on equine tendon cells in 3-dimensional (3D) cultures.

Here, we tested three inhibitors of NF-κB signalling (Bortezomib, BAY11-7082 and Wedelolactone) along withTJ-M2010-5, an inhibitor of MyD88, which is a critical adaptor protein for mediating IL-1β signalling. None of these inhibitors were able to rescue gel contraction by equine tenocytes exposed to IL-1β in 3D culture. However, the daily application of the interleukin-1 receptor-associated kinase (IRAK)−4 inhibitor PF-06650833 resulted in a partial rescue of collagen contraction and interleukin-6 (IL-6) production by equine tenocytes in 3D culture. Global gene expression using RNA sequencing also revealed a partial rescue, although this was not as complete as that achieved using interleukin-1 receptor antagonist protein (IL1Ra), with many inflammatory pathways remaining upregulated. ENPP2 expression was significantly increased by IL-1β and rescued by both IL1Ra and PF-06650833 suggesting ENPP2 may be involved in collagen contraction. However, direct ENPP2 inhibition does not rescue IL-1β mediated inhibition of contraction and ENPP2 inhibition alone reduces collagen contraction.

Together, this data demonstrates that IL-1β has a broad mechanism of action on tendon cells which cannot be fully mediated by targeting specific parts of the signalling pathway.

The online version contains supplementary material available at 10.1007/s11033-025-11219-2.