Tumour necrosis factor alpha, interleukin 1 beta and interferon gamma have detrimental effects on equine tenocytes that cannot be rescued by IL-1RA or mesenchymal stromal cell-derived factors.

Tendon injuries occur commonly in both human and equine athletes, and poor tendon regeneration leads to functionally deficient scar tissue and an increased frequency of re-injury. Despite evidence suggesting inadequate resolution of inflammation leads to fibrotic healing, our understanding of the inflammatory pathways implicated in tendinopathy remains poorly understood, meaning successful targeted treatments are lacking. Here, we demonstrate IL-1ß, TNFα and IFN-γ work synergistically to induce greater detrimental consequences for equine tenocytes than when used individually. This includes altering tendon associated and matrix metalloproteinase gene expression and impairing the cells' ability to contract a 3-D collagen gel, a culture technique which more closely resembles the in vivo environment. Moreover, these adverse effects cannot be rescued by direct suppression of IL-1ß using IL-1RA or factors produced by BM-MSCs. Furthermore, we provide evidence that NF-κB, but not JNK, P38 MAPK or STAT 1, is translocated to the nucleus and able to bind to DNA in tenocytes following TNFα and IL-1ß stimulation, suggesting this signalling cascade may be responsible for the adverse downstream consequences of these inflammatory cytokines. We suggest a superior approach for treatment of tendinopathy may therefore be to target specific signalling pathways such as NF-κB.

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-1ß) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF-KB) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1ß for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF-KB inhibitors on gel contraction and IL6 secretion were measured in 3D culture, with NF-KB-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-1ß increased NF-KB-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-KB signaling. Administering direct pharmacological inhibitors of NF-KB did reduce NF-KB-P65 nuclear translocation, but had no effect on 3D gel contraction or IL6 secretion in the presence of IL-1ß. However, IL1Ra restored 3D gel contraction and partially rescued global gene expression. Tenocyte 3D gel contraction and gene expression is adversely impacted by IL-1ß which can only be rescued by blockade of interleukin 1 receptor, but not NF-KB, signalling.

Chronic ingestion of a low dose of caffeine induces tolerance to the performance benefits of caffeine.

This study examined effects of 4 weeks of caffeine supplementation on endurance performance. Eighteen low-habitual caffeine consumers (<75 mg · day-1) were randomly assigned to ingest caffeine (1.5-3.0 mg · kg-1day-1; titrated) or placebo for 28 days. Groups were matched for age, body mass, V̇O2peak and Wmax (P > 0.05). Before supplementation, all participants completed one V̇O2peak test, one practice trial and 2 experimental trials (acute 3 mg · kg-1 caffeine [precaf] and placebo [testpla]). During the supplementation period a second V̇O2peak test was completed on day 21 before a final, acute 3 mg · kg-1 caffeine trial (postcaf) on day 29. Trials consisted of 60 min cycle exercise at 60% V̇O2peak followed by a 30 min performance task. All participants produced more external work during the precaf trial than testpla, with increases in the caffeine (383.3 ± 75 kJ vs. 344.9 ± 80.3 kJ; Cohen's d effect size [ES] = 0.49; P = 0.001) and placebo (354.5 ± 55.2 kJ vs. 333.1 ± 56.4 kJ; ES = 0.38; P = 0.004) supplementation group, respectively. This performance benefit was no longer apparent after 4 weeks of caffeine supplementation (precaf: 383.3 ± 75.0 kJ vs. postcaf: 358.0 ± 89.8 kJ; ES = 0.31; P = 0.025), but was retained in the placebo group (precaf: 354.5 ± 55.2 kJ vs. postcaf: 351.8 ± 49.4 kJ; ES = 0.05; P > 0.05). Circulating caffeine, hormonal concentrations and substrate oxidation did not differ between groups (all P > 0.05). Chronic ingestion of a low dose of caffeine develops tolerance in low-caffeine consumers. Therefore, individuals with low-habitual intakes should refrain from chronic caffeine supplementation to maximise performance benefits from acute caffeine ingestion.

Equine Embryonic Stem Cell-Derived Tenocytes are Insensitive to a Combination of Inflammatory Cytokines and Have Distinct Molecular Responses Compared to Primary Tenocytes.

Tissue fibrosis following tendon injury is a major clinical problem due to the increased risk of re-injury and limited treatment options; however, its mechanism remains unclear. Evidence suggests that insufficient resolution of inflammation contributes to fibrotic healing by disrupting tenocyte activity, with the NF-κB pathway being identified as a potential mediator. Equine embryonic stem cell (ESC) derived tenocytes may offer a potential cell-based therapy to improve tendon regeneration, but how they respond to an inflammatory environment is largely unknown. Our findings reveal for the first time that, unlike adult tenocytes, ESC-tenocytes are unaffected by IFN-γ, TNFα, and IL-1ß stimulation; producing minimal changes to tendon-associated gene expression and generating 3-D collagen gel constructs indistinguishable from unstimulated controls. Inflammatory pathway analysis found these inflammatory cytokines failed to activate NF-κB in the ESC-tenocytes. However, NF-κB could be activated to induce changes in gene expression following stimulation with NF-κB pharmaceutical activators. Transcriptomic analysis revealed differences between cytokine and NF-κB signalling components between adult and ESC-tenocytes, which may contribute to the mechanism by which ESC-tenocytes escape inflammatory stimuli. Further investigation of these molecular mechanisms will help guide novel therapies to reduce fibrosis and encourage superior tendon healing.

Effect of a moderate caffeine dose on endurance cycle performance and thermoregulation during prolonged exercise in the heat.

OBJECTIVES: This study investigated the influence of a moderate caffeine dose on endurance cycle performance and thermoregulation during prolonged exercise in high ambient temperature. DESIGN: Double-blind cross-over study. METHODS: Eight healthy, recreationally active males (mean±SD; age: 22±1 years; body mass: 71.1±8.5kg; VO2peak: 55.9±5.8mLkg-1min-1; Wmax: 318±37W) completed one VO2peak test, one familiarisation trial and two experimental trials. After an overnight fast, participants ingested a placebo or a 6mgkg-1 caffeine dose 60min before exercise. The exercise protocol consisted of 60min of cycle exercise at 55% Wmax, followed by a 30min performance task (total kJ produced) in 30°C and 50% RH. RESULTS: Performance was enhanced (Cohen's d effect size=0.22) in the caffeine trial (363.8±47.6kJ) compared with placebo (353.0±49.0kJ; p=0.004). Caffeine did not influence core (p=0.188) or skin temperature (p=0.577) during exercise. Circulating prolactin (p=0.572), cortisol (p=0.842) and the estimated rates of fat (p=0.722) and carbohydrate oxidation (p=0.454) were also similar between trial conditions. Caffeine attenuated perceived exertion during the initial 60min of exercise (p=0.033), with no difference in thermal stress across trials (p=0.911). CONCLUSIONS: Supplementation with 6mgkg-1 caffeine improved endurance cycle performance in a warm environment, without differentially influencing thermoregulation during prolonged exercise at a fixed work-rate versus placebo. Therefore, moderate caffeine doses which typically enhance performance in temperate environmental conditions also appear to benefit endurance performance in the heat.

Supplementation with a low-dose of octopamine does not influence endurance cycling performance in recreationally active men.

OBJECTIVES: The aim of this study was to examine the influence of octopamine supplementation on endurance performance and exercise metabolism. DESIGN: Double-blind cross-over study. METHODS: Ten healthy, recreationally active men (Mean±SD; age: 24±2 years; body mass: 78.4±8.7kg; VO2peak: 50.5±6.8 mLkg-1min-1) completed one VO2peak test, one familiarisation trial and two experimental trials. After an overnight fast, participants ingested either a placebo or 150mg of octopamine 60min prior to exercise. Trials consisted of 30min of cycle exercise at 55% peak power output, followed by a 30min performance task whereby participants completed as much work (kJ) as possible. RESULTS: Performance was similar between the experimental trials (placebo: 352.8±39kJ; octopamine: 350.9±38.3kJ; Cohen's d effect size=0.05; p=0.380). Substrate oxidation and circulating concentrations of free fatty acids, prolactin and cortisol were similar between trial conditions (all p>0.05). There were also no differences across trials for heart rate or perceived exertion during exercise (both p>0.05). CONCLUSIONS: Acute supplementation with a low dose of octopamine did not influence endurance cycle performance, substrate oxidation or circulating hormonal concentrations, which could be due to the low serum octopamine concentrations observed. Future studies should investigate the influence of larger doses of octopamine in recreationally active and well-trained individuals during prolonged exercise in temperate and high ambient conditions.