Evaluating ex vivo toll-like receptor agonist response as a potential biomarker in equine gastric ulcer syndrome.

Equine gastric ulcer syndrome (EGUS) is highly prevalent in horses. Despite its presumed painful nature, evaluation of objective biomarkers associated with EGUS presence and severity remains limited. The objective of this study was to investigate the potential of interleukin-1 beta (IL-1β) release by peripheral blood mononuclear cells (PBMCs) following ex vivo stimulation of toll-like receptors 2 and 4 (TLR2 and TLR4) as a potential biomarker for EGUS status, with gastric lesions used as a proxy for presumed EGUS-associated pain. Venous blood was collected from 77 horses, PBMCs were isolated and stimulated with increasing concentrations of TLR2 and TLR4 agonists (Pam3CSK4 and lipopolysaccharide (LPS)) to assess IL-1β production. Fisher's exact test compared IL-1β responder proportion between horses with and without EGUS. Cochrane-Armitage test assessed trends across severity groups, and diagnostic potential was assessed via Receiver Operating Characteristic (ROC) curve analysis. Statistical significance was set at p < 0.05. Stimulation of TLR2 with Pam3CSK4 was unsuccessful (all <LLOQ) in all horses and was removed from final analysis. After exclusions for lameness and systemic inflammation, 25% of horses were LPS responders and EGUS was present in 92.2% of horses. No associations were identified between IL-1β response and EGUS presence (OR (95% CI) = 0.47 (0.049-6.2); p = 0.6). IL-1β AUC lacked diagnostic ability to differentiate between horses with and without EGUS (area under the ROC curve [AUROC] (95% CI) = 0.39 (0.11-0.67)), Equine Squamous Gastric Disease (ESGD) (AUROC = 0.30 (0.10-0.49)) and Equine Glandular Gastric Disease (EGGD) (AUROC = 0.56 (0.43-0.68)). The tested LPS-induced PBMC IL-1β release protocol was not associated with EGUS status in this cohort.