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

Overview

• This study assessed whether the release of the inflammatory molecule interleukin-1 beta (IL-1β) from horse immune cells after stimulation with certain immune system activators could serve as a biomarker for equine gastric ulcer syndrome (EGUS), a common painful stomach condition in horses.
• The results showed no significant association between this immune response and the presence or severity of EGUS, indicating that this approach may not be useful for diagnosing or monitoring the disease.

Background

• Equine gastric ulcer syndrome (EGUS) is a widespread condition in horses characterized by painful ulcers in the stomach lining.
• Despite its suspected painful nature, there is a lack of objective biomarkers to indicate the presence and severity of EGUS.
• Biomarkers could help diagnose the condition and assess treatment effectiveness objectively.

Objective

• To investigate whether IL-1β release by peripheral blood mononuclear cells (PBMCs) after stimulation with toll-like receptor (TLR) agonists—specifically TLR2 and TLR4—could serve as a biomarker for EGUS status.
• Gastric lesions observed via endoscopy were used as a proxy for presumed pain associated with EGUS.

Methods

• Venous blood was collected from 77 horses with various EGUS statuses.
• Peripheral blood mononuclear cells (PBMCs) from the blood samples were isolated.
• These PBMCs were stimulated ex vivo with increasing concentrations of:
• Pam3CSK4 to activate TLR2
• Lipopolysaccharide (LPS) to activate TLR4
• IL-1β production in response to these stimulations was measured.
• Statistical tests used included:
• Fisher’s exact test to compare the proportion of IL-1β responders among horses with and without EGUS.
• Cochrane-Armitage test for trends across severity groups.
• Receiver Operating Characteristic (ROC) curve analysis to assess diagnostic ability.
• Statistical significance was determined at p < 0.05.
• Horses with lameness or systemic inflammation were excluded from final analyses to eliminate confounding factors.

Key Findings

• The TLR2 agonist Pam3CSK4 failed to induce measurable IL-1β release in all horses and was therefore excluded from further analysis.
• Only 25% of the horses responded with IL-1β release to the TLR4 agonist LPS.
• EGUS was present in 92.2% of the horses studied, demonstrating the high prevalence of the condition in the cohort.
• No statistically significant association was found between IL-1β responder status and the presence or absence of EGUS (Odds Ratio = 0.47, p = 0.6).
• ROC curve analysis showed poor diagnostic accuracy for IL-1β levels in differentiating horses with EGUS or its subtypes:
  • Overall EGUS: AUROC = 0.39 (near chance level)
  • Equine Squamous Gastric Disease (ESGD) subtype: AUROC = 0.30
  • Equine Glandular Gastric Disease (EGGD) subtype: AUROC = 0.56

Interpretation

• The inability of Pam3CSK4 to stimulate IL-1β suggests that TLR2 activation in PBMCs may not be a viable pathway for assessing inflammation related to EGUS.
• Only a minority of horses responded to TLR4 stimulation, and this response was not linked with EGUS presence or severity.
• The measured IL-1β release thus did not correlate with gastric disease status and cannot be recommended as a biomarker for EGUS based on this study.
• This suggests that EGUS-associated pain and inflammation may not be reflected by systemic TLR-induced IL-1β production from circulating immune cells.

Study Limitations and Future Directions

• The study focused solely on IL-1β production from PBMCs after ex vivo stimulation, which may not fully capture complex in vivo inflammatory responses.
• Only two TLR pathways (TLR2 and TLR4) were tested; other pathways or inflammatory markers may be more relevant.
• Further research is needed to identify reliable objective biomarkers that correlate with the severity and presence of gastric ulcers in horses.
• Exploring other immune cells, different cytokines, or in vivo inflammatory markers could yield more promising diagnostic tools.

Conclusion

• The tested ex vivo protocol measuring LPS-induced IL-1β release from horse PBMCs was not effective as a biomarker for diagnosing or assessing equine gastric ulcer syndrome.
• This highlights the ongoing challenge of objectively measuring EGUS-associated pain and encourages continued biomarker discovery efforts in equine medicine.