Targeting the cytokine-redox axis: dose-dependent immunomodulation by methotrexate in a biomarker-guided ovine endotoxaemia model.

Background: Oxidative stress and excessive inflammation are central to the pathophysiology of sepsis and its large-animal analogue, endotoxaemia. Current anti-inflammatory treatments in ruminants lack efficacy in modulating redox balance or acute-phase responses. Low-dose methotrexate (MTX), a known immunometabolic modulator, may offer targeted attenuation of these processes.

Objective: To evaluate the anti-inflammatory, antioxidant, and organ-sparing effects of two low-dose MTX regimens in a controlled ovine model of lipopolysaccharide (LPS)-induced endotoxaemia.

Methods: 20 clinically healthy ewes were randomised into four groups (n = 5/group): Ctrl - (saline), Ctrl + (LPS), LD-MTX1 (LPS + MTX 0.45 mg/kg), and LD-MTX2 (LPS + MTX 0.90 mg/kg). Endotoxaemia was induced via intravenous Escherichia coli O55:B5 LPS, and MTX was administered intramuscularly 1 h post-challenge. Serum biomarkers (TAC, GPx, MDA, haptoglobin, ALT, AST, urea, creatinine and CK-MB), clinical parameters and behavioural scores were assessed at five time points over 24 h. Data were analysed using mixed-effects models with adjusted post hoc contrasts.

Results: Both MTX regimens improved TAC within 6 h; LD-MTX2 showed a higher 6 h peak vs Ctrl + (p = 0.0006) and LD-MTX1 increased TAC AUC₀-6 h (p = 0.048). GPx displayed a Group × Time interaction: LD-MTX1 rebounded by 4-6 h, whereas LD-MTX2 recovered later by 24 h. HP was non-monotonic: AUC₀-6 h was lower with LD-MTX1 vs Ctrl + (p = 0.045) but higher with LD-MTX2 vs Ctrl + (p = 0.0002). LD-MTX2 markedly suppressed fever (+ 0.36 °C vs + 1.88 °C in Ctrl + ; p = 0.0011). The organ signals were dose-specific; LD-MTX1 showed no biochemical organ stress, and LD-MTX2 caused transient ALT/AST rises (p ≤ 0.006) with renal indices trending lower and no CK-MB increase. The clinical and behavioural metrics mirrored these effects.

Conclusion: Low-dose MTX at 0.45 mg kg⁻¹ produced early redox benefit with partial GPx recovery and lower HP AUC, without biochemical organ stress. The 0.90-mg kg⁻¹ dose improved temperature control and peak TAC but increased HP AUC and caused transient ALT/AST elevations, indicating a narrow therapeutic window. MTX, therefore, warrants evaluation as a precision, time-limited adjunct in early endotoxaemia/sepsis in ruminants, with dose selection guided by time-integrated biomarkers and early liver-enzyme monitoring.