In Vitro Treatment of Equine Peripheral Blood Mononuclear Cells with Lactate, Heat, and Lipopolysaccharide

Abstract

Peripheral blood mononuclear cells (PBMCs) play a central role in immune regulation and mediating inflammation through pro- and anti-inflammatory cytokine secretion in response to physiological stressors such as exercise. Isolated PBMCs provide a controlled in vitro model to investigate exercise-induced byproducts independent of confounding systemic variables. We examined whether heat and lactate are additive or synergistic for cytokine gene expression in PBMCs. Fresh PBMC isolates were treated with lactate (0, 2, 4, or 20 mM) or lipopolysaccharide (LPS; E. coli 026:B6, 5 ug/mL final concentration) as a positive control for 1 or 4 hours at 37 °C. Heat exposure experiments incubated cells at 37 °C or 42 °C for 20 minutes or 1 hour, with additional trials combining heat and lactate. Following treatment, RNA or protein was isolated for the quantification of IL-1β, IL-6, IL-8, IL-10, and TNF-α gene expression, which was quantified using RT-qPCR, and activation of NF-κB and ERK 1/2 was assessed by Western blot. Lactate had a main effect (P ≤ 0.05), with the greatest expression of pro-inflammatory TNF-ɑ, IL-1β, and IL-8, following incubation with 20 mM lactate; the same concentration reduced expression of anti-inflammatory IL-6 and IL-10. Lactate had no effect on pS536-NF-κB, pS276-NF-κB, or pERK1/2, whereas LPS significantly increased them. RT-qPCR experiments showed that LPS signaling is 50-300 fold more potent than lactate as a PBMC stimulant. Lactate pre-treatment at 20 mM did not affect subsequent LPS-driven cytokine gene expression (P ≥ 0.05), indicating that physiological concentrations of lactate following exhaustive exercise do not seem to prime horses for a bacterial infection.