Systemic inflammation and priming of peripheral blood leukocytes persist during clinical remission in horses with heaves

Abstract

Objective: To compare innate immune responses of peripheral blood leukocytes from healthy and asymptomatic heaves-affected horses.

Animals: Heaves-affected horses (n=5-6) and healthy controls (n=4-5) kept under low dust environments (pasture or shavings and pellets).

Methods: Blood neutrophil and neutrophil-depleted cell populations were isolated using MACS system. Cells were incubated with or without bacterial products (lipopolysaccharide (LPS), 100 ng/mL and fMLP, 5 ng/mL, 5 h). Cytokine (IL-1β, IL-8, TNF, IL-4, INFγ and IL-10) and receptor (TLR4) mRNA expression was assessed by qPCR. TNF concentration in culture supernatants and serum samples was assessed using equine specific ELISA. Apoptotic rate of resting and stimulated neutrophils was assessed by flow cytometry using AnnexinV and 7-AAD (18 h) and correlated with early pro-inflammatory cytokine expression in the same cells (5 h).

Results: Stimulation with bacterial-derived products resulted in overexpression of pro-inflammatory cytokines in both neutrophils (IL-1β and TNF) and neutrophil-depleted leukocytes (IL-1β and IL-8) from heaves-affected horses. Neutrophil survival (18 h) was associated with their early TNF expression, but not IL-8. Neutrophil-depleted leukocytes from these horses also had significantly increased basal TNF mRNA levels. Serum TNF concentration was also significantly higher in heaves-affected horses compared to healthy horses kept in similar environment.

Conclusions: Altered innate immune response to bacterial products is observable ex vivo in peripheral blood leukocytes from asymptomatic heaves-susceptible horses and is associated with high serum TNF concentration. It remains to be determined if this phenomenon is caused by intrinsic differences in innate immune responses or to cellular priming caused by systemic inflammation.