Positive correlation between interleukin (IL) 1 beta to IL-1 receptor antagonist levels in Standardbred racehorses prior to racing

Abstract

Interleukin 1 beta (IL-1β) and IL-1 receptor antagonist (IL-1RA) are both upregulated following traumatic injury. As IL-1RA blocks inflammatory signaling by IL-1β, overexpression of IL-1β relative to IL-1RA may drive inflammatory diseases. As such, determination of the relationship between IL-1β to IL-1RA expression levels in horses may provide insight into disease states or serve as a therapeutic readout of response to medical interventions. As techniques to detect plasma concentrations of IL-1β and IL-1RA in horses lack sensitivity, we developed and validated novel enzyme-linked immunosorbent assays (ELISAs) to assess the levels of these cytokines in Standardbred racehorses prior to racing. The sandwich ELISAs we developed used analyte-specific polyclonal antibodies (PAb) for capture and their biotinylated conjugates for increased sensitivity of detection. Recombinant proteins were used to generate standard curves for calibration and quantification. During assay validation for linearity, specificity, precision, and accuracy, we did not observe any significant cross-reactivity with other proteins tested and serial dilution of plasma samples led to a proportional decrease in signal intensity. Finally, replacement of the detection Ab by capture Ab led to a proportional decrease in signal intensity. Using these ELISAs, we demonstrated that both IL-1β and IL-1RA concentrations increased significantly when whole blood was treated with lipopolysaccharide (p < 0.01). Moreover, we show that while plasma IL-1β and IL-1RA concentrations varied greatly in a Standardbred racehorse population (n = 312) at rest, ranging from 0 ∼ 48 ng/mL and 0 ∼ 112 ng/mL, respectively, they were positively correlated (rho_c = 0.875, Pearson’s r = 0.911, p < 0.001), with data points arranged symmetrically along a line of perfect concordance for the majority of samples. However, a few outliers (n = 7) were identified that deviated from this concordance and had plasma concentrations exceeding the upper limit of the standard curve (6000 pg/mL for IL-1β and 2000 pg/mL for IL-1RA), potentially identifying horses undergoing an inflammatory response. This study identified useful assays to quantify IL-1β and IL-1RA concentrations in equine plasma and suggests that an altered ratio of these cytokines in Standardbred racehorses may be worthy of further investigation.