2016 ACVIM Forum Research Abstract Program

Peripheral blood mononuclear cells were isolated from four healthy adult horses. Cells were cultured with LPS (10 ng/mL) or control for 0, 2 and 4 hours. Tumor necrosis factor alpha (TNFa) was measured in culture supernatants by ELISA. RNA was extracted from the cells and the miRNA transcriptome was sequenced using an Illumina HiSeq 2500 analyzer after size selection. High quality sequence data were mapped to the equine genome and annotated using the miRNA database miRBase 21. Differential expression was analyzed with generalized linear models, using the Benjamini-Hochberg procedure to control the false discovery rate at 0.1. 327 mature miRNAs were detected, with 191 present in all samples. Basal expression was highly consistent between horses. The most abundant miRNAs in baseline samples were miR-21, let-7 g and miR-150, miRNAs associated with cell cycle regulation and both innate and adaptive immunity in other species. TNFa expression was significantly higher in the supernatants from LPStreated cells than controls at both 2 and 4 hours (P = 0.016 and 0.0003 respectively). After correction for multiple comparisons, only miR-155 was significantly upregulated by LPS (P = 0.00018, 1.5 to 1.6 fold change versus controls). 9 miRNAs showed statistically significant expression changes with time. These included miR-146a and miR-146b, which are induced by LPS in other species but had non-significant upregulation by LPS here. MiR-155 expression was significantly correlated to supernatant TNFa (R 2 =0.78).