1072 Evaluation of immune function of circulating leukocytes during the transition period in dairy cows

Abstract

The aims of the study were to determine immune function circulating leukocytes using an ex vivowhole blood stimulation assay (WBA) with lipopolysaccharide (LPS) and assess gene expression profiles by RNA sequencing in the transition period of dairy cows. The WBA was performed on whole blood of 6 Holstein multiparous cows at −20, −3, 3, 7 d from parturition (DFP) using 0, 0.01 and 5 µg LPS/mL. The plasma collected after stimulation was used to analyze IL-1β and IL-6 concentration via ELISA. The data were analyzed as a factorial design with repeated measures, using PROC MIXED in SAS. At the same day of the WBA test, RNA was isolated from whole blood and sequenced on a Hiseq1000 (Illumina, USA). Differential gene expression analysis was conducted with the edgeR package, and a general linear model was applied considering −20 DFP as the baseline. A threshold of 1.5-fold change and P < 0.05 were used to define differentially expressed genes (DEG), which were subsequently analyzed through the Dynamic Impact Approach (DIA) using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The IL-1b and IL-6 released after LPS stimulation was higher at −3 DFP (P < 0.05) in comparison with −20 DFP. After calving, the response of IL-1β to stimulation of LPS decreased markedly, while the IL-6 response was unchanged up to 3 DFP (P < 0.01 vs. -20 DFP) and then declined at 7 DFP. The most-impacted and activated KEGG pathways highlighted by the DIA analysis at −3 vs. −20 DFP were: PPAR signaling, adipocytokine signaling, hematopoietic cell lineage, ECM-receptor interaction and phagosome. After calving (3 and 7 DFP) the impact and activation of the above listed pathways was strongly increased, but there also was a strong inhibition of arachidonic acid metabolism (in particular enzymes regulating leukotrienes synthesis) as well as glycine, serine and threonine metabolism. Overall, the WBA and transcriptomic data confirmed changes in immune-competence of the circulating leukocytes around calving and, in particular, indicate mainly an increase of their activity and function. These data support the idea that the dairy cow's immune system is dysfunctional but not immunosuppressed around calving.