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MiRNAseq of neutrophils during the transition period in cows with divergent metabolic phenotypes
Several adaptations in leukocytes, i.e. neutrophils, are required for a successful transition to lactation in dairy cows. Micro RNA (miRNA) molecules are small non-coding RNAs that regulate gene expression; their importance in immune cell function has been well documented. We characterized the miRNA of neutrophils isolated from dairy cows divergent in their risk of infection or metabolic dysfunction, at three time points over the transition period: day of calving, 1 wk, and 4 wk post-calving. From a total of 150 cows, 10 cows were selected with high (n = 5; High Risk) and low (n = 5; Low Risk) concentrations of non-esterified fatty acids, β-hydroxybutyrate, and liver triacylglycerol during wk 1 and 2 post-calving. Neutrophils were isolated from whole blood using differential centrifugation. Flow cytometric analysis of these isolates revealed a median of 75% ± 2% neutrophils (± SEM). Total RNA was extracted from neutrophils using TRIzol®, size selected for miRNA, and sequenced on the Illumina HiSeq. The miRNA reads were mapped to the Bos taurus 6 genome (UMD 3.1) using Bowtie 2 and counted. Differential expression analysis was conducted using the limma/voom R-package and pair-wise analyses were conducted to assess differential expression between risk categories and across time points, with a false discovery rate set at 0.05. There was no effect of risk category on miRNA expression on the day of calving or 4 wk post-calving. However, expression of mir-19b, mir-148a, and mir-21 in the Low Risk cows tended (adj P = 0.1) to be greater at 1 wk post-calving. When assessed for the effect of time relative to the day of calving, regardless of risk, expression of miR-150 and -486 increased (P ≤ 0.05) at 1 wk post-calving and eight miRNA genes were differentially expressed at 4 wk post-calving (P ≤ 0.05): miR-150 and -30c were greater, and miR-19b, -19a, -30d, -101-1, and -106b were lower. The results indicate that the divergent metabolic phenotype did not significantly alter miRNA in neutrophils during early lactation. However, the altered miRNA profile in neutrophils over time indicates an important role for miRNA in the regulation of immune cell function during the peripartum period.
Keywords: MicroRNA, dairy cows, transition