Browsing by Author "Parkinson, Nicholas J."

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    Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing
    Parkinson, Nicholas J.; Buechner-Maxwell, Virginia A.; Witonsky, Sharon G.; Pleasant, R. Scott; Werre, Stephen R.; Ahmed, Sattar Ansar (PLOS, 2017-05-26)
    The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces differential microRNA expression in equine peripheral blood mononuclear cells in a manner comparable to humans. Illumina Next Generation Sequencing was used to characterize the basal microRNA transcriptome in isolated peripheral blood mononuclear cells from healthy adult horses, and to evaluate LPS-induced changes in microRNA expression in cells cultured for up to four hours. Selected expression changes were validated using quantitative reverse-transcriptase PCR. Only miR-155 was significantly upregulated by LPS, changing in parallel with supernatant tumor necrosis factor-α concentration. Eight additional microRNAs, including miR-146a and miR-146b, showed significant expression change with time in culture without a clear LPS effect. Target predictions indicated a number of potential immunity-associated targets for miR-155 in the horse, including SOCS1, TAB2 and elements of the PI3K signaling pathway, suggesting that it is likely to influence the acute inflammatory response to LPS. Gene alignment showed extensive conservation of the miR-155 precursor gene and associated promoter regions between horses and humans. The basal and LPS-stimulated microRNA expression pattern characterized here were similar to those described in human leukocytes. As well as providing a resource for further research into the roles of microRNAs in immune responses in horses, this will facilitate inter-species comparative study of the role of microRNAs in the inflammatory cascade during endotoxemia and sepsis.
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    Endotoxin-induced microRNA expression in equine peripheral blood mononuclear cells
    Parkinson, Nicholas J. (Virginia Tech, 2016-07-22)
    The innate immune response to lipopolysaccharide (LPS) mediated by toll-like receptor 4 (TLR4) contributes substantially to the morbidity of equine gastrointestinal disease, neonatal sepsis and other diseases. MicroRNAs (miRNAs), small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in TLR4 signaling regulation in other species. The central hypothesis of this study was that LPS induces differential expression of miRNAs in equine peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy adult horses and cultured with LPS or medium only for 2, 4 and 8 hours. Concentrations of inflammatory cytokines were measured in supernatants by immunoassay. Illumina Next-Generation Sequencing of the miRNA transcriptome was performed in PBMCs at 0, 2 and 4 hours. Selected expression changes were verified by qRT-PCR. 327 mature miRNAs were detected in equine PBMCs. Only miR-155 was significantly upregulated by LPS. 9 miRNAs showed statistically significant expression changes with time. Tumor necrosis factor-α concentration was significantly higher in supernatants from LPS-treated cells than controls from 2 hours, while interleukin-10 and interferon-γ were increased at 8 hours. miR-155 expression was correlated to all three cytokines. These data provide a foundation for future research into miRNA involvement in equine inflammatory responses. miR-155 is the principal LPS-induced miRNA in horses. Bioinformatic target predictions support roles in regulation of innate and adaptive immune responses including TLR4 signaling, as in humans. It is thus likely to influence the acute inflammatory response to LPS. Further research will be necessary to establish its role in naturally occurring disease.