Abstract
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.
Highlights
Excessive activation of the innate immune system can be as damaging to the host as the insult that triggered it
Improved understanding of the regulation of this system could facilitate the design of therapeutic interventions that could limit the damaging consequences of the inflammatory response without inducing a deleterious state of immunosuppression, potentially improving outcomes for intensive care patients
We aimed to characterize the pattern of LPS-induced expression change in equine peripheral blood mononuclear cells (PBMCs), with a view to establishing areas of common ground between equine endotoxemia and the human response to endotoxin exposure and subsequent septic shock
Summary
Excessive activation of the innate immune system can be as damaging to the host as the insult that triggered it. One aspect of TLR signaling regulation that is gaining increasing attention is the role of post-transcriptional regulation by microRNAs (miRNAs) [1]. These are a recently discovered class of non-coding RNA, 17–25 nucleotides in length, that function via interactions with messenger RNA (mRNA), predominantly acting to suppress protein production. A number of miRNAs have been shown to regulate elements of the TLR4 signaling cascade. MiR-146a suppresses a number of intermediate signaling proteins, resulting in reduced activity of the transcription factor nuclear factor-κB (NFκB) and reduced production of inflammatory cytokines [4]. MicroRNA-16, miR-125b and miR-187 all reduce TNFα expression by reducing mRNA stability, while miR-181a targets IL-1α mRNA in a similar manner [5,6,7,8]
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