Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing

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.

Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus

“…From a confirmative viewpoint, these findings are similar to those reported for human SEs (34), suggesting that these miRNAs can be involved in similar modulatory processes in both species. Furthermore, the let-7 family of miRNAs as well as mir-21 are highly abundant in equine serum and plasma and also expressed in diverse tissue types (59,(75)(76)(77)(78)(79)(80). Validation of the miRNA sequencing data by RT-qPCR confirmed specific downregulation of eca-mir-128 in long-term persistently infected stallions, an intronic miRNA encoded by two distinct genes, eca-mir-128-1 and eca-mir-128-2, located on (81).…”

Immunohistochemical study of morphology and distribution of CD163+ve macrophages in the normal adult equine gastrointestinal tract

“…For example, in response to LPS equine bone marrow-derived macrophages do not produce nitric oxide unlike rodents which do (Young et al, 2018). Several studies have highlighted that the horse immune response is more similar to that of human than mouse (Karagianni et al, 2017;Parkinson et al, 2017). Additionally, sequence analysis of human, rodent and horse genes involved in immunity have demonstrated greater synteny between horse and human than rodent and human (Hudgens et al, 2011;Tompkins et al, 2010).…”

The equine mononuclear phagocyte system: The relevance of the horse as a model for understanding human innate immunity

“…As demonstrated in other mammalian‐derived cells, TLR2 ligands induce a mild inflammatory response in equine PBMC; this is in comparison to the much greater response to TLR4 agonists 9,89 . Recent transcriptomic studies have reported that LPS induces differential microRNA expression in equine PBMC in a manner comparable to humans, thus facilitating interspecies comparative study of the role of microRNAs in the inflammatory cascade during endotoxaemia and sepsis 90 . Equine monocytes are also responsive to TLR3 stimulation with double‐stranded RNA (polyinosinic polycytidylic acid [Poly IC]), 91 which is dependent upon the TRIF adaptor.…”