Coordinated circulating cell-free miRNA and cytokine shifts during obesity-induced leptin resistance in sheep

Abstract Background Circulating cell-free microRNAs (miRNAs) are emerging mediators of inter-organ communication and biomarkers of metabolic and inflammatory regulation; however, the relationship between circulating miRNA dynamics and inflammatory cytokine responses during the development of obesity-induced leptin resistance remains incompletely characterised. Thus, this study investigated the longitudinal interplay between circulating miRNAs and cytokine profiles in sheep with diet-induced leptin resistance. Results Obese ewes developed persistent hyperleptinemia and progressive insulin resistance, consistent with leptin resistance. Cytokine profiling revealed a two-phase inflammatory response, with sustained TNF-α and IL-1α elevation from week seven and a transient acute episode at week ten marked by IL-1β and IL-6 peaks. IL-10 was consistently lower in obese ewes, and the rise in IL-36RA seen in controls by week fourteen was absent in obese animals. Plasma small RNA sequencing identified ten differentially expressed miRNAs with predicted origins in immune cells (oar-miR-150, oar-miR-17-5p), and adipose and metabolic organs (oar-miR-30b, oar-miR-29a), alongside 257 novel ovine miRNA candidates, of which 16 were differentially expressed at padj < 0.10, including a homolog of miR-342-3p linked to adipogenesis. Plasma miR-150 concentrations were highest at week ten, when they correlated positively with IL-1β and IL-6 in the validation cohort, though this increase was not significant relative to baseline after correction for multiple testing. Members of the miR-30 family declined progressively and correlated negatively with IL-6 and IL-8, a relationship that persisted after accounting for repeated animal sampling. Plasma and CSF concentrations of the four miRNAs were not significantly correlated, although with three animals per group this does not establish independent central and peripheral compartments. oar-let-7c concentrations were markedly higher in CSF of obese animals at week fourteen but were unchanged in plasma. Conclusions Plasma miRNA and cytokine profiles undergo coordinated temporal changes during obesity-induced leptin resistance in sheep, with specific miRNA subsets tracking acute and chronic inflammatory phases. These findings position circulating miRNAs as candidate biomarkers of the inflammatory response accompanying obesity-induced leptin resistance, warranting future mechanistic investigation of their functional role. De novo discovery of differentially expressed novel ovine miRNA candidates broadens the annotated ovine miRNome and opens new lines of investigation.