Stage-dependent changes in prostaglandin E₂ in tick saliva determine its immunosuppressive effects

Tick saliva contains bioactive molecules that can modulate host immune responses and facilitate blood feeding. Among these, prostaglandin E₂ (PGE₂) has been implicated in host immunosuppression, but its temporal dynamics during feeding and functional relevance remain unclear. In this study, we quantified PGE₂ concentrations in Rhipicephalus microplus saliva across different feeding stages and assessed its immunomodulatory effects on bovine macrophages. Salivary PGE₂ levels showed stage-dependent changes, with the highest concentrations detected during the mid-feeding phase. PGE₂ suppressed TNF-α production from macrophages stimulated with LPS and IFN-γ in a concentration-dependent manner. Notably, saliva samples with higher endogenous PGE₂ concentrations induced stronger suppression of TNF-α. Furthermore, the immunosuppressive effect of PGE₂ was replicated by selective agonists against the two PGE₂ receptors EP2 and EP4, suggesting a receptor-mediated mechanism. In addition, the saliva-mediated suppression of TNF-α production was significantly reversed by pharmacological blockade of EP receptors. Our findings reveal that the temporal increase in salivary PGE₂ during tick feeding is functionally linked to suppression of host macrophage activation. These results suggest that PGE₂ contributes to the immunomodulatory activity of tick saliva during blood feeding.