Equine monocyte-derived macrophages revisited: isolation and comprehensive characterization of pro- versus anti-inflammatory polarisation

Macrophages play key roles in tissue homeostasis and regeneration-associated inflammation. Unlike humans, a reliable protocol to obtain and polarise equine monocyte-derived macrophages is lacking. In this study the polarisation of equine macrophages, derived from CD172a⁺ peripheral blood monocytes is described. After differentiation, IFN-γ/LPS or IL-4 were used to induce pro- and anti-inflammatory phenotypes, respectively. Evaluation criteria included morphology, mRNA (RT-qPCR) and protein expression (flow cytometry, immunofluorescence), nitric oxide and arginase production, cytokine secretion (multiplex), and functional effects of conditioned medium (CM). IFN-γ/LPS-stimulated cells exhibited a rounded morphology with cytoplasmic extensions, while IL-4 stimulation induced spindle-shaped and multinucleated giant cells. IFN-γ/LPS upregulated <i>CXCL8, CD86</i>, <i>IL10</i> and <i>TGFB1</i> mRNA, whereas IL-4 upregulated <i>CXCL8</i>, <i>MRC1</i> and <i>TGFB1</i>. Polarisation was confirmed with IFN-γ/LPS-stimulated macrophages expressing CD86 and secreting TNFα and IL-1β, while IL-4-stimulation increased CD206 positivity and VEGFα expression. Increased proliferation and altered mRNA expression in tendon cells treated with 50% CM further validate the functional impact of macrophage polarisation. In summary, a robust protocol to obtain equine macrophages was developed, followed by in-depth characterization of their pro- and anti-inflammatory polarisation. Given the horse's increasing relevance as large animal model, this research holds both a strong species-specific and translational value.