Macrophages have a key role in the initiation and progress of atherosclerosis. Both, classically activated (M1) and alternatively activated (M2) macrophages, were detected within atherosclerotic lesions. M1 macrophages show a pro-inflammatory and M2 macrophages a more anti-inflammatory phenotype. Furthermore, cell mechanic functions like migration and efferocytosis, the phagocytosis of apoptotic cells, strongly affect the progression of atherosclerosis. The Eph/ephrin-system as the largest system of receptor tyrosine kinases influences a wide range of cellular functions like proliferation, survival, motility, differentiation and migration. Up to now, little is known about the regulation and impact of the Eph/ephrin-system on macrophage polarization and their cellular properties. Therefore, the study aimed to address these questions.

Human monocyte-derived macrophages (M0) were polarized into M1 and M2a phenotype using IFN‑γ/LPS or IL-4 respectively. First, the expression of all Eph-receptors and ephrin-ligands was measured in these three phenotypes. The most pronounced effect was seen in M2a macrophages, which showed a massive induction of ephrin-A1 expression. Using ChIP and siRNA-silencing the transcription factor STAT6 was detected as a mediator of IL-4 induced ephrin-A1 expression. Interestingly, we could show that ephrin-A1 influences M2a polarization itself. Inhibition of ephrin-A1 in M2a macrophages strongly reduced expression of M2 genes. By investigating cell mechanic functions of macrophages, we detected that polarization and ephrin-A1 expression affected migration, efferocytosis and their deformability. In comparison to M0 macrophages, M2a macrophages showed a higher migration potential and inhibition of ephrin-A1 reduced their migration. Similar results could be shown in case of efferocytosis. M2a macrophages revealed a higher capacity of efferocytosis compared to M0 macrophages and silencing of ephrin-A1 significantly reduced it. Beside others, the ability of migration and efferocytosis depends on their deformability. Using real-time deformability cytometry (RT-DC) we could show that M2a polarization resulted in an increase and silencing of ephrin-A1 in a lowering of the deformability, which might explain the demonstrated changings in migration and efferocytosis of M2a macrophages. 

In conclusion, ephrin-A1 is upregulated in M2a macrophages, influences M2 polarization itself and strongly affects the cytomechanical phenotype of human macrophages. In addition, macrophage migration and efferocytosis are pivotal protective functions of M2 macrophages in atherosclerotic plaques, which makes ephrin-A1 to a potential target to analyze for its atherogenic potential and may open new perspectives for the understanding of this chronic-inflammatory disease.