Anti-inflammatory macrophages limit endothelial inflammation and restrict neutrophil recruitment through Gas6/AXL signaling

Neutrophil extravasation requires coordinated interactions between the endothelium and surrounding cells, including tissue resident macrophages. Macrophages adopt distinct polarization states in the tissue environment, positioning them as regulators of vascular inflammation. However, how these states influence barrier function and neutrophil extravasation remains unclear. Here, we polarized macrophages in the matrix surrounding an endothelial vessel, in a three-dimensional inflammation-on-a-chip model developed only from primary human cells. We found anti-inflammatory macrophages limited endothelial inflammation, reducing ICAM-1 upregulation, maintaining VE-Cadherin expression, halving permeability relative to pro-inflammatory devices, and reducing neutrophil extravasation and migration relative to macrophage-free devices. Secretome profiling identified Gas6, an AXL ligand, as a potential regulator of these effects. Inhibiting AXL increased extravasation in devices with anti-inflammatory macrophages, and soluble Gas6 reduced extravasation in macrophage-free devices, implicating Gas6 as a mediator of this regulation. This indicates macrophages regulate endothelial inflammation and the neutrophil response via Gas6 signaling.