Endothelial Robo4 regulates IL-6 production by endothelial cells and monocytes via a crosstalk mechanism in inflammation.

Roundabout4 (Robo4) is an endothelial cell-specific receptor that stabilizes vasculature in pathological angiogenesis. Previous studies have shown that Robo4 is a potential therapeutic target for inflammatory diseases, but its precise roles in inflammation remain unclear. To investigate physiological Robo4 functions in inflammation, we performed a loss-of-function study in vitro and in vivo using lipopolysaccharide (LPS)-induced endotoxemia models. Subcutaneous injection of LPS into Robo4-knockout mice reduced circulating IL-6 levels. siRNA-mediated Robo4 knockdown suppressed IL-6 production induced by LPS, IL-1β, and TNFα, in human umbilical vein endothelial cells (HUVECs). Coculture experiments with HUVECs and a monocytic cell line, U937 cells, demonstrated that Robo4 knockdown suppresses IL-6 production by both endothelial cells and U937 cells. Further coculture experiments demonstrated that Robo4 knockdown inhibited a novel IL-6 amplification mechanism mediated by crosstalk between endothelial cells and U937 cells via direct interactions and two mediators, GM-CSF and IL-1β. Taken together, we demonstrated novel Robo4 functions in inflammation, i.e., it promotes IL-6 production by endothelial cells and immune cells via crosstalk.