Small Molecule Inhibitor Screen to Identify Mechanisms Controlling Selective Clearance of Hemoglobin By Liver Sinusoidal Endothelial Cells

Hemolysis and accumulation of cell free hemoglobin (Hb) is a major pathophysiological complication associated with hemolytic disorders such as sickle cell disease (SCD). In the absence of a functional spleen, the hepatic Kupffer cells play a large role in Hb clearance. We recently demonstrated that along with Kupffer cells, liver sinusoidal endothelial cells (LSECs) are also capable of Hb clearance. Based on this finding, we hypothesized that LSECs promote HbS clearance via endocytic trafficking. To evaluate the mechanism of HbS internalization, we performed an in vitro screen in primary LSECs using small molecule inhibitors or drugs of endocytic pathway blockers. Our in vitro screen results demonstrated that LSEC induced HbS clearance occurs through multiple endocytic routes simultaneously. Whereas micropinocytosis or fluid phase endocytosis is the predominant method of HbS clearance, blocking clathrin mediated endocytosis, receptor recycling, lipid vesicle formation as well as low pH can significantly impede HbS clearance by LSECs. Our screen also identified mannose receptor CD206 as a potent inhibitor of HbS clearance. We further demonstrated that blocking CD206 in vivo in SCD mice caused significant amelioration of hepatic Hb-heme-iron accumulation as well as hemolysis induced liver damage. Remarkably, this effect was also accompanied by a strong increase in Hb scavenger receptor CD163 in vivo in hepatic Kupffer cells. In conclusion, we identified various alternate methods of LSEC mediated HbS internalization that might provide useful therapeutic implications to combat hemolysis related liver damage in SCD and other hemolytic disorders. These findings also implicate a novel role of mannose receptor CD206 in mediating hepatic Hb clearance.

No relevant conflicts of interest to declare.