Increased Release of Soluble MD-2 in Sickle Cell Disease and Its Role in Pro-Inflammatory Signaling in Endothelial Cells

Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide, resulting from a mutation in the beta globin gene. SCD has significant pathophysiological consequences – hemolysis, inflammation, oxidative stress, hypercoagulability, endothelial dysfunction and painful vaso-occlusive crises. The latter can be precipitated by infection or other metabolic stressors. Hemolysis chronically exposes endothelial cells, leukocytes, and platelets to hemoglobin and heme that promote pro-inflammatory and prothrombotic phenotypes. We previously demonstrated that toll-like receptor 4 (TLR4) signaling is required for microvascular stasis induced by hemoglobin, heme, or lipopolysaccharide (LPS) in sickle mice. MD-2 is a glycoprotein, co-expressed with TLR4 at the surface of various cell types, principally myeloid and endothelial lineages. MD-2 also exists as a soluble plasma protein (sMD-2), mainly as a large disulfide-bound multimeric glycoprotein, as well as oligomers and monomers. sMD-2 binds LPS and confers TLR4 sensitivity to LPS . A marked increase in sMD-2 has been reported in plasma from patients with sepsis and rheumatoid arthritis. sMD-2 in SCD plasma has not been studied. Since SCD has a pro-inflammatory phenotype, we hypothesized that sMD-2 is increased in SCD plasma and promotes pro-inflammatory signaling of endothelial cells. We assessed plasma levels of sMD-2 by Western blot and found that sMD-2 was increased 1.7-fold in SS human plasma (n=8) compared to healthy AA plasma (p To determine if sMD-2 in plasma could activate TLR4 signaling in endothelial cells, we incubated HUVEC with 2% SS or AA human plasma for 18 hours and measured IL-8 in the media by ELISA. Media IL-8 concentration was 2.6-fold higher in HUVEC incubated with SS plasma compared to AA plasma (p Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti: Mitobridge, an Astellas Company: Consultancy, Research Funding.