Increased Release of Soluble MD-2 in Sickle Cell Disease and Its Role in Pro-Inflammatory Signaling in Endothelial Cells
Authors: Zhang Ping , Belcher John D , Nguyen Julia , Abdulla Fuad , Vercellotti Gregory M 
Published: February 2020

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&lt;0.05, n=7). In mice, plasma sMD-2 was increased 7.6-fold in Townes-SS sickle mice (n=9) compared to control Townes-AA mice (p&lt;0.0002, n=7). In contrast, plasma CD14, another required component of LPS-TLR4 signaling, was not significantly different in SS humans (n=8) and SS mice (n=9) compared to AA controls (p&lt;0.05). The liver is one potential source of sMD-2 in plasma. In mice, hepatic MD-2 mRNA was increased 2.1-fold in SS compared to AA (p&lt;0.05, n=6). Activated vascular endothelium is another potential source and target of sMD-2 in plasma. It has been reported by other groups and confirmed by us that LPS induces sMD-2 secretion by human umbilical vein endothelial cells (HUVEC). To determine whether heme can induce sMD-2 secretion from endothelial cells, we treated HUVEC with heme (0-30 μM) for 18 hours and found heme increased sMD-2 in media in a dose-responsive manner.
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&lt;0.02, n=4). Tak242, a TLR4 signaling inhibitor, blocked IL-8 secretion by HUVEC + SS plasma. Since heme has been shown to activate TLR4 signaling, we examined whether heme could bind to sMD-2 in plasma using a heme-agarose pull-down assay. Human plasma was incubated with heme-agarose to pull down heme binding proteins, followed by Western blot for sMD-2 protein in the pellet. The blot confirmed that sMD-2 in plasma bound specifically to heme. When sMD-2 was removed from SS plasma using an anti-MD-2 affinity column, the sMD-2-depleted plasma reduced IL-8 secretion by HUVEC by 34.3% (p&lt;0.002, n=4). Furthermore, when the high-affinity heme-binding protein hemopexin (10 μM) was added to SS plasma, IL-8 secretion by HUVEC was reduced by 31.6% (p&lt;0.01, n=7). Next, we made recombinant human sMD-2 in CHO cells with protein-free ProCHO medium. UV/Vis absorption spectra (250-600 nm) and heme-agarose pull-down assays found there was heme bound to recombinant sMD-2 in the ProCHO medium. When recombinant sMD-2-heme was added to human AA plasma and incubated with HUVEC, IL-8 secretion increased 2.2-fold (p&lt;0.004, n=3). TLR4 inhibitor Tak242 blocked this increase in IL-8 secretion. When hemopexin was added to the recombinant sMD-2-heme before adding it to AA plasma, IL-8 production was reduced 38% compared to non-hemopexin treated (p&lt;0.01, n=7). In conclusion, these data indicate that sMD-2 is increased in SCD plasma, binds heme, and can stimulate endothelial cell IL-8 production through a TLR4-dependent mechanism. We speculate that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD.

Disclosures
Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.