Abstract 44: Reactive Oxygen Species Mediate TLR4 MyD88 Independent Signaling in Human Hemoglobin-Associated Macrophages Through TLR4 Lipid Raft Interactions

Objectives: Experimental data indicate an important role for Toll-like Receptor 4 (TLR4) MyD88 independent signaling in upregulating Interferon β (IFN-β) production and driving atherosclerosis. We recently identified a distinct non-foam cell macrophage (M(Hb) or Hb-associated macrophage) in areas of intraplaque hemorrhage characterized by reduced reactive oxygen species (ROS) and pro-inflammatory cytokines. In this study, we investigated the role of iron and ROS in mediating TLR4 MyD88 independent signaling in these cells. Methods and Results: Areas rich in M(Hb) in atherosclerotic plaques demonstrated significantly reduced IFN-β expression compared to foam cell areas by immunostaining and quantitative PCR. M(Hb) did not upregulate IFN-β when exposed to ox LDL in contrast to control macrophages, a response which was inhibited in the presence of a TLR4 blocking antibody. To further investigate TLR4 responses in M(Hb), we used the TLR4 activator LPS. LPS produced significant increases in IFN-β in control macrophages but had no effect in M(Hb). This defect could be corrected by raising intracellular iron by pretreating M(Hb) with hepcidin prior to LPS treatment, suggesting redox state mediates this effect. The interaction of TLR4 with TRIF was examined by immunoprecipitation of lysates from control or M(Hb) cells treated with LPS using a TLR4 antibody and immunoblotting for TRIF. LPS treatment of control but not M(Hb) cells resulted in an increase in TRIF. Hepcidin pretreatment of M(Hb) corrected this interaction in response to LPS while differentiating monocytes in superoxide dismutase prevented it. Lastly, the interaction between lipid rafts and TLR4 was examined using FITC-cholera toxin (CTx) and a TLR4 antibody. In control cells the distribution of CTx on the plasma membrane was homogeneous and TLR4 localized to both the membrane and intracellular compartment. After LPS, a large fraction of TLR4 translocated to the plasma membrane, and colocalization of TLR4 and CTx was observed. In M(Hb) the LPS- induced translocation of TLR4 to the membrane rafts was inhibited. Conclusion: M(Hb) cells modulate TLR4 MyD88 independent signaling through reducing ROS which inhibits TLR4 lipid raft interactions.