Miltefosine increases macrophage cholesterol efflux and inhibits NLRP3-inflammasome assembly and IL-1β release.

We tested Miltefosine, an FDA approved drug, for multiple effects in macrophages including cholesterol efflux, phospholipid trafficking, autophagy, and inflammation. Miltefosine increased ABCA1 mediated cholesterol efflux in RAW264.7 macrophages in absence of any cholesterol acceptor. Miltefosine increased lipid-raft disruption and cell surface phosphatidylserine (PS) exposure via decreased flip of PS from the cell-surface. Miltefosine treatment induced redistribution of phosphatidylinositol 4,5-bisphosphate from the plasma membrane to the actin rich regions in the cells. Miltefosine induced basal autophagy as indicated by increased p62 and LC3-II puncta formation. Free cholesterol levels were increased via degradation of lipid-droplets in Miltefosine treated cells. TLR4 signaling pathway was blunted by Miltefosine in mouse bone marrow derived macrophages, leading to ~75% reduction in pro-IL-1b mRNA levels. Miltefosine potently inhibited NLRP3 speck formation and the release of mature IL1-b. However, Miltefosine did not alter AIM2 inflammasome activity, indicating specific targeting of NLRP3 inflammasome pathway by Miltefosine. Endotoxin induced reactive oxygen species generation was significantly blunted by Miltefosine. Miltefosine pretreatment also prevented endotoxin mediated loss of mitochondrial membrane potential. Overall, Miltefosine targets multiple pathways in macrophages that are involved in atherosclerosis, inflammation, and metabolic diseases. The detailed investigation of mechanisms involved in Miltefosine9s action may led to novel therapeutic targets for treating inflammatory disease such as atherosclerosis.