Miltefosine increased cholesterol efflux, induced autophagy and inhibited NLRP3-inflammasome assembly and IL-1β release.

In advanced human plaques and aged patients, athero-protective pathways such as autophagy and reverse cholesterol transport (RCT) become dysfunctional, while atherogenic pathways such as NLRP3 inflammasome and TLR2/4 are induced. Here, we report that Miltefosine, an FDA approved drug for treating leishmaniasis, increased cholesterol efflux, induced membrane remodeling, and induced autophagy in macrophages. Macrophages treated with Miltefosine exhibited markedly increased ABCA1 mediated cholesterol efflux and decreased phosphatidylserine flip from the cell-surface. Miltefosine treatment of macrophages led to redistribution of phosphatidylinositol 4,5-bisphosphate (PIP2) from plasma membrane to actin rich regions of the cell. RAW264.7 macrophages treated with Miltefosine showed marked increase in p62 and LC3 puncta staining vs. control cells. The Lipid droplet degradation was induced by Miltefosine leading to ~ 50% decrease in the CE:FC (cholesterol ester: free cholesterol) ratio. The TLR4 signaling pathway in LPS primed bone marrow derived macrophages was blunted by Miltefosine treatment, leading to ~75% reduction in pro-IL-1b; mRNA levels. Miltefosine pretreatment of macrophages potently inhibited NLRP3 inflammasome assembly induced by LPS/ATP treatment, exhibiting ~70% reduction in ASC1 speck forming cells. Gasdermin D mediated release of mature IL-1b; was reduced by ~80% in Miltefosine treated vs. control cells. The qRT-PCR and western blot analysis showed no changes in basal or LPS induced levels of inflammasome components (NLRP3, ASC1, procaspase1), while the LPS mediated induction in ROS levels was significantly blunted in Miltefosine treated vs. control macrophages. Miltefosine did not alter the AIM2 inflammasome activity indicating specific targeting of Nlrp3 inflammasome pathway. Overall, this study showed that Miltefosine targets lipid trafficking, cell migration, autophagy, and Nlrp3 inflammasome activity in macrophages.