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, 9 (1), 11128

Miltefosine Increases Macrophage Cholesterol Release and Inhibits NLRP3-inflammasome Assembly and IL-1β Release

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Miltefosine Increases Macrophage Cholesterol Release and Inhibits NLRP3-inflammasome Assembly and IL-1β Release

Amanda J Iacano et al. Sci Rep.

Abstract

Miltefosine is an FDA approved oral drug for treating cutaneous and visceral leishmaniasis. Leishmania is a flagellated protozoa, which infects and differentiates in macrophages. Here, we studied the effects of Miltefosine on macrophage's lipid homeostasis, autophagy, and NLRP3 inflammasome assembly/activity. Miltefosine treatment conferred multiple effects on macrophage lipid homeostasis leading to increased cholesterol release from cells, increased lipid-raft disruption, decreased phosphatidylserine (PS) flip from the cell-surface, and redistribution of phosphatidylinositol 4,5-bisphosphate (PIP2) from the plasma membrane to actin rich regions in the cells. Enhanced basal autophagy, lipophagy and mitophagy was observed in cells treated with Miltefosine vs. control. Miltefosine treated cells showed marked increased in phosphorylation of kinases involved in autophagy induction such as; Adenosine monophosphate-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase (ULK1). The Toll like receptor (TLR) signaling pathway was blunted by Miltefosine treatment, resulting in decreased TLR4 recruitment to cell-surface and ~75% reduction in LPS induced pro-IL-1β mRNA levels. Miltefosine reduced endotoxin-mediated mitochondrial reactive oxygen species and protected the mitochondrial membrane potential. Miltefosine treatment induced mitophagy and dampened NLRP3 inflammasome assembly. Collectively, our data shows that Miltefosine induced ABCA1 mediated cholesterol release, induced AMPK phosphorylation and mitophagy, while dampening NLRP3 inflammasome assembly and IL-1β release.

Conflict of interest statement

A patent application related to this work has been filed by the Cleveland Clinic that lists K.G. and J.D.S. as inventors. Authors declare no non-financial competing interests.

Figures

Figure 1
Figure 1
Miltefosine increases ABCA1 mediated cholesterol release. (A) RAW264.7 macrophages were labeled with 3H-cholesterol and pretreated with or without 300 μM 8Br-cAMP to induce ABCA1 expression. Cholesterol release to chase media was performed for 4 h at 37 °C in serum-free DMEM without addition of acceptor containing either vehicle or 7.5 μM Miltefosine. Values are % cholesterol release mean ± SD, N = 5, different letters above the bars show p < 0.01 by ANOVA Bonferroni posttest, with separate analyses for ± ABCA1 induction. (B) Western blot analysis of 8Br-cAMP induced RAW264.7 cell total and cell-surface ABCA1 ± 7.5 μM Miltefosine treatment for 4 h. (C) Cholesterol release (4 h at 37 °C) from ABCA1 stably transfected and control HEK293 to serum-free DMEM without addition of acceptor containing either vehicle or 7.5 μM Miltefosine. Values are % cholesterol release mean ± SD, N = 3–5, different letters above the bars show p < 0.01 by ANOVA Bonferroni posttest, with separate analyses for ± ABCA1 induction. (D) Cholesterol release (4 h at 37 °C) from ABCA1 and ABCA1 (W590S, C1477R) double mutant (DM) stably transfected HEK293 to serum-free DMEM ±7.5 μM Miltefosine. Values are % cholesterol release mean ± SD, N = 3, different letters above the bars show p < 0.01 by ANOVA Bonferroni posttest.
Figure 2
Figure 2
Miltefosine disrupts lipid-rafts and inhibits PS flip across plasma membrane. (A) GM1 levels assessed by binding of cholera toxin B (CTB) in live RAW macrophages ±7.5 µM Miltefosine for 16 h at 37 °C. (B) Flow cytometry quantification of CTB binding of RAW cells treated ±7.5 µM Miltefosine for 16 h at 37 °C. Values are the mean ± SD of the median fluorescence from 3 independent wells (**p < 0.01 by two-tailed t-test). (C) RAW macrophages were incubated with or without 8Br-cAMP to induce ABCA1 and ±7.5 μM Miltefosine for 16 hrs. PS exposure was determined Annexin V binding via flow cytometry (different letters above the bars show p < 0.01 by ANOVA Bonferroni posttest). (D) Cells were pretreated ±7.5 µM Miltefosine and incubated with 25 µM NBD-PS at RT for 15 min to assess cellular association of PS. (E) Quantification of NBD-PS translocated inside the cells. RAW macrophages were pretreated ±7.5 µM Miltefosine and incubated with 25 µM NBD-PS at 37 °C for 15 min in phen°l red free DMEM. The cells were subjected to flow cytometry analysis ± sodium dithionite to quench extracellular NBD fluorescence, yielding only intracellular NBD fluorescence (mean ± SD % NBD-PS translocated into the cells; N = 3; ***p < 0.001 by two-tailed t-test).
Figure 3
Figure 3
Miltefosine alters plasma membrane PIP2 localization. (A) RAW264.7 cells were stably transfected with 2X-PH-PLCeGFP reporter plasmid and treated ±7.5 µM Miltefosine for 12 h at 37 °C and the localization of the GFP-tagged PIP2 reporter was observed. (B) RAW264.7 cells treated as in (A) were fixed, permeabilized, and stained with mouse anti β-actin antibody to observe colocalization of actin with the PIP2 reporter. (C) RAW264.7 cells stably transfected with 2X-PH-PLCeGFP reporter plasmid ±7.5 µM Miltefosine treatment, stained for β-actin with Phalloidin and DAPI counterstained.
Figure 4
Figure 4
Miltefosine induces autophagy. (A) RAW264.7 cells or LC3-GFP transfected RAW264.7 cells (lower panel) were treated with ±7.5 µM Miltefosine, followed by staining with p62 antibody (red, upper panel). LC3-GFP protein (green) localization is shown in the lower panel. (B,C) RAW264.7 cells were treated ±7.5 µM Miltefosine and autophagic flux was assessed in presence of 30 μM chloroquine for the last 2 h of incubation. The amount of LC3-II was assessed by western blot and densitometry analysis relative to GAPDH (mean ± SD, N = 3, different letters above the bars represent p < 0.001 by ANOVA with Bonferroni posttest). (D) Cellular CE/FC ratio in RAW264.7 cells loaded with 100 µg/ml acetylated low-density lipoprotein (AcLDL) and addition of 2 µg/ml ACAT inhibitor (ACATi) and/or 7.5 µM Miltefosine as indicated (N = 3, mean ± SD; *p < 0.05 vs. control; ***p < 0.001 vs. control by ANOVA Bonferroni posttest). (E) Cellular neutral lipids assayed by Nile red staining and flow cytometry ± AcLDL loading, ±4 h chase ±7.5 µM Miltefosine ±2 µg/ml ACATi, as indicated (mean ± SD, N = 4, different letters above the bars represent p < 0.0001 by ANOVA with Bonferroni posttest). The densitometry analysis of p-AMPK (F) or p-ULK1 (G) relative to AMPK or ULK1 (mean ± SD, N = 3; ***p < 0.001, *p < 0.05, by two-tailed t-test).
Figure 5
Figure 5
Miltefosine dampens TLR4 signaling in bone marrow derived macrophages. (A) Mouse BMDMs were treated with ±5 µM Miltefosine for 16 h at 37 °C and primed by incubation with ±1 μg/ml LPS at 37 °C for 4 hrs, followed by analysis of TLR4 antibody binding by flow cytometry (N = 3, different letters above the bars show p < 0.01 by ANOVA Bonferroni posttest. (B) Mouse BMDMs were treated as in (A), followed by analysis of pro-IL-1β mRNA with β-actin mRNA used as a control (N = 6, mean ± SD; different letters above the bars represent p < 0.01 by ANOVA with Bonferroni posttest). (B) Mouse BMDMs were treated with ±5 µM Miltefosine and primed by incubation with ±1 mg/ml LPS followed by western blot analysis for IL-1β with GAPDH used as loading control.
Figure 6
Figure 6
Miltefosine inhibits Nlrp3 inflammasome assembly and IL-1β release. (A) Mouse BMDMs were pretreated with ±5 µM Miltefosine, primed by incubation with ±1 μg/ml LPS, and induced for NLRP3 inflammasome assembly by incubation with 1 mM ATP for 20 min. Cells were fixed and stained with anti-ASC with DAPI. (B) IL-1β levels in media from cells treated with LPS and ATP ±5 µM Miltefosine pretreatment (N = 4, mean ± SD; **p < 0.01 by two tailed t-test). (C) qRT-PCR analysis of NLRP3, caspase 1, and Gasdermin D (GsdmD) mRNAs, relative to β-actin mRNA, from mouse BMDMs ±5 µM Miltefosine pretreatment, ± LPS treatment (N = 6, mean ± SD; ****p < 0.0001 by ANOVA with Bonferroni posttest). (D) IL-1β released from mouse BMDMs treated with ±5 µM Miltefosine and primed with 1 μg/ml LPS, followed by transfection with 2 μg of poly (dA-dT) for 3 h to induce the AIM2 inflammasome. (E) Cellular total cholesterol levels were determined in mouse BMDMs treated with ±5 µM Miltefosine for 16 h or 1 mM cyclodextrin for 2 h. (F) IL-1β released from mouse BMDMs treated ±1 mM cyclodextrin for 2 h or ±5 µM Miltefosine for 16 h followed by LPS and ATP treatment (mean ± SD, N = 3, different letters above the bars represent p < 0.05 by ANOVA with Bonferroni posttest).
Figure 7
Figure 7
Miltefosine alters mitochondrial homeostasis and induces mitophagy. (A) BMDMs pretreated ±5 µM Miltefosine and treated ±1 μg/ml LPS were stained with MitoSox to observe mitochondrial ROS in live cells. (B) Flow-cytometry analysis showing quantification of MitoSox staining of live BMDMs treated as indicated (mean ± SD, N = 3, different letters above the bars represent p < 0.01 by ANOVA with Bonferroni posttest). (C) Live cell flow cytometry of mitochondrial membrane potential detected by TMRM staining of BMDMs pretreated ±5 µM Miltefosine and treated ±1 μg/ml LPS (mean ± SD, N = 3, different letters above the bars represent p < 0.05 by ANOVA with Bonferroni posttest). (D) RAW264.7 cells treated ±7.5 µM Miltefosine and live cells were stained for mitophagy and lysosomes.

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