Activation of inflammasomes, such as Nlrp3 and AIM2, can exacerbate atherosclerosis in mice and humans. Gasdermin D (GsdmD) serves as a final executor of inflammasome activity, by generating membrane pores for the release of mature Interleukin-1beta (IL-1β). Inflammation dampens reverse cholesterol transport (RCT) and promotes atherogenesis, while anti-IL-1β antibodies were shown to reduce cardiovascular disease in humans. Though Nlrp3/AIM2 and IL-1β nexus is an emerging atherogenic pathway, the direct role of GsdmD in atherosclerosis is not yet fully clear. Here, we used in vivo Nlrp3 inflammasome activation to show that the GsdmD–/– mice release ∼80% less IL-1β vs. Wild type (WT) mice. The GsdmD–/– macrophages were more resistant to Nlrp3 inflammasome mediated reduction in cholesterol efflux, showing ∼26% decrease vs. ∼60% reduction in WT macrophages. GsdmD expression in macrophages exacerbated foam cell formation in an IL-1β dependent fashion. The GsdmD–/– mice were resistant to Nlrp3 inflammasome mediated defect in RCT, with ∼32% reduction in plasma RCT vs. ∼57% reduction in WT mice, ∼17% reduction in RCT to liver vs. 42% in WT mice, and ∼37% decrease in RCT to feces vs. ∼61% in WT mice. The LDLr antisense oligonucleotides (ASO) induced hyperlipidemic mouse model showed the role of GsdmD in promoting atherosclerosis. The GsdmD–/– mice exhibit ∼42% decreased atherosclerotic lesion area in females and ∼33% decreased lesion area in males vs. WT mice. The atherosclerotic plaque-bearing sections stained positive for the cleaved N-terminal fragment of GsdmD, indicating cleavage of GsdmD in atherosclerotic plaques. Our data show that GsdmD mediates inflammation-induced defects in RCT and promotes atherosclerosis.

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.

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.3 Significance Statement:Atherosclerosis is driven by cholesterol accumulation and inflammation, and the arterial macrophage is a key cell type in both of these processes. The macrophage characteristics that protect against atherosclerosis include increased cholesterol efflux/reverse cholesterol transport, increased autophagy, and deceased inflammatory cytokine production and signaling.Here, we show that one single orally available compound, Miltefosine, can target multiple macrophage pathways involved in lipid homeostasis and inflammation. Miltefosine activated cholesterol release and autophagy while inhibiting pro IL-1β gene expression and NLRP3 inflammasome assembly. Miltefosine activated AMPK signaling pathway and mitophagy, leading to reduced NLRP3 inflammasome assembly and IL-1β release.4 Introduction:

Nlrp3 inflammasome is activated in advanced human atherosclerotic plaques. Gasdermin D (GsdmD) serves as a final executor of Nlrp3 inflammasome activity, by generating membrane pores for the release of mature Interleukin-1beta (IL-b). Inflammation dampens reverse cholesterol transport (RCT) and promotes atherogenesis, while anti-IL-1b; antibodies were shown to reduce cardiovascular disease in humans. Though Nlrp3/IL-1b; nexus is an emerging atherogenic pathway, the direct role of GsdmD in atherosclerosis is not yet clear. Here, we used in-vivo Nlrp3 inflammasome activation to show that the GsdmD-/- mice release ~80% less IL-1b; vs WT mice. The GsdmD-/- macrophages were more resistant to Nlrp3 inflammasome mediated reduction in cholesterol efflux, showing ~26% decrease vs. ~60% reduction in WT macrophages. GsdmD expression in macrophages exacerbated foam cell formation in an IL-1b; dependent fashion. The GsdmD-/- mice were resistance to Nlrp3 inflammasome mediated defect in RCT, with ~32% reduction in plasma RCT vs. ~ 57% reduction in WT mice, ~ 17% reduction in RCT to liver vs. 42% in WT mice, and ~ 37% decrease in RCT to feces vs. ~ 61% in WT mice. The LDLr anti-sense oligonucleotides (ASO) induced hyperlipidemic mouse model showed role of GsdmD in promoting atherosclerosis. The GsdmD-/- mice exhibit ~42% decreased atherosclerotic lesion area in females and ~33% decreased lesion area in males vs. WT mice. The atherosclerotic plaque-bearing WT mice showed the presence of cleaved N-terminal fragment of GsdmD, indicating cleavage of GsdmD during atherosclerosis. Our data show that GsdmD mediates inflammation-induced defect in RCT and promotes atherosclerosis.

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