Disulfiram Reduces Atherosclerosis and Enhances Efferocytosis, Autophagy, and Atheroprotective Gut Microbiota in Hyperlipidemic Mice.

BACKGROUND: Pyroptosis executor GsdmD (gasdermin D) promotes atherosclerosis in mice and humans. Disulfiram was recently shown to potently inhibit GsdmD, but the in vivo efficacy and mechanism of disulfiram's antiatherosclerotic activity is yet to be explored. METHODS AND RESULTS: We used human/mouse macrophages, endothelial cells, and smooth muscle cells and a hyperlipidemic mouse model of atherosclerosis to determine disulfiram antiatherosclerotic efficacy and mechanism. The effects of disulfiram on several atheroprotective pathways such as autophagy, efferocytosis, phagocytosis, and gut microbiota were determined. Atomic force microscopy was used to determine the effects of disulfiram on the biophysical properties of the plasma membrane of macrophages. Disulfiram-fed hyperlipidemic apolipoprotein E-/- mice showed significantly reduced interleukin-1ß release upon in vivo Nlrp3 (NLR family pyrin domain containing 3) inflammasome activation. Disulfiram-fed mice showed smaller atherosclerotic lesions (~27% and 29% reduction in males and females, respectively) and necrotic core areas (~50% and 46% reduction in males and females, respectively). Disulfiram induced autophagy in macrophages, smooth muscle cells, endothelial cells, hepatocytes/liver, and atherosclerotic plaques. Disulfiram modulated other atheroprotective pathways (eg, efferocytosis, phagocytosis) and gut microbiota. Disulfiram-treated macrophages showed enhanced phagocytosis/efferocytosis, with the mechanism being a marked increase in cell-surface expression of efferocytic receptor MerTK. Atomic force microscopy analysis revealed altered biophysical properties of disulfiram-treated macrophages, showing increased order-state of plasma membrane and increased adhesion strength. Furthermore, 16sRNA sequencing of disulfiram-fed hyperlipidemic mice showed highly significant enrichment in atheroprotective gut microbiota Akkermansia and a reduction in atherogenic Romboutsia species. CONCLUSIONS: Taken together, our data show that disulfiram can simultaneously modulate several atheroprotective pathways in a GsdmD-dependent as well as GsdmD-independent manner.

Disulfiram reduces atherosclerosis and enhances efferocytosis, autophagy, and atheroprotective gut microbiota in hyperlipidemic mice.

Pyroptosis executor Gasdermin (GsdmD) promotes atherosclerosis in mice and humans. Disulfiram (DSF) was recently shown to potently inhibit GsdmD, but the in-vivo efficacy and mechanism of DSF's anti-atherosclerotic activity is yet to be explored. We used human/mouse macrophages and a hyperlipidemic mouse model of atherosclerosis to determine DSF anti-atherosclerotic efficacy and mechanism. DSF-fed hyperlipidemic apoE -/- mice showed significantly reduced IL-1ß release upon in-vivo Nlrp3 inflammasome assembly and showed smaller atherosclerotic lesions (∼27% and 29% reduction in males and females, respectively). The necrotic core area was also smaller (∼50% and 46% reduction in DSF-fed males and females, respectively). DSF induced autophagy in macrophages, hepatocytes/liver, and in atherosclerotic plaques. DSF modulated other atheroprotective pathways such as efferocytosis, phagocytosis, and gut microbiota. DSF-treated macrophages showed enhanced phagocytosis/efferocytosis, with a mechanism being a marked increase in cell-surface expression of efferocytic receptor MerTK. Atomic-force microscopy analysis revealed altered biophysical membrane properties of DSF treated macrophages, showing increased ordered-state of the plasma membrane and increased adhesion strength. Furthermore, the 16sRNA sequencing of DSF-fed hyperlipidemic mice showed highly significant enrichment in atheroprotective gut microbiota Akkermansia and a reduction in atherogenic Romboutsia species. Taken together, our data shows that DSF can simultaneously modulate multiple atheroprotective pathways, and thus may serve as novel adjuvant therapeutic to treat atherosclerosis.

Impavido attenuates inflammation, reduces atherosclerosis, and alters gut microbiota in hyperlipidemic mice.

Impavido (Miltefosine) is an FDA-approved drug for treating leishmaniasis and primary amebic meningoencephalitis. We have shown previously that Miltefosine increased cholesterol release and dampened Nlrp3 inflammasome assembly in macrophages. Here, we show that Miltefosine reduced LPS-induced choline uptake by macrophages, and attenuated Nlrp3 inflammasome assembly in mice. Miltefosine-fed mice showed reduced plasma IL-1ß in a polymicrobial cecal slurry model of systemic inflammation. Miltefosine-fed mice showed increased reverse cholesterol transport to the plasma, liver, and feces. Hyperlipidemic apoE-/- mice fed with WTD + Miltefosine showed significantly reduced weight gain and markedly reduced atherosclerotic lesions versus mice fed with WTD. The 16S rDNA sequencing and analysis of gut microbiota showed marked alterations in the microbiota profile of Miltefosine-fed hyperlipidemic apoE-/- versus control, with the most notable changes in Romboutsia and Bacteriodes species. Taken together, these data indicate that Miltefosine causes pleiotropic effects on lipid metabolism, inflammasome activity, atherosclerosis, and the gut microbiota.

Myeloid-cell-specific role of Gasdermin D in promoting lung cancer progression in mice.

The activities of the NLRP3 and AIM2 inflammasomes and Gasdermin D (GsdmD) are implicated in lung cancer pathophysiology but it's not clear if their contributions promote or retard lung cancer progression. Using a metastatic Lewis lung carcinoma (LLC) cell model, we show that GsdmD knockout (GsdmD-/-) mice form significantly fewer cancer foci in lungs, exhibit markedly decreased lung cancer metastasis, and show a significant ∼50% increase in median survival rate. The cleaved forms of GsdmD and IL-1ß were detected in lung tumor tissue, indicating inflammasome activity in lung tumor microenvironment (TME). Increased migration and growth of LLC cells was observed upon exposure to the conditioned media derived from inflammasome-induced wild type, but not the GsdmD-/-, macrophages. Using bone marrow transplantations, we show a myeloid-specific contribution of GsdmD in lung cancer metastasis. Taken together, our data show that GsdmD plays a myeloid-specific role in lung cancer progression.