Loss of MLKL Decreases Necrotic Core but Increases Macrophage Lipid Accumulation In Atherosclerosis

Abstract

Objectives -During the advancement of atherosclerosis, the cellularity of the plaque is governed by the influx of monocyte-derived macrophages and their turnover via apoptotic and non-apoptotic forms of cell death. Previous reports have demonstrated that programmed necrosis, or necroptosis, of macrophages within the plaque contribute to necrotic core formation. Knockdown or inhibition of the necrosome components RIPK1 and RIPK3 slow the progression of atherosclerosis, and activation of the terminal step of necroptosis, MLKL, has been demonstrated in advanced human atherosclerotic plaques. However, whether MLKL directly contributes to lesion development and necrotic core formation has not been investigated.Approaches and Results -MLKL expression was knocked down in atherogenic Apoeknockout mice via subcutaneous administration of antisense oligonucleotides (ASO). During advanced atherogenesis, Mlkl knockdown potently reduced cell death in the plaque, with a significant reduction in the necrotic core. However, total lesion area in the aortic sinus remained unchanged. Furthermore, treatment with the MLKL ASO unexpectedly reduced circulating cholesterol levels compared to control ASO, while staining for lipids within the plaque was significantly increased. Peritoneal macrophages transfected with the MLKL ASO showed increased lipid loading upon incubation with modified cholesterol-rich lipoproteins. In lipid-loaded macrophages, MLKL co-localized with Rab7, a marker of the late endosome.Conclusions -These studies confirm the requirement for MLKL as the executioner of necroptosis, and as such a significant contributor to the necrotic core during atherogenesis. We also identified a previously unknown role for MLKL in interacting with endosomal trafficking components to regulate lipid uptake in macrophages during atherogenesis. ABBREVIATIONS acLDL: acetylated low-density lipoprotein agLDL: aggregated low-density lipoprotein ApoA1: apolipoprotein A1 ASO: antisense oligonucleotide DAMP: damage-associated molecular patterns HDL: high-density lipoprotein MLKL: mixed lineage kinase-like domain protein ORO: Oil Red O oxLDL: oxidized low density-lipoprotein RIPK1: receptor interacting protein kinase 1 RIPK3: receptor interacting protein kinase 3