Abstract Body: Objectives: Examine transcriptional and bioenergetic responses sensitive to THP1-IL4-EVs among Ly-6C^hi monocytes and macrophages that improve their ability to drive the resolution of inflammation and atherosclerosis in mice with dyslipidemia.
Methods: THP-1 macrophages were stimulated with IL-4 and EVs purified from conditioned medium using ultracentrifugation. LDLR-deficient mice fed a high fat diet (HFD) for 12 weeks were infused triweekly with 10⁹ THP1-IL4-EVs for 6 weeks while maintained on the HFD. Ly-6C^hi monocytes were isolated and RNA examined using NanoString Autoimmune and Metabolic gene panels. Plasma cytokines were quantified with V-PLEX assays. Atherosclerosis was assessed by detecting myeloid cell subsets in aortae and ORO-positive lesions in the aortic root. Sections were also stained with Sirius Red for collagen deposition, TMR red for DNA strand breaks, MOMA2 for macrophages, and ACTA2 for fibrous cap. Bioenergetic metabolism and continual efferocytosis were examined in vitro and ex vivo using THP1-IL4-EV-treated primary and peritoneal macrophages derived from the cohorts of mice using a Seahorse XFe-analyzer and by exposing the cells to CFSE- and PKH26-labeled Jurkat cells.
Results: THP1-IL4-EVs reduced the number of Ly-6C^hi monocytes and caused profound transcriptional reprogramming. This included reduced signaling pathways controlled by MAPK, PI3K, TLR, and inflammasomes, as well as pathways involved in glucose transport, hypoxia, and tryptophan metabolism. In contrast, THP1-IL4-EVs upregulated metabolic pathways involved in arginine metabolism, IDH1/2 activity, redox stress control and mitochondrial respiration. This led to reduced levels of IL-1b, IL-6, IFNg, and TNFa in plasma, CD45⁺ aortic myeloid cells, as well as MOMA2- and ORO-positive aortic lesions. Plaque stabilization was further noted by increased collagen deposition and a thicker ACTA2-positive fibrous cap, reduced necrotic core volumes. Elevated expression of MertK-positive macrophages with increased apoptotic cell co-localization, supporting improvements in efferocytosis. In vitro and ex vivo studies of macrophages exposed to THP1-IL4-EVs confirmed their improved capacity for continual efferocytosis that derived from improved OxPHOS and tempered glycolytic metabolism.
Conclusions: THP1-IL4-EVs offer new therapeutic opportunities to stabilize atheroma beyond plasma lipid correction by communicating profound bioenergetic transcriptional signaling to monocytes and macrophages.