Objective—: Although the role of monocytes in the pathogenesis of atherosclerosis is well established, the persistent vascular inflammation remains largely unexplained. Recently, our group reported that stimulation of monocytes with various microbial products can induce a long-lasting proinflammatory phenotype via epigenetic reprogramming, a process termed trained immunity. We now hypothesize that oxidized low-density lipoprotein (oxLDL) also induces a long-lasting proinflammatory phenotype in monocytes, which accelerates atherosclerosis by proinflammatory cytokine production and foam cell formation. Approach and Results—: Isolated human monocytes were exposed for 24 hours to medium or oxLDL. After washing and resting for 6 days, the cells were exposed to toll-like receptor 2 and 4 agonists. Pre-exposure to oxLDL increased mRNA expression and protein formation on toll-like receptor 2 and 4 stimulation of several proatherogenic proteins, including interleukin-6, interleukin-18, interleukin-8, tumor necrosis factor-α, monocyte chemoattractant protein 1, and matrix metalloproteinase 2 and 9. In addition, foam cell formation was enhanced after oxLDL exposure, which was associated with an upregulation of scavenger receptors CD36 and scavenger receptor-A and downregulation of ATP-binding cassette transporters, ABCA1 and ABCG1. Chromatin immunoprecipitation performed 6 days after oxLDL stimulation demonstrated increased trimethylation of lysine 4 at histone 3 in promoter regions of tnfα , il-6 , il-18 , mcp-1 , mmp2 , mmp9 , cd36 , and sr-a. Finally, pretreatment of the monocytes with the histone methyltransferase inhibitor methylthioadenosine completely prevented the oxLDL-induced long-lasting proinflammatory phenotype. Conclusions—: Brief exposure of monocytes to a low concentration of oxLDL induces a long-lasting proatherogenic macrophage phenotype via epigenetic histone modifications, characterized by increased proinflammatory cytokine production and foam cell formation.

Traditionally, the innate and adaptive immune systems are differentiated by their specificity and memory capacity. In recent years, however, this paradigm has shifted: Cells of the innate immune system appear to be able to gain memory characteristics after transient stimulation, resulting in an enhanced response upon secondary challenge. This phenomenon has been called trained immunity. Trained immunity is characterized by nonspecific increased responsiveness, mediated via extensive metabolic and epigenetic reprogramming. Trained immunity explains the heterologous effects of vaccines, which result in increased protection against secondary infections. However, in chronic inflammatory conditions, trained immunity can induce maladaptive effects and contribute to hyperinflammation and progression of cardiovascular disease, autoinflammatory syndromes, and neuroinflammation. In this review we summarize the current state of the field of trained immunity, its mechanisms, and its roles in both health and disease.

Background: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. Methods: Bone marrow–derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr ^{−/ −} mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. Results: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow–derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr ^{−/ −} mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow–derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. Conclusions: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

We have recently reported that monocytes can undergo functional and transcriptional reprogramming towards a long-term pro-inflammatory phenotype after brief in vitro exposure to atherogenic stimuli such as oxidized LDL. This process is termed 'trained immunity', and is mediated by epigenetic remodeling and a metabolic switch towards increased aerobic glycolysis. We hypothesize that trained immunity contributes to atherogenesis. Therefore, we investigated the inflammatory phenotype and epigenetic remodeling of monocytes from patients with and without established atherosclerosis. Monocytes were isolated from 20 patients with severe symptomatic coronary atherosclerosis (total plaque score >4 on coronary computed tomography angiography) and 17 patients with asymptomatic carotid atherosclerosis and matched controls for both groups. Ex vivo stimulation, RNA analysis and chromatin immunoprecipitation were performed. Monocytes from patients with symptomatic atherosclerosis have a higher production of pro-inflammatory cytokines upon LPS stimulation than healthy controls (TNFα 499 ± 102 vs. 267 ± 45 pg/ml, p = 0.01). This was associated with lower histone 3 lysine 4 trimethylation (H3K4me3) (19% vs. 33%, p = 0.002), and lower H3K27me3 (0.005% vs. 0.8%, p < 0.0001) on the TNFα promoter. Furthermore, relative mRNA expression of the glycolytic rate limiting enzymes hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 was higher in patients (0.7 ± 0.2 vs. 0.3 ± 0.1 resp. 1.7 ± 0.2 vs. 1.0 ± 0.1, p = 0.007 resp. 0.003) compared to control individuals. Interestingly, this pro-inflammatory phenotype was only present in patients with symptomatic atherosclerosis, and not in patients with asymptomatic carotid atherosclerosis. Circulating monocytes of patients with symptomatic, but not asymptomatic, atherosclerosis have a pro-inflammatory phenotype and increased expression of glycolytic enzymes, associated with epigenetic remodeling at the level of histone methylation.