Patients with hypercholesterolemia have an increased risk for atherosclerotic cardiovascular disease, and despite lipid lowering with statins, an important residual risk remains (Benn et al., 2012). We hypothesize that this is due to persistent pro-inflammatory reprogramming of circulating monocytes. Monocytederived macrophages are key components of atherosclerotic plaques. These cells can build a long-term pro-inflammatory phenotype after brief exposure to atherogenic compounds, such as oxidized low-density lipoprotein (oxLDL)(Bekkering et al., 2014). This immune memory is mediated by metabolic and epigenetic reprogramming and is termed trained immunity (Netea et al., 2016). In Ldlr À/À mice, a Western-type diet induces trained innate immunity, which persists despite switching back to chow diet, due to reprogramming of myeloid progenitor cells (Christ et al., 2018). We propose that monocytes from FH patients have a trained immune phenotype, which persists after treatment with statins despite normalization of circulating cholesterol. In a prospective cohort study in 25 statin-naive patients with definitive or probable familial hypercholesterolemia (FH), we studied the functional and transcriptional reprogramming of circulating monocytes compared to 20 normocholesterolemic individuals and the effect of three months statin treatment (Table S1). See Methods S1 for methodological details. First, flow cytometry revealed a similar distribution of classical, non-classical, and intermediate monocytes in both groups, but monocytes from FH patients had a higher expression of the activation markers CCR2, CD11b, CD11c, and CD29 (Figure S1). Statin treatment reduced CCR2 and CD29 expression without affecting CD11b and CD11c (Figure S1). Cytokine production capacity is the major defining characteristic of trained immunity. Peripheral blood mononuclear cells from patients had a higher production of pro-inflammatory (TNFa, IL-6, and IL-1b) and anti-inflammatory (IL-10 and IL-1Ra) cytokines after Tolllike receptor stimulation (Figure S2), comparable to the oxLDL-induced trained monocytes in vitro (Bekkering et al., 2014). This hyperresponsiveness persisted during statin treatment, although production of the anti-inflammatory IL-1Ra after Candida stimulation was reduced (Figure S2). To investigate the transcriptional reprogramming underlying this functional hyper-responsiveness, we performed RNA sequencing analysis (RNA-seq) of unstimulated monocytes of 5 FH patients with the largest LDL-c reduction by statin treatment and 5 normocholesterolemic controls, matched for age and sex. RNA-seq identified 117 differentially upregulated and 153 downregulated genes in the patients (Figure S3). Pathway analysis of the upregulated genes revealed a significant enrichment of pathways involving activation of the immune system (Figure S3). Unsupervised principle component analysis of all expressed genes showed a separation of patients and controls in PC1 ($31% of all variance explained; Figure S3). Pathway enrichment analysis of the top and bottom 200 contributors to PC1 revealed an enrichment in the FH patients of metabolic pathways (oxidative phosphorylation, glycolysis, and amino acid synthesis) and inflammatory pathways (eg, NF-kB activation and the TNF signaling pathway), which are important in facilitating trained immunity (Arts et al., 2016; Bekkering et al., 2018; Cheng et al., 2014). Importantly, statin treatment did not change the RNA expression profile to a great extent, with only 17 genes downregulated by statin treatment, resulting in a persistent clustering in the PCA plot with the patients before statin treatment and not with the …