PROJECT SUMMARY In the last decade, emerging evidence has unveiled that the innate immune system retains long- term epigenetic and metabolic changes after infection or vaccination. This de facto innate immune memory has been termed ‘trained immunity’ and is characterized by myeloid cells’ hyper-responsiveness following a subsequent stimulus. Recent work has shown that sterile atherogenic/inflammatory triggers, such as oxidized LDL or catecholamines, similarly induce a trained immunity phenotype through epigenetic and metabolic reprogramming of the myeloid compartment. The long-term persistence of trained immunity in vivo is due to the reprogramming of hematopoietic stem and progenitor cells (HSPC) in the bone marrow. We have recently reported that monocytes isolated from patients with risk factors for CVD, such as familial hypercholesterolemia or pheochromocytoma, display a ‘trained’ phenotype. Importantly, in patients with established coronary artery disease, we found HSPCs reprogrammed towards a pro-inflammatory myeloid lineage. Project 2 will focus on stress-induced trained immunity’s mechanistic aspects in cardiovascular disease patients and mouse models. Our central hypothesis is that chronic stress induces trained immunity via HSPC reprogramming, which exacerbates the development of atherosclerosis and worsens the outcome of cardiovascular events. In Aim 1, we will use deep phenotyping and imaging to study patients at high risk for cardiovascular events in order to obtain an integrated view of stress-induced reprogramming of the myeloid cell compartment. In Aim 2, we will study mice that were exposed to chronic mild psychosocial stress or to key hormonal signals that promote peripheral effects on stress, followed by a rest period of 4 weeks. After the rest period, stressed cohorts and non-stressed controls will undergo coronary artery ligation or induction of atherosclerosis to test the hypothesis that preceding stress or exposure to stress hormones activates trained immunity, thus increasing myelopoiesis and consequently worsening cardiovascular disease. Our unique ability to profile patients’ HSPCs at Radboudumc, the human models, and the mouse models that mimic them will yield critical insights into the relationship between cardiovascular disease and psychosocial stress.