The goal of this randomized controlled cross-over trial is to investigate whether a single high-fat challenge can induce trained innate immunity in healthy volunteers. The main question it aims to answer is: Can a single high-fat challenge induce a persistent pro-inflammatory and pro-atherogenic monocyte phenotype, as detected by an augmented cytokine production capacity? To study this, participants will receive an oral high-fat and reference shake in a cross-over design and blood will be drawn before and at 1, 2, 4, 6, 24, and 72 hours after the shakes.

Rationale: Atherosclerosis is characterized by a persistent inflammation of the arterial wall.

Monocyte-derived macrophages are the most abundant immune cells in atherosclerotic plaques. It has recently been shown that not only immune cells of the adaptive immune system, but innate immune cells as well are able to adopt a long-term pro-inflammatory phenotype upon stimulation. This nonspecific memory of innate immune cells is mediated by epigenetic and metabolic reprogramming and is termed "trained innate immunity." Previous findings from our lab have shown that not only bacterial components such as LPS, but also pro-atherogenic particles such as oxidized LDL can induce trained immunity in monocytes. Interestingly, this memory-effect of trained immunity indicates that even temporary triggers could induce the persistent inflammation in atherosclerosis.

Triglyceride-rich lipoproteins (TRL) have been identified as an important independent risk factor for atherosclerosis. Moreover, elevated plasma levels of these lipoproteins are associated with increased pro-inflammatory markers. TRLs, however, are characterized by alternating plasma levels, with brief elevations following (fat containing) meals. Notably, a high-fat meal not only contributes to the transient increase of TRL plasma levels, but also induces a brief elevation in LPS levels by briefly increasing the permeability of the gut.

We now aim to investigate whether a single high-fat meal can induce trained innate immunity, since this concept might explain how brief postprandial effects can translate into a long-term pro-inflammatory and pro-atherogenic monocyte phenotype.

Objective: The primary objective is to determine whether a high-fat meal can induce a persistent pro-inflammatory monocyte phenotype, characterized by an increased cytokine production capacity upon ex vivo stimulation. Secondary objectives are metabolic and epigenetic reprogramming of monocytes at these time points as well as the capacity of serum, isolated before and 1-6h after an oral fat load, to induce an increased cytokine production in healthy human monocytes.

Study design: Randomized cross-over high-fat challenge intervention study.

Study population: Healthy human volunteers, aged between 18 and 40 years.

Intervention: A single high-fat challenge (milkshake containing 95g of fat) and 'control' shake (comparable to an average breakfast).

Main study parameters/endpoints: Blood will be drawn at t=0h (before) and at t=1h, t=2h, t=4h, t=6h, t=24 and t=72h after an oral fat load and at the same time points after a 'control' shake. The primary endpoint is the monocyte TNFα production upon ex vivo stimulation with LPS at the 72h time point. Additional secondary endpoints are the production of other cytokines and chemokines upon ex vivo stimulation at t=0h, t=4h, t=24h and t=72h, the monocytes' inflammatory phenotype as assessed by flowcytometry analysis and serum induced persistent cytokine production capacity.

Inclusion Criteria:

• LDL cholesterol < 3.5 mmol/l, fasting triglycerides < 2 mmol/l
• No previous cardiovascular events

Exclusion Criteria:

• Smoking within the year before study entry
• Diagnosed with any long-term medical condition that can interfere with the study (i.e.

gallbladder disease, renal failure, cardiovascular disease, diabetes, rheumatoid arthritis etc.)

• Medication (with the exception of oral contraceptives) or supplement use (i.e. omega3)
• BMI < 18 or > 27 kg/m2
• Previous vaccination within 3 months prior to study entry
• Current infection or clinically significant infections within 1 month before study entry (defined as fever > 38.5°C)
• Allergic to cow milk/dairy products
• Pregnancy/lactation
• Abuse of drugs or alcohol
• Vegetarian diet