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Major surgery such as coronary artery bypass grafting (CABG) is associated with an increased post-operative risk of atherosclerotic cardiovascular events. Cells of the innate immune system can adopt a long-lasting pro-inflammatory and atherogenic phenotype after brief exposure to exogenous or endogenous inflammatory stimuli, a process called "trained immunity". We hypothesized that the surgery-induced inflammation leads to sustained alterations in monocyte function, which promote the subsequent occurrence of cardiovascular events. Blood from 13 patients undergoing elective CABG was obtained before, 3-7 days (median 4) after, and 6-8 weeks (median 6) weeks after surgery. At 3-7 days postoperatively, circulating C-reactive protein (CRP) concentration, leukocyte counts and ex vivo Peripheral Blood Mononuclear Cell (PBMC) IL-6, TNFα and IL-1Ra production after stimulation (with various inflammatory stimuli) were significantly increased. Simultaneously, there was a reduction in monocyte HLA-DR expression. 6-8 weeks after CABG there was an ongoing systemic pro-inflammatory state with higher CRP concentrations, increased stimulated ex vivo PBMC IL-6 production, changes in monocytes subsets, and a higher expression of CCR2 on monocytes compared to baseline. In conclusion, CABG induces a persistent systemic inflammatory reaction with a sustained activated monocyte phenotype. This might contribute to the increased atherosclerotic cardiovascular event risk observed in cardiac surgery patients., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Wilson F. Abdo reports financial support was provided by Netherlands Organisation for Health Research and Development. Mihai Netea reports financial support was provided by European Research Council. Mihai Netea reports financial support was provided by Dutch Research Council. Niels Riksen reports financial support was provided by Netherlands Heart Foundation. Siroon Bekkering reports financial support was provided by Netherlands Heart Foundation. Mihai Netea reports a relationship with TTxD that includes: equity or stocks. Mihai netea reports a relationship with Lemba that includes: equity or stocks. Mihai Netea reports a relationship with Biotrip that includes: equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. MGN is scientific founder of TTxD, Lemba and Biotrip., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Recent landmark trials showed that colchicine provides a substantial benefit in reducing major cardiovascular events in patients with coronary artery disease. Yet, its exact mechanism of action is still poorly understood. This study aimed to unravel the effect of colchicine on monocyte and neutrophil phenotype and function. A randomized double-blind placebo-controlled cross-over intervention study was executed in patients with a history of myocardial infarction. In neutrophils, colchicine treatment decreased CD62L expression and NGAL release upon ex vivo stimulation and increased PMA-induced ROS production. The effects of colchicine on monocytes were limited to a decrease in HLA-DR expression in the intermediate and nonclassical monocytes. Also, on the level of RNA expression, colchicine did not affect monocyte phenotype, while affecting various immunomodulating genes in neutrophils. Overall, our study suggests that treatment with colchicine affects neutrophil function, particularly by reducing neutrophil recruitment, lowering concentrations of NGAL, and changing the expression of various genes with immunomodulatory potential, whereas the effect on monocytes is limited., (© 2024 The Author(s). Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Brief exposure of monocytes to atherogenic molecules, such as oxidized lipoproteins, triggers a persistent pro-inflammatory phenotype, named trained immunity. In mice, transient high-fat diet leads to trained immunity, which aggravates atherogenesis. We hypothesized that a single high-fat challenge in humans induces trained immunity. In a randomized controlled cross-over study, 14 healthy individuals received a high-fat or reference shake, and blood was drawn before and after 1, 2, 4, 6, 24, and 72 h. Incubation of donor monocytes with the post-high-fat-shake serum induced trained immunity, regulated via Toll-like receptor 4. This was not mediated via triglyceride-rich lipoproteins, C12, 14, and 16, or metabolic endotoxemia. In vivo , however, the high-fat challenge did not affect monocyte phenotype and function. We conclude that a high-fat challenge leads to alterations in the serum composition that have the potential to induce trained immunity in vitro . However, this does not translate into a (persistent) hyperinflammatory monocyte phenotype in vivo ., Competing Interests: M.G.N. and L.A.B.J. are scientific founders of TTxD and Lemba Therapeutics., (© 2024 The Author(s).)

Innate immune cells can develop a long-lasting hyperresponsive phenotype, termed trained immunity, mediated by epigenetic and metabolic reprogramming. In mice, exposure to Bacille Calmette-Guérin (BCG), β-glucan, or Western diet induces trained immunity by reprogramming hematopoietic progenitor cells (HPCs), through interleukin-1β (IL-1β) signaling in the bone marrow (BM). We investigated whether IL-1β induces trained immunity in primary human BM-derived HPCs in vitro. We exposed human BM-derived HPCs to IL-1β for 4 h. HPCs were expanded and differentiated into monocytes followed by functional and transcriptomic characterization. IL-1β-exposed HPCs showed higher granulocyte-macrophage colony-forming units. The monocyte offspring produced more tumor necrosis factor (TNF) and IL-1β after restimulation with lipopolysaccharide (LPS) and Pam3Cys and is metabolically more active. Transcriptomic analysis showed upregulation of key atherogenic and inflammatory pathways. In conclusion, brief exposure of human BM-derived HPCs to IL-1β in vitro induces a trained immunity phenotype., Competing Interests: Declaration of interests M.G.N. and L.A.B.J. are scientific founders of TTxD and Lemba TX. M.G.N. is scientific founder of Biotrip. W.H.C.R. is a consultant for Stryker for educational purposes only., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cardiometabolic risk accrues across the life course and childhood and adolescence are key periods for effective prevention. Obesity is associated with inflammation in adults, but pediatric data are scarce. In a cross-sectional and longitudinal study, we investigated immune cell composition and activation in 31 adolescents with obesity (41.9% male, BMIz>2.5, 14.4 years) and 22 controls with healthy weight (45.1% male, -1.5

Background: Traditional risk stratification modestly predicts adverse cardiovascular events in patients with coronary artery disease (CAD). Our aim was to investigate the association between monocyte subsets numbers and function, and the first major adverse cardiovascular event (MACE) in patients with symptomatic stable CAD and angiographically documented coronary atherosclerosis., Methods: Patients with stable CAD were screened for inclusion. Using flow cytometry, we identified classical, intermediate, and non-classical monocyte subsets and we assessed cytokine production capacity after ex-vivo stimulation of peripheral blood mononuclear cells. Clinical follow-up was performed after four years. The endpoint was the composite of cardiovascular death, acute myocardial infarction, and ischemic stroke., Results: A cohort of 229 patients was recruited. The percentage of intermediate monocytes was positively associated with adverse cardiovascular events at follow-up (HR 1.09; 95%CI 1.02-1.16; p = 0.006), while the percentage of classical monocytes was identified as a protective factor for adverse outcomes (HR 0.96; 95%CI 0.94-0.99; p = 0.02). The percentage of intermediate monocytes remained independently associated with outcomes after adjusting for age, systolic blood pressure, and left ventricular ejection fraction (HR 1.07; 95% CI 1.01-1.14; p = 0.04). Several correlations were identified between monocyte subsets and stimulated cytokine production, but cytokine production capacity was not associated with adverse outcomes., Conclusions: In patients with stable CAD, intermediate monocytes were associated with MACE at follow-up. The association was not due to an increased cytokine production capacity. Novel biomarkers could improve risk stratification in patients with stable CAD and could represent new pharmacological targets against atherosclerosis progression., Competing Interests: Declaration of competing interest The authors report no relationships that could be construed as a conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Clonal hematopoiesis (CH) is a risk factor for atherosclerotic cardiovascular disease, but the impact of smaller clones and the effect on inflammatory parameters is largely unknown. Using ultrasensitive single-molecule molecular inversion probe sequencing, we evaluated the association between CH and a first major adverse cardiovascular event (MACE) in patients with angiographically documented stable coronary artery disease (CAD) and no history of acute ischemic events. CH was associated with an increased rate of MACE at four years follow-up. The size of the clone predicted MACE at an optimal cut-off value of 1.07% variant allele frequency (VAF). Mutation carriers had no change in monocytes subsets or cytokine production capacity but had higher levels of circulating tissue factor, matrilysin, and proteinase-activated receptor-1. Our study identified CH driver mutations with a VAF as small as 1.07% as a residual cardiovascular risk factor and identified potential biomarkers and therapeutic targets for patients with stable CAD., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Low-grade systemic inflammation is a key pathophysiological component of atherosclerotic cardiovascular disease (CVD), and long-term activation of myeloid cells is thought to be crucial for these effects. Obesity and associated metabolic complications including hyperglycaemia and dyslipoproteinaemia can induce long-lasting inflammatory reprogramming of the innate immune cells and their bone marrow progenitors, which in turn contributes to atherosclerosis. In this review, we discuss the mechanisms through which innate immune cells undergo long-term changes in their functional, epigenetic, and metabolic characteristics upon even short-term exposure to endogenous ligands, a process also termed 'trained immunity'. Inappropriate induction of trained immunity leads to the development of long-lasting hyperinflammatory and proatherogenic changes in monocytes and macrophages, an important factor in the development of atherosclerosis and CVDs. Knowledge of the specific immune cells and the distinct intracellular molecular pathways involved in the induction of trained immunity will reveal novel pharmacological targets that could be used to prevent or treat CVDs in the future., Competing Interests: Conflict of interest: M.G.N. is scientif founder of TTxD and Lemba Therapeutics., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Leptin is associated with cardiometabolic complications of obesity, such as metabolic syndrome and atherosclerosis. In obese men, the presence of metabolic syndrome is associated with higher circulating leptin and interleukin (IL)-6 concentrations and increased monocyte cytokine production capacity. Here, we investigated the effects of leptin on monocyte function and systemic inflammatory markers in obese individuals. We specifically explored whether leptin can induce long-term changes in innate immune function by inducing innate immune memory (also called trained immunity). We exposed human primary monocytes for 24 h to relevant leptin concentrations in vitro and measured cytokine production. In addition, after removing leptin, we incubated monocytes for 5 d in culture medium, and we restimulated them on day 6 to assess cytokine production capacity, phagocytosis, and foam cell formation. Direct stimulation with leptin did not induce cytokine production, but exposure to 50 ng/mL leptin augmented lipopolysaccharide- and R848-induced tumor necrosis factor α (TNF-α) production after 1 wk. In a separate in vivo study in a cohort of 302 obese subjects (body mass index [BMI] >27 kg/m2, 55 to 81 yr), we measured circulating leptin, inflammatory markers, and cytokine production upon ex vivo stimulation of isolated peripheral blood mononuclear cells. Circulating leptin concentrations positively correlated with circulating IL-1β and IL-6, which was more pronounced in men than in women. Four single nucleotide polymorphisms in the leptin gene influenced circulating IL-6 concentrations in men, suggesting a direct effect of leptin on IL-6. In conclusion, in vitro, leptin does not directly stimulate monocytes to produce cytokines, yet induces long-term monocyte hyperresponsiveness, i.e. trained immunity. In obese subjects, leptin is associated with circulating IL-6 in a sex-dependent manner. The underlying mechanisms of the sex-specific effect of leptin on innate immune cells remain to be further investigated., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

Background: Obesity is accompanied by dysregulated inflammation, which can contribute to vasculometabolic complications including metabolic syndrome and atherosclerosis. Recently, clonal hematopoiesis of indeterminate potential (CHIP) has emerged as a risk factor for cardiovascular diseases. We aimed to determine how CHIP is related to immune cell function, systemic inflammation, and vasculometabolic complications in obese individuals., Methods and Results: Two hundred ninety-seven individuals with overweight and obesity, between the ages of 54 and 81 years, were recruited in a cross-sectional study. Clonal hematopoiesis driver mutations (CHDMs) were identified with an ultrasensitive targeted assay. Assessment of carotid artery atherosclerosis was performed with ultrasound. Detailed immunological parameters, including cytokine production capacity of peripheral blood mononuclear cells, and targeted plasma proteomics analysis, were studied. Adipose tissue inflammation was determined in subcutaneous fat biopsies. Individuals with CHIP had higher concentrations of circulating IL (interleukin)-6. Total number of leukocytes and neutrophils were higher in individuals with CHIP. In contrast, ex vivo cytokine production capacity of peripheral blood mononuclear cells was significantly lower in individuals with CHIP. Sex-stratified analysis showed that men with CHDMs had significantly higher leukocyte and neutrophil counts, and ex vivo cytokine production capacity was lower in women with CHDMs. Surprisingly, the presence of atherosclerotic plaques was significantly lower in individuals with CHDMs. There was no relation between CHIP and metabolic syndrome., Conclusions: In individuals with overweight or obesity, CHDMs are not associated with vasculometabolic complications, but rather with a lower presence of carotid plaques. CHDMs associate with increased circulating inflammatory markers and leukocyte numbers, but a lower peripheral blood mononuclear cell cytokine production capacity.

Trained immunity, also known as innate immune memory, is a persistent hyper-responsive functional state of innate immune cells. Accumulating evidence implicates trained immunity as an underlying mechanism of chronic inflammation in atherosclerotic cardiovascular disease. In this context, trained immunity is induced by endogenous atherosclerosis-promoting factors, such as modified lipoproteins or hyperglycaemia, causing broad metabolic and epigenetic reprogramming of the myeloid cell compartment. In addition to traditional cardiovascular risk factors, lifestyle factors, including unhealthy diets, sedentary lifestyle, sleep deprivation and psychosocial stress, as well as inflammatory comorbidities, have been shown to activate trained immunity-like mechanisms in bone marrow haematopoietic stem cells. In this Review, we discuss the molecular and cellular mechanisms of trained immunity, its systemic regulation through haematopoietic progenitor cells in the bone marrow, and the activation of these mechanisms by cardiovascular disease risk factors. We also highlight other trained immunity features that are relevant for atherosclerotic cardiovascular disease, including the diverse cell types that show memory characteristics and transgenerational inheritance of trained immunity traits. Finally, we propose potential strategies for the therapeutic modulation of trained immunity to manage atherosclerotic cardiovascular disease., (© 2023. Springer Nature Limited.)

Background: Obesity-associated chronic inflammation mediates the development of adverse cardiometabolic outcomes. There are sparse data on associations between severe obesity and inflammatory biomarkers in adolescence; most are cross-sectional and limited to acute phase reactants. Here, we investigate associations between adiposity measures and inflammatory biomarkers in children and adolescents with severe obesity both cross-sectionally and longitudinally., Methods: From the Childhood Overweight Biorepository of Australia (COBRA) study, a total of n = 262 participants, mean age 11.5 years (SD 3.5) with obesity had measures of adiposity (body mass index, BMI; % above the 95th BMI-centile, %>95th BMI-centile; waist circumference, WC; waist/height ratio, WtH; % total body fat, %BF; % truncal body fat, %TF) and inflammation biomarkers (glycoprotein acetyls, GlycA; high-sensitivity C-Reactive Protein, hsCRP; white blood cell count, WBC; and neutrophil/lymphocyte ratio, NLR) assessed at baseline. Ninety-eight individuals at mean age of 15.9 years (3.7) participated in a follow-up study 5.6 (2.1) years later. Sixty-two individuals had longitudinal data. Linear regression models, adjusted for age and sex for cross-sectional analyses were applied. To estimate longitudinal associations between change in adiposity measures with inflammation biomarkers, models were adjusted for baseline measures of adiposity and inflammation., Results: All adiposity measures were cross-sectionally associated with GlycA, hsCRP and WBC at both time points. Change in BMI, %>95th BMI-centile, WC, WtH and %TF were associated with concomitant change in GlycA and WBC, but not in hsCRP and NLR., Conclusion: GlycA and WBC but not hsCRP and NLR may be useful in assessing adiposity-related severity of chronic inflammation over time., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An increasing number of patients develop an atherothrombotic myocardial infarction (MI) in the absence of standard modifiable risk factors (SMuRFs). Monocytes and macrophages regulate the development of atherosclerosis, and monocytes can adopt a long-term hyperinflammatory phenotype by epigenetic reprogramming, which can contribute to atherogenesis (called "trained immunity"). We assessed circulating monocyte phenotype and function and specific histone marks associated with trained immunity in SMuRFless patients with MI and matched healthy controls. Even in the absence of systemic inflammation, monocytes from SMuRFless patients with MI had an increased overall cytokine production capacity, with the strongest difference for LPS-induced interleukin-10 production, which was associated with an enrichment of the permissive histone marker H3K4me3 at the promoter region. Considering the lack of intervenable risk factors in these patients, trained immunity could be a promising target for future therapy., Competing Interests: M.N. is scientific founder of Trained Therapeutic Discovery, Inc., (© 2023 The Authors.)

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, where persistent presence of antiphospholipid antibodies (aPL) leads to thrombotic and obstetric complications. APS is a paradigmatic thromboinflammatory disease. Thromboinflammation is a pathophysiological mechanism coupling inflammation and thrombosis, which contributes to the pathophysiology of cardiovascular disease. APS can serve as a model to unravel mechanisms of thromboinflammation and the relationship between innate immune cells and thrombosis. Monocytes are activated by aPL into a proinflammatory and procoagulant phenotype, producing proinflammatory cytokines such as tumor necrosis factor α, interleukin 6, as well as tissue factor. Important cellular signaling pathways involved are the NF-κB-pathway, mammalian target of rapamycin (mTOR) signaling, and the NOD-, LRR-, and pyrin domain-containing protein 3 inflammasome. All of these may serve as future therapeutic targets. Neutrophils produce neutrophil extracellular traps in response to aPL, and this leads to thrombosis. Thrombosis in APS also stems from increased interaction of neutrophils with endothelial cells through P-selectin glycoprotein ligand-1. NETosis can be targeted not only with several experimental therapeutics, such as DNase, but also through the redirection of current therapies such as defibrotide and the antiplatelet agent dipyridamole. Activation of platelets by aPL leads to a procoagulant phenotype. Platelet-leukocyte interactions are increased, possibly mediated by increased levels of soluble P-selectin and soluble CD40-ligand. Platelet-directed future treatment options involve the inhibition of several platelet receptors activated by aPL, as well as mTOR inhibition. This review discusses mechanisms underlying thromboinflammation in APS that present targetable therapeutic options, some of which may be generalizable to other thromboinflammatory diseases., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The mechanisms underlying innate immune memory have been extensively explored in the last decades but are in fact largely unknown. Although the specificity of adaptive immune memory in vertebrates is ensured through the recombination of immunoglobulin family genes and clonal expansion, the basic mechanisms of innate immune cells' nonspecific increased responsiveness rely on epigenetic, transcriptional, and metabolic programs after transient stimulation. Changes in these programs result in enhanced responsiveness to secondary challenges with a wide variety of stimuli. This phenomenon is termed "trained immunity" or "innate immune memory." On one hand, trained immunity improves the response to infections and vaccination, facilitating stronger innate immune responses and enhanced protection against a variety of microbial stimuli. Conversely, trained immunity may contribute to the pathophysiology of cardiovascular, autoinflammatory, and neurodegenerative diseases. In this review, we gather the current body of knowledge in this field and summarize the foundations and mechanisms of trained immunity, the different cell types involved, its consequences for health and disease, and the potential of its modulation as a therapeutic tool.

Bacille-Calmette Guérin (BCG) modulates atherosclerosis development in experimental animals, but it remains unclear whether neonatal BCG vaccination is pro- or anti-atherogenic. Many animal models differ fundamentally from BCG administration to human infants in terms of age, vaccine preparation, dosing schedule, and route of administration. We aimed to elucidate the effect of neonatal subcutaneous BCG vaccination—analogous to human BCG vaccination—on atherosclerosis development in ApoE−/− mice. At 2 days of age, a total of 40 ApoE−/− mice received either a weight-equivalent human dose of BCG, or saline, subcutaneously. From 4 weeks onwards, the mice were fed a Western-type diet containing 22% fat. At 16 weeks of age, mice were sacrificed for the assessment of atherosclerosis. Body weight, plasma lipids, atherosclerosis lesion size and collagen content were similar in both groups. Atherosclerosis lesion number was lower in mice that received BCG. Macrophage content was 20% lower in the BCG-vaccinated mice (p < 0.05), whereas plaque lipid content was increased by 25% (p < 0.01). In conclusion, neonatal BCG vaccination reduces atherosclerosis plaque number and macrophage content but increases lipid content in a murine model of atherosclerosis. Human epidemiological and mechanistic studies are warranted to investigate whether neonatal BCG vaccination is potentially atheroprotective.

Background: Emotional and behavioral problems (EBP) are common in children. Environmental factors like socioeconomic disadvantage influence EBP pathogenesis and can trigger inflammation. However, the link between early inflammation-EBP in children is unclear. We investigated the associations between i) infant inflammatory biomarkers and subsequent EBP and ii) early life environmental factors and EBP and assessed whether infant inflammation mediated these associations., Methods: Inflammatory biomarkers glycoprotein acetyls (GlycA) and high-sensitivity C-reactive protein (hsCRP) were quantified at birth and 12 months in a population-derived birth cohort, the Barwon Infant Study. Early life factors including demographic, prenatal, and perinatal factors were collected from antenatal to the two-year period. Internalizing and externalizing problems at age two were measured by the Child Behavior Checklist. Prospective associations were examined by multivariable regression analyses adjusted for potential confounders. Indirect effects of early life factors on EBP through inflammation were identified using mediation analyses., Results: Elevated GlycA levels at birth (GlycA birth ) were associated with greater internalizing problems at age two (β = 1.32 per SD increase in GlycA; P = 0.001). Inflammation at birth had a stronger magnitude of effect with later EBP than at 12 months. GlycA birth partially mediated the associations between lower household income (6%), multiparity (12%) and greater number of older siblings (13%) and EBP. Patterns were less evident for hsCRP or externalizing problems., Conclusions: GlycA birth was positively associated with EBP at age two and partially mediated the association between several indicators of socioeconomic disadvantage and EBP. Prenatal and perinatal inflammation may be relevant to early neurodevelopment and emotional health., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Trained immunity - or innate immune memory - can be described as the long-term reprogramming of innate immune cells towards a hyperresponsive state which involves intracellular metabolic changes. Trained immunity has been linked to atherosclerosis. A subgroup of patients with primary Sjögren's syndrome (pSS) exhibits systemic type I interferon (IFN) pathway activation, indicating innate immune hyperactivation. Here, we studied the link between type I IFNs and trained immunity in an in vitro monocytic cell model and peripheral blood mononuclear cells (PBMCs) from pSS patients., Methods: The training stimuli heat killed Candida albicans , muramyl dipeptide, IFNβ, and patient serum were added to THP-1 cells for 24 hours, after which the cells were washed, rested for 48 hours and subsequently re-stimulated with LPS, Pam3Cys, poly I:C, IFNβ or oxLDL for 4-24 hours. PBMCs from pSS patients and healthy controls were stimulated with LPS, Pam3Cys, poly I:C or IFNβ for 0.5-24 hours., Results: Training with IFNβ induced elevated production of pro-atherogenic cytokines IL-6, TNFα and CCL2 , differential cholesterol- and glycolysis-related gene expression, and increased glucose consumption and oxLDL uptake upon re-stimulation. Type I IFN production was increased in Candida albicans - and IFNβ-trained cells after LPS re-stimulation, but was reduced after poly I:C re-stimulation. Training with muramyl dipeptide and IFNβ, but not Candida albicans , affected the IFN-stimulated gene expression response to IFNβ re-stimulation. PBMCs from pSS patients consumed more glucose compared with healthy control PBMCs and tended to produce more TNFα and type I IFNs upon LPS stimulation, but less type I IFNs upon poly I:C stimulation., Conclusions: Type I IFN is a trainer inducing a trained immunity phenotype with pro-atherogenic properties in monocytes. Conversely, trained immunity also affects the production of type I IFNs and transcriptional response to type I IFN receptor re-stimulation. The phenotype of pSS PBMCs is consistent with trained immunity. This connection between type I IFN, trained immunity and cholesterol metabolism may have important implications for pSS and the pathogenesis of (subclinical) atherosclerosis in these patients., Competing Interests: LJ is scientific founder of Trained Therapeutix Discovery (TTxD). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Huijser, van Helden-Meeuwsen, Grashof, Tarn, Brkic, Huisman, Wahadat, van de Werken, Lopes, van Roon, van Daele, Kamphuis, Ng, Bekkering, Joosten, Dik and Versnel.)

Calcific aortic valve disease (CAVD) is the most common valvular disease in the developed world with currently no effective pharmacological treatment available. CAVD results from a complex, multifactorial process, in which valvular inflammation and fibro-calcific remodelling lead to valve thickening and cardiac outflow obstruction. The exact underlying pathophysiology of CAVD is still not fully understood, yet the development of CAVD shows many similarities with the pathophysiology of atherosclerotic cardiovascular disease (ASCVD), such as coronary artery disease. Innate immune cells play a crucial role in ASCVD and might also play a pivotal role in the development of CAVD. This review summarizes the current knowledge on the role of innate immune cells, both in the circulation and in the aortic valve, in the development of CAVD and the similarities and differences with ASCVD. Trained immunity and clonal haematopoiesis of indeterminate potential are proposed as novel immunological mechanisms that possibly contribute to the pathophysiology of CAVD and new possible treatment targets are discussed., (© 2022. The Author(s).)

Background: The risk of adult onset cardiovascular and metabolic (cardiometabolic) disease accrues from early life. Infection is ubiquitous in infancy and induces inflammation, a key cardiometabolic risk factor, but the relationship between infection, inflammation, and metabolic profiles in early childhood remains unexplored. We investigated relationships between infection and plasma metabolomic and lipidomic profiles at age 6 and 12 months, and mediation of these associations by inflammation., Methods: Matched infection, metabolomics, and lipidomics data were generated from 555 infants in a pre-birth longitudinal cohort. Infection data from birth to 12 months were parent-reported (total infections at age 1, 3, 6, 9, and 12 months), inflammation markers (high-sensitivity C-reactive protein [hsCRP]; glycoprotein acetyls [GlycA]) were quantified at 12 months. Metabolic profiles were 12-month plasma nuclear magnetic resonance metabolomics (228 metabolites) and liquid chromatography/mass spectrometry lipidomics (776 lipids). Associations were evaluated with multivariable linear regression models. In secondary analyses, corresponding inflammation and metabolic data from birth (serum) and 6-month (plasma) time points were used., Results: At 12 months, more frequent infant infections were associated with adverse metabolomic (elevated inflammation markers, triglycerides and phenylalanine, and lower high-density lipoprotein [HDL] cholesterol and apolipoprotein A1) and lipidomic profiles (elevated phosphatidylethanolamines and lower trihexosylceramides, dehydrocholesteryl esters, and plasmalogens). Similar, more marked, profiles were observed with higher GlycA, but not hsCRP. GlycA mediated a substantial proportion of the relationship between infection and metabolome/lipidome, with hsCRP generally mediating a lower proportion. Analogous relationships were observed between infection and 6-month inflammation, HDL cholesterol, and apolipoprotein A1., Conclusions: Infants with a greater infection burden in the first year of life had proinflammatory and proatherogenic plasma metabolomic/lipidomic profiles at 12 months of age that in adults are indicative of heightened risk of cardiovascular disease, obesity, and type 2 diabetes. These findings suggest potentially modifiable pathways linking early life infection and inflammation with subsequent cardiometabolic risk., Funding: The establishment work and infrastructure for the BIS was provided by the Murdoch Children's Research Institute (MCRI), Deakin University, and Barwon Health. Subsequent funding was secured from National Health and Medical Research Council of Australia (NHMRC), The Shepherd Foundation, The Jack Brockhoff Foundation, the Scobie & Claire McKinnon Trust, the Shane O'Brien Memorial Asthma Foundation, the Our Women's Our Children's Fund Raising Committee Barwon Health, the Rotary Club of Geelong, the Minderoo Foundation, the Ilhan Food Allergy Foundation, GMHBA, Vanguard Investments Australia Ltd, and the Percy Baxter Charitable Trust, Perpetual Trustees. In-kind support was provided by the Cotton On Foundation and CreativeForce. The study sponsors were not involved in the collection, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication. Research at MCRI is supported by the Victorian Government's Operational Infrastructure Support Program. This work was also supported by NHMRC Senior Research Fellowships to ALP (1008396); DB (1064629); and RS (1045161) , NHMRC Investigator Grants to ALP (1110200) and DB (1175744), NHMRC-A*STAR project grant (1149047). TM is supported by an MCRI ECR Fellowship. SB is supported by the Dutch Research Council (452173113)., Competing Interests: TM TM has received a postdoctoral fellowship from MCRI, is supported by NHMRC funding, has received travel support from MCRI and the University of Melbourne, and received a PhD scholarship from the University of Melbourne, RS, SB, AS, RR, PS, FC, PM No competing interests declared, AP ALP is an unpaid scientific advisor for, and has shares in, Dysrupt Labs. ALP has shares in Prevatex Pty Ltd, MT MLKT has received funding paid to Murdoch Childen's Research Institute (MCRI) from NHMRC, Prota Theraputics, Abbott Nutrition, the Allergy and Immunology Foundation of Australasia, and the National Children's Research Centre of Ireland, and has received internal research funding from MCRI. MLKT is inventor of 2 patents owned by MCRI relating to allergy treatment and a method to induce tolerance to an allergen. MLKT is a member of the Advisory Boards for Pfizer (has received personal fee) and Anaphylaxis & Anaphylaxis Australia, and of allergy/anaphylaxis-related Committees for the World Allergy Organisation, the International Union of Immunological Societies, the Asia Pacific Association of Allergy Asthma and Clinical Immunology, the American Academy of Allergy Asthma and Immunology, and the Australasian Society of Clinical Immunology and Allergy. MLKT is employee of, and has share options in, Prota Therapeutics. MLKT is an Associate Editor for the Journal of Allergy and Clinical Immunology: Global, MO MOH has stocks in Prevatex Pty Ltd, SB S Bekkering has received postdoctoral grants from the Dutch Heart Foundation and the Dutch Research Council, and travel support from the European Society for Atherosclerosis, SR SR is Director of the Lung Foundation Australia. SR has stocks/options in Prevatex Pty Ltd, PV PV is an inventor on a patent relating to the relationship between maternal carriage of Prevotella. copri and offspring allergic disease, and has stocks/options in Prevatex Pty Ltd, DB DB has received an Investigator Grant and Project Grant from the Australian National Health and Medical Research Council (NHMRC), (© 2022, Mansell et al.)

Background: Obesity in childhood is associated with metabolic dysfunction, adverse subclinical cardiovascular phenotypes and adult cardiovascular disease. Longitudinal studies of youth with obesity investigating changes in severity of obesity with metabolomic profiles are sparse. We investigated associations between (i) baseline body mass index (BMI) and follow-up metabolomic profiles; (ii) change in BMI with follow-up metabolomic profiles; and (iii) change in BMI with change in metabolomic profiles (mean interval 5.5 years)., Methods: Participants (n = 98, 52% males) were recruited from the Childhood Overweight Biorepository of Australia study. At baseline and follow-up, BMI and the % >95th BMI-centile (percentage above the age-, and sex-specific 95th BMI-centile) indicate severity of obesity, and nuclear magnetic resonance spectroscopy profiling of 72 metabolites/ratios, log-transformed and scaled to standard deviations (SD), was performed in fasting serum. Fully adjusted linear regression analyses were performed., Results: Mean (SD) age and % >95th BMI-centile were 10.3 (SD 3.5) years and 134.6% (19.0) at baseline, 15.8 (3.7) years and 130.7% (26.2) at follow-up. Change in BMI over time, but not baseline BMI, was associated with metabolites at follow-up. Each unit (kg/m 2 ) decrease in sex- and age-adjusted BMI was associated with change (SD; 95% CI; p value) in metabolites of: alanine (-0.07; -0.11 to -0.04; p < 0.001), phenylalanine (-0.07; -0.10 to -0.04; p < 0.001), tyrosine (-0.07; -0.10 to -0.04; p < 0.001), glycoprotein acetyls (-0.06; -0.09 to -0.04; p < 0.001), degree of fatty acid unsaturation (0.06; 0.02 to 0.10; p = 0.003), monounsaturated fatty acids (-0.04; -0.07 to -0.01; p = 0.004), ratio of ApoB/ApoA1 (-0.05; -0.07 to -0.02; p = 0.001), VLDL-cholesterol (-0.04; -0.06 to -0.01; p = 0.01), HDL cholesterol (0.05; 0.08 to 0.1; p = 0.01), pyruvate (-0.08; -0.11 to -0.04; p < 0.001), acetoacetate (0.07; 0.02 to 0.11; p = 0.005) and 3-hydroxybuturate (0.07; 0.02 to 0.11; p = 0.01). Results using the % >95th BMI-centile were largely consistent with age- and sex-adjusted BMI measures., Conclusions: In children and young adults with obesity, decreasing the severity of obesity was associated with changes in metabolomic profiles consistent with lower cardiovascular and metabolic disease risk in adults., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Early childhood is characterised by repeated infectious exposures that result in inflammatory responses by the innate immune system. In addition, this inflammatory response to infection is thought to contribute to the epidemiological evidence linking childhood infection and adult non-communicable diseases. Consequently, the relationship between innate immune responses and inflammation during early life may inform prevention of NCDs later in life. In adults, non-genetic host factors such as age, sex, and obesity, strongly impact cytokine production and circulating mediators, but data in children are lacking. Here, we assessed cytokine responses and inflammatory markers in a population of healthy preschool children (mean age 4.2 years). We studied associations between cytokines, plasma inflammatory markers and non-genetic host factors, such as sex, age, adiposity, season, and immune cell composition. Similar to adults, boys had a higher inflammatory response than girls, with IL-12p70 and IL-10 upregulated following TLR stimulation. Adiposity and winter season were associated with increased circulating inflammatory markers but not cytokine production. The inflammatory markers GlycA and hsCRP were positively associated with production of a number of cytokines and may therefore reflect innate immune function and inflammatory potential. This dataset will be informative for future prospective studies relating immune parameters to preclinical childhood NCD phenotypes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Collier, Chau, Mansell, Faye-Chauhan, Vuillermin, Ponsonby, Saffery, Tang, O’Hely, Carlin, Gray, Bekkering, Burgner and the Barwon Infant Study Investigator Group.)

Objective: Despite the advances in the control of traditional risk factors, coronary artery disease (CAD) remains the greatest cause of morbidity and mortality. Our aim was to establish the relation between plasma proteomics analysis and the risk of cardiovascular events in patients with stable CAD., Materials and Methods: Patients with stable CAD and documented coronary atherosclerosis were screened for inclusion. Using proximity extension assays, 177 plasma proteins were simultaneously measured. The endpoint consisted of the first major adverse cardiovascular event (MACE) and was the composite of cardiovascular death, acute coronary syndrome, stroke, transient ischemic attack, or acute limb ischemia at 18 months follow-up. Cox proportional-hazards regression with adjustment for multiple comparisons was used to identify biomarkers for the outcomes of interest., Results: The cohort consisted of 229 patients. Six mediators were associated with MACE ( p < 0.001). For these markers, the risk of MACE was calculated: tumor necrosis factor receptor superfamily member 13B (HR = 1.65; 95% CI: 1.30-2.10), C-C motif chemokine-3 (HR = 1.57; 95% CI: 1.23-1.98), decorin (HR = 1.65; 95% CI: 1.26-2.16), fibroblast growth factor-23 (HR = 1.56; 95% CI: 1.23-1.99), tumor necrosis factor-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2) (HR = 1.61; 95% CI: 1.23-2.11), and tumor necrosis factor receptor superfamily member 10A (HR = 1.69; 95% CI: 1.25-2.29). Except for TRAIL-R2, the other proteins were associated with MACE independent of age, sex, diabetes mellitus, or estimated glomerular filtration rate., Conclusions: In patients with stable CAD, five novel biomarkers were identified as independent risk factors for adverse outcomes. Novel biomarkers could represent pharmacological targets for the prevention of adverse cardiovascular events., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dregoesc, Ţigu, Bekkering, van der Heijden, Bolboacǎ, Joosten, Visseren, Netea, Riksen and Iancu.)

Background and Aims: Childhood obesity is associated with cardiovascular risk factors (CVRF), subclinical cardiovascular phenotypes (carotid intima-media thickness, cIMT; pulse-wave velocity, PWV; and carotid elasticity), and adult cardiovascular disease (CVD) mortality. In youth with obesity (body mass index, BMI ≥95th centile), we investigated associations between changes in adiposity and CVRF in early adolescence and subclinical cardiovascular phenotypes in late adolescence., Methods: Participants had adiposity measures (the severity of obesity in percentage >95th BMI-centile (%>95th BMI-centile)), waist circumference (WC), percentage total body fat (%BF) and CVRF (systolic blood pressure, SBP; glycoprotein acetyls, GlycA; and low-density lipoprotein cholesterol) assessed in early (mean age 10.2 ± 3.5y) and late (15.7 ± 3.7y) adolescence. Subclinical cardiovascular phenotypes were assessed in late adolescence. Multivariable regression analysis was performed., Results: Decreasing the %>95th BMI-centile was associated with carotid elasticity (0.945%/10 mmHg, p = 0.002) in females, and with PWV in males (-0.75 m/s, p < 0.001). Changes in all adiposity measures (per 1-unit increase) were associated with carotid elasticity (-0.020 to -0.063%/10 mmHg, p < 0.005), and PWV (0.011-0.045 m/s, p < 0.005). Changes in GlycA (per 50μmol-increase) were associated with elasticity (-0.162%/10 mmHg, p = 0.042), and changes in SBP (per 10 mmHg-increase) were associated with PWV (0.260 m/s, p < 0.001). Adjusted for change in BMI, the coefficient for GlycA was reduced by 46% and for SBP by 12%. Only male sex was associated with cIMT (+34 μm, p = 0.006)., Conclusions: In youth with obesity, decreasing or maintaining the severity of obesity, and decreasing the levels of SBP and GlycA from early to late adolescence was associated with low arterial stiffness., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Preterm birth is associated with increased risk of cardiovascular disease (CVD). This may reflect a legacy of inflammatory exposures such as chorioamnionitis which complicate pregnancies delivering preterm, or recurrent early-life infections, which are common in preterm infants. We previously reported that experimental chorioamnionitis followed by postnatal inflammation has additive and deleterious effects on atherosclerosis in ApoE-/- mice. Here, we aimed to investigate whether innate immune training is a contributory inflammatory mechanism in this murine model of atherosclerosis., Methods: Bone marrow-derived macrophages and peritoneal macrophages were isolated from 13-week-old ApoE-/- mice, previously exposed to prenatal intra-amniotic (experimental choriomanionitis) and/or repeated postnatal (peritoneal) lipopolysaccharide (LPS). Innate immune responses were assessed by cytokine responses following ex vivo stimulation with toll-like receptor (TLR) agonists (LPS, Pam3Cys) and RPMI for 24-h. Bone marrow progenitor populations were studied using flow cytometric analysis., Results: Following postnatal LPS exposure, bone marrow-derived macrophages and peritoneal macrophages produced more pro-inflammatory cytokines following TLR stimulation than those from saline-treated controls, characteristic of a trained phenotype. Cytokine production ex vivo correlated with atherosclerosis severity in vivo. Prenatal LPS did not affect cytokine production capacity. Combined prenatal and postnatal LPS exposure was associated with a reduction in populations of myeloid progenitor cells in the bone marrow., Conclusions: Postnatal inflammation results in a trained phenotype in atherosclerosis-prone mice that is not enhanced by prenatal inflammation. If analogous mechanisms occur in humans, then there may be novel early life opportunities to reduce CVD risk in infants with early life infections., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Aims: Periodontitis is an independent risk factor for cardiovascular disease, but the mechanistic link is not fully understood. In atherosclerotic cardiovascular disease, monocytes can adopt a persistent hyperresponsive phenotype, termed trained immunity. We hypothesized that periodontitis-associated bacteria can induce trained immunity in monocytes, which subsequently accelerate atherosclerosis development., Materials and Methods: We combined in vitro experiments on human primary monocytes and in vivo techniques in patients with periodontitis to test this hypothesis. Adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with lipopolysaccharide (LPS) or Pam3CysK4 (P3C) six days later, to measure interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) production. In an exploratory observational study, patients with severe periodontitis (63 ± 6 years, n=14) and control subjects with no-to-mild periodontitis (54 ± 10 years, n=14) underwent venipuncture and 2'-deoxy-2'-[ 18 F]fluoro-D-glucose positron-emission-tomography ([ 18 F]FDG PET/CT) scanning., Results: When adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with LPS or P3C six days later, IL-6 and TNFα production was significantly increased (TNFα/P3C, p<0.01). Circulating leukocytes, IL-6 and interleukin-1 receptor antagonist (IL-1Ra) concentrations were generally higher in patients compared to controls (leukocytes: p<0.01; IL-6: p=0.08; IL-1Ra: p=0.10). Cytokine production capacity in PBMCs after 24h stimulation revealed no differences between groups. [ 18 F]FDG PET/CT imaging showed a trend for increased [ 18 F]FDG-uptake in the periodontium [mean standard uptake value (SUV mean ), p=0.11] and in femur bone marrow (SUV mean , p=0.06), but no differences were observed for vascular inflammation. Positive correlations between severity of periodontitis, measured by The Dutch Periodontal Screening Index and pocket depth, with circulating inflammatory markers and tissue inflammation were found., Conclusions: P. gingivalis induces long-term activation of human monocytes in vitro (trained immunity). Patients with severe periodontitis did have signs of increased systemic inflammation and hematopoietic tissue activation. However, their circulating monocytes did not show a hyperresponsive phenotype. Together we suggest that trained immunity might contribute to local periodontal inflammation which warrants further investigation., Competing Interests: LJ and MGN are scientific founders of TTxD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Noz, Plachokova, Smeets, Aarntzen, Bekkering, Vart, Joosten, Netea and Riksen.)

Objective: Obesity is an established risk factor for severe coronavirus disease 2019 (COVID-19), but the contribution of overweight and/or diabetes remains unclear. In a multicenter, international study, we investigated if overweight, obesity, and diabetes were independently associated with COVID-19 severity and whether the BMI-associated risk was increased among those with diabetes., Research Design and Methods: We retrospectively extracted data from health care records and regional databases of hospitalized adult patients with COVID-19 from 18 sites in 11 countries. We used standardized definitions and analyses to generate site-specific estimates, modeling the odds of each outcome (supplemental oxygen/noninvasive ventilatory support, invasive mechanical ventilatory support, and in-hospital mortality) by BMI category (reference, overweight, obese), adjusting for age, sex, and prespecified comorbidities. Subgroup analysis was performed on patients with preexisting diabetes. Site-specific estimates were combined in a meta-analysis., Results: Among 7,244 patients (65.6% overweight/obese), those with overweight were more likely to require oxygen/noninvasive ventilatory support (random effects adjusted odds ratio [aOR], 1.44; 95% CI 1.15-1.80) and invasive mechanical ventilatory support (aOR, 1.22; 95% CI 1.03-1.46). There was no association between overweight and in-hospital mortality (aOR, 0.88; 95% CI 0.74-1.04). Similar effects were observed in patients with obesity or diabetes. In the subgroup analysis, the aOR for any outcome was not additionally increased in those with diabetes and overweight or obesity., Conclusions: In adults hospitalized with COVID-19, overweight, obesity, and diabetes were associated with increased odds of requiring respiratory support but were not associated with death. In patients with diabetes, the odds of severe COVID-19 were not increased above the BMI-associated risk., (© 2021 by the American Diabetes Association.)

Background: The etiology of cerebral small vessel disease (SVD) remains elusive, though evidence is accumulating that inflammation contributes to its pathophysiology. We recently showed retrospectively that pro-inflammatory monocytes are associated with the long-term progression of white matter hyperintensities (WMHs). In this prospective high-frequency imaging study, we hypothesize that the incidence of SVD progression coincides with a pro-inflammatory monocyte phenotype. Methods: Individuals with SVD underwent monthly magnetic resonance imaging (MRI) for 10 consecutive months to detect SVD progression, defined as acute diffusion-weighted imaging-positive (DWI+) lesions, incident microbleeds, incident lacunes, and WMH progression. Circulating inflammatory markers were measured, cytokine production capacity of monocytes was assessed after ex vivo stimulation, and RNA sequencing was performed on isolated monocytes in a subset of participants. Results: 13 out of 35 individuals developed SVD progression (70 ± 6 years, 54% men) based on incident lesions ( n = 7) and/or upper quartile WMH progression ( n = 9). Circulating E-selectin concentration ( p < 0.05) and the cytokine production capacity of interleukin (IL)-1β and IL-6 ( p < 0.01) were higher in individuals with SVD progression. Moreover, RNA sequencing revealed a pro-inflammatory monocyte signature including genes involved in myelination, blood-brain barrier, and endothelial-leukocyte interaction. Conclusions: Circulating monocytes of individuals with progressive SVD have an inflammatory phenotype, characterized by an increased cytokine production capacity and a pro-inflammatory transcriptional signature., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Noz, Telgte, Wiegertjes, Tuladhar, Kaffa, Kersten, Bekkering, van der Heijden, Hoischen, Joosten, Netea, Duering, de Leeuw and Riksen.)

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.

Macrophages play a central role in the pathogenesis of atherosclerosis. The inflammatory properties of these cells are dictated by their metabolism, of which the mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator. Using myeloid cell-specific nanobiologics in apolipoprotein E-deficient ( Apoe -/- ) mice, we found that targeting the mTOR and ribosomal protein S6 kinase-1 (S6K1) signaling pathways rapidly diminished plaque macrophages' inflammatory activity. By investigating transcriptome modifications, we identified Psap , a gene encoding the lysosomal protein prosaposin, as closely related with mTOR signaling. Subsequent in vitro experiments revealed that Psap inhibition suppressed both glycolysis and oxidative phosphorylation. Transplantation of Psap -/- bone marrow to low-density lipoprotein receptor knockout ( Ldlr -/- ) mice led to a reduction in atherosclerosis development and plaque inflammation. Last, we confirmed the relationship between PSAP expression and inflammation in human carotid atherosclerotic plaques. Our findings provide mechanistic insights into the development of atherosclerosis and identify prosaposin as a potential therapeutic target., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

A growing number of studies show that innate immune cells can undergo functional reprogramming, facilitating a faster and enhanced response to heterologous secondary stimuli. This concept has been termed "trained immunity." We outline here a protocol to recapitulate this in vitro using adherent monocytes from consecutive isolation of peripheral blood mononuclear cells. The induction of trained immunity and the associated functional reprogramming of monocytes is described in detail using β-glucan (from Candida albicans ) and Bacillus Calmette-Guérin as examples. For complete details on the use and execution of this protocol, please refer to Repnik et al. (2003) and Bekkering et al. (2016)., Competing Interests: The authors declare no competing interests, (© 2021 The Author(s).)