Host genetic regulation of human gut microbial structural variation.

Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established1-6, little is known about how host genetics regulates the genetic diversity of gut microorganisms. Here we conducted a meta-analysis of associations between human genetic variation and gut microbial structural variation in 9,015 individuals from four Dutch cohorts. Strikingly, the presence rate of a structural variation segment in Faecalibacterium prausnitzii that harbours an N-acetylgalactosamine (GalNAc) utilization gene cluster is higher in individuals who secrete the type A oligosaccharide antigen terminating in GalNAc, a feature that is jointly determined by human ABO and FUT2 genotypes, and we could replicate this association in a Tanzanian cohort. In vitro experiments demonstrated that GalNAc can be used as the sole carbohydrate source for F. prausnitzii strains that carry the GalNAc-metabolizing pathway. Further in silico and in vitro studies demonstrated that other ABO-associated species can also utilize GalNAc, particularly Collinsella aerofaciens. The GalNAc utilization genes are also associated with the host's cardiometabolic health, particularly in individuals with mucosal A-antigen. Together, the findings of our study demonstrate that genetic associations across the human genome and bacterial metagenome can provide functional insights into the reciprocal host-microbiome relationship.

Sex-specific association of visceral and subcutaneous adipose tissue volumes with systemic inflammation and innate immune cells in people living with obesity.

BACKGROUND AND AIMS: Obesity predisposes to metabolic and cardiovascular diseases. Adipose tissue inflammation and systemic inflammation contribute to these complications. There are strong sex differences in adipose tissue distribution and in systemic inflammation. Women have more subcutaneous adipose tissue (SAT) and less visceral adipose tissue (VAT) than men. We explored the sex differences in the association between the different adipose compartments and inflammatory markers that are important in cardiometabolic disease pathophysiology. METHODS: Single-center observational cohort study with 302 individuals with a BMI ≥ 27 kg/m2. We were unable to acquire MRI data from seven individuals and from another 18 the MRI data were not usable, resulting in 277 people (155 men, 122 women), aged 55-81 years. INTERVENTION: We performed the following measurements: abdominal magnetic resonance imaging to measure VAT, and SAT (deep and superficial) volumes; circulating leukocyte counts and cytokine production capacity of peripheral blood mononuclear cells (PBMCs), circulating cytokines, adipokines, and targeted proteomics; abdominal sSAT biopsies for histology and gene expression. RESULTS: Only in women, (s)SAT volume was associated with circulating leukocytes, monocytes, and neutrophils. Circulating IL-6 and IL-18BP were associated with SAT volume in women and VAT in men. Several circulating proteins, including monocyte-colony-stimulating factor 1 and hepatocyte growth factor, are associated with sSAT in women and VAT in men. Only in women, SAT volume is associated with SAT expression of inflammatory proteins, including leptin, CD68, TNFα and IL-1α. CONCLUSION: In women living with obesity, abdominal SAT volume, especially sSAT, is associated with circulating leukocytes and inflammatory proteins. In men, these parameters mainly show associations with VAT volume. This could be because only in women, sSAT volume is associated with sSAT expression of inflammatory proteins. These findings underscore that future research on adipose tissue in relation to cardiometabolic and cardiovascular disease should take sex differences into account.

No association between markers of systemic inflammation and endothelial dysfunction with Alzheimer's disease progression: a longitudinal study.

INTRODUCTION: Systemic inflammation and endothelial dysfunction are potentially modifiable factors implicated in Alzheimer's disease (AD), which offer potential therapeutic targets to slow disease progression. METHODS: We investigated the relationship between baseline circulating levels of inflammatory (TNF-α, IL-1ß) and endothelial cell markers (VCAM-1, ICAM-1, E-selectin) and 18-month cognitive decline (ADAS-cog12) in 266 mild-to-moderate AD patients from the NILVAD study. We employed individual growth models to examine associations, potential mediation, and interaction effects while adjusting for confounders. RESULTS: The average increase in ADAS-cog12 scores over all patients was 8.1 points in 18 months. No significant association was found between the markers and the rate of cognitive decline. Mediation analysis revealed no mediating role for endothelial cell markers, and interaction effects were not observed. DISCUSSION: Our results do not support the role of systemic inflammation or endothelial dysfunction in progression in persons with AD.

Association Between Clonal Hematopoiesis Driver Mutations, Immune Cell Function, and the Vasculometabolic Complications of Obesity.

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.

Clonal hematopoiesis is associated with cardiovascular events in patients with stable coronary artery disease.

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.

Increased innate immune responses in adolescents with obesity and its relation to subclinical cardiovascular measures: An exploratory study.

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

Intermediate monocytes are associated with the first major adverse cardiovascular event in patients with stable coronary artery disease.

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.

The Effect of Low-Dose Colchicine on the Phenotype and Function of Neutrophils and Monocytes in Patients with Chronic Coronary Artery Disease: A Double-Blind Randomized Placebo-Controlled Cross-Over Study.

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.

Relationship of neutrophil-to-lymphocyte ratio, in addition to C-reactive protein, with cardiovascular events in patients with type 2 diabetes.

AIM: To quantify the relationship of neutrophil-to-lymphocyte ratio (NLR) with cardiovascular events and all-cause mortality in patients with type 2 diabetes (T2D), independent of C-reactive protein (CRP). METHODS: Patients with T2D from the UCC-SMART-cohort were studied using multivariable-adjusted Cox regression. The relationship of NLR and CRP with vascular events (cerebrovascular events, myocardial infarction and vascular death) and all-cause mortality was quantified. RESULTS: During 10,833 person-years, 232 vascular events and 302 deaths occurred in 1,239 patients with T2D. Risk of vascular events and all-cause mortality increased per standard deviation (SD) in NLR (hazard ratio (HR) 1.27; 95 % confidence interval (CI):1.11-1.46) and 1.15; 95 % CI:1.02-1.30) after adjustment for CRP. CRP was not associated with vascular events after adjustment for NLR, (HR per SD 1.03; 95 % CI: 0.90-1.19), but was associated with all-cause mortality (HR per SD 1.18; 95 % CI: 1.04-1.33). Notably, NLR was related to vascular events in patients with CRP < 2 mg/L (HR per unit 1.45; 95 % CI: 1.19-1.77). CONCLUSION: In patients with T2D, NLR is related to higher risk of CVD and all-cause mortality, independently from CRP. NLR is related to CVD even when CRP is low, indicating that NLR is a marker of CVD-risk in addition to CRP. Both NLR and CRP are independently related to all-cause mortality in T2D patients.

Immune regulation and blood-brain barrier permeability in cerebral small vessel disease: study protocol of the INflammation and Small Vessel Disease (INSVD) study - a multicentre prospective cohort study.

INTRODUCTION: The INflammation and Small Vessel Disease (INSVD) study aims to investigate whether peripheral inflammation, immune (dys)regulation and blood-brain barrier (BBB) permeability relate to disease progression in cerebral small vessel disease (SVD). This research aims to pinpoint specific components of the immune response in SVD relating to disease progression. This could identify biomarkers of SVD progression, as well as potential therapeutic targets to inform the development and repurposing of drugs to reduce or prevent SVD, cognitive decline and vascular dementia. METHODS AND ANALYSIS: INSVD is a prospective observational multicentre cohort study in individuals with symptomatic SVD. This longitudinal study combines comprehensive immunophenotyping of the peripheral blood immune compartment with advanced neuroimaging markers of SVD and BBB permeability. The main SVD marker of interest is white matter microstructure as determined by diffusion tensor imaging, a valuable marker of disease progression owing to its sensitivity to early alterations to white matter integrity. The research is being conducted in two sites-in the UK (Cambridge) and the Netherlands (Nijmegen)-with each site recruiting 100 participants (total n=200). Participants undergo clinical and cognitive assessments, blood draws, and brain MRI at baseline and 2-year follow-up. ETHICS AND DISSEMINATION: This study received ethical approval from the local ethics boards (UK: East of England-Cambridge Central Research Ethics Committee (REC) ref: 22/EE/00141, Integrated Research Application System (IRAS) ID: 312 747. Netherlands: Medical Research Ethics Committee (MREC) Oost-Nederland, ref: 2022-13623, NL-number: NL80258.091.22). Written informed consent was obtained from all subjects before the study. Any participant-derived benefits resulting from this research, such as new insights into disease mechanisms or possible novel therapies, will be disseminated to study participants, patient groups and members of the public. TRIAL REGISTRATION NUMBER: NCT05746221.

Cardiometabolic Differences in People Living with HIV Receiving Integrase Strand Transfer Inhibitors Compared to Non-nucleoside Reverse Transcriptase Inhibitors: Implications for Current ART Strategies.

In people living with HIV (PLHIV), integrase strand transfer inhibitors (INSTIs) are part of the first-line combination antiretroviral therapy (cART), while non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens are alternatives. Distinct cART regimens may variably influence the risk for non-AIDS comorbidities. We aimed to compare the metabolome and lipidome of INSTI and NNRTI-based regimens. The 2000HIV study includes asymptomatic PLHIV (n = 1646) on long-term cART, separated into a discovery cohort with 730 INSTI and 617 NNRTI users, and a validation cohort encompassing 209 INSTI and 90 NNRTI users. Baseline plasma samples from INSTI and NNRTI users were compared using mass spectrometry-based untargeted metabolomic (n = 500) analysis. Perturbed metabolic pathways were identified using MetaboAnalyst software. Subsequently, nuclear magnetic resonance spectroscopy was used for targeted lipoprotein and lipid (n = 141) analysis. Metabolome homogeneity was observed between the different types of INSTI and NNRTI. In contrast, higher and lower levels of 59 and 45 metabolites, respectively, were found in the INSTI group compared to NNRTI users, of which 77.9% (81/104) had consistent directionality in the validation cohort. Annotated metabolites belonged mainly to 'lipid and lipid-like molecules', 'organic acids and derivatives' and 'organoheterocyclic compounds'. In pathway analysis, perturbed 'vitamin B1 (thiamin) metabolism', 'de novo fatty acid biosynthesis', 'bile acid biosynthesis' and 'pentose phosphate pathway' were detected, among others. Lipoprotein and lipid levels in NNRTIs were heterogeneous and could not be compared as a group. INSTIs compared to individual NNRTI types showed that HDL cholesterol was lower in INSTIs compared to nevirapine but higher in INSTIs compared to doravirine. In addition, LDL size was lower in INSTIs and nevirapine compared to doravirine. NNRTIs show more heterogeneous cardiometabolic effects than INSTIs, which hampers the comparison between these two classes of drugs. Targeted lipoproteomic and lipid NMR spectroscopy showed that INSTI use was associated with a more unfavorable lipid profile compared to nevirapine, which was shifted to a more favorable profile for INSTI when substituting nevirapine for doravirine, with evidently higher fold changes. The cardiovascular disease risk profile seems more favorable in INSTIs compared to NNRTIs in untargeted metabolomic analysis using mass-spectrometry.

Common and distinct metabolomic markers related to immune aging in Western European and East African populations.

In old age, impaired immunity causes high susceptibility to infections and cancer, higher morbidity and mortality, and poorer vaccination efficiency. Many factors, such as genetics, diet, and lifestyle, impact aging. This study aimed to investigate how immune responses change with age in healthy Dutch and Tanzanian individuals and identify common metabolites associated with an aged immune profile. We performed untargeted metabolomics from plasma to identify age-associated metabolites, and we correlated their concentrations with ex-vivo cytokine production by immune cells, DNA methylation-based epigenetic aging, and telomere length. Innate immune responses were impacted differently by age in Dutch and Tanzanian cohorts. Age-related decline in steroid hormone precursors common in both populations was associated with higher systemic inflammation and lower cytokine responses. Hippurate and 2-phenylacetamide, commonly more abundant in older individuals, were negatively correlated with cytokine responses and telomere length and positively correlated with epigenetic aging. Lastly, we identified several metabolites that might contribute to the stronger decline in innate immunity with age in Tanzanians. The shared metabolomic signatures of the two cohorts suggest common mechanisms of immune aging, revealing metabolites with potential contributions. These findings also reflect genetic or environmental effects on circulating metabolites that modulate immune responses.

Novel Proteome Targets Marking Insulin Resistance in Metabolic Syndrome.

CONTEXT/OBJECTIVE: In order to better understand which metabolic differences are related to insulin resistance in metabolic syndrome (MetSyn), we used hyperinsulinemic-euglycemic (HE) clamps in individuals with MetSyn and related peripheral insulin resistance to circulating biomarkers. DESIGN/METHODS: In this cross-sectional study, HE-clamps were performed in treatment-naive men (n = 97) with MetSyn. Subjects were defined as insulin-resistant based on the rate of disappearance (Rd). Machine learning models and conventional statistics were used to identify biomarkers of insulin resistance. Findings were replicated in a cohort with n = 282 obese men and women with (n = 156) and without (n = 126) MetSyn. In addition to this, the relation between biomarkers and adipose tissue was assessed by nuclear magnetic resonance imaging. RESULTS: Peripheral insulin resistance is marked by changes in proteins related to inflammatory processes such as IL-1 and TNF-receptor and superfamily members. These proteins can distinguish between insulin-resistant and insulin-sensitive individuals (AUC = 0.72 ± 0.10) with MetSyn. These proteins were also associated with IFG, liver fat (rho 0.36, p = 1.79 × 10-9) and visceral adipose tissue (rho = 0.35, p = 6.80 × 10-9). Interestingly, these proteins had the strongest association in the MetSyn subgroup compared to individuals without MetSyn. CONCLUSIONS: MetSyn associated with insulin resistance is characterized by protein changes related to body fat content, insulin signaling and pro-inflammatory processes. These findings provide novel targets for intervention studies and should be the focus of future in vitro and in vivo studies.

Unraveling interindividual variation of trimethylamine N-oxide and its precursors at the population level.

Trimethylamine N-oxide (TMAO) is a circulating microbiome-derived metabolite implicated in the development of atherosclerosis and cardiovascular disease (CVD). We investigated whether plasma levels of TMAO, its precursors (betaine, carnitine, deoxycarnitine, choline), and TMAO-to-precursor ratios are associated with clinical outcomes, including CVD and mortality. This was followed by an in-depth analysis of their genetic, gut microbial, and dietary determinants. The analyses were conducted in five Dutch prospective cohort studies including 7834 individuals. To further investigate association results, Mendelian Randomization (MR) was also explored. We found only plasma choline levels (hazard ratio [HR] 1.17, [95% CI 1.07; 1.28]) and not TMAO to be associated with CVD risk. Our association analyses uncovered 10 genome-wide significant loci, including novel genomic regions for betaine (6p21.1, 6q25.3), choline (2q34, 5q31.1), and deoxycarnitine (10q21.2, 11p14.2) comprising several metabolic gene associations, for example, CPS1 or PEMT. Furthermore, our analyses uncovered 68 gut microbiota associations, mainly related to TMAO-to-precursors ratios and the Ruminococcaceae family, and 16 associations of food groups and metabolites including fish-TMAO, meat-carnitine, and plant-based food-betaine associations. No significant association was identified by the MR approach. Our analyses provide novel insights into the TMAO pathway, its determinants, and pathophysiological impact on the general population.