The gut microbiota in cardiovascular disease:Interactions between the diet, microbiota and the gut immune barrier

Cardiovascular disease (CVD) causes 17.7 million deaths annually worldwide and is therefore a major health burden to our society. Over the last century, CVD treatment has greatly improved. The discovery of cholesterol lowering drugs has led to a strongly improved treatment of CVD. However, atherosclerosis, the main underlying cause of CVD, is not solely a cholesterol-driven disease. Recently a large human study showed that inflammation is also an important factor in the development of atherosclerosis. Understanding the factors that contribute to inflammation is therefore of great importance. The gut microbiota is one such factor that has recently been linked to cardiovascular disease and inflammation. However, it is unknown whether alterations in microbiota composition can contribute to CVD development and if interactions between the gut immune barrier, diet and gut microbiota can affect systemic inflammation and atherosclerosis. In this thesis, we used mouse models to understand how the interaction between the diet, gut microbiota and intestinal immune barrier contributes to systemic inflammation and atherosclerosis. Our studies show a causal role of the gut microbiota in the development of systemic inflammation and atherosclerosis. Furthermore, our studies indicate a protective role of the gut immune system and in particular of antimicrobial against development of CVD. Thus our studies indicate that the gut microbiota and the gut immune system may be interesting future targets for the treatment of CVD

The Gut Microbiome Contributes to a Substantial Proportion of the Variation in Blood Lipids

Rationale: Evidence suggests that the gut microbiome is involved in the development of cardiovascular disease, with the host–microbe interaction regulating immune and metabolic pathways. However, there was no firm evidence for associations between microbiota and metabolic risk factors for cardiovascular disease from large-scale studies in humans. In particular, there was no strong evidence for association between cardiovascular disease and aberrant blood lipid levels. Objectives: To identify intestinal bacteria taxa, whose proportions correlate with body mass index and lipid levels, and to determine whether lipid variance can be explained by microbiota relative to age, sex, and host genetics. Methods and Results: We studied 893 subjects from the LifeLines-DEEP population cohort. After correcting for age and sex, we identified 34 bacterial taxa associated with body mass index and blood lipids; most are novel associations. Cross-validation analysis revealed that microbiota explain 4.5% of the variance in body mass index, 6% in triglycerides, and 4% in high-density lipoproteins, independent of age, sex, and genetic risk factors. A novel risk model, including the gut microbiome explained ≤25.9% of high-density lipoprotein variance, significantly outperforming the risk model without microbiome. Strikingly, the microbiome had little effect on low-density lipoproteins or total cholesterol. Conclusions: Our studies suggest that the gut microbiome may play an important role in the variation in body mass index and blood lipid levels, independent of age, sex, and host genetics. Our findings support the potential of therapies altering the gut microbiome to control body mass, triglycerides, and high-density lipoproteins

Antigen-Independent Restriction of Pneumococcal Density by Mucosal Adjuvant Cholera Toxin Subunit B

For many bacterial respiratory infections, development of (severe) disease is preceded by asymptomatic colonization of the upper airways. For Streptococcus pneumoniae, the transition to severe lower respiratory tract infection is associated with an increase in nasopharyngeal colonization density. Insight into how the mucosal immune system restricts colonization may provide new strategies to prevent clinical symptoms. Several studies have provided indirect evidence that the mucosal adjuvant cholera toxin subunit B (CTB) may confer nonspecific protection against respiratory infections. Here, we show that CTB reduces the pneumococcal load in the nasopharynx, which required activation of the caspase-1/11 inflammasome, mucosal T cells, and macrophages. Our findings suggest that CTB-dependent activation of the local innate response synergizes with noncognate T cells to restrict bacterial load. Our study not only provides insight into the immunological components required for containment and clearance of pneumococcal carriage, but also highlights an important yet often understudied aspect of adjuvants

Gut microbiota dysbiosis augments atherosclerosis in LDLR-/- mice

Aim: In recent years the gut microbiome has been recognized as an important participant in the etiology of obesity and -associated comorbidities. Moreover, recent studies have implicated an involvement of the gut microbiota in both cardiovascular health and disease. Although the gut microbiome has been suggested as a contributor to atherosclerosis, firm evidence to support a causal role for the gut microbiota in atherosclerosis is limited. Methods: To investigate the effect of the gut microbiota in atherosclerosis, we performed fecal microbiota transplantation to transplant the gut microbiota of caspase1-/- mice, an established model for dysbiosis, into LDLR-/- mice (LDLR-/-[casp1-/-]). Fecal microbiota transplantation of the gut microbiota of LDLR-/- mice into LDLR-/- mice (LDLR-/-[LDLR-/-]) served as control. Mice were fed a chow or high-fat cholesterol diet (HFC, 0,21% cholesterol) for 13 weeks and fecal samples were collected to determine microbiota composition by 16S rRNA sequencing. Plaque size was analyzed in the aortic root and immune cell subsets were analyzed by flow cytometry. Results: 16S rRNA sequencing of fecal samples confirmed the induction of dysbiosis in LDLR-/-[casp1-/-] compared to LDLR-/-[LDLR-/-] mice. Body weight, plasma triglyceride and cholesterol levels were significantly increased by HFC-feeding in LDLR-/-[casp1-/-] and LDLR-/-[LDLR-/-] mice. However, dysbiosis did not affect these parameters. In contrast, plaque size was significantly increased in HFC-fed LDLR-/-[casp1-/-] compared to HFC-fed (LDLR-/-[LDLR-/-] mice. Furthermore, dysbiosis in LDLR-/- mice was associated with a moderate increase in circulatory levels of Ly6C-high monocytes. Conclusions: Our data shows that gut microbiota dysbiosis augments atherosclerosis, possibly by exacerbating low-grade systemic inflammation

Lipid sorting by ceramide structure from plasma membrane to ER for the cholera toxin receptor ganglioside GM1.

The glycosphingolipid GM1 binds cholera toxin (CT) on host cells and carries it retrograde from the plasma membrane (PM) through endosomes, the trans-Golgi (TGN), and the endoplasmic reticulum (ER) to induce toxicity. To elucidate how a membrane lipid can specify trafficking in these pathways, we synthesized GM1 isoforms with alternate ceramide domains and imaged their trafficking in live cells. Only GM1 with unsaturated acyl chains sorted efficiently from PM to TGN and ER. Toxin binding, which effectively crosslinks GM1 lipids, was dispensable, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. The results implicate a protein-dependent mechanism of lipid sorting by ceramide structure and provide a molecular explanation for the diversity and specificity of retrograde trafficking by CT in host cells

A Pro-Inflammatory Gut Microbiota Increases Systemic Inflammation and Accelerates Atherosclerosis

RATIONALE: Several studies have suggested a role for the gut microbiota in inflammation and atherogenesis. A causal relation relationship between gut microbiota, inflammation and atherosclerosis has not been explored previously. OBJECTIVE: Here, we investigated whether a pro-inflammatory microbiota from Caspase1-/- ( Casp1-/-) mice accelerates atherogenesis in Ldlr-/- mice. METHODS AND RESULTS: We treated female Ldlr-/- mice with antibiotics and subsequently transplanted them with fecal microbiota from Casp1-/- mice based on a co-housing approach. Autologous transplantation of fecal microbiota of Ldlr-/- mice served as control. Mice were co-housed for 8 or 13 weeks and fed chow or a high-fat cholesterol-rich (HFC) diet. Fecal samples were collected, and factors related to inflammation, metabolism, intestinal health and atherosclerotic phenotypes were measured. Unweighted Unifrac distances of 16S rDNA sequences confirmed the introduction of the Casp1-/- and Ldlr-/- microbiota into Ldlr-/- mice (referred to as Ldlr-/-( Casp1-/-) or Ldlr-/-( Ldlr-/-) mice). Analysis of atherosclerotic lesion size in the aortic root demonstrated a significant 29% increase in plaque size in 13-week HFC-fed Ldlr-/-( Casp1-/-) mice compared to Ldlr-/-( Ldlr-/-) mice. We found increased numbers of circulating monocytes and neutrophils and elevated pro-inflammatory cytokine levels in plasma in HFC-fed Ldlr-/-( Casp1-/-) compared to Ldlr-/-( Ldlr-/-) mice. Neutrophil accumulation in the aortic root of Ldlr-/-( Casp1-/-) mice was enhanced compared to Ldlr-/-( Ldlr-/-) mice. 16S-rDNA-encoding sequence analysis in feces identified a significant reduction in the short-chain fatty acid (SCFA)-producing taxonomies Akkermansia, Christensenellaceae, Clostridium and Odoribacter in Ldlr-/-( Casp1-/-) mice. Consistent with these findings, cumulative concentrations of the anti-inflammatory SCFAs proprionate, acetate and butyrate in the cecum were significantly reduced in 13-week HFC-fed Ldlr-/-( Casp1-/-) compared to Ldlr-/-( Ldlr-/-) mice. CONCLUSIONS: Introduction of the pro-inflammatory Casp1-/- microbiota into Ldlr-/- mice enhances systemic inflammation and accelerates atherogenesis

A Pro-Inflammatory Gut Microbiota Increases Systemic Inflammation and Accelerates Atherosclerosis

RATIONALE: Several studies have suggested a role for the gut microbiota in inflammation and atherogenesis. A causal relation relationship between gut microbiota, inflammation and atherosclerosis has not been explored previously. OBJECTIVE: Here, we investigated whether a pro-inflammatory microbiota from Caspase1-/- ( Casp1-/-) mice accelerates atherogenesis in Ldlr-/- mice. METHODS AND RESULTS: We treated female Ldlr-/- mice with antibiotics and subsequently transplanted them with fecal microbiota from Casp1-/- mice based on a co-housing approach. Autologous transplantation of fecal microbiota of Ldlr-/- mice served as control. Mice were co-housed for 8 or 13 weeks and fed chow or a high-fat cholesterol-rich (HFC) diet. Fecal samples were collected, and factors related to inflammation, metabolism, intestinal health and atherosclerotic phenotypes were measured. Unweighted Unifrac distances of 16S rDNA sequences confirmed the introduction of the Casp1-/- and Ldlr-/- microbiota into Ldlr-/- mice (referred to as Ldlr-/-( Casp1-/-) or Ldlr-/-( Ldlr-/-) mice). Analysis of atherosclerotic lesion size in the aortic root demonstrated a significant 29% increase in plaque size in 13-week HFC-fed Ldlr-/-( Casp1-/-) mice compared to Ldlr-/-( Ldlr-/-) mice. We found increased numbers of circulating monocytes and neutrophils and elevated pro-inflammatory cytokine levels in plasma in HFC-fed Ldlr-/-( Casp1-/-) compared to Ldlr-/-( Ldlr-/-) mice. Neutrophil accumulation in the aortic root of Ldlr-/-( Casp1-/-) mice was enhanced compared to Ldlr-/-( Ldlr-/-) mice. 16S-rDNA-encoding sequence analysis in feces identified a significant reduction in the short-chain fatty acid (SCFA)-producing taxonomies Akkermansia, Christensenellaceae, Clostridium and Odoribacter in Ldlr-/-( Casp1-/-) mice. Consistent with these findings, cumulative concentrations of the anti-inflammatory SCFAs proprionate, acetate and butyrate in the cecum were significantly reduced in 13-week HFC-fed Ldlr-/-( Casp1-/-) compared to Ldlr-/-( Ldlr-/-) mice. CONCLUSIONS: Introduction of the pro-inflammatory Casp1-/- microbiota into Ldlr-/- mice enhances systemic inflammation and accelerates atherogenesis

Population-based metagenomics analysis reveals markers for gut microbiome composition and diversity

Deep sequencing of the gut microbiomes of 1135 participants from a Dutch population-based cohort shows relations between the microbiome and 126 exogenous and intrinsic host factors, including 31 intrinsic factors, 12 diseases, 19 drug groups, 4 smoking categories, and 60 dietary factors. These factors collectively explain 18.7% of the variation seen in the interindividual distance of microbial composition. We could associate 110 factors to 125 species and observed that fecal chromogranin A (CgA), a protein secreted by enteroendocrine cells, was exclusively associated with 61 microbial species whose abundance collectively accounted for 53% of microbial composition. Low CgA concentrations were seen in individuals with a more diverse microbiome. These results are an important step toward a better understanding of environment-diet-microbe-host interactions.status: publishe