The long-term genetic stability and individual specificity of the human gut microbiome.

By following up the gut microbiome, 51 human phenotypes and plasma levels of 1,183 metabolites in 338 individuals after 4 years, we characterize microbial stability and variation in relation to host physiology. Using these individual-specific and temporally stable microbial profiles, including bacterial SNPs and structural variations, we develop a microbial fingerprinting method that shows up to 85% accuracy in classifying metagenomic samples taken 4 years apart. Application of our fingerprinting method to the independent HMP cohort results in 95% accuracy for samples taken 1 year apart. We further observe temporal changes in the abundance of multiple bacterial species, metabolic pathways, and structural variation, as well as strain replacement. We report 190 longitudinal microbial associations with host phenotypes and 519 associations with plasma metabolites. These associations are enriched for cardiometabolic traits, vitamin B, and uremic toxins. Finally, mediation analysis suggests that the gut microbiome may influence cardiometabolic health through its metabolites.

Phage display sequencing reveals that genetic, environmental, and intrinsic factors influence variation of human antibody epitope repertoire.

Phage-displayed immunoprecipitation sequencing (PhIP-seq) has enabled high-throughput profiling of human antibody repertoires. However, a comprehensive overview of environmental and genetic determinants shaping human adaptive immunity is lacking. In this study, we investigated the effects of genetic, environmental, and intrinsic factors on the variation in human antibody repertoires. We characterized serological antibody repertoires against 344,000 peptides using PhIP-seq libraries from a wide range of microbial and environmental antigens in 1,443 participants from a population cohort. We detected individual-specificity, temporal consistency, and co-housing similarities in antibody repertoires. Genetic analyses showed the involvement of the HLA, IGHV, and FUT2 gene regions in antibody-bound peptide reactivity. Furthermore, we uncovered associations between phenotypic factors (including age, cell counts, sex, smoking behavior, and allergies, among others) and particular antibody-bound peptides. Our results indicate that human antibody epitope repertoires are shaped by both genetics and environmental exposures and highlight specific signatures of distinct phenotypes and genotypes.

Phage-display immunoprecipitation sequencing of the antibody epitope repertoire in inflammatory bowel disease reveals distinct antibody signatures.

Inflammatory bowel diseases (IBDs), e.g., Crohn's disease (CD) and ulcerative colitis (UC), are chronic immune-mediated inflammatory diseases. A comprehensive overview of an IBD-specific antibody epitope repertoire is, however, lacking. Using high-throughput phage-display immunoprecipitation sequencing (PhIP-Seq), we identified antibodies against 344,000 antimicrobial, immune, and food antigens in 497 individuals with IBD compared with 1,326 controls. IBD was characterized by 373 differentially abundant antibody responses (202 overrepresented and 171 underrepresented), with 17% shared by both IBDs, 55% unique to CD, and 28% unique to UC. Antibody reactivities against bacterial flagellins dominated in CD and were associated with ileal involvement, fibrostenotic disease, and anti-Saccharomyces cerevisiae antibody positivity, but not with fecal microbiome composition. Antibody epitope repertoires accurately discriminated CD from controls (area under the curve [AUC] = 0.89), and similar discrimination was achieved when using only ten antibodies (AUC = 0.87). Individuals with IBD thus show a distinct antibody repertoire against selected peptides, allowing clinical stratification and discovery of immunological targets.

Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity.

Gut microbial dysbioses are linked to aberrant immune responses, which are often accompanied by abnormal production of inflammatory cytokines. As part of the Human Functional Genomics Project (HFGP), we investigate how differences in composition and function of gut microbial communities may contribute to inter-individual variation in cytokine responses to microbial stimulations in healthy humans. We observe microbiome-cytokine interaction patterns that are stimulus specific, cytokine specific, and cytokine and stimulus specific. Validation of two predicted host-microbial interactions reveal that TNFα and IFNγ production are associated with specific microbial metabolic pathways: palmitoleic acid metabolism and tryptophan degradation to tryptophol. Besides providing a resource of predicted microbially derived mediators that influence immune phenotypes in response to common microorganisms, these data can help to define principles for understanding disease susceptibility. The three HFGP studies presented in this issue lay the groundwork for further studies aimed at understanding the interplay between microbial, genetic, and environmental factors in the regulation of the immune response in humans. PAPERCLIP.

Genome-wide characterization of circulating metabolic biomarkers.

Genome-wide association analyses using high-throughput metabolomics platforms have led to novel insights into the biology of human metabolism1-7. This detailed knowledge of the genetic determinants of systemic metabolism has been pivotal for uncovering how genetic pathways influence biological mechanisms and complex diseases8-11. Here we present a genome-wide association study for 233 circulating metabolic traits quantified by nuclear magnetic resonance spectroscopy in up to 136,016 participants from 33 cohorts. We identify more than 400 independent loci and assign probable causal genes at two-thirds of these using manual curation of plausible biological candidates. We highlight the importance of sample and participant characteristics that can have significant effects on genetic associations. We use detailed metabolic profiling of lipoprotein- and lipid-associated variants to better characterize how known lipid loci and novel loci affect lipoprotein metabolism at a granular level. We demonstrate the translational utility of comprehensively phenotyped molecular data, characterizing the metabolic associations of intrahepatic cholestasis of pregnancy. Finally, we observe substantial genetic pleiotropy for multiple metabolic pathways and illustrate the importance of careful instrument selection in Mendelian randomization analysis, revealing a putative causal relationship between acetone and hypertension. Our publicly available results provide a foundational resource for the community to examine the role of metabolism across diverse diseases.

Structural variation in the gut microbiome associates with host health.

Differences in the presence of even a few genes between otherwise identical bacterial strains may result in critical phenotypic differences. Here we systematically identify microbial genomic structural variants (SVs) and find them to be prevalent in the human gut microbiome across phyla and to replicate in different cohorts. SVs are enriched for CRISPR-associated and antibiotic-producing functions and depleted from housekeeping genes, suggesting that they have a role in microbial adaptation. We find multiple associations between SVs and host disease risk factors, many of which replicate in an independent cohort. Exploring genes that are clustered in the same SV, we uncover several possible mechanistic links between the microbiome and its host, including a region in Anaerostipes hadrus that encodes a composite inositol catabolism-butyrate biosynthesis pathway, the presence of which is associated with lower host metabolic disease risk. Overall, our results uncover a nascent layer of variability in the microbiome that is associated with microbial adaptation and host health.

Environment dominates over host genetics in shaping human gut microbiota.

Human gut microbiome composition is shaped by multiple factors but the relative contribution of host genetics remains elusive. Here we examine genotype and microbiome data from 1,046 healthy individuals with several distinct ancestral origins who share a relatively common environment, and demonstrate that the gut microbiome is not significantly associated with genetic ancestry, and that host genetics have a minor role in determining microbiome composition. We show that, by contrast, there are significant similarities in the compositions of the microbiomes of genetically unrelated individuals who share a household, and that over 20% of the inter-person microbiome variability is associated with factors related to diet, drugs and anthropometric measurements. We further demonstrate that microbiome data significantly improve the prediction accuracy for many human traits, such as glucose and obesity measures, compared to models that use only host genetic and environmental data. These results suggest that microbiome alterations aimed at improving clinical outcomes may be carried out across diverse genetic backgrounds.

Systematic analysis of relationships between plasma branched-chain amino acid concentrations and cardiometabolic parameters: an association and Mendelian randomization study.

BACKGROUND: Branched-chain amino acids (BCAAs; valine, leucine, and isoleucine) are essential amino acids that are associated with an increased risk of cardiometabolic diseases (CMD). However, there are still only limited insights into potential direct associations between BCAAs and a wide range of CMD parameters, especially those remaining after correcting for covariates and underlying causal relationships. METHODS: To shed light on these relationships, we systematically characterized the associations between plasma BCAA concentrations and a large panel of 537 CMD parameters (including atherosclerosis-related parameters, fat distribution, plasma cytokine concentrations and cell counts, circulating concentrations of cardiovascular-related proteins and plasma metabolites) in 1400 individuals from the Dutch population cohort LifeLines DEEP and 294 overweight individuals from the 300OB cohort. After correcting for age, sex, and BMI, we assessed associations between individual BCAAs and CMD parameters. We further assessed the underlying causality using Mendelian randomization. RESULTS: A total of 838 significant associations were detected for 409 CMD parameters. BCAAs showed both common and specific associations, with the most specific associations being detected for isoleucine. Further, we found that obesity status substantially affected the strength and direction of associations for valine, which cannot be corrected for using BMI as a covariate. Subsequent univariable Mendelian randomization (UVMR), after removing BMI-associated SNPs, identified seven significant causal relationships from four CMD traits to BCAA levels, mostly for diabetes-related parameters. However, no causal effects of BCAAs on CMD parameters were supported. CONCLUSIONS: Our cross-sectional association study reports a large number of associations between BCAAs and CMD parameters. Our results highlight some specific associations for isoleucine, as well as obesity-specific effects for valine. MR-based causality analysis suggests that altered BCAA levels can be a consequence of diabetes and alteration in lipid metabolism. We found no MR evidence to support a causal role for BCAAs in CMD. These findings provide evidence to (re)evaluate the clinical importance of individual BCAAs in CMD diagnosis, prevention, and treatment.

Yoghurt consumption is associated with changes in the composition of the human gut microbiome and metabolome.

BACKGROUND: Yoghurt contains live bacteria that could contribute via modulation of the gut microbiota to its reported beneficial effects such as reduced body weight gain and lower incidence of type 2 diabetes. To date, the association between yoghurt consumption and the composition of the gut microbiota is underexplored. Here we used clinical variables, metabolomics, 16S rRNA and shotgun metagenomic sequencing data collected on over 1000 predominantly female UK twins to define the link between the gut microbiota and yoghurt-associated health benefits. RESULTS: According to food frequency questionnaires (FFQ), 73% of subjects consumed yoghurt. Consumers presented a healthier diet pattern (healthy eating index: beta = 2.17 ± 0.34; P = 2.72x10-10) and improved metabolic health characterised by reduced visceral fat (beta = -28.18 ± 11.71 g; P = 0.01). According to 16S rRNA gene analyses and whole shotgun metagenomic sequencing approach consistent taxonomic variations were observed with yoghurt consumption. More specifically, we identified higher abundance of species used as yoghurt starters Streptococcus thermophilus (beta = 0.41 ± 0.051; P = 6.14x10-12) and sometimes added Bifidobacterium animalis subsp. lactis (beta = 0.30 ± 0.052; P = 1.49x10-8) in the gut of yoghurt consumers. Replication in 1103 volunteers from the LifeLines-DEEP cohort confirmed the increase of S. thermophilus among yoghurt consumers. Using food records collected the day prior to faecal sampling we showed than an increase in these two yoghurt bacteria could be transient. Metabolomics analysis revealed that B. animalis subsp. lactis was associated with 13 faecal metabolites including a 3-hydroxyoctanoic acid, known to be involved in the regulation of gut inflammation. CONCLUSIONS: Yoghurt consumption is associated with reduced visceral fat mass and changes in gut microbiome including transient increase of yoghurt-contained species (i.e. S. thermophilus and B. lactis).

Long-term dietary patterns are associated with pro-inflammatory and anti-inflammatory features of the gut microbiome.

OBJECTIVE: The microbiome directly affects the balance of pro-inflammatory and anti-inflammatory responses in the gut. As microbes thrive on dietary substrates, the question arises whether we can nourish an anti-inflammatory gut ecosystem. We aim to unravel interactions between diet, gut microbiota and their functional ability to induce intestinal inflammation. DESIGN: We investigated the relation between 173 dietary factors and the microbiome of 1425 individuals spanning four cohorts: Crohn's disease, ulcerative colitis, irritable bowel syndrome and the general population. Shotgun metagenomic sequencing was performed to profile gut microbial composition and function. Dietary intake was assessed through food frequency questionnaires. We performed unsupervised clustering to identify dietary patterns and microbial clusters. Associations between diet and microbial features were explored per cohort, followed by a meta-analysis and heterogeneity estimation. RESULTS: We identified 38 associations between dietary patterns and microbial clusters. Moreover, 61 individual foods and nutrients were associated with 61 species and 249 metabolic pathways in the meta-analysis across healthy individuals and patients with IBS, Crohn's disease and UC (false discovery rate<0.05). Processed foods and animal-derived foods were consistently associated with higher abundances of Firmicutes, Ruminococcus species of the Blautia genus and endotoxin synthesis pathways. The opposite was found for plant foods and fish, which were positively associated with short-chain fatty acid-producing commensals and pathways of nutrient metabolism. CONCLUSION: We identified dietary patterns that consistently correlate with groups of bacteria with shared functional roles in both, health and disease. Moreover, specific foods and nutrients were associated with species known to infer mucosal protection and anti-inflammatory effects. We propose microbial mechanisms through which the diet affects inflammatory responses in the gut as a rationale for future intervention studies.

Whole exome sequencing analyses reveal gene-microbiota interactions in the context of IBD.

OBJECTIVE: Both the gut microbiome and host genetics are known to play significant roles in the pathogenesis of IBD. However, the interaction between these two factors and its implications in the aetiology of IBD remain underexplored. Here, we report on the influence of host genetics on the gut microbiome in IBD. DESIGN: To evaluate the impact of host genetics on the gut microbiota of patients with IBD, we combined whole exome sequencing of the host genome and whole genome shotgun sequencing of 1464 faecal samples from 525 patients with IBD and 939 population-based controls. We followed a four-step analysis: (1) exome-wide microbial quantitative trait loci (mbQTL) analyses, (2) a targeted approach focusing on IBD-associated genomic regions and protein truncating variants (PTVs, minor allele frequency (MAF) >5%), (3) gene-based burden tests on PTVs with MAF <5% and exome copy number variations (CNVs) with site frequency <1%, (4) joint analysis of both cohorts to identify the interactions between disease and host genetics. RESULTS: We identified 12 mbQTLs, including variants in the IBD-associated genes IL17REL, MYRF, SEC16A and WDR78. For example, the decrease of the pathway acetyl-coenzyme A biosynthesis, which is involved in short chain fatty acids production, was associated with variants in the gene MYRF (false discovery rate <0.05). Changes in functional pathways involved in the metabolic potential were also observed in participants carrying rare PTVs or CNVs in CYP2D6, GPR151 and CD160 genes. These genes are known for their function in the immune system. Moreover, interaction analyses confirmed previously known IBD disease-specific mbQTLs in TNFSF15. CONCLUSION: This study highlights that both common and rare genetic variants affecting the immune system are key factors in shaping the gut microbiota in the context of IBD and pinpoints towards potential mechanisms for disease treatment.

An association between chronic widespread pain and the gut microbiome.

OBJECTIVES: Chronic widespread musculoskeletal pain (CWP) is a characteristic symptom of fibromyalgia, which has been shown to be associated with an altered gut microbiome. Microbiome studies to date have not examined the milder CWP phenotype specifically nor have they explored the role of raised BMI. The aim of this study was to investigate whether the microbiome is abnormal in CWP. METHODS: CWP was assessed using a standardized screening questionnaire in female volunteers from the TwinsUK cohort including 113 CWP cases and 1623 controls. The stool microbiome was characterized using 16S rRNA amplicon sequencing and amplicon sequence variants, and associations with CWP examined using linear mixed-effects models adjusting for BMI, age, diet, family relatedness and technical factors. RESULTS: Alpha diversity was significantly lower in CWP cases than controls (Mann-Whitney test, P-values 2.3e-04 and 1.2e-02, for Shannon and Simpson indices respectively). The species Coprococcus comes was significantly depleted in CWP cases (Padj = 3.04e-03). A genome-wide association study (GWAS) performed for C. comes in TwinsUK followed by meta-analysis with three Dutch cohorts (total n = 3521) resulted in nine suggestive regions, with the most convincing on chromosome 4 near the TRAM1L1 gene (rs76957229, P = 7.4e-8). A Mendelian randomization study based on the results of the GWAS did not support a causal role for C. comes on the development of CWP. CONCLUSIONS: We have demonstrated reduced diversity in the microbiome in CWP, indicating an involvement of the gut microbiota in CWP; prospectively the microbiome may offer therapeutic opportunities for this condition.

Gut Microbial Associations to Plasma Metabolites Linked to Cardiovascular Phenotypes and Risk.

RATIONALE: Altered gut microbial composition has been linked to cardiovascular diseases (CVDs), but its functional links to host metabolism and immunity in relation to CVD development remain unclear. OBJECTIVES: To systematically assess functional links between the microbiome and the plasma metabolome, cardiometabolic phenotypes, and CVD risk and to identify diet-microbe-metabolism-immune interactions in well-documented cohorts. METHODS AND RESULTS: We assessed metagenomics-based microbial associations between 231 plasma metabolites and microbial species and pathways in the population-based LLD (Lifelines DEEP) cohort (n=978) and a clinical obesity cohort (n=297). After correcting for age, sex, and body mass index, the gut microbiome could explain ≤11.1% and 16.4% of the variation in plasma metabolites in the population-based and obesity cohorts, respectively. Obese-specific microbial associations were found for lipid compositions in the VLDL, IDL, and LDL lipoprotein subclasses. Bacterial L-methionine biosynthesis and a Ruminococcus species were associated to cardiovascular phenotypes in obese individuals, namely atherosclerosis and liver fat content, respectively. Integration of microbiome-diet-inflammation analysis in relation to metabolic risk score of CVD in the population cohort revealed 48 microbial pathways associated to CVD risk that were largely independent of diet and inflammation. Our data also showed that plasma levels rather than fecal levels of short-chain fatty acids were relevant to inflammation and CVD risk. CONCLUSIONS: This study presents the largest metagenome-based association study on plasma metabolism and microbiome relevance to diet, inflammation, CVD risk, and cardiometabolic phenotypes in both population-based and clinical obesity cohorts. Our findings identified novel bacterial species and pathways that associated to specific lipoprotein subclasses and revealed functional links between the gut microbiome and host health that provide a basis for developing microbiome-targeted therapy for disease prevention and treatment.

Gut Microbiota Composition of Biliary Atresia Patients Before Kasai Portoenterostomy Associates With Long-term Outcome.

BACKGROUND AND AIMS: Biliary atresia (BA) is a cholestatic, fibro-obliterative cholangiopathy of unknown etiology. BA is primarily treated by a surgical approach, that is, the Kasai portoenterostomy (KPE), to obtain clearance of jaundice (COJ). The gut microbiota (GM) composition has been associated with the course of several cholestatic liver diseases. It is largely unknown, however, whether GM composition associates with the outcome of KPE. We compared the GM composition of BA patients and controls and assessed if GM composition before KPE was related to COJ after KPE. METHODS: We compared feces of term-born BA patients before KPE and controls (patients undergoing inguinal hernia repair) by 16S rRNA sequencing. Composition and alpha diversity of the GM were compared between BA and controls before KPE and after KPE, between patients with COJ versus without COJ (total serum bilirubin < or ≥20 µmol/L <6 months post-KPE). RESULTS: Alpha diversity was comparable between BA (n = 12, age 1.6 [1.3-1.8] months) and controls (n = 6, age 2.0 [1.4-2.1] months; P = 0.22). Compared with controls, BA patients had lower abundances of Bifidobacteriaceae (ß = -1.98, P < 0.001) and Lachnospiraceae (ß = -1.84, P = 0.007), and higher abundances of Streptococcus (ß = -1.13, P = 0.003). The alpha diversity before KPE correlated negatively with COJ (R = -0.63, P = 0.03). Lower alpha diversity pre-KPE was associated with COJ [+] (ßlogit = -0.64, P = 0.04). We observed greater abundances of genus Acinetobacter (ß = 1.27, P = 0.03) and family Clostridiaceae (ß = 1.45, P = 0.03) and lower abundances of the family Enterobacteriaceae (genera Klebsiella (ß = -1.21, P = 0.01), Salmonella (ß = -1.57, P = 0.02)) in COJ [+] versus COJ [-]. CONCLUSIONS: The GM of BA patients before Kasai portoenterostomy associates with outcome, clearance of jaundice, suggestive of predictive, and mechanistic roles of the gut microbiota composition in bile homeostasis.

Host Genetics and Gut Microbiome: Challenges and Perspectives.

The mammalian gut is colonized by trillions of microorganisms collectively called the microbiome. It is increasingly clear that this microbiome has a critical role of in many aspects of health including metabolism and immunity. While environmental factors such as diet and medications have been shown to influence the microbiome composition, the role of host genetics has only recently emerged in human studies and animal models. In this review, we summarize the current state of microbiome research with an emphasis on the effect of host genetics on the gut microbiome composition. We focus particularly on genetic determinants of the host immune system that help shape the gut microbiome and discuss avenues for future research.

Studying the gut virome in the metagenomic era: challenges and perspectives.

The human gut harbors a complex ecosystem of microorganisms, including bacteria and viruses. With the rise of next-generation sequencing technologies, we have seen a quantum leap in the study of human-gut-inhabiting bacteria, yet the viruses that infect these bacteria, known as bacteriophages, remain underexplored. In this review, we focus on what is known about the role of bacteriophages in human health and the technical challenges involved in studying the gut virome, of which they are a major component. Lastly, we discuss what can be learned from studies of bacteriophages in other ecosystems.

Shared gut, but distinct oral microbiota composition in primary Sjögren's syndrome and systemic lupus erythematosus.

OBJECTIVE: Alterations in the microbiota composition of the gastro-intestinal tract are suspected to be involved in the etiopathogenesis of two closely related systemic inflammatory autoimmune diseases: primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE). Our objective was to assess whether alterations in gut and oral microbiota compositions are specific for pSS and SLE. METHODS: 16S ribosomal RNA gene sequencing was performed on fecal samples from 39 pSS patients, 30 SLE patients and 965 individuals from the general population, as well as on buccal swab and oral washing samples from the same pSS and SLE patients. Alpha-diversity, beta-diversity and relative abundance of individual bacteria were used as outcome measures. Multivariate analyses were performed to test associations between individual bacteria and disease phenotype, taking age, sex, body-mass index, proton-pump inhibitor use and sequencing-depth into account as possible confounding factors. RESULTS: Fecal microbiota composition from pSS and SLE patients differed significantly from population controls, but not between pSS and SLE. pSS and SLE patients were characterized by lower bacterial richness, lower Firmicutes/Bacteroidetes ratio and higher relative abundance of Bacteroides species in fecal samples compared with population controls. Oral microbiota composition differed significantly between pSS patients and SLE patients, which could partially be explained by oral dryness in pSS patients. CONCLUSIONS: pSS and SLE patients share similar alterations in gut microbiota composition, distinguishing patients from individuals in the general population, while oral microbiota composition shows disease-specific differences between pSS and SLE patients.