Analysis of strain, sex, and diet-dependent modulation of gut microbiota reveals candidate keystone organisms driving microbial diversity in response to American and ketogenic diets.

BACKGROUND: The gut microbiota is modulated by a combination of diet, host genetics, and sex effects. The magnitude of these effects and interactions among them is important to understanding inter-individual variability in gut microbiota. In a previous study, mouse strain-specific responses to American and ketogenic diets were observed along with several QTLs for metabolic traits. In the current study, we searched for genetic variants underlying differences in the gut microbiota in response to American and ketogenic diets, which are high in fat and vary in carbohydrate composition, between C57BL/6 J (B6) and FVB/NJ (FVB) mouse strains. RESULTS: Genetic mapping of microbial features revealed 18 loci under the QTL model (i.e., marginal effects that are not specific to diet or sex), 12 loci under the QTL by diet model, and 1 locus under the QTL by sex model. Multiple metabolic and microbial features map to the distal part of Chr 1 and Chr 16 along with eigenvectors extracted from principal coordinate analysis of measures of ß-diversity. Bilophila, Ruminiclostridium 9, and Rikenella (Chr 1) were identified as sex- and diet-independent QTL candidate keystone organisms, and Parabacteroides (Chr 16) was identified as a diet-specific, candidate keystone organism in confirmatory factor analyses of traits mapping to these regions. For many microbial features, irrespective of which QTL model was used, diet or the interaction between diet and a genotype were the strongest predictors of the abundance of each microbial trait. Sex, while important to the analyses, was not as strong of a predictor for microbial abundances. CONCLUSIONS: These results demonstrate that sex, diet, and genetic background have different magnitudes of effects on inter-individual differences in gut microbiota. Therefore, Precision Nutrition through the integration of genetic variation, microbiota, and sex affecting microbiota variation will be important to predict response to diets varying in carbohydrate composition. Video Abstract.

Precision pharmacological reversal of strain-specific diet-induced metabolic syndrome in mice informed by epigenetic and transcriptional regulation.

Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat, high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genome-wide DNA methylation analyses for each strain and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects revealed a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by epigenetic and transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the farnesoid X receptor pathway, and found that GW4064 exerts strain-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis. Furthermore, GW4064 treatment induced inflammatory-related gene expression changes in NOD, indicating a strain-specific effect in its associated toxicities as well as its therapeutic efficacy. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention and a mouse platform for guiding this approach.

Integrative analysis of hepatic transcriptional profiles reveals genetic regulation of atherosclerosis in hyperlipidemic Diversity Outbred-F1 mice.

Atherogenesis is an insipidus but precipitating process leading to serious consequences of many cardiovascular diseases (CVD). Numerous genetic loci contributing to atherosclerosis have been identified in human genome-wide association studies, but these studies have limitations in the ability to control environmental factors and to decipher cause/effect relationships. To assess the power of hyperlipidemic Diversity Outbred (DO) mice in facilitating quantitative trait loci (QTL) analysis of complex traits, we generated a high-resolution genetic panel of atherosclerosis susceptible (DO-F1) mouse cohort by crossing 200 DO females with C57BL/6J males carrying two human genes: encoding apolipoprotein E3-Leiden and cholesterol ester transfer protein. We examined atherosclerotic traits including plasma lipids and glucose in the 235 female and 226 male progeny before and after 16 weeks of a high-fat/cholesterol diet, and aortic plaque size at 24 weeks. We also assessed the liver transcriptome using RNA-sequencing. Our QTL mapping for atherosclerotic traits identified one previously reported female-specific QTL on Chr10 with a narrower interval of 22.73 to 30.80 Mb, and one novel male-specific QTL at 31.89 to 40.25 Mb on Chr19. Liver transcription levels of several genes within each QTL were highly correlated with the atherogenic traits. A majority of these candidates have already known atherogenic potential in humans and/or mice, but integrative QTL, eQTL, and correlation analyses further pointed Ptprk as a major candidate of the Chr10 QTL, while Pten and Cyp2c67 of the Chr19 QTL in our DO-F1 cohort. Finally, through additional analyses of RNA-seq data we identified genetic regulation of hepatic transcription factors, including Nr1h3, contributes to atherogenesis in this cohort. Thus, an integrative approach using DO-F1 mice effectively validates the influence of genetic factors on atherosclerosis in DO mice and suggests an opportunity to discover therapeutics in the setting of hyperlipidemia.

Precision pharmacological reversal of genotype-specific diet-induced metabolic syndrome in mice informed by transcriptional regulation.

Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genomic DNA methylation analyses and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects reveals a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the Farnesoid X receptor pathway, and found that GW4064 exerts genotype-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis, as well as increased inflammatory-related gene expression changes in NOD. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention, and a mouse platform for guiding this approach.

Analysis of strain, sex, and diet-dependent modulation of gut microbiota reveals candidate keystone organisms driving microbial diversity in response to American and ketogenic diets.

Background The gut microbiota is modulated by a combination of diet, host genetics, and sex effects. The magnitude of these effects and interactions among them is important to understanding inter-individual variability in gut microbiota. In a previous study, mouse strain-specific responses to American and ketogenic diets were observed along with several QTL for metabolic traits. In the current study, we searched for genetic variants underlying differences in the gut microbiota in response to American and ketogenic diets, which are high in fat and vary in carbohydrate composition, between C57BL/6J (B6) and FVB/NJ (FVB) mouse strains. Results Genetic mapping of microbial features revealed 18 loci under the QTL model (i.e., marginal effects that are not specific to diet or sex), 12 loci under the QTL by diet model, and 1 locus under the QTL by sex model. Multiple metabolic and microbial features map to the distal part of Chr 1 and Chr 16 along with eigenvectors extracted from principal coordinate analysis of measures of ß-diversity. Bilophila , Ruminiclostridium 9 , and Rikenella (Chr 1) were identified as sex and diet independent QTL candidate keystone organisms and Rikenelleceae RC9 Gut Group (Chr 16) was identified as a diet-specific, candidate keystone organism in confirmatory factor analyses of traits mapping to these regions. For many microbial features, irrespective of which QTL model was used, diet or the interaction between diet and a genotype were the strongest predictors of the abundance of each microbial trait. Sex, while important to the analyses, was not as strong of a predictor for microbial abundances. Conclusions These results demonstrate that sex, diet, and genetic background have different magnitudes of effects on inter-individual differences in gut microbiota. Therefore, Precision Nutrition through the integration of genetic variation, microbiota, and sex affecting microbiota variation will be important to predict response to diets varying in carbohydrate composition.

Chronic Kidney Disease is Associated With Attenuated Plasma Metabolome Response to Oral Glucose Tolerance Testing.

OBJECTIVE: Chronic kidney disease (CKD) is associated with decreased anabolic response to insulin contributing to protein-energy wasting. Targeted metabolic profiling of oral glucose tolerance testing (OGTT) may help identify metabolic pathways contributing to disruptions to insulin response in CKD. METHODS: Using targeted metabolic profiling, we studied the plasma metabolome response in 41 moderate-to-severe nondiabetic CKD patients and 20 healthy controls at fasting and 2 hours after an oral glucose load. We used linear mixed modeling with random intercepts, adjusting for age, gender, race/ethnicity, body weight, and batch to assess heterogeneity in response to OGTT by CKD status. RESULTS: Mean estimated glomerular filtration rate among CKD participants was 38.9 ± 12.7 mL/min per 1.73 m2 compared to 87.2 ± 17.7 mL/min per 1.73 m2 among controls. Glucose ingestion induced an anabolic response resulting in increased glycolysis products and a reduction in a wide range of metabolites including amino acids, tricarboxylic acid cycle intermediates, and purine nucleotides compared to fasting. Participants with CKD demonstrated a blunted anabolic response to OGTT evidenced by significant changes in 13 metabolites compared to controls. The attenuated metabolome response predominant involved mitochondrial energy metabolism, vitamin B family, and purine nucleotides. Compared to controls, CKD participants had elevated lactate:pyruvate (L:P) ratio and decreased guanosine diphosphate:guanosine triphosphate ratio during OGTT. CONCLUSION: Metabolic profiling of OGTT response suggests a broad disruption of mitochondrial energy metabolism in CKD patients. These findings motivate further investigation into the impact of insulin sensitizers and mitochondrial targeted therapeutics on energy metabolism in patients with nondiabetic CKD.

Gut microbiota and host genetics modulate the effect of diverse diet patterns on metabolic health.

Metabolic diseases are major public health issues worldwide and are responsible for disproportionately higher healthcare costs and increased complications of many diseases including SARS-CoV-2 infection. The Western Diet (WD) specifically is believed to be a major contributor to the global metabolic disease epidemic. In contrast, the Mediterranean diet (MeD), Ketogenic diet (KD), and Japanese diet (JD) are often considered beneficial for metabolic health. Yet, there is a growing appreciation that the effect of diet on metabolic health varies depending on several factors including host genetics. Additionally, poor metabolic health has also been attributed to altered gut microbial composition and/or function. To understand the complex relationship between host genetics, gut microbiota, and dietary patterns, we treated four widely used metabolically diverse inbred mouse strains (A/J, C57BL/6J, FVB/NJ, and NOD/ShiLtJ) with four human-relevant diets (MeD, JD, KD, WD), and a control mouse chow from 6 weeks to 30 weeks of age. We found that diet-induced alteration of gut microbiota (α-diversity, ß-diversity, and abundance of several bacteria including Bifidobacterium, Ruminococcus, Turicibacter, Faecalibaculum, and Akkermansia) is significantly modified by host genetics. In addition, depending on the gut microbiota, the same diet could have different metabolic health effects. Our study also revealed that C57BL/6J mice are more susceptible to altered gut microbiota compared to other strains in this study indicating that host genetics is an important modulator of the diet-microbiota-metabolic health axis. Overall, our study demonstrated complex interactions between host genetics, gut microbiota, and diet on metabolic health; indicating the need to consider both host genetics and the gut microbiota in the development of new and more effective precision nutrition strategies to improve metabolic health.

Bifidobacterium Species Colonization in Infancy: A Global Cross-Sectional Comparison by Population History of Breastfeeding.

Bifidobacterium species are beneficial and dominant members of the breastfed infant gut microbiome; however, their health benefits are partially species-dependent. Here, we characterize the species and subspecies of Bifidobacterium in breastfed infants around the world to consider the potential impact of a historic dietary shift on the disappearance of B. longum subsp. infantis in some populations. Across populations, three distinct patterns of Bifidobacterium colonization emerged: (1) The dominance of Bifidobacterium longum subspecies infantis, (2) prevalent Bifidobacterium of multiple species, and (3) the frequent absence of any Bifidobacterium. These patterns appear related to a country's history of breastfeeding, with infants in countries with historically high rates of long-duration breastfeeding more likely to be colonized by B. longum subspecies infantis compared with infants in countries with histories of shorter-duration breastfeeding. In addition, the timing of infant colonization with B. longum subsp. infantis is consistent with horizontal transmission of this subspecies, rather than the vertical transmission previously reported for other Bifidobacterium species. These findings highlight the need to consider historical and cultural influences on the prevalence of gut commensals and the need to understand epidemiological transmission patterns of Bifidobacterium and other major commensals.

Genetic architecture modulates diet-induced hepatic mRNA and miRNA expression profiles in Diversity Outbred mice.

Genetic approaches in model organisms have consistently demonstrated that molecular traits such as gene expression are under genetic regulation, similar to clinical traits. The resulting expression quantitative trait loci (eQTL) have revolutionized our understanding of genetic regulation and identified numerous candidate genes for clinically relevant traits. More recently, these analyses have been extended to other molecular traits such as protein abundance, metabolite levels, and miRNA expression. Here, we performed global hepatic eQTL and microRNA expression quantitative trait loci (mirQTL) analysis in a population of Diversity Outbred mice fed two different diets. We identified several key features of eQTL and mirQTL, namely differences in the mode of genetic regulation (cis or trans) between mRNA and miRNA. Approximately 50% of mirQTL are regulated by a trans-acting factor, compared to ∼25% of eQTL. We note differences in the heritability of mRNA and miRNA expression and variance explained by each eQTL or mirQTL. In general, cis-acting variants affecting mRNA or miRNA expression explain more phenotypic variance than trans-acting variants. Finally, we investigated the effect of diet on the genetic architecture of eQTL and mirQTL, highlighting the critical effects of environment on both eQTL and mirQTL. Overall, these data underscore the complex genetic regulation of two well-characterized RNA classes (mRNA and miRNA) that have critical roles in the regulation of clinical traits and disease susceptibility.

Modulating the Microbiota as a Therapeutic Intervention for Type 2 Diabetes.

Mounting evidence suggested that the gut microbiota has a significant role in the metabolism and disease status of the host. In particular, Type 2 Diabetes (T2D), which has a complex etiology that includes obesity and chronic low-grade inflammation, is modulated by the gut microbiota and microbial metabolites. Current literature supports that unbalanced gut microbial composition (dysbiosis) is a risk factor for T2D. In this review, we critically summarize the recent findings regarding the role of gut microbiota in T2D. Beyond these associative studies, we focus on the causal relationship between microbiota and T2D established using fecal microbiota transplantation (FMT) or probiotic supplementation, and the potential underlying mechanisms such as byproducts of microbial metabolism. These microbial metabolites are small molecules that establish communication between microbiota and host cells. We critically summarize the associations between T2D and microbial metabolites such as short-chain fatty acids (SCFAs) and trimethylamine N-Oxide (TMAO). Additionally, we comment on how host genetic architecture and the epigenome influence the microbial composition and thus how the gut microbiota may explain part of the missing heritability of T2D found by GWAS analysis. We also discuss future directions in this field and how approaches such as FMT, prebiotics, and probiotics supplementation are being considered as potential therapeutics for T2D.

Hepatic transcriptional profile reveals the role of diet and genetic backgrounds on metabolic traits in female progenitor strains of the Collaborative Cross.

Mice have provided critical mechanistic understandings of clinical traits underlying metabolic syndrome (MetSyn) and susceptibility to MetSyn in mice is known to vary among inbred strains. We investigated the diet- and strain-dependent effects on metabolic traits in the eight Collaborative Cross (CC) founder strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). Liver transcriptomics analysis showed that both atherogenic diet and host genetics have profound effects on the liver transcriptome, which may be related to differences in metabolic traits observed between strains. We found strain differences in circulating trimethylamine N-oxide (TMAO) concentration and liver triglyceride content, both of which are traits associated with metabolic diseases. Using a network approach, we identified a module of transcripts associated with TMAO and liver triglyceride content, which was enriched in functional pathways. Interrogation of the module related to metabolic traits identified NADPH oxidase 4 (Nox4), a gene for a key enzyme in the production of reactive oxygen species, which showed a strong association with plasma TMAO and liver triglyceride. Interestingly, Nox4 was identified as the highest expressed in the C57BL/6J and NZO/HILtJ strains and the lowest expressed in the CAST/EiJ strain. Based on these results, we suggest that there may be genetic variation in the contribution of Nox4 to the regulation of plasma TMAO and liver triglyceride content. In summary, we show that liver transcriptomic analysis identified diet- or strain-specific pathways for metabolic traits in the Collaborative Cross (CC) founder strains.

Indole-3-lactic acid associated with Bifidobacterium-dominated microbiota significantly decreases inflammation in intestinal epithelial cells.

BACKGROUND: Bifidobacterium longum subsp. infantis (B. infantis) is a commensal bacterium that colonizes the gastrointestinal tract of breast-fed infants. B. infantis can efficiently utilize the abundant supply of oligosaccharides found in human milk (HMO) to help establish residence. We hypothesized that metabolites from B. infantis grown on HMO produce a beneficial effect on the host. RESULTS: In a previous study, we demonstrated that B. infantis routinely dominated the fecal microbiota of a breast fed Bangladeshi infant cohort (1). Characterization of the fecal metabolome of binned samples representing high and low B. infantis populations from this cohort revealed higher amounts of the tryptophan metabolite indole-3-lactic acid (ILA) in feces with high levels of B. infantis. Further in vitro analysis confirmed that B. infantis produced significantly greater quantities of the ILA when grown on HMO versus lactose, suggesting a growth substrate relationship to ILA production. The direct effects of ILA were assessed in a macrophage cell line and intestinal epithelial cell lines. ILA (1-10 mM) significantly attenuated lipopolysaccharide (LPS)-induced activation of NF-kB in macrophages. ILA significantly attenuated TNF-α- and LPS-induced increase in the pro-inflammatory cytokine IL-8 in intestinal epithelial cells. ILA increased mRNA expression of the aryl hydrogen receptor (AhR)-target gene CYP1A1 and nuclear factor erythroid 2-related factor 2 (Nrf2)-targeted genes glutathione reductase 2 (GPX2), superoxide dismutase 2 (SOD2), and NAD(P) H dehydrogenase (NQO1). Pretreatment with either the AhR antagonist or Nrf-2 antagonist inhibited the response of ILA on downstream effectors. CONCLUSIONS: These findings suggest that ILA, a predominant metabolite from B. infantis grown on HMO and elevated in infant stool high in B. infantis, and protects gut epithelial cells in culture via activation of the AhR and Nrf2 pathway.

High-Dose Neonatal Vitamin A Supplementation to Bangladeshi Infants Increases the Percentage of CCR9-Positive Treg Cells in Infants with Lower Birthweight in Early Infancy, and Decreases Plasma sCD14 Concentration and the Prevalence of Vitamin A Deficiency at Two Years of Age.

BACKGROUND: Vitamin A (VA) stores are low in early infancy and may impair development of the immune system. OBJECTIVE: This study determined if neonatal VA supplementation (VAS) affects the following: 1) development of regulatory T (Treg) cells; 2) chemokine receptor 9 (CCR9) expression, which directs mucosal targeting of immune cells; and 3) systemic endotoxin exposure as indicated by changed plasma concentrations of soluble CD14 (sCD14). Secondarily, VA status, growth, and systemic inflammation were investigated. METHODS: In total, 306 Bangladeshi infants were randomly assigned to receive 50,000 IU VA or placebo (PL) within 48 h of birth, and immune function was assessed at 6 wk, 15 wk, and 2 y. Primary outcomes included the following: 1) peripheral blood Treg cells; 2) percentage of Treg, T, and B cells expressing CCR9; and 3) plasma sCD14. Secondary outcomes included the following: 4) VA status measured using the modified relative dose-response (MRDR) test and plasma retinol; 5) infant growth; and 6) plasma C-reactive protein (CRP). Statistical analysis identified group differences and interactions with sex and birthweight. RESULTS: VAS increased (P = 0.004) the percentage of CCR9+ Treg cells (13.2 ± 1.37%) relative to PL (9.17 ± 1.15%) in children below the median birthweight but had the opposite effect (P = 0.04) in those with higher birthweight (VA, 9.13 ± 0.89; PL, 12.1 ± 1.31%) at 6 and 15 wk (values are combined mean ± SE). VAS decreased (P = 0.003) plasma sCD14 (1.56 ± 0.025 mg/L) relative to PL (1.67 ± 0.032 mg/L) and decreased (P = 0.034) the prevalence of VA deficiency (2.3%) relative to PL (9.2%) at 2 y. CONCLUSIONS: Neonatal VAS enhanced mucosal targeting of Treg cells in low-birthweight infants. The decreased systemic exposure to endotoxin and improved VA status at 2 y may have been due to VA-mediated improvements in gut development resulting in improved barrier function and nutrient absorption. This trial was registered at clinicaltrials.gov as NCT01583972 and NCT02027610.

Genetic Architecture Modulates Diet-Induced Hepatic mRNA and miRNA Expression Profiles in Diversity Outbred Mice.

Genetic approaches in model organisms have consistently demonstrated that molecular traits such as gene expression are under genetic regulation, similar to clinical traits. The resulting expression quantitative trait loci (eQTL) have revolutionized our understanding of genetic regulation and identified numerous candidate genes for clinically relevant traits. More recently, these analyses have been extended to other molecular traits such as protein abundance, metabolite levels, and miRNA expression. Here, we performed global hepatic eQTL and microRNA expression quantitative trait loci (mirQTL) analysis in a population of Diversity Outbred mice fed two different diets. We identified several key features of eQTL and mirQTL, namely differences in the mode of genetic regulation (cis or trans) between mRNA and miRNA. Approximately 50% of mirQTL are regulated by a trans-acting factor, compared to ∼25% of eQTL. We note differences in the heritability of mRNA and miRNA expression and variance explained by each eQTL or mirQTL. In general, cis-acting variants affecting mRNA or miRNA expression explain more phenotypic variance than trans-acting variants. Lastly, we investigated the effect of diet on the genetic architecture of eQTL and mirQTL, highlighting the critical effects of environment on both eQTL and mirQTL. Overall, these data underscore the complex genetic regulation of two well-characterized RNA classes (mRNA and miRNA) that have critical roles in the regulation of clinical traits and disease susceptibility.

Dissecting the Genetic Architecture of Cystatin C in Diversity Outbred Mice.

Plasma concentration of Cystatin C (CysC) level is a biomarker of glomerular filtration rate in the kidney. We use a Systems Genetics approach to investigate the genetic determinants of plasma CysC concentration. To do so we perform Quantitative Trait Loci (QTL) and expression QTL (eQTL) analysis of 120 Diversity Outbred (DO) female mice, 56 weeks of age. We performed network analysis of kidney gene expression to determine if the gene modules with common functions are associated with kidney biomarkers of chronic kidney diseases. Our data demonstrates that plasma concentrations and kidney mRNA levels of CysC are associated with genetic variation and are transcriptionally coregulated by immune genes. Specifically, Type-I interferon signaling genes are coexpressed with Cst3 mRNA levels and associated with CysC concentrations in plasma. Our findings demonstrate the complex control of CysC by genetic polymorphisms and inflammatory pathways.

High-Dose Neonatal Vitamin A Supplementation Transiently Decreases Thymic Function in Early Infancy.

BACKGROUND: Vitamin A deficiency (VAD) impairs T-cell-mediated immunity. In regions where VAD is prevalent, vitamin A supplementation (VAS) reduces child mortality, perhaps by improving immune function. OBJECTIVE: Our objective was to determine if neonatal VAS would improve thymic function in Bangladeshi infants, and to determine if such effects differed by sex or nutritional status (i.e., birth weight above/below the median). METHODS: Three hundred and six infants were randomly assigned to 50,000 IU vitamin A (VA) or placebo (PL) within 48 h of birth. Primary outcomes were measured at multiple ages and included 1) thymic index (TI) at 1, 6, 10, and 15 wk; 2) T-cell receptor excision circles (TREC), an index of thymic output of naïve T cells; and 3) total/naïve T cells in peripheral blood at 6 wk, 15 wk, and 2 y. A mixed linear model for repeated measures was used to assess group differences at each age and identify interactions with sex and birth weight. RESULTS: VAS did not significantly (P = 0.21) affect TI overall (i.e., at all ages) but decreased TI by 7.8% (P = 0.029) at 6 wk: adjusted TI means for the PL and VA groups at 1, 6, 10, and 15 wk were 4.09 compared with 3.80 cm2, 7.78 compared with 7.18 cm2, 8.11 compared with 7.84 cm2, and 7.91 compared with 7.97 cm2, respectively. VAS did not significantly (P = 0.25) affect TREC overall but decreased TREC by 19% (P = 0.029) at 15 wk: adjusted TREC means for the PL and VA groups at 6 wk, 15 wk, and 2 y were 13.6 compared with 16.1 copies/pg DNA, 19.4 compared with 15.7 copies/pg DNA, and 11.8 compared with 10.0 copies/pg DNA, respectively. VAS did not significantly affect overall total (P = 0.10) or naïve (P = 0.092) T cells: adjusted naïve T-cell means for the PL and VA groups at 6 wk, 15 wk, and 2 y were 3259 compared with 3109 cells/µL, 3771 compared with 3487 cells/µL, and 1976 compared with 1898 cells/µL, respectively. CONCLUSION: In contrast to our hypothesis, VAS decreased thymic function early in infancy but health effects are presumably negligible owing to the transience and small magnitude of this effect. This trial was registered at clinicaltrials.gov as NCT01583972 and NCT02027610.

Neonatal Vitamin A Supplementation and Vitamin A Status Are Associated with Gut Microbiome Composition in Bangladeshi Infants in Early Infancy and at 2 Years of Age.

BACKGROUND: Infancy is a crucial period for establishing the intestinal microbiome. This process may be influenced by vitamin A (VA) status because VA affects intestinal immunity and epithelial integrity, factors that can, in turn, modulate microbiome development. OBJECTIVES: The aim of this study was to determine if neonatal VA supplementation (VAS) affected the abundance of Bifidobacterium, a beneficial commensal, or of Proteobacteria, a phylum containing enteric pathogens, in early (6-15 wk) or late (2 y) infancy. Secondary objectives were to determine if VAS affected the abundance of other bacterial taxa, and to determine if VA status assessed by measuring plasma retinol was associated with bacterial abundance. METHODS: Three hundred and six Bangladeshi infants were randomized by sex and birthweight status (above/below median) to receive 1 VA dose (50,000 IU) or placebo within 48 h of birth. Relative abundance at the genus level and above was assessed by 16S rRNA gene sequencing. A terminal restriction fragment-length polymorphism assay was used to identify Bifidobacterium species and subspecies at 6 wk. RESULTS: Linear regression showed that Bifidobacterium abundance in early infancy was lower in boys (median, 1st/3rd quartiles; 0.67, 0.52/0.78) than girls (0.73, 0.60/0.80; P = 0.003) but that boys receiving VAS (0.69, 0.55/0.78) had higher abundance than boys receiving placebo (0.65, 0.44/0.77; P = 0.039). However this difference was not seen in girls (VAS 0.71, 0.54/0.80; placebo 0.75, 0.63/0.81; P = 0.25). VAS did not affect Proteobacteria abundance. Sex-specific associations were also seen for VA status, including positive associations of plasma retinol with Actinobacteria (the phylum containing Bifidobacterium) and Akkermansia, another commensal with possible health benefits, for girls in late infancy. CONCLUSIONS: Better VA status in infancy may influence health both in infancy and later in life by promoting the establishment of a healthy microbiota. This postulated effect of VA may differ between boys and girls. This trial was registered at clinicaltrials.gov as NCT02027610.

Bifidobacterium Abundance in Early Infancy and Vaccine Response at 2 Years of Age.

BACKGROUND: The intestinal microbiome in early infancy affects immunologic development and thus may affect vaccine memory, though few prospective studies have examined such associations. We examined the association of Bifidobacterium levels in early infancy with memory responses to early vaccination measured at 2 years of age. METHODS: In this prospective observational study, we examined the association of Bifidobacterium abundance in the stool of healthy infants at 6 to 15 weeks of age, near the time of vaccination, with T-cell and antibody responses measured at 6 weeks, 15 weeks, and 2 years of age. Infants were vaccinated with Bacillus Calmette-Guérin (BCG) (at birth), oral polio virus (at birth and at 6, 10, and 14 weeks), tetanus toxoid (TT) (at 6, 10, and 14 weeks), and hepatitis B virus (at 6, 10, and 14 weeks). Fecal Bifidobacterium was measured at 6, 11, and 15 weeks. Bifidobacterium species and subspecies were measured at 6 weeks. RESULTS: Mean Bifidobacterium abundance in early infancy was positively associated with the CD4 T-cell responses to BCG, TT, and hepatitis B virus at 15 weeks, with CD4 responses to BCG and TT at 2 years, and with plasma TT-specific immunoglobulin G and stool polio-specific immunoglobulin A at 2 years. Similar associations were seen for the predominant subspecies, Bifidobacterium longum subspecies infantis. CONCLUSIONS: Bifidobacterium abundance in early infancy may increase protective efficacy of vaccines by enhancing immunologic memory. This hypothesis could be tested in clinical trials of interventions to optimize Bifidobacterium abundance in appropriate populations.

Infant cortisol stress-response is associated with thymic function and vaccine response.

Stress can impair T cell-mediated immunity. To determine if infants with high stress responses had deficits in T-cell mediated immunity, we examined the association of pain-induced cortisol responsiveness with thymic function and vaccine responses in infants. This study was performed among 306 (male = 153 and female = 153) participants of a randomized, controlled trial examining the effect of neonatal vitamin A supplementation on immune function in Bangladesh (NCT01583972). Salivary cortisol was measured before and 20 min after a needle stick (vaccination) at 6 weeks of age. The thymic index (TI) was determined by ultrasonography at 1, 6, 10 and 15 weeks. T-cell receptor excision circle and blood T-cell concentrations were measured at 6 and 15 weeks. Responses to Bacillus Calmette-Guérin (BCG), tetanus toxoid, hepatitis B virus and oral poliovirus vaccination were assayed at 6 and 15 weeks. Cortisol responsiveness was negatively associated with TI at all ages (p < .01) in boys only, was negatively associated with naïve helper T-cell concentrations in both sexes at both 6 (p = .0035) and 15 weeks (p = .0083), and was negatively associated with the delayed-type hypersensitivity (DTH) skin test response to BCG vaccination at 15 weeks (p = .034) in both sexes. Infants with a higher cortisol response to pain have differences in the T-cell compartment and a lower DTH response to vaccination. Sex differences in the immune system were seen as early as 6 weeks of age in these healthy infants.

Bifidobacterial Dominance of the Gut in Early Life and Acquisition of Antimicrobial Resistance.

Bifidobacterium species are important commensals capable of dominating the infant gut microbiome, in part by producing acids that suppress growth of other taxa. Bifidobacterium species are less prone to possessing antimicrobial resistance (AMR) genes (ARGs) than other taxa that may colonize infants. Given that AMR is a growing public health crisis and ARGs are present in the gut microbiome of humans from early life, this study examines the correlation between a Bifidobacterium-dominated infant gut microbiome and AMR levels, measured by a culture-independent metagenomic approach both in early life and as infants become toddlers. In general, Bifidobacterium dominance is associated with a significant reduction in AMR in a Bangladeshi cohort, both in the number of acquired AMR genes present and in the abundance of AMR genes. However, by year 2, Bangladeshi infants had no significant differences in AMR related to their early-life Bifidobacterium levels. A generalized linear model including all infants in a previously published Swedish cohort found a significant negative association between log-transformed total AMR and Bifidobacterium levels, thus confirming the relationship between Bifidobacterium levels and AMR. In both cohorts, there was no change between early-life and later-life AMR abundance in high-Bifidobacterium infants but a significant reduction in AMR abundance in low-Bifidobacterium infants. These results support the hypothesis that early Bifidobacterium dominance of the infant gut microbiome may help reduce colonization by taxa containing ARGs.IMPORTANCE Infants are vulnerable to an array of infectious diseases, and as the gut microbiome may serve as a reservoir of AMR for pathogens, reducing the levels of AMR in infants is important to infant health. This study demonstrates that high levels of Bifidobacterium are associated with reduced levels of AMR in early life and suggests that probiotic interventions to increase infant Bifidobacterium levels have the potential to reduce AMR in infants. However, this effect is not sustained at year 2 of age in Bangladeshi infants, underscoring the need for more detailed studies of the biogeography and timing of infant AMR acquisition.