Gut microbiota promotes stem cell differentiation through macrophage and mesenchymal niches in early postnatal development.

Intestinal stem cell maturation and development coincide with gut microbiota exposure after birth. Here, we investigated how early life microbial exposure, and disruption of this process, impacts the intestinal stem cell niche and development. Single-cell transcriptional analysis revealed impaired stem cell differentiation into Paneth cells and macrophage specification upon antibiotic treatment in early life. Mouse genetic and organoid co-culture experiments demonstrated that a CD206+ subset of intestinal macrophages secreted Wnt ligands, which maintained the mesenchymal niche cells important for Paneth cell differentiation. Antibiotics and reduced numbers of Paneth cells are associated with the deadly infant disease, necrotizing enterocolitis (NEC). We showed that colonization with Lactobacillus or transfer of CD206+ macrophages promoted Paneth cell differentiation and reduced NEC severity. Together, our work defines the gut microbiota-mediated regulation of stem cell niches during early postnatal development.

Intestinal Microbial Composition of Children in a Randomized Controlled Trial of Probiotics to Treat Acute Gastroenteritis.

Compositional analysis of the intestinal microbiome in pre-schoolers is understudied. Effects of probiotics on the gut microbiota were evaluated in children under 4-years-old presenting to an emergency department with acute gastroenteritis. Included were 70 study participants (n=32 placebo, n=38 probiotics) with stool specimens at baseline (day 0), day 5, and after a washout period (day 28). Microbiota composition and deduced functions were profiled using 16S ribosomal RNA sequencing and predictive metagenomics, respectively. Probiotics were detected at day 5 of administration but otherwise had no discernable effects, whereas detection of bacterial infection (P<0.001) and participant age (P<0.001) had the largest effects on microbiota composition, microbial diversity, and deduced bacterial functions. Participants under 1 year had lower bacterial diversity than older aged pre-schoolers; compositional changes of individual bacterial taxa were associated with maturation of the gut microbiota. Advances in age were associated with differences in gut microbiota composition and deduced microbial functions, which have the potential to impact health later in life. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT01853124.

Structure-Function Relationships of Human Milk Oligosaccharides on the Intestinal Epithelial Transcriptome in Caco-2 Cells and a Murine Model of Necrotizing Enterocolitis.

SCOPE: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency affecting preterm infants. Breastmilk protects against NEC, partly due to human milk oligosaccharides (HMOs). HMO compositions are highly diverse, and it is unclear if anti-NEC properties are specific to carbohydrate motifs. Here, this study compares intestinal epithelial transcriptomes of five synthetic HMOs (sHMOs) and examines structure-function relationships of HMOs on intestinal signaling. METHODS AND RESULTS: This study interrogates the transcriptome of Caco-2Bbe1 cells in response to five synthetic HMOs (sHMOs) using RNA sequencing: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3FL), 6'-siallyllactose (6'-SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT). Protection against intestinal barrier dysfunction and inflammation occurred in an HMO-dependent manner. Each sHMO exerts a unique set of host transcriptome changes and modulated unique signaling pathways. There is clustering between HMOs bearing similar side chains, with little overlap in gene regulation which is shared by all sHMOs. Interestingly, most sHMOs protect pups against NEC, exerting divergent mechanisms on intestinal cell morphology and inflammation. CONCLUSIONS: These results demonstrate that while structurally distinct HMOs impact intestinal physiology, their mechanisms of action differ. This finding establishes the first structure-function relationship of HMOs in the context of intestinal cell signaling responses and offers a functional framework by which to screen and design HMO-like compounds.

Antimicrobial susceptibilities and comparative whole genome analysis of two isolates of the probiotic bacterium Lactiplantibacillus plantarum, strain ATCC 202195.

A synbiotic containing Lactiplantibacillus plantarum [American Type Culture Collection (ATCC) strain identifier 202195] and fructooligosaccharide was reported to reduce the risk of sepsis in young infants in rural India. Here, the whole genome of two isolates of L. plantarum ATCC 202195, which were deposited to the ATCC approximately 20 years apart, were sequenced and analyzed to verify their taxonomic and strain-level identities, identify potential antimicrobial resistant genes and virulence factors, and identify genetic characteristics that may explain the observed clinical effects of L. plantarum ATCC 202195. Minimum inhibitory concentrations for selected antimicrobial agents were determined using broth dilution and gradient strip diffusion techniques. The two L. plantarum ATCC 202195 isolates were genetically identical with only three high-quality single nucleotides polymorphisms identified, and with an average nucleotide identity of 99.99%. In contrast to previously published reports, this study determined that each isolate contained two putative plasmids. No concerning acquired or transferable antimicrobial resistance genes or virulence factors were identified. Both isolates were sensitive to several clinically important antibiotics including penicillin, ampicillin and gentamicin, but resistant to vancomycin. Genes involved in stress response, cellular adhesion, carbohydrate metabolism and vitamin biosynthesis are consistent with features of probiotic organisms.

Probiotic stool secretory immunoglobulin A modulation in children with gastroenteritis: a randomized clinical trial.

BACKGROUND: We previously conducted the Probiotic Regimen for Outpatient Gastroenteritis Utility of Treatment (PROGUT) study, which identified no improvements in children with acute gastroenteritis (AGE) administered a probiotic. However, the aforementioned study did not evaluate immunomodulatory benefits. OBJECTIVES: The object of this study was to determine if stool secretory immunoglobulin A (sIgA) concentrations in children with AGE increase more among participants administered a Lactobacillus rhamnosus/helveticus probiotic compared with those administered placebo. METHODS: This a priori planned multicenter, randomized, double-blinded, placebo-controlled ancillary study enrolled children presenting for emergency care who received a 5-d probiotic or placebo course. Participants submitted stool specimens on days 0, 5, and 28. The primary endpoint was the change in stool sIgA concentrations on day 5 compared with baseline. RESULTS: A total of 133 (n = 66 probiotic, 67 placebo) of 886 PROGUT participants (15.0%) provided all 3 specimens. Median stool sIgA concentrations did not differ between the probiotic and placebo groups at any of the study time points: day 0 median (IQR): 1999 (768, 4071) compared with 2198 (702, 5278) (P = 0.27, Cohen's d = 0.17); day 5: 2505 (1111, 5310) compared with 3207 (982, 7080) (P = 0.19, Cohen's d = 0.16); and day 28: 1377 (697, 2248) compared with 1779 (660, 3977) (P = 0.27, Cohen's d = 0.19), respectively. When comparing measured sIgA concentrations between days 0 and 5, we found no treatment allocation effects [ß: -0.24 (-0.65, 0.18); P = 0.26] or interaction between treatment and specimen collection day [ß: -0.003 (-0.09, 0.09); P = 0.95]. Although stool sIgA decreased between day 5 and day 28 within both groups (P < 0.001), there were no differences between the probiotic and placebo groups in the median changes in sIgA concentrations when comparing day 0 to day 5 median (IQR) [500 (-1135, 2362) compared with 362 (-1122, 4256); P = 0.77, Cohen's d = 0.075] and day 5 to day 28 [-1035 (-3130, 499) compared with -1260 (-4437, 843); P = 0.70, Cohen's d = 0.067], respectively. CONCLUSIONS: We found no effect of an L. rhamnosus/helveticus probiotic, relative to placebo, on stool IgA concentrations. This trial was registered at clinicaltrials.gov as NCT01853124.

Microbiota-immune alterations in adolescents following early life adversity: A proof of concept study.

Early adverse care has long-term impacts on physical and mental health. The influence of rearing conditions on the infant's gut microbiota and its relationship with developmental health has become more evident. The microbiome is essential for normal growth and metabolism, and the signaling from the gut to the brain may underlie individual differences in resilience later in life. Microbial diversity and composition were determined using 16S rRNA gene amplicon sequencing in fecal samples from 17 adolescents adopted internationally from orphanages into the United States and 18 adolescents reared in birth families who had similar educational and income levels. Analyses focused on diversity of the microbial community structure and differences in the abundance of specific bacterial taxa. Blood samples were used to immunophenotype the numbers of several T-cell subsets and cytomegalovirus (CMV) seropositivity. Negative binomial regression analysis revealed several operational taxonomic units that were significantly different based on early rearing conditions and CMV seropositivity. There were significant associations between the relative abundance of certain taxa, the percentages of T-cell subsets in circulation, and CMV seropositivity. These findings demonstrate a possible link between the gut microbiota and associations with immune alterations initiated by early life adversity.

High Fat-High Fructose Diet-Induced Changes in the Gut Microbiota Associated with Dyslipidemia in Syrian Hamsters.

Aim: The objective of this study was to characterize the early effects of high fructose diets (with and without high fat) on both the composition of the gut microbiota and lipid metabolism in Syrian hamsters, a reproducible preclinical model of diet-induced dyslipidemia. Methods: Eight-week-old male hamsters were fed diets consisting of high-fat/high-fructose, low-fat/high-fructose or a standard chow diet for 14 days. Stool was collected at baseline (day 0), day 7 and day 14. Fasting levels of plasma triglycerides and cholesterol were monitored on day 0, day 7 and day 14, and nonfasting levels were also assayed on day 15. Then, 16S rRNA sequencing of stool samples was used to determine gut microbial composition, and predictive metagenomics was performed to evaluate dietary-induced shifts in deduced microbial functions. Results: Both high-fructose diets resulted in divergent gut microbiota composition. A high-fat/high-fructose diet induced the largest shift in overall gut microbial composition, with dramatic shifts in the Firmicute/Bacteroidetes ratio, and changes in beta diversity after just seven days of dietary intervention. Significant associations between genus level taxa and dietary intervention were identified, including an association with Ruminococceace NK4A214 group in high-fat/high-fructose fed animals and an association with Butryimonas with the low-fat/high-fructose diet. High-fat/high-fructose feeding induced dyslipidemia with increases in plasma triglycerides and cholesterol, and hepatomegaly. Dietary-induced changes in several genus level taxa significantly correlated with lipid levels over the two-week period. Differences in microbial metabolic pathways between high-fat/high-fructose and low-fat/high-fructose diet fed hamsters were identified, and several of these pathways also correlated with lipid profiles in hamsters. Conclusions: The high-fat/high-fructose diet caused shifts in the host gut microbiota. These dietary-induced alterations in gut microbial composition were linked to changes in the production of secondary metabolites, which contributed to the development of metabolic syndrome in the host.

Human Milk Oligosaccharides Protect against Necrotizing Enterocolitis by Activating Intestinal Cell Differentiation.

SCOPE: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency and currently the leading cause of mortality in preterm infants. Recent studies show that human milk oligosaccharides (HMOs) reduce the frequency and incidence of NEC; however, the molecular mechanisms for their protection are largely unexplored. METHODS AND RESULTS: To address this gap, a genome-wide profiling of the intestinal epithelial transcriptome in response to HMOs using RNA-sequencing is performed. It is found that HMOs alter the host transcriptome in 225 unique target genes pertaining to cell proliferation and differentiation, including upregulation of stem cell differentiation marker HMGCS2. To validate these results, differentiation in Caco-2Bbe1 (Caco-2) intestinal cells is verified by Alcian Blue staining and transepithelial electrical resistance (TER) recordings. Furthermore, an in vivo model of NEC is also employed whereby neonatal pups are gavage fed HMOs. Interestingly, HMOs-fed pups show enhanced cell MUC2 differentiation and HMGCS2 expression. CONCLUSIONS: These findings demonstrate HMOs protect against NEC in part by altering the differentiation of the crypt-villus axis. In addition, this study suggests that pooled HMOs directly induce a series of biological processes, which provide mechanistic insights to how HMOs protect the host intestine.

Vitamin B12 Deficiency Alters the Gut Microbiota in a Murine Model of Colitis.

Purpose: Inflammatory bowel disease (IBD) refers to a spectrum of autoimmune diseases, which result in chronic intestinal inflammation. Previous findings suggest a role for diet, nutrition and dysbiosis of the gut microbiota in both the development and progression of the condition. Vitamin B12 is a key cofactor of methionine synthase and is produced solely by microbes. Previous work links increased levels of homocysteine, a substrate of methionine synthase, MetH, to IBD indicating a potential role for vitamin B12 deficiency in intestinal injury and inflammation. This study assessed the role of vitamin B12 in shaping the gut microbiota and determining responses to intestinal injury using a reproducible murine model of colitis. Methods: The effects of vitamin B12 supplementation and deficiency were assessed in vivo; 3-week-old post-weanling C57Bl/6 mice were divided into three dietary treatment groups: (1) sufficient vitamin B12 (50 mg/Kg), (2) deficient vitamin B12 (0 mg/Kg) and (3) supplemented vitamin B12 (200 mg/Kg) for a period of 4 weeks. Intestinal injury was induced with 2% dextran sodium sulphate (DSS) via drinking water for 5 days. The impact of varying levels of dietary vitamin B12 on gut microbiota composition was assessed using 16S rRNA gene sequencing from fecal samples collected at day 0 and day 28 of the dietary intervention, and 7 days following induction of colitis on day 38, when blood and colonic tissues were also collected. Results: No significant alterations were found in the gut microbiota composition of disease-free animals in response to dietary interventions. By contrast, after DSS-induced colitis, >30 genera were significantly altered in vitamin B12 deficient mice. Altered B12 levels produced no significant effect on composite disease-activity scores; however, administration of a B12 deficient diet resulted in reduced DSS-induced epithelial tissue damage. Conclusions: Vitamin B12 supplementation does not alter the gut microbiota composition under healthy conditions, but does contribute to differential microbial responses and intestinal dysbiosis following the induction of experimental colitis.

Microbe and host interaction in gastrointestinal homeostasis.

RATIONALE: Researchers in psychiatry and neuroscience are increasingly recognizing the importance of gut-brain communication in mental health. Both genetics and environmental factors influence gut microbiota composition and function. This study examines host-microbe signaling at the gastrointestinal barrier to identify bottom-up mechanisms of microbiota-brain communication. OBJECTIVES: We examined differences in gut microbiota composition and fecal miRNA profiles in BALB/c and C57BL/6 mice, in relation to gastrointestinal homeostasis and evaluated the response to perturbation of the gut microbiota by broad-spectrum antibiotic treatment. METHODS AND RESULTS: Differences in the gut microbiota composition between BALB/c and C57BL/6 mice, evaluated by fecal 16S rRNA gene sequencing, included significant differences in genera Prevotella, Alistipes, Akkermansia, and Ruminococcus. Significant differences in fecal miRNA profiles were determined using the nCounter NanoString platform. A BLASTn analysis identified conserved fecal miRNA target regions in bacterial metagenomes with 14 significant correlations found between fecal miRNA and predicted taxa relative abundance in our dataset. Treatment with broad-spectrum antibiotics for 2 weeks resulted in a host-specific physiological response at the gastrointestinal barrier including a decrease in barrier permeability in BALB/c mice and alterations in the expression of barrier regulating genes in both strains. Genera Parabacteroides and Bacteroides were associated with changes in barrier function. CONCLUSIONS: The results of this study provide insight into how specific taxa influence gut barrier integrity and function. More generally, these data in the context of recent published studies makes a significant contribution to our understanding of host-microbe interactions providing new knowledge that can be harnessed by us and others in future mechanistic studies.

Metabolic and Microbiota Measures as Peripheral Biomarkers in Major Depressive Disorder.

Advances in understanding the role of the microbiome in physical and mental health are at the forefront of medical research and hold potential to have a direct impact on precision medicine approaches. In the past 7 years, we have studied the role of microbiota-brain communication on behavior in mouse models using germ-free mice, mice exposed to antibiotics, and healthy specific pathogen free mice. Through our work and that of others, we have seen an amazing increase in our knowledge of how bacteria signal to the brain and the implications this has for psychiatry. Gut microbiota composition and function are influenced both by genetics, age, sex, diet, life experiences, and many other factors of psychiatric and bodily disorders and thus may act as potential biomarkers of the gut-brain axis that could be used in psychiatry and co-morbid conditions. There is a particular need in major depressive disorder and other mental illness to identify biomarkers that can stratify patients into more homogeneous groups to provide better treatment and for development of new therapeutic approaches. Peripheral outcome measures of host-microbe bidirectional communication have significant translational value as biomarkers. Enabling stratification of clinical populations, based on individual biological differences, to predict treatment response to pharmacological and non-pharmacological interventions. Here we consider the links between co-morbid metabolic syndrome and depression, focusing on biomarkers including leptin and ghrelin in combination with assessing gut microbiota composition, as a potential tool to help identify individual differences in depressed population.