Germ-free Controls Research Articles

Comparison of gnotobiotic communities reveals milk-adapted metabolic functions and unexpected amino acid metabolism by the pre-weaning microbiome

ABSTRACT The intestinal microbiome during infancy and childhood has distinct metabolic functions and microbial composition compared to adults. We recently published a gnotobiotic mouse model of the pre-weaning microbiome (PedsCom), which retains a pre-weaning configuration during the transition from a milk-based diet to solid foods, leads to a stunted immune system, and increases susceptibility to enteric infection. Here, we compared the phylogenetic and metabolic relationships of the PedsCom consortium to two adult-derived gnotobiotic communities, Altered Schaedler Flora and Oligo-Mouse Microbiota 12 (Oligo-MM12). We find that PedsCom contains several unique functions relative to these adult-derived mouse consortia, including differences in carbohydrate and lipid metabolism genes. Notably, amino acid degradation metabolic modules are more prevalent among PedsCom isolates, which is in line with the ready availability of these nutrients in milk. Indeed, metabolomic analysis revealed significantly lower levels of total free amino acids and lower levels of specific amino acids abundant in milk (e.g. glutamine and glutamic acid) in the intestinal contents of adult PedsCom colonized mice compared to Oligo-MM12 controls. Metabolomic analysis of pre-weaning intestinal contents also showed lower levels of amino acids that are replete in milk compared to germ-free controls. Thus, enhanced amino acid metabolism is a prominent feature of the pre-weaning microbiome that may facilitate design of early-life microbiome interventions.

Luminal Polyamines Disrupt Intestinal Barrier and Undergo Complex Regulation by Host and Commensal Microbiota

Background: Polyamines produced by host cells and intestinal microbiota and are critical to overall health. Studies have shown putrescine, a polyamine, can strengthen, or in some instances disrupt, gastrointestinal (GI) barrier integrity, yet the regulation of polyamine concentrations in the GI tract is unknown. Host colonocytes and microbiota both metabolize polyamines, and in GI diseases like irritable bowel syndrome (IBS) where intestinal microbiota is significantly altered, polyamine metabolism may be affected. Aim: Determine regulation of luminal polyamine concentration and its effects on intestinal barrier integrity. Hypothesis: Luminal polyamine concentration is suppressed by commensal intestinal microbiota and increasing concentration of putrescine results in intestinal barrier disruption. Methods: Microbiota effects on luminal polyamine concentrations were assessed by humanizing germ-free (GF) mice with a single oral gavage of commensal human microbiota. Polyamine effect on barrier integrity was assessed in vitro using Caco-2 cells and ex vivo on Ussing using mouse colonic tissue with 300Da fluorescein and 4kDa FITC-Dextran tracers. Changes in transepithelial resistance (TER) and rate of tracer flux was recorded. Untargeted metabolomics with shotgun metagenomics was completed on human fecal samples and sigmoid colonic biopsies were collected for bulk RNAseq. Bray-Curtis distance was determined at the species level using the PERMANOVA method and used to test relationships between beta-diversity and fecal polyamine concentration. Results: Humanization with human commensal microbiota (1.2% C. aerofaciens) lead to a 20-fold increase in cecal putrescine and a 20-fold decrease in fecal putrescine compared to GF controls. In vitro addition of putrescine (100mM) caused increased fluorescein flux (3.50±0.24 vs 1.44±0.04 mg/mL) and a rapid loss of transepithelial resistance compared to controls (% of Baseline TER, 36.6±8.2 vs 102.8±5.4, 3h postexposure). Ex vivo addition of putrescine caused a loss of TER (57% vs 89% of baseline by 3h) and an increased rate of 4kDa FITC-Dextran flux compared to untreated controls (1236±131 vs 562±70 ng/hr/cm2). Transcriptomic profiles show decreased expression of ornithine decarboxylase antienzyme (OAZ1, FDR=0.018) in IBS which inhibits the rate limiting enzyme for putrescine production. Increased lactulose excretion (2-24h), suggestive of increased distal colonic permeability, correlated with N-acetylputrescine concentration (p=0.07). N-acetylputrescine also correlated positively with increased stool frequency (p=0.009) and looser consistency (p=0.013). C. aerofaciens, a polyamine consumer, negatively correlated with n-acetylputrescine (FDR=0.02) suggesting a loss of specific taxa may lead to insuffcient polyamine metabolism. Conclusion: Polyamines, namely putrescine, disrupts intestinal barrier integrity, correlate with in vivo permeability, and are decreased in the colon by commensal microbiota. These observations identify putrescine as a negative effector of barrier function and suggest a novel role for the commensal microbiome in regulating barrier integrity through polyamine metabolism. Further work investigating host and microbiota mechanisms of polyamine metabolism, signaling and the specific taxa involved is needed to better understand the effects polyamines have on GI physiology. NIH DK 127998 (MG), support from Mayo Clinic Center for Individualized Medicine (CIM), and Mayo Clinic Center for Cell Signalling in Gastroenterology (C-Sig) P30DK084567. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enterococcus Induces MHC-II Expression in the Colonic Epithelium to Aggravate Graft-Versus-Host Disease

Intestinal domination by Enterococcus, a genus of Gram-positive facultative bacteria, has been associated with an increased risk of GVHD mortality in allo-HCT patients and in preclinical models. However, the immunological mechanisms underlying this association remain unclear. Several preclinical studies have demonstrated that MHC-II expression by intestinal epithelial cells (IECs) is involved in allo-antigen presentation and GVHD and can be modulated by the intestinal microbiota. MHC-II genes are constitutively expressed by professional antigen-presenting cells (APCs) but can be induced in non-professional APCs by IFN-γ. Here we reported that Enterococcus could potentially aggravate GVHD through upregulation of MHC-II expression by IECs in an IFN-γ-dependent manner. Using an MHC-disparate mouse model of GVHD (C57BL/6J into BALB/cJ), we profiled the fecal microbiome of GVHD mice by 16S rRNA sequencing and the intestinal epithelium by flow cytometry on day 7 post-transplant. We found that endogenous Enterococcus relative abundance was positively correlated with MHC-II expression by colonic IECs (CD45- CD31- EpCAM+) during GVHD ( Fig. 1a; p=0.0004; 2 replicates; n=19). To determine whether this phenotype occurs in the absence of systemic inflammation, we utilized gnotobiotic mice treated with either E. faecalis, B. producta (a bacteria genus previously associated with improved GVHD outcomes)or PBS via oral gavage. Monocolonization with E. faecalis was sufficient to upregulate MHC-II expression by colonic IECs in non-transplanted (steady-state) animals compared to PBS-treated germfree controls ( Fig. 1b; p=0.0027; 3 replicates; n=8-12/group). On the contrary, monocolonization by B. producta did not induce MHC-II expression by colonic IECs compared to germfree controls ( p=0.96). We also observed that MHC-II upregulation was promoted by specific species within the genus Enterococcus, such that compared to PBS-treated germfree controls, only E. faecalis ( p=0.0007; 3 replicates; n=8-12/group) was able to induce MHC-II expression by colonic IECs, but not E. durans ( p=0.06) , E. gallinarium ( p=0.98) and E. hirae ( p=0.99). Using a Villin-Cre IFN- γ R fl/flmouse model with genetic ablation of the IFN-γ receptor specifically in epithelial cells, we investigated whether MHC-II expression induced by Enterococcus was dependent on IFN-γ signaling. We colonized Villin-Cre pos and Villin-Cre neg littermate controls with E. faecalis via oral gavage after antibiotics decontamination (ampicillin/enrofloxacin) and profiled the intestinal epithelium. We observed that Villin-Cre posmice exhibited a significantly lower MHC-II expression in the small intestine compared to Villin-Cre negmice on day 3 post- Enterococcus colonization ( p=0.012; 2 replicates; n=7/group), suggesting that IFN-γ signaling is necessary to mediate Enterococcus interaction with the intestinal epithelium. Finally, to explore a potential intervention to reduce intestinal inflammation upon Enterococcus domination, we treated GVHD mice with either butyrate-producing B. producta or E. faecalis. Colonization with B. producta reduced fecal Enterococcus burden on day 7 post-transplant ( p=0.007; 2 replicates; n=8/group) and improved survival in GVHD mice, compared to the E. faecalis-colonized group ( p=0.002; 2 replicates; n=18-19/group). We and others have previously shown that butyrate administration can alleviate GVHD through its effects on epithelial homeostasis. To determine whether butyrate could regulate allo-antigen presentation by the intestinal epithelium, we employed the human colorectal adenocarcinoma HT-29 cell culture. We pre-treated the cells with IFNγ (100U/ml) to induce MHC-II expression, then added butyrate (5mM) to the culture for 24 hours. Butyrate treatment was sufficient to suppress MHC-II expression by HT-29 cells, compared to DMSO-treated controls ( p=0.006; 3 replicates; n=6/group). Altogether, our results suggest that one mechanism by which Enterococcus can aggravate GVHD involves the upregulation of MHC-II expression by colonic IECs, and treatment with butyrate might serve as an intervention to prevent intestinal inflammation. Further identification of the upstream mediators involved will provide additional mechanistic insight into the interaction between Enterococcus and the intestinal epithelium at steady state and during GVHD.

Absence of gut microbiota impairs depletion of Paneth cells but not goblet cells in germ-free Atoh1lox/lox VilCreERT2 mice.

Mouse atonal homolog 1 (Math1/Atoh1) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency on Math1lox/loxVilCreERT2 (Math1ΔIEC) mice treatment with tamoxifen in the presence or absence of intestinal microbiota showed a failure on Paneth cell depletion in germ-free mice as compared with specific pathogen-free (SPF) mice. However, goblet cells were efficiently depleted in Math1ΔIEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1ΔIEC mice 5 days after tamoxifen injection as compared with germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts and apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1ΔIEC mice for the first 7 wk after the last tamoxifen injection, far already from the 5 days time-laps observed in SPF conditions, an incomplete depletion of Paneth cells was observed 14 wk after the last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1ΔIEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells.NEW & NOTEWORTHY Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.

Live probiotic bacteria administered in a pathomimetic Leaky Gut Chip ameliorate impaired epithelial barrier and mucosal inflammation

Here, we report a pathomimetic Leaky Gut Chip that recapitulates increased epithelial permeability and intestinal inflammation to assess probiotic intervention as live biotherapeutics. We leveraged a mechanodynamic human gut-on-a-chip (Gut Chip) that recreates three-dimensional epithelial layers in a controlled oxygen gradient and biomechanical cues, where the addition of a cocktail of pro-inflammatory cytokines, TNF-α and IL-1β, reproducibly induced impaired epithelial barrier followed by intestinal inflammation. This inflamed leaky epithelium was not recovered for up to 3 days, although the cytokine treatment ceased. However, when probiotic bacteria, either Lactobacillus rhamnosus GG or a multi-species mixture (VSL#3), were respectively administered on the leaky epithelium, bacterial cells colonized mucosal surface and significantly improved barrier function, enhanced the localization of tight junction proteins such as ZO-1 and occludin, and elevated mucus production. In addition, inflammatory markers, including p65, pSTAT3, and MYD88, that were highly expressed in the germ-free control were significantly reduced when probiotic bacteria were co-cultured in a Leaky Gut Chip. Probiotic treatment also significantly reduced the production of secretory pro-inflammatory cytokines. Hence, our pathomimetic Leaky Gut Chip may offer a translational strategy to dissect the therapeutic mechanism of live biotherapeutic products and validate their clinical potential by incorporating patient-derived organoids.

Microbial-induced Redox Imbalance in the Neonatal Lung Is Ameliorated by Live Biotherapeutics.

Bronchopulmonary dysplasia (BPD) is a common lung disease of premature infants. Hyperoxia exposure and microbial dysbiosis are contributors to BPD development. However, the mechanisms linking pulmonary microbial dysbiosis to worsening lung injury are unknown. Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that regulates oxidative stress responses and modulates hyperoxia-induced lung injury. We hypothesized that airway dysbiosis would attenuate Nrf2-dependent antioxidant function, resulting in a more severe phenotype of BPD. Here, we show that preterm infants with a Gammaproteobacteria-predominant dysbiosis have increased endotoxin in tracheal aspirates, and mice monocolonized with the representative Gammaproteobacteria Escherichia coli show increased tissue damage compared with germ-free (GF) control mice. Furthermore, we show Nrf2-deficient mice have worse lung structure and function after exposure to hyperoxia when the airway microbiome is augmented with E. coli. To confirm the disease-initiating potential of airway dysbiosis, we developed a novel humanized mouse model by colonizing GF mice with tracheal aspirates from human infants with or without severe BPD, producing gnotobiotic mice with BPD-associated and non-BPD-associated lung microbiomes. After hyperoxia exposure, BPD-associated mice demonstrated a more severe BPD phenotype and increased expression of Nrf2-regulated genes, compared with GF and non-BPD-associated mice. Furthermore, augmenting Nrf2-mediated antioxidant activity by supporting colonization with Lactobacillus species improved dysbiotic-augmented lung injury. Our results demonstrate that a lack of protective pulmonary microbiome signature attenuates an Nrf2-mediated antioxidant response, which is augmented by a respiratory probiotic blend. We anticipate antioxidant pathways will be major targets of future microbiome-based therapeutics for respiratory disease.

Dynamics of the intestinal bacterial community in black soldier fly larval guts and its influence on insect growth and development.

Black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is a prominent insect for the bioconversion of various organic wastes. As a saprotrophic insect, the BSF inhabits microbe-rich environments. However, the influences of the intestinal microorganisms on BSF growth and development are not very clear. In this study, the dynamics of the intestinal bacterial community of BSF larvae (BSFL) were analyzed using pyrosequencing. Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were the most prevalent bacterial phyla in the intestines of all larval instars. The dynamic changes in bacterial community compositions among different larval instars were striking at the genus level. Klebsiella, Clostridium, Providencia, and Dysgonomonas were the relatively most abundant bacteria in the 1st- to 4th-instar BSFL, respectively. Dysgonomonas and Providencia also dominated the 5th- and 6th-instar larvae, at ratios of 31.1% and 47.2%, respectively. In total, 148 bacterial strains affiliated with 20 genera were isolated on different media under aerobic and anaerobic conditions. Among them, 6 bacteria, BSF1-BSF6, were selected for further study. The inoculation of the 6 isolates independently into germ-free BSFL feeding on an artificial diet showed that all the bacteria, except BSF4, significantly promoted BSF growth and development compared with the germ-free control. Citrobacter, Dysgonomonas, Klebsiella, Ochrobactrum, and Providencia promoted BSF development significantly by increasing the weight gains of larvae and pupae, as well as increasing the prepupae and eclosion rates. In addition, Citrobacter, Klebsiella and Providencia shortened the BSF life cycle significantly. The results illustrate the promotive effects of intestinal bacteria on BSF growth and development.

Bacteroides ovatus colonization influences the abundance of intestinal short chain fatty acids and neurotransmitters

SummaryGut microbes can synthesize multiple neuro-active metabolites. We profiled neuro-active compounds produced by the gut commensal Bacteroides ovatus in vitro and in vivo by LC-MS/MS. We found that B. ovatus generates acetic acid, propionic acid, isobutyric acid, and isovaleric acid. In vitro, B. ovatus consumed tryptophan and glutamate and synthesized the neuro-active compounds glutamine and GABA. Consistent with our LC-MS/MS-based in vitro data, we observed elevated levels of acetic acid, propionic acid, isobutyric acid, and isovaleric acid in the intestines of B. ovatus mono-associated mice compared with germ-free controls. B. ovatus mono-association also increased the concentrations of intestinal GABA and decreased the concentrations of tryptophan and glutamine compared with germ-free controls. Computational network analysis revealed unique links between SCFAs, neuro-active compounds, and colonization status. These results highlight connections between microbial colonization and intestinal neurotransmitter concentrations, suggesting that B. ovatus selectively influences the presence of intestinal neurotransmitters.

Intestinal microbiota-mediated biotransformations alter the pharmacokinetics of the major metabolites of azathioprine in rats after oral administration

Adverse reactions to azathioprine (AZA) vary greatly among individuals, which is associated with the variable levels of its major metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). The intestinal microbiota has been proven to contain AZA-metabolizing enzymes, although the explicit role of the intestinal microbiota in AZA metabolism in vivo remains poorly comprehended. In this study, the pharmacokinetic behaviours of 6-TGN and 6-MMP were assessed in the pseudo germ-free (PGF) group and control group following oral administration of AZA. The AUC0-t and Cmax of 6-TGN in the PGF group were significantly decreased by 34.0% and 35.0% (P < 0.05) compared with those in the control group. Additionally, the AUC0-t and Cmax of 6-MMP were reduced by 27.9% and 34.2% in the PGF group, although the differences were not significant. The TPMT and NUDT15 genotypes of rats in the two groups were genetically identical. The expression levels of key AZA-metabolizing enzymes in liver were not different between two groups. Furthermore, the major metabolites of AZA in the incubation system with intestinal microbial enzymes were identified. In summary, shifts in the composition of the intestinal microbiota may regulate the exposure of 6-TGN in vivo by altering the gut microbial metabolism of AZA.

Dietary Modulation Alters Susceptibility to Listeria monocytogenes and Salmonella Typhimurium with or without a Gut Microbiota.

ABSTRACTFood safety has considerably improved worldwide, yet infections with foodborne human enteric pathogens, such as Listeria spp. and Salmonella spp., still cause numerous hospitalizations and fatalities. Since dietary alterations, including fiber deficiency, might impact the colonization resistance mediated by the gut microbiome, studying the diet–microbiome–pathogen axis holds promise in further understanding the pathogenesis mechanisms. Using a gnotobiotic mouse model containing a 14-member synthetic human gut microbiota (14SM), we have previously shown that dietary fiber deprivation promotes proliferation of mucin-degrading bacteria, leading to a microbiome-mediated erosion of the colonic mucus barrier, which results in an increased susceptibility toward the rodent enteric pathogen Citrobacter rodentium. Here, we sought to understand how a low-fiber diet affects susceptibility to Listeria monocytogenes and Salmonella enterica serovar Typhimurium by using our 14SM gnotobiotic mouse model in BALB/c and C57BL/6 mouse backgrounds, respectively. Intriguingly, and in contrast to our results with C. rodentium, we observed that depriving mice of dietary fiber protected them from infections with both pathogens, compared to mice fed a standard chow. The microbiome delayed the overall pathogenicity compared to the onset of disease observed in germfree control mice. Nevertheless, we observed the same effect of diet on germfree mice, suggesting that the susceptibility is directly driven by the diet itself even in the absence of the gut microbiome. Our study points out an important observation, namely, that dietary fiber plays a crucial role in either the host’s susceptibility, the virulence of these pathogens, or both. It would be judicious to design and interpret future studies on this basis.IMPORTANCE The human enteric pathogens Listeria monocytogenes and Salmonella Typhimurium are employed as classical models in rodent hosts to understand the pathogenesis mechanisms of foodborne pathogens. Research in the past decade has stressed the importance of the gut microbial composition in modulating susceptibility to these pathogens. The results of our study—using gnotobiotic mice and germfree control animals—additionally suggest that the dietary fiber components can dominate the impact of enteropathogenic virulence over the pathogenicity-modulating properties of the gut microbiome. The significance of our research is that there is a need to carefully choose a certain chow when performing the enteropathogen-associated mouse experiments and to cautiously match the rodent diets when trying to replicate experiments across different laboratories. Finally, our data underscore the importance of using germfree control animals to study these pathogens, as our findings would have been prone to misinterpretation in the absence of these controls.

Neurotransmitter Profiles Are Altered in the Gut and Brain of Mice Mono-Associated with Bifidobacterium dentium.

Background: Accumulating evidence indicates that the gut microbiota can synthesize neurotransmitters as well as impact host-derived neurotransmitter levels. In the past, it has been challenging to decipher which microbes influence neurotransmitters due to the complexity of the gut microbiota. Methods: To address whether a single microbe, Bifidobacterium dentium, could regulate important neurotransmitters, we examined Bifidobacteria genomes and explored neurotransmitter pathways in secreted cell-free supernatant using LC-MS/MS. To determine if B. dentium could impact neurotransmitters in vivo, we mono-associated germ-free mice with B. dentium ATCC 27678 and examined fecal and brain neurotransmitter concentrations. Results: We found that B. dentium possessed the enzymatic machinery to generate γ-aminobutyric acid (GABA) from glutamate, glutamine, and succinate. Consistent with the genome analysis, we found that B. dentium secreted GABA in a fully defined microbial media and elevated fecal GABA in B. dentium mono-associated mice compared to germ-free controls. We also examined the tyrosine/dopamine pathway and found that B. dentium could synthesize tyrosine, but could not generate L-dopa, dopamine, norepinephrine, or epinephrine. In vivo, we found that B. dentium mono-associated mice had elevated levels of tyrosine in the feces and brain. Conclusions: These data indicate that B. dentium can contribute to in vivo neurotransmitter regulation.

Bacteroides ovatus Influences the Levels of Intestinal Neurotransmitters in a Gnotobiotic Model

Background Microbial metabolites and colonization have been associated with the levels and turn-over of a wide range of host neurotransmitters. Although animal models have been paramount in linking microbes to fundamental gastrointestinal and neural processes, the majority of current work has focused on complete microbial communities. The complex nature of these communities’ obscures which species drive neurotransmitter changes and makes it challenging to identify specific gut-brain-axis pathways. Previous studies have identified that Bacteroides, a dominant member of the human gut microbiome, can influence the gut-brain-axis; although the exact pathways of communication remain unclear. We hypothesized that Bacteroides species could influence gut neurotransmitter levels, initiating communication between distant organs. Methods & Result We grew B. ovatus, a representative human gut commensal, in a fully defined bacteria media, ZMBI, and examined short chain fatty acids and neurotransmitters by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that B. ovatus generates high levels of acetic acid and propionic acid, and relatively low concentrations of isobutyric acid and isovaleric acid. Pathway analysis revealed that B. ovatus consumed tryptophan and glutamate, and synthesized glutamine and GABA, potent neurotransmitters. We did not observe serotonin, 5-HIAA, melatonin, 5-hydroxytryptophan, dopamine, norepinephrine, or epinephrine in any of the samples. To determine how B. ovatus secreted products influenced neurotransmitter release from the intestinal epithelium, we microinjected intestinal organoids with B. ovatus supernatant. B. ovatus injected organoids were also found to have increased concentrations of tyrosine and tyramine compared to media control. Interestingly, B. ovatus was not found to stimulate host serotonin, melatonin, 5-HIAA or 5-hydroxytryptophan. Finally, we mono-associated germ-free adult C57BL/6J mice with the commensal B. ovatus for 17 days and examined colonic short chain fatty acids and neurotransmitters levels. Similar to B. ovatus supernatant, we observed acetic acid, propionic acid, isobutyric acid and isovaleric acid in gnotobiotic intestines. Complementing our organoid experiments, the concentrations of serotonin, melatonin, 5-HIAA and 5-hydroxytryptophan were not changed in B. ovatus mono-associated mice. However, B. ovatus mono-associated mice did increase intestinal tyrosine, glutamate, glutamine and GABA concentrations compared to germ-free controls. Conclusions These results highlight novel connections between microbial colonization and neurotransmitters. Our data indicates that B. ovatus selectively influences intestinal neurotransmitters levels, particular those of the GABA/Glu/Gln pathway, which likely has far reaching implications on human health.

Bifidobacterium dentium-derived y-glutamylcysteine suppresses ER-mediated goblet cell stress and reduces TNBS-driven colonic inflammation

ABSTRACT Endoplasmic reticulum (ER) stress compromises the secretion of MUC2 from goblet cells and has been linked with inflammatory bowel disease (IBD). Although Bifidobacterium can beneficially modulate mucin production, little work has been done investigating the effects of Bifidobacterium on goblet cell ER stress. We hypothesized that secreted factors from Bifidobacterium dentium downregulate ER stress genes and modulates the unfolded protein response (UPR) to promote MUC2 secretion. We identified by mass spectrometry that B. dentium secretes the antioxidant γ-glutamylcysteine, which we speculate dampens ER stress-mediated ROS and minimizes ER stress phenotypes. B. dentium cell-free supernatant and γ-glutamylcysteine were taken up by human colonic T84 cells, increased glutathione levels, and reduced ROS generated by the ER-stressors thapsigargin and tunicamycin. Moreover, B. dentium supernatant and γ-glutamylcysteine were able to suppress NF-kB activation and IL-8 secretion. We found that B. dentium supernatant, γ-glutamylcysteine, and the positive control IL-10 attenuated the induction of UPR genes GRP78, CHOP, and sXBP1. To examine ER stress in vivo, we first examined mono-association of B. dentium in germ-free mice which increased MUC2 and IL-10 levels compared to germ-free controls. However, no changes were observed in ER stress-related genes, indicating that B. dentium can promote mucus secretion without inducing ER stress. In a TNBS-mediated ER stress model, we observed increased levels of UPR genes and pro-inflammatory cytokines in TNBS treated mice, which were reduced with addition of live B. dentium or γ-glutamylcysteine. We also observed increased colonic and serum levels of IL-10 in B. dentium- and γ-glutamylcysteine-treated mice compared to vehicle control. Immunostaining revealed retention of goblet cells and mucus secretion in both B. dentium- and γ-glutamylcysteine-treated animals. Collectively, these data demonstrate positive modulation of the UPR and MUC2 production by B. dentium-secreted compounds.

Human-Derived Bifidobacterium dentium Modulates the Mammalian Serotonergic System and Gut-Brain Axis.

Background & AimsThe human gut microbiota can regulate production of serotonin (5-hydroxytryptamine [5-HT]) from enterochromaffin cells. However, the mechanisms underlying microbial-induced serotonin signaling are not well understood.MethodsAdult germ-free mice were treated with sterile media, live Bifidobacterium dentium, heat-killed B dentium, or live Bacteroides ovatus. Mouse and human enteroids were used to assess the effects of B dentium metabolites on 5-HT release from enterochromaffin cells. In vitro and in vivo short-chain fatty acids and 5-HT levels were assessed by mass spectrometry. Expression of tryptophan hydroxylase, short-chain fatty acid receptor free fatty acid receptor 2, 5-HT receptors, and the 5-HT re-uptake transporter (serotonin transporter) were assessed by quantitative polymerase chain reaction and immunostaining. RNA in situ hybridization assessed 5-HT–receptor expression in the brain, and 5-HT–receptor–dependent behavior was evaluated using the marble burying test.ResultsB dentium mono-associated mice showed increased fecal acetate. This finding corresponded with increased intestinal 5-HT concentrations and increased expression of 5-HT receptors 2a, 4, and serotonin transporter. These effects were absent in B ovatus-treated mice. Application of acetate and B dentium–secreted products stimulated 5-HT release in mouse and human enteroids. In situ hybridization of brain tissue also showed significantly increased hippocampal expression of 5-HT–receptor 2a in B dentium–treated mice relative to germ-free controls. Functionally, B dentium colonization normalized species-typical repetitive and anxiety-like behaviors previously shown to be linked to 5-HT–receptor 2a.ConclusionsThese data suggest that B dentium, and the bacterial metabolite acetate, are capable of regulating key components of the serotonergic system in multiple host tissues, and are associated with a functional change in adult behavior.

Bifidobacteria shape host neural circuits during postnatal development by promoting synapse formation and microglial function

We hypothesized that early-life gut microbiota support the functional organization of neural circuitry in the brain via regulation of synaptic gene expression and modulation of microglial functionality. Germ-free mice were colonized as neonates with either a simplified human infant microbiota consortium consisting of four Bifidobacterium species, or with a complex, conventional murine microbiota. We examined the cerebellum, cortex, and hippocampus of both groups of colonized mice in addition to germ-free control mice. At postnatal day 4 (P4), conventionalized mice and Bifidobacterium-colonized mice exhibited decreased expression of synapse-promoting genes and increased markers indicative of reactive microglia in the cerebellum, cortex and hippocampus relative to germ-free mice. By P20, both conventional and Bifidobacterium-treated mice exhibited normal synaptic density and neuronal activity as measured by density of VGLUT2+ puncta and Purkinje cell firing rate respectively, in contrast to the increased synaptic density and decreased firing rate observed in germ-free mice. The conclusions from this study further reveal how bifidobacteria participate in establishing functional neural circuits. Collectively, these data indicate that neonatal microbial colonization of the gut elicits concomitant effects on the host CNS, which promote the homeostatic developmental balance of neural connections during the postnatal time period.

Genetic and metabolic links between the murine microbiome and memory

BackgroundRecent evidence has linked the gut microbiome to host behavior via the gut–brain axis [1–3]; however, the underlying mechanisms remain unexplored. Here, we determined the links between host genetics, the gut microbiome and memory using the genetically defined Collaborative Cross (CC) mouse cohort, complemented with microbiome and metabolomic analyses in conventional and germ-free (GF) mice.ResultsA genome-wide association analysis (GWAS) identified 715 of 76,080 single-nucleotide polymorphisms (SNPs) that were significantly associated with short-term memory using the passive avoidance model. The identified SNPs were enriched in genes known to be involved in learning and memory functions. By 16S rRNA gene sequencing of the gut microbial community in the same CC cohort, we identified specific microorganisms that were significantly correlated with longer latencies in our retention test, including a positive correlation with Lactobacillus. Inoculation of GF mice with individual species of Lactobacillus (L. reuteri F275, L. plantarum BDGP2 or L. brevis BDGP6) resulted in significantly improved memory compared to uninoculated or E. coli DH10B inoculated controls. Untargeted metabolomics analysis revealed significantly higher levels of several metabolites, including lactate, in the stools of Lactobacillus-colonized mice, when compared to GF control mice. Moreover, we demonstrate that dietary lactate treatment alone boosted memory in conventional mice. Mechanistically, we show that both inoculation with Lactobacillus or lactate treatment significantly increased the levels of the neurotransmitter, gamma-aminobutyric acid (GABA), in the hippocampus of the mice.ConclusionTogether, this study provides new evidence for a link between Lactobacillus and memory and our results open possible new avenues for treating memory impairment disorders using specific gut microbial inoculants and/or metabolites.8Wgvn7aSo1UtQ-gTeXXmu8Video

Microbial Regulation of Enteric Eosinophils and Its Impact on Tissue Remodeling and Th2 Immunity.

Eosinophils have emerged as multifaceted cells that contribute to tissue homeostasis. However, the impact of the microbiota on their frequency and function at mucosal sites remains unclear. Here, we investigated the role of the microbiota in the regulation of enteric eosinophils. We found that small intestinal (SI) eosinophilia was significantly greater in germ-free (GF) mice compared to specific pathogen free (SPF) controls. This was associated with changes in the production of enteric signals that regulate eosinophil attraction and survival, and was fully reversed by complex colonization. Additionally, SI eosinophils of GF mice exhibited more cytoplasmic protrusions and less granule content than SPF controls. Lastly, we generated a novel strain of eosinophil-deficient GF mice. These mice displayed intestinal fibrosis and were less prone to allergic sensitization as compared to GF controls. Overall, our study demonstrates that commensal microbes regulate intestinal eosinophil frequency and function, which impacts tissue repair and allergic sensitization to food antigens. These data support a critical interplay between the commensal microbiota and intestinal eosinophils in shaping homeostatic, innate, and adaptive immune processes in health and disease.

Effects of microbiota-directed foods in gnotobiotic animals and undernourished children.

To examine the contributions of impaired gut microbial community development to childhood undernutrition, we combined metabolomic and proteomic analyses of plasma samples with metagenomic analyses of fecal samples to characterize the biological state of Bangladeshi children with severe acute malnutrition (SAM) as they transitioned, after standard treatment, to moderate acute malnutrition (MAM) with persistent microbiota immaturity. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes targeting weaning-phase bacterial taxa underrepresented in SAM and MAM microbiota were characterized in gnotobiotic mice and gnotobiotic piglets colonized with age- and growth-discriminatory bacteria. A randomized, double-blind controlled feeding study identified a lead MDCF that changes the abundances of targeted bacteria and increases plasma biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function in children with MAM.INTRODUCTIONThere is a dimension to post-natal human development that involves assembly of microbial communities in different body habitats, including the gut. Children with acute malnutrition have impaired development of their gut microbiota, leaving them with communities that appear younger (more immature) than those of chronologically age-matched healthy individuals. Current therapeutic foods given to children with acute malnutrition have not been formulated based on knowledge of how they affect the developmental biology of the gut microbiota. Moreover, they are largely ineffective in ameliorating the long-term sequelae of malnutrition that include persistent stunting, neurodevelopmental abnormalities, and immune dysfunction.RATIONALERepairing microbiota immaturity and determining the degree to which such repair restores healthy growth requires identification of microbial targets that are not only biomarkers of community assembly but also mediators of various aspects of growth. Identifying ingredients in complementary foods, consumed during the transition from exclusive milk feeding to a fully weaned state, that increase the representation and expressed beneficial functions of growth-promoting bacterial taxa in the developing microbiota could provide an effective, affordable, culturally acceptable, and sustainable approach to treatment.RESULTSMetabolomic and proteomic analyses of serially collected plasma samples were combined with metagenomic analyses of serially collected fecal samples from Bangladeshi children with severe acute malnutrition (SAM) treated with standard therapy. The results provided a readout of their biological features as they transitioned from SAM to a state of persistent moderate acute malnutrition (MAM) with accompanying persistent microbiota immaturity. Significant correlations were identified between levels of plasma proteins, anthropometry, plasma metabolites, and the representation of bacteria in their microbiota. Gnotobiotic mice were subsequently colonized with a defined consortium of bacterial strains that represent various phases of microbiota development in healthy Bangladeshi children. Administration of different combinations of Bangladeshi complementary food ingredients to colonized mice and germ-free controls revealed diet-dependent increases in the abundance and changes in the metabolic activities of targeted weaning-phase strains as well as diet- and colonization-dependent augmentation of growth-promoting host signaling pathways. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes were subsequently examined in gnotobiotic mice colonized with immature microbiota from children with post-SAM MAM and in gnotobiotic piglets colonized with a defined consortium of targeted age- and growth-discriminatory taxa. A randomized, double-blind study of standard therapy versus various MDCF prototypes emerging from these preclinical models, conducted in Bangladeshi children with MAM, identified a lead MDCF that increased levels of biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function toward a state resembling healthy children. Using an approach inspired by statistical methods applied to financial markets, we show in the accompanying paper by Raman et al. that this lead MDCF was most effective in repairing the microbiota.CONCLUSIONThese findings demonstrate the translatability of results obtained from pre-clinical gnotobiotic animal models to humans, directly support the hypothesis that healthy microbiota development is causally linked to healthy growth, illustrate an approach for treating childhood undernutrition, and with the capacity to deliberately reconfigure immature microbiota, suggest a means to decipher how elements of the gut microbial community operate to regulate various host systems involved in healthy growth.Overview of therapeutic food discovery and testing. The approach used for integrating preclinical gnotobiotic animal models with human studies to understand the contributions of perturbed gut microbiota development to childhood malnutrition and to identify MDCFs.

Gallotannins and Lactobacillus plantarum WCFS1 Mitigate High-Fat Diet-Induced Inflammation and Induce Biomarkers for Thermogenesis in Adipose Tissue in Gnotobiotic Mice.

Intestinal microbial metabolites from gallotannins (GT), including gallic acid (GA) and pyrogallol (PG), may possess potential anti-obesogenic properties. Lactobacillus plantarum (L. plantarum) found in the intestinal microbiome encodes for enzymatic activities that metabolize GT into GA and PG. Anti-obesogenic activities of orally administered GT in the presence or absence of L. plantarum is examined in gnotobiotic mice fed a high-fat diet (HFD). Germ-free (GF) C57BL/6J mice are divided into three groups, GF control, GF gavaged with GT, and mice colonized with L. plantarum and gavaged with GT. Compared to the control, GT decreases the expressions of lipogenic genes (e.g., fatty acid synthase (FAS)) in epididymal white adipose tissue and increases thermogenic genes (e.g., nuclear factor erythroid-2-like 1 (Nfe2l1)) in interscapular brown adipose tissue. Intestinal colonization with L. plantarum enhances these effects, and mice colonized with L. plantarum exhibit lower levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), leptin and plasma insulin. Results indicate that GT and L. plantarum reduce HFD-induced inflammation, insulin resistance, and promote thermogenesis in adipose tissue potentially through the activity of GT-metabolizing bacterial enzymes yielding absorbable bioactive GT metabolites. These findings imply the potential role of prebiotic-probiotic interactions in the prevention of diet-induced metabolic disorders.

Fusobacteria is Associated with a Th1 and Th17 Immune Microenvironment in Colon Cancer Patients and Germ‐Free Mice

Sporadic colorectal cancer (CRC) remains a global health burden with a rising incidence in younger patients (&lt;50 years), enhancing the need to identify novel targets for diagnosis and treatment. Certain T cell subsets appear to be associated with CRC prognosis; Th1 subsets, marked by interferon gamma (IFNγ), with better prognoses, and Th17, marked by interleukin‐17 (IL‐17), with worse prognoses. Several studies have shown that Fusobacteria (Fuso), specifically Fusobacterium nucleatum (Fn), are prevalent in CRC tissues despite their predominant location in the oral microbiome. Herein, we tested the hypothesis that Fuso are associated with colonic mucosal Th17 responses. First, using tissues from surgically‐resected CRC patients (tumor and paired normal flanking tissue, n=19 tissues) that were positive for Fuso by 16S ribosomal RNA gene (16S) sequencing, we analyzed tissue‐associated Th1 and Th17 cell populations by flow cytometry. Fuso 16S reads positively correlated with both IL‐17+ and IFNγ+ cells in these patient tissues (R2=0.3, p=0.03). We next tested whether subspecies (subsp.) of Fn isolated from CRC patients would differentially impact colonic mucosal immune responses in germ‐free (GF) mice. GF mice (n=4 GF controls; n=5/subsp.) were inoculated with Fn subsp. animalis, nucleatum, vincentii, or polymorphum for 2 weeks before tissue harvest. Colonization of GF mice with bacteria typically results in decreased ceca weights, however all subsp. resulted in increased ceca weights compared to GF mice (Kruskal‐Wallis 1way ANOVA, p=0.0148). Stools (n=2/subsp.) were positive for Fuso colonization by fluorescence in situ hybridization (FISH), except for mice colonized with subsp. polymorphum. Fuso‐specific fecal 16S rRNA qPCR and colon FISH are in progress. Mice (n=3/subsp.) were analyzed for expression of inflammatory cytokines associated with CRC and/or Fn. Compared to the GF control, subsp. animalis and vincentii induced IL‐17a expression by an average fold‐change (ΔΔCt) of 1.6 and 4.1, respectively; subsp. nucleatum and polymorphum did not differ from the GF mouse control. All subsp. induced an increase in IL‐6 expression that contributes to IL‐17 T cell polarization. Similar to observations in our patient cohort, all subspecies increased IFNγ expression. In summary, these findings suggest human CRC tissue Th1 and Th17 immune responses correlate with Fuso, specifically, Fn, tissue abundance. This dual response was mimicked by select Fn subsp. in GF mice, and certain CRC‐derived Fn subsp. may be more proinflammatory than others. Further investigations into the immune‐modulating and tumorigenic potential of CRC‐associated Fn subsp. are needed.Support or Funding InformationBloomberg~Kimmel Institute for Cancer Immunotherapy; Cancer Research InstituteThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.