Gut Microbiome In Mice Research Articles

Caffeine-Induced Sleep Restriction Alters the Gut Microbiome and Fecal Metabolic Profiles in Mice

Insufficient sleep is becoming increasingly common and contributes to many health issues. To combat sleepiness, caffeine is consumed daily worldwide. Thus, caffeine consumption and sleep restriction often occur in succession. The gut microbiome can be rapidly affected by either one's sleep status or caffeine intake, whereas the synergistic effects of a persistent caffeine-induced sleep restriction remain unclear. In this study, we investigated the impact of a chronic caffeine-induced sleep restriction on the gut microbiome and its metabolic profiles in mice. Our results revealed that the proportion of Firmicutes and Bacteroidetes was not altered, while the abundance of Proteobacteria and Actinobacteria was significantly decreased. In addition, the content of the lipids was abundant and significantly increased. A pathway analysis of the differential metabolites suggested that numerous metabolic pathways were affected, and the glycerophospholipid metabolism was most significantly altered. Combined analysis revealed that the metabolism was significantly affected by variations in the abundance and function of the intestinal microorganisms and was closely relevant to Proteobacteria and Actinobacteria. In conclusion, a long-term caffeine-induced sleep restriction affected the diversity and composition of the intestinal microbiota in mice, and substantially altered the metabolic profiles of the gut microbiome. This may represent a novel mechanism by which an unhealthy lifestyle such as mistimed coffee breaks lead to or exacerbates disease.

A prebiotic diet modulates microglial states and motor deficits in α-synuclein overexpressing mice.

Parkinson's disease (PD) is a movement disorder characterized by neuroinflammation, α-synuclein pathology, and neurodegeneration. Most cases of PD are non-hereditary, suggesting a strong role for environmental factors, and it has been speculated that disease may originate in peripheral tissues such as the gastrointestinal (GI) tract before affecting the brain. The gut microbiome is altered in PD and may impact motor and GI symptoms as indicated by animal studies, although mechanisms of gut-brain interactions remain incompletely defined. Intestinal bacteria ferment dietary fibers into short-chain fatty acids, with fecal levels of these molecules differing between PD and healthy controls and in mouse models. Among other effects, dietary microbial metabolites can modulate activation of microglia, brain-resident immune cells implicated in PD. We therefore investigated whether a fiber-rich diet influences microglial function in α-synuclein overexpressing (ASO) mice, a preclinical model with PD-like symptoms and pathology. Feeding a prebiotic high-fiber diet attenuates motor deficits and reduces α-synuclein aggregation in the substantia nigra of mice. Concomitantly, the gut microbiome of ASO mice adopts a profile correlated with health upon prebiotic treatment, which also reduces microglial activation. Single-cell RNA-seq analysis of microglia from the substantia nigra and striatum uncovers increased pro-inflammatory signaling and reduced homeostatic responses in ASO mice compared to wild-type counterparts on standard diets. However, prebiotic feeding reverses pathogenic microglial states in ASO mice and promotes expansion of protective disease-associated macrophage (DAM) subsets of microglia. Notably, depletion of microglia using a CSF1R inhibitor eliminates the beneficial effects of prebiotics by restoring motor deficits to ASO mice despite feeding a prebiotic diet. These studies uncover a novel microglia-dependent interaction between diet and motor symptoms in mice, findings that may have implications for neuroinflammation and PD.

Lactobacillus paracasei IMC 502 ameliorates type 2 diabetes by mediating gut microbiota-SCFA-hormone/inflammation pathway in mice.

Type 2 diabetes mellitus (T2DM) is a complex and prevalent metabolic disease that seriously threatens human health. Numerous studies have shown that probiotics as dietary supplements have the potential to prevent and treat T2DM. However, the ability of various strains to improve diabetes symptoms and corresponding mechanisms are different. Thus, mechanistic investigation is required to validate the pharmacology of each probiotic strain for T2DM treatment. Lactobacillus paracasei IMC 502 was originally isolated from Italian elderly human feces and its probiotic attributes have been demonstrated. Here, the antidiabetic pharmacodynamics of L. paracasei IMC 502 on T2DM mice was explored. Lactobacillus paracasei IMC 502 significantly decreased blood glucose, HbA1c and lipid levels, improved insulin resistance and glucose intolerance, regulated the mRNA/protein expression of key hepatic enzymes associated with gluconeogenesis, de novo lipogenesis and PI3K/Akt pathway, and repaired pancreatic and hepatic tissue damage. This probiotic conferred beneficial outcomes in the gut microbiome of diabetic mice, which induced transformation of short-chain fatty acids (SCFAs) and further enhanced the secretion of downstream hormones, and ultimately ameliorated the inflammatory response. Lactobacillus paracasei IMC 502 prevents and alleviates T2DM by mediating the gut microbiota-SCFA-hormone/inflammation pathway. © 2022 Society of Chemical Industry.

Impact of Germinated Brown Rice and Brown Rice on Metabolism, Inflammation, and Gut Microbiome in High Fat Diet-Induced Insulin Resistant Mice.

The constituents of germinated brown rice (GBR), brown rice (BRR), and white rice (WHR) and their impact on metabolism, inflammation, and gut microbiota in high fat (HF) diet-fed mice were examined. The contents of total fiber and γ-aminobutyric acid in BRR and GBR were higher than that in WHR (p < 0.05). Male C57 BL/6J mice received HF diet+26 g% of WHR, BRR, or GBR for 12 weeks. BRR and GBR comparably reduced HF diet-induced increases in fasting plasma glucose, lipids, insulin resistance, and inflammatory markers compared to WHR (p < 0.01). The abundance of fecal Bacteroidetes in mice fed HF+GBR or HF+BRR was higher than in HF+WHR-fed mice (p < 0.05). The abundance of fecal Lactobacillus gasseri in GBR-fed mice was greater than that in WHR- or BRR-fed mice (p < 0.05). The results indicated that GBR or BRR attenuated hyperglycemia, insulin resistance, and inflammation in mice. HF+GBR, but not HF+BRR, increased a probiotic bacteria in the gut.

The effect of Porphyromonas gingivalis on the gut microbiome of mice in relation to aging.

The translocation of oral bacteria, including Porphyromonas gingivalis, to the gut has been shown to alter gut microbiome. However, the effect of P.gingivalis on gut microbiome in relation to aging has not been demonstrated. We hypothesize that P.gingivalis has more detrimental effect on gut environment with increased age. The objective of this study is to investigate the effect of P.gingivalis on gut environment using aged mice. C57BL/6J mice aged 4 weeks (young) or 76 weeks (old) were divided into four groups: control-young, control-old, P.gingivalis-administered young, and P.gingivalis-administered old. P. gingivalis was orally administered thrice weekly for 5 weeks. At 30 days after the last P.gingivalis administration, 16S rRNA sequencing was performed to study the gut microbiome. The mRNA and protein expression of intestinal junctional barrier molecules and the levels of the inflammatory cytokines IL-1β and TNF-α in the serum were evaluated. Significant differences in the gut microbiomes between the groups, in terms of taxonomic abundance, bacterial diversity, and predicted metagenome function, were observed. A significant reduction in the alpha diversity and in the abundance of beneficial bacteria, such as Akkermansia and Clostridiaceae, in the P.gingivalis-administered old mice was observed. The mRNA and protein levels of Claudin-1 and Claudin-2 in the intestine were significantly elevated, while E-cadherin was significantly downregulated in the P.gingivalis-administered old mice, as were the serum levels of IL-1β and TNF-α. The effect of P.gingivalis on the gut environment is more pronounced in old mice than in young mice.

Mining the microbiota to identify gut commensals modulating neuroinflammation in a mouse model of multiple sclerosis

BackgroundThe gut microbiome plays an important role in autoimmunity including multiple sclerosis and its mouse model called experimental autoimmune encephalomyelitis (EAE). Prior studies have demonstrated that the multiple sclerosis gut microbiota can contribute to disease, hence making it a potential therapeutic target. In addition, antibiotic treatment has been shown to ameliorate disease in the EAE mouse model of multiple sclerosis. Yet, to this date, the mechanisms mediating these antibiotic effects are not understood. Furthermore, there is no consensus on the gut-derived bacterial strains that drive neuroinflammation in multiple sclerosis.ResultsHere, we characterized the gut microbiome of untreated and vancomycin-treated EAE mice over time to identify bacteria with neuroimmunomodulatory potential. We observed alterations in the gut microbiota composition following EAE induction. We found that vancomycin treatment ameliorates EAE, and that this protective effect is mediated via the microbiota. Notably, we observed increased abundance of bacteria known to be strong inducers of regulatory T cells, including members of Clostridium clusters XIVa and XVIII in vancomycin-treated mice during the presymptomatic phase of EAE, as well as at disease peak. We identified 50 bacterial taxa that correlate with EAE severity. Interestingly, several of these taxa exist in the human gut, and some of them have been implicated in multiple sclerosis including Anaerotruncus colihominis, a butyrate producer, which had a positive correlation with disease severity. We found that Anaerotruncus colihominis ameliorates EAE, and this is associated with induction of RORγt+ regulatory T cells in the mesenteric lymph nodes.ConclusionsWe identified vancomycin as a potent modulator of the gut-brain axis by promoting the proliferation of bacterial species that induce regulatory T cells. In addition, our findings reveal 50 gut commensals as regulator of the gut-brain axis that can be used to further characterize pathogenic and beneficial host-microbiota interactions in multiple sclerosis patients. Our findings suggest that elevated Anaerotruncus colihominis in multiple sclerosis patients may represent a protective mechanism associated with recovery from the disease.9r9NT9qYv7iDsLZaQd6sb5Video

Comprehensive in silico analysis of the probiotics, and preparation of compound probiotics-Polygonatum sibiricum saponin with hypoglycemic properties

In this study, a combined application of Polygonatum sibiricum saponin (PSS) with probiotics was developed as a new formulation that could be a candidate for a dietary supplement. The properties of nine probiotics were evaluated by principal component and heatmap analysis. And the hypoglycemic properties of compound probiotics were compared with single strains. The results showed the inhibition (%) of α-amylase was higher when L. casei ATCC393 was used synergistically with L. Bulgaricus 1.1480 compared with single strains as well as other strain combinations. And it was also found that the inhibition (%) of α-amylase was higher as 70.35 % after PSS and the compound probiotic were compounded as a ratio of 2:1. Furturemore, PSS-compound probiotics could regulate the composition of the gut microbiome of T2DM mice and enhance the metabolic capacity. In conclusion, the combination of PSS and compound probiotics has shown massive potential as management nutraceuticals of T2DM.

Regular fecal microbiota transplantation to Senescence Accelerated Mouse-Prone 8 (SAMP8) mice delayed the aging of locomotor and exploration ability by rejuvenating the gut microbiota.

Recent evidence points out the role of the gut microbiota in the aging process. However, the specific changes and relevant interventions remain unclear. In this study, Senescence Accelerated Mouse-Prone 8 (SAMP8) mice were divided into four groups; young-FMT-group transplanted fecal microbiota from young donors (2–3°months old) and old-FMT-group transplanted from old donors (10–11°months old); additionally, other two groups either adult mice injected with saline solution or untreated mice served as the saline and blank control groups, respectively. All mice were intervened from their 7-months-old until 13-months-old. The open field test at 9 and 11°months of age showed that the mice transplanted with gut microbiota from young donors had significantly better locomotor and exploration ability than those of transplanted with old-donors gut microbiota and those of saline control while was comparable with the blank control. 16S rRNA gene sequencing showed that the gut microbiome of recipient mice of young donors was altered at 11°months of age, whereas the alternation of the gut microbiome of old-donor recipient mice was at 9°months. For comparison, the recipient mice in the blank and saline control groups exhibited changes in the gut microbiome at 10°months of age. The hallmark of aging-related gut microbiome change was an increase in the relative abundance of Akkermansia, which was significantly higher in the recipients transplanted with feces from older donors than younger donors at 9°months of age. This study shows that fecal microbiota transplantation from younger donors can delay aging-related declines in locomotor and exploration ability in mice by changing the gut microbiome.

Tianhuang formula reduces the oxidative stress response of NAFLD by regulating the gut microbiome in mice

BackgroundThe gut microbiome affects the occurrence and development of NAFLD, but its mechanism has not yet been fully elucidated. Chinese medicine is a new treatment strategy to improve NAFLD by regulating the gut microbiome. Tianhuang formula (TH) has been proved to have a lipid-lowering effect in which constituents of ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rb, ginsenoside Re, and ginsenoside R1 from Panax notoginseng and berberine, palmatine, and coptisine from Coptis chinensis have low drug permeability, which results in poor intestinal absorption into the human body, and are thus able to come into contact with the gut microflora for a longer time. Therefore, it might be able to influence the gut microbial ecosystem, but it still needs to be investigated.MethodThe characteristics of the gut microbiome were represented by 16S rRNA sequencing, and the metabolites in intestinal contents and liver were discovered by non-targeted metabolomics. Correlation analysis and fermentation experiments revealed the relationship between the gut microbiome and metabolites. Blood biochemical indicators, liver function indicators, and oxidation-related indicators were assayed. H&E staining and Oil Red O staining were used to analyze the characteristics of hepatic steatosis. RT-qPCR and western blotting were used to detect the expression of genes and proteins in liver tissues, and fecal microbial transplantation (FMT) was performed to verify the role of the gut microbiome.ResultsGut microbiome especially Lactobacillus reduced, metabolites such as 5-Methoxyindoleacetate (5-MIAA) significantly reduced in the liver and intestinal contents, the level of hepatic GSH and SOD reduced, MDA increased, and the protein expression of Nrf2 also reduced in NAFLD mice induced by high-fat diet (HFD). The normal diet mice transplanted with NAFLD mice feces showed oxidative liver injury, indicating that the NAFLD was closely related to the gut microbiome. TH and TH-treated mice feces both can reshape the gut microbiome, increase the abundance of Lactobacillus and the content of 5-MIAA in intestinal contents and liver, and improve oxidative liver injury. This indicated that the effect of TH improving NAFLD was related to the gut microbiome, especially Lactobacillus. 5-MIAA, produced by Lactobacillus, was proved with fermentation experiments in vitro. Further experiments proved that 5-MIAA activated the Nrf2 pathway to improve oxidative stress in NAFLD mice induced by HFD. TH reshaped the gut microbiome, increased the abundance of Lactobacillus and its metabolite 5-MIAA to alleviate oxidative stress, and improved NAFLD.ConclusionThe study has demonstrated a mechanism by which the gut microbiome modulated oxidative stress in NAFLD mice induced by HFD. The traditional Chinese medicine TH improved NAFLD by regulating the gut microbiome, and its mechanism was related to the “Lactobacillus-5-MIAA-Nrf2” pathway. It provided a promising way for the intervention of NAFLD.

Design, construction, and in vivo augmentation of a complex gut microbiome

Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize invitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.

Effects Of Broad-spectrum Antibiotic Administration And Germ-free Status On Biochemical And Endurance Adaptations To Regular Exercise In Mouse Muscle Tissue

PURPOSE: The gut microbiota modulates a variety of physiologic processes. Recent studies from our group have shown that regular endurance exercise alters the composition of the gut microbiome and metabolome in mice and humans, and we hypothesized that the presence of microbes would affect endurance training adaptations in muscular endurance capacity, mitochondrial activity and gene expression in mice. METHODS: To discern differences in adaptations from 6 wks of voluntary wheel running (VWR), we depleted microbes with antibiotics (ABX) and used germ free (GF) mice to compare to control (CON). Male and female C57Bl/6 mice of all groups underwent daily VWR or sedentary (SED) conditions in a 2 x 3 design (VWR/SED, CON/ABX/GF, n = 56). After the intervention, treadmill endurance was assessed and gastrocnemius and soleus tissue were harvested and analyzed for activity of key Krebs cycle enzyme Citrate Synthase (CS) and expression of genes indicating mitochondrial adaptations respectively. RESULTS: Two way ANOVA revealed that VWR increased treadmill endurance, ABX had no effect, and that GF status significantly reduced performance. Additionally, VWR increased Citrate Synthase enzyme activity in gastrocnemius muscle tissue in all groups, and ABX and GF status did not reduce VWR’s effect or have an effect of their own. VWR also tended to increase expression of genes associated with increased mitochondrial activity (PGC-1α, CS, Succinate Dehydrogenase) in soleus tissue, but ABX had no effect on these observations. CONCLUSIONS: We conclude that ABX treatment and GF status do not affect VWR induced adaptations in endurance capacity, gastrocnemius mitochondrial activity or expression of metabolic genes in soleus tissue, but that GF status significantly hinders endurance capacity. This indicates that removing gut microbes does not inhibit muscular endurance training adaptations, but that germ free mice possess hindered endurance exercise capacity.

Gut Microbiome and Mycobiome Alterations in an In Vivo Model of Alzheimer's Disease.

Gut microbiota has emerged as an important key regulator of health and disease status. Indeed, gut microbial dysbiosis has been identified in an increasing number of diseases, including neurodegenerative disorders. Accordingly, microbial alterations have been reported also in Alzheimer’s disease (AD), suggesting possible pathogenetic mechanisms contributing to the development of specific AD hallmarks and exacerbating metabolic alterations and neuroinflammation. The identification of these mechanisms is crucial to develop novel, targeted therapies and identify potential biomarkers for diagnostic purposes. Thus, the possibility to have AD in vivo models to study this microbial ecosystem represents a great opportunity for translational applications. Here, we characterized both gut microbiome and mycobiome of 3xTg-AD mice, one of the most widely used AD models, to identify specific microbial alterations with respect to the wild-type counterpart. Interestingly, we found a significant reduction of the Coprococcus and an increased abundance of Escherichia_Shigella and Barnesiella genera in the AD mice compatible with a pro-inflammatory status and the development of AD-related pathogenetic features. Moreover, the fungal Dipodascaceae family was significantly increased, thus suggesting a possible contribution to the metabolic alterations found in AD. Our data point out the strict connection between bacterial dysbiosis and AD and, even if further studies are required to clarify the underlining mechanisms, it clearly indicates the need for extensive metagenomic studies over the bacterial counterpart.

Metabolic protection by the dietary flavonoid 7,8-dihydroxyflavone requires an intact gut microbiome

Background7,8-dihydroxyflavone (DHF) is a naturally occurring flavonoid found in Godmania, Tridax, and Primula species that confers protection against high-fat diet (HFD) induced metabolic pathologies selectively in female mice. We have previously reported that this metabolic protection is associated with early and stable remodeling of the intestinal microbiome, evident in female but not male DHF-supplemented mice. Early changes in the gut microbiome in female DHF-fed mice were highly predictive of subsequent metabolic protection, suggesting a causative association between the gut microbiome and the metabolic effects of DHF.ObjectiveTo investigate a causal association between the gut microbiome and the metabolic effects of DHF using a model of antibiotic-induced gut microbiome ablation.Materials and methodsAge-matched male and female C57Bl6/J mice were given ad libitum access to HFD and drinking water containing vehicle or DHF for 12 weeks. For antibiotic (Abx) treatment, female mice were given drinking water containing a cocktail of antibiotics for 2 weeks prior to HFD feeding and throughout the feeding period. Metabolic phenotyping consisted of longitudinal assessments of body weights, body composition, food, and water intake, as well as measurement of energy expenditure, glucose tolerance, and plasma and hepatic lipids. Protein markers mediating the cellular effects of DHF were assessed in brown adipose tissue (BAT) and skeletal muscle.ResultsMetabolic protection conferred by DHF in female HFD-fed mice was only apparent in the presence of an intact gut microbiome. Abx-treated mice were not protected from HFD-induced obesity by DHF administration. Further, tissue activation of the tropomyosin-related kinase receptor B (TrkB) receptor, which has been attributed to the biological activity of DHF, was lost upon gut microbiome ablation, indicating a requirement for microbial “activation” of DHF for its systemic effects. In addition, we report for the first time that DHF supplementation significantly activates TrkB in BAT of female, but not male, mice uncovering a novel target tissue of DHF. DHF supplementation also increased uncoupling protein 1 (UCP1) and AMP-activated protein kinase (AMPK) protein in BAT, consistent with protection from diet-induced obesity.ConclusionThese results establish for the first time a requirement for the gut microbiome in mediating the metabolic effects of DHF in female mice and uncover a novel target tissue that may mediate these sexually-dimorphic protective effects.

Klebsiella pneumoniae infection is associated with alterations in the gut microbiome and lung metabolome

Gut microbiota is involved in maintaining homeostasis, and intestinal dysbiosis may lead to opportunistic infections and diseases. Pathogens can disrupt the gut homeostasis and establish colonization, but how they modulate the microbiome and metabolome along the gut-lung axis warrants further investigation. In the present study, we used a classical low virulence Klebsiella pneumoniae (cKp) strain to address this question. We assessed the gut microbiome and lung metabolome in cKp-infected mice by 16S rRNA sequencing and untargeted liquid chromatography-mass spectrometry, respectively. Our data revealed that cKp infection reduced gut microbiota diversity and altered microbiome composition. Specifically, cKp infection increased the abundance of MWH-CFBk5 and Actinomadura and reduced the abundance of Lachnospiraceae_NK4A136_group, Clostridium sensu_stricto 1, Bifidobacterium, and Intestinimonas at the genus level. Notably, caffeine and caffeine metabolism were significantly affected in the lung by cKp infection. Moreover, Spearman correlation analysis revealed remarkable correlations of specific lung metabolites and bacteria species at the genus level. These findings suggest that cKp infection is linked to gut dysbiosis and alterations in the lung metabolome. This study is of significance for developing innovative gut microbiota-directed therapy for respiratory diseases.

Key features of the genetic architecture and evolution of host-microbe interactions revealed by high-resolution genetic mapping of the mucosa-associated gut microbiome in hybrid mice.

Determining the forces that shape diversity in host-associated bacterial communities is critical to understanding the evolution and maintenance of metaorganisms. To gain deeper understanding of the role of host genetics in shaping gut microbial traits, we employed a powerful genetic mapping approach using inbred lines derived from the hybrid zone of two incipient house mouse species. Furthermore, we uniquely performed our analysis on microbial traits measured at the gut mucosal interface, which is in more direct contact with host cells and the immune system. Several mucosa-associated bacterial taxa have high heritability estimates, and interestingly, 16S rRNA transcript-based heritability estimates are positively correlated with cospeciation rate estimates. Genome-wide association mapping identifies 428 loci influencing 120 taxa, with narrow genomic intervals pinpointing promising candidate genes and pathways. Importantly, we identified an enrichment of candidate genes associated with several human diseases, including inflammatory bowel disease, and functional categories including innate immunity and G-protein-coupled receptors. These results highlight key features of the genetic architecture of mammalian host-microbe interactions and how they diverge as new species form.

Bioaccessibility of arsenic from contaminated soils and alteration of the gut microbiome in an in vitro gastrointestinal model

Arsenic exposure has been reported to alter the gut microbiome in mice. Activity of the gut microbiome derived from fecal microbiota has been found to affect arsenic bioaccessibility in an in vitro gastrointestinal (GI) model. Only a few studies have explored the relation between arsenic exposure and changes in the composition of the gut microbiome and in arsenic bioaccessibility. Here, we used simulated GI model system (GIMS) containing a stomach, small intestine, colon phases and microorganisms obtained from mouse feces (GIMS-F) and cecal contents (GIMS-C) to assess whether exposure to arsenic-contaminated soils affect the gut microbiome and whether composition of the gut microbiome affects arsenic bioaccessibility. Soils contaminated with arsenic did not alter gut microbiome composition in GIMS-F colon phase. In contrast, arsenic exposure resulted in the decline of bacteria in GIMS-C, including members of Clostridiaceae, Rikenellaceae, and Parabacteroides due to greater diversity and variability in microbial sensitivity to arsenic exposure. Arsenic bioaccessibility was greatest in the acidic stomach phase of GIMS (pH 1.5–1.7); except for GIMS-C colon phase exposed to mining-impacted soil in which greater levels of arsenic solubilized likely due to microbiome effects. Physicochemical properties of different test soils likely influenced variability in arsenic bioaccessibility (GIMS-F bioaccessibility range: 8–37%, GIMS-C bioaccessibility range: 2–18%) observed in this study.

Gut microbiome and circulating bacterial DNA ("blood microbiome") in a mouse model of total parenteral nutrition: Evidence of two distinct separate microbiotic compartments.

Total parenteral nutrition (TPN) causes gut atrophy, dysbiosis and leakage of the gut barrier. This study aimed to characterize the gut microbiome in response to different TPNs and tested the hypothesis whether increased gut permeability in TPN would lead to changes in the circulating bacterial DNA ("blood microbiome"). Male C57BL/6J mice were randomly allocated to the following groups for seven days (1) chow-fed control (C) without jugular vein catheter (JVC, n=6) (2) chow-fed with JVC and infusion of saline (S) (n=6) (3) Intralipid-based TPN (n-6:n-3 ratio 7:1) (IL, n=6) (4) Omegaven-based TPN (n-6:n-3 ratio 1:8) (OV, n=6). Blood was collected by cardiac puncture and feces (stool pellet) were collected from the colon. Blood and stool samples were analyzed by 16S rRNA gene sequencing. TPN administration was associated with a compositional shift in the gut microbial community that involved the expansion of Bacteroidota along with a decrease in gut bacteria belonging to the Firmicutes phylum as compared to chow-fed mice. Gram-negative Verrucomicrobiota and Proteobacteria were also increased in the gut microbiome of mice receiving TPN. Gammaproteobacteria, namely Burkholderiales, were specifically increased in Intralipid-based TPN. On the other hand, Proteobacteria and Actinobacteriota were the dominant taxa in blood samples. The families Comamonadaceae and Burkholderiaceae (both from Burkholderiales order) were increased in the "blood microbiome" of mice with indwelling JVC when compared with chow-fed mice without JVC. The increase in Burkholderiaceae was more pronounced in Intralipid-based TPN. Profound changes in the gut microbiome of mice subjected to TPN occurred, which were not reflected in the "blood microbiome" suggesting that the gut and "blood microbiome" represent two rather distinct separate microbiotic compartments. The parenteral provision of n-3 fatty acids appears to protect against proinflammatory bacteria in the gut and against the increased presence of JVC-associated bacteria as measured by circulating bacterial DNA.

Glypican-3-specific CAR-NKT cells overexpressing BATF3 mediate potent antitumor activity against hepatocellular carcinoma.

e14521 Background: Hepatocellular carcinoma (HCC), the fourth leading cause of cancer-related deaths worldwide, is amenable to immunotherapies that target glypican-3 (GPC3), a protein expressed by over 75% of HCCs but not by healthy tissues. The invariant natural killer T cells (NKTs) prevent HCC tumorogenesis through interactions with the gut microbiome in mice, and the presence of tumor-infiltrating NKTs is associated with improved clinical outcomes. We previously tested GPC3-specific chimeric antigen receptors (CARs) and selected a receptor with both CD28 and 41BB costimulatory endodomains based on its ability to mediate superior antitumor activity of transduced NKTs in vivo. Co-expression of interleukin-15 (IL15), a cytokine that supports NKT expansion and persistence, with the chosen GPC3-CAR further enhanced this activity. GPC3-CAR NKTs co-expressing IL15 (15.GPC3-CAR) overexpressed the Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF3), a member of the AP-1 family. Since AP-1 family proteins are master regulators of cell survival and proliferation, we hypothesized that overexpressing BATF3 in GPC3-CAR NKTs (BATF3.GPC3-CAR) will lead to superior expansion, persistence, and antitumor activity compared to 15.GPC3-CAR NKTs. Methods: We used RNAseq and flow cytometry to define the transcriptomic and phenotypic characteristics, respectively, of BATF3-overexpressing NKTs at baseline and after tumor cell killing in co-culture. We also used co-culture assays to evaluate the ability of BATF3- or IL15-expressing NKTs to proliferate, produce effector cytokines, and kill tumor cells after multiple rounds of exposure. Finally, we evaluated the ability of BATF3.GPC3-CAR and IL15.GPC3-CAR NKTs to expand, persist, and target tumors in an HCC xenograft murine model. Results: BATF3 overexpression in NKTs induced significant differences in gene expression profiles compared to a GFP control at baseline (DEGs up: 20; down: 26, p &lt; 0.01) and after tumor cell killing (DEGs up: 19; down: 32; p &lt; 0.01; fold change &gt; 2 or &lt; 0.5, respectively) corresponding to enrichment of gene sets related to metabolic fitness. Importantly, CD62L+ NKTs, which have enhanced antitumor properties, were enriched in BATF3-expressing NKTs (27.6% vs 8.2%, p &lt; 0.05, versus GFP control). Further, BATF3 significantly enhanced NKT proliferation (area under the curve of absolute NKT cell counts: 5x1011 vs 1.1x1011, p = 0.001; fold expansion: 6.12x104 vs 1.2x104, p = 0.001, versus IL15 NKTs). Finally, BATF3.GPC3-CAR NKTs induced long-term tumor control and significantly improved survival of HCC xenograft-bearing mice compared to 15.GPC3-CAR NKTs (p = 0.0002, Mantel-Cox analysis). Conclusions: BATF3.GPC3-CAR NKTs induce robust antitumor activity in pre-clinical models of HCC. These results provide a foundation for evaluating BATF3.GPC3-CAR NKTs in patients with relapsed/refractory HCC.

Short-chain fatty acids promote the effect of environmental signals on the gut microbiome and metabolome in mice

Gut microorganisms and the products of their metabolism thoroughly affect host brain development, function and behavior. Since alterations of brain plasticity and cognition have been demonstrated upon motor, sensorial and social enrichment of the housing conditions, we hypothesized that gut microbiota and metabolome could be altered by environmental stimuli, providing part of the missing link among environmental signals and brain effects. In this preliminary study, metagenomic and metabolomic analyses of mice housed in different environmental conditions, standard and enriched, identify environment-specific microbial communities and metabolic profiles. We show that mice housed in an enriched environment have distinctive microbiota composition with a reduction in gut bacterial richness and biodiversity and are characterized by a metabolomic fingerprint with the increase of formate and acetate and the decrease of bile salts. We demonstrate that mice treated with a mixture of formate and acetate recapitulate some of the brain plasticity effects modulated by environmental enrichment, such as hippocampal neurogenesis, neurotrophin production, short-term plasticity and cognitive behaviors, that can be further exploited to decipher the mechanisms involved in experience-dependent brain plasticity.

Intestine-Specific NHE3 Deletion in Adulthood Causes Microbial Dysbiosis.

In the intestine, the Na+/H+ exchanger 3 (NHE3) plays a critical role for Na+ and fluid absorption. NHE3 deficiency predisposes patients to inflammatory bowel disease (IBD). In mice, selective deletion of intestinal NHE3 causes various local and systemic pathologies due to dramatic changes in the intestinal environment, which can influence microbiota colonization. By using metagenome shotgun sequencing, we determined the effect of inducible intestinal epithelial cell-specific deletion of NHE3 (NHE3IEC-KO) in adulthood on the gut microbiome in mice. Compared with control mice, NHE3IEC-KO mice show a significantly different gut microbiome signature, with an unexpected greater diversity. At the phylum level, NHE3IEC-KO mice showed a significant expansion in Proteobacteria and a tendency for lower Firmicutes/Bacteroidetes (F/B) ratio, an indicator of dysbiosis. At the family level, NHE3IEC-KO mice showed significant expansions in Bacteroidaceae, Rikenellaceae, Tannerellaceae, Flavobacteriaceae and Erysipelotrichaceae, but had contractions in Lachnospiraceae, Prevotellaceae and Eubacteriaceae. At the species level, after removing those with lowest occurrence and abundance, we identified 23 species that were significantly expanded (several of which are established pro-inflammatory pathobionts); whereas another 23 species were found to be contracted (some of which are potential anti-inflammatory probiotics) in NHE3IEC-KO mice. These results reveal that intestinal NHE3 deletion creates an intestinal environment favoring the competitive advantage of inflammophilic over anti-inflammatory species, which is commonly featured in conventional NHE3 knockout mice and patients with IBD. In conclusion, our study emphasizes the importance of intestinal NHE3 for gut microbiota homeostasis, and provides a deeper understanding regarding interactions between NHE3, dysbiosis, and IBD.