Germ-free Mice Research Articles

Metabolomic and transcriptomic correlative analyses in germ free mice link probiotic-associated metabolites to host intestinal fatty acid metabolism and b-oxidation

While a transcriptomics-led examination of the effects of probiotics offers valuable insights into their influence on host gene regulation, the dynamic interactions between host metabolism and probiotics require an integrated analysis and multiomics approach to provide deeper insight into the underlying molecular mechanisms governing these associations. Here, we developed a high throughput bioinformatic tool ‘metabolome-transcriptome correlation analysis’ (METRCA, in press) that can determine whether a certain gene-metabolite association is up or downregulated and predict whether a metabolite’s or gene’s tissue location impacts its interaction with one another. To test this, we analyzed by LCMS and bulk-RNA sequencing then assessed linkages between serum as well as fecal metabolites and disease- or metabolism-associated genes in the ileum of gnotobiotic mice fed either a tryptophan (trp)-deficient or -suffcient diet then mono-associated with Lactobacillus rhamnosus GG (LGG) or administered PBS (control). We discovered significant associations, mainly in LGG mice fed trp, between metabolites and ileal genes involved in fatty acid metabolism (FAM) and oxidation (FAO). This association was specific and not observed in GF mice gavaged with PBS, monocolonized with R. gnavus, or fed trp- diets, and in SPF mice. Many fecal trp metabolites like indole-3-acetamide and serum trp metabolites like indole acetonitrile were significantly, positively correlated with important intestinal FAM genes Dgat1 and Mgat2. Likewise, numerous fecal metabolites like 5-hydroytrp and several serum trp metabolites like serotonin emerged as positively correlated metabolites with the rate-limiting FAO genes Acaa2 and Cpt2. Serum indoles were more strongly associated with both FAM and FAO genes than fecal indoles. Few fecal and serum metabolites were found negatively correlated with FAM and FAO genes. Since most are positive correlators, these findings suggest a testable hypothesis that LGG- and trp-dependent metabolites increase the intestinal metabolism of dietary lipids, providing a mechanism to previous findings that LGG supplementation in mice protected against diet-induced obesity and fatty liver disease. Analytical approaches like METRCA can thus be used to extract valuable information from metabolite and RNA-Seq datasets, generating novel hypotheses that may discover new interactions between microbe- or nutrient-associated metabolites and host metabolic or disease-related pathways. NIHR01-AT010243 (NG,RF), R01DK119198 (NG), NSF 1754783 (RF)). 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.

Unveiling the roles of dietary Tryptophan and Arginine metabolism in Lactobacillus rhamnosus GG-mediated barrier protection

Inflammatory bowel disease (IBD) is often characterized by increased intestinal paracellular permeability and abnormalities in tight junctions (TJ). IBD patients commonly exhibit reduced serum tryptophan (TRP) levels. Furthermore, animal models on low-TRP diets are more susceptible to chemically induced intestinal inflammation, while those on high-TRP diets show reduced inflammation. The interaction between TRP and gut microbiota in reinforcing the intestinal barrier warrants further investigation. To address this, we introduced Lactobacillus rhamnosus GG (LGG), known for enhancing the gut barrier, and PBS as a control into germ-free mice fed TRP-suffcient or -deficient diets. Our analysis of the mouse ileal transcriptome revealed dramatic upregulation of key genes in arginine metabolism only in LGG-TRP mice, particularly of argininosuccinate lyase ( Asl) that catabolizes argininosuccinate (ASA). Interestingly, our in-silico analysis of human IBD data GEO 179285 transcriptome showed marked dysregulation of similar genes, including inappropriate increases in ASA synthetase ( Ass1). Moreover, this unique expression pattern in the mouse ileum was TRP-dependent and specific to LGG, and not observed in mice monocolonized with pathobiont Ruminococcus gnavus or specific pathogen-free mice. LC-MS analysis of fecal and serum metabolites in LGG-TRP mice revealed elevated levels of fecal arginine, glutamine, and aspartate, suggesting their involvement in host arginine biosynthesis, along with significant reductions in serum ASA levels. ASA was highly negatively correlated with numerous TJ genes, suggesting its potential to compromise barrier function. Indeed, treating mouse enteroids with ASA enhanced dextran permeability, exacerbated LPS disruption of the gut barrier, and decreased expression of TJ genes Ocln and Tjp1. Furthermore, elevated serum ASA and decreased colonic ASL levels were observed in colitis-afflicted mice, perturbing gut homeostasis, as indicated by increased fecal lipocalin-2 and delayed colitis recovery after intraperitoneal ASA administration. We propose that LGG-TRP stimulates gut-mediated ASA turnover as evidenced by substantial increases in Asl levels. Interestingly, LGG alone reduced ASA concentration in the culture medium under anaerobic and aerobic conditions. Collectively, our study identifies ASA as a potential biomarker of gut inflammation and enhanced permeability. Moreover, we highlight the potential of LGG-TRP therapy for IBD-associated leaky gut conditions, offering novel insights for gut health interventions. NIHR01-AT010243 (NG,RF), R01DK119198 (NG), NSF 1754783 (RF). 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.

Exploring a Role for Gut Microbiota to Modulate Aldosterone

Hypertension is associated with an increased risk of mortality for numerous conditions including heart disease, diabetes, and renal disease (D. Aune, Biomed Res Int, 2021). Hypertension is often viewed as idiopathic and treatment is seldom aimed at addressing an underlying mechanism; however, a recent study indicates that primary aldosteronism is an underappreciated cause of idiopathic hypertension (J. Brown, Ann Intern Med, 2020). Here, we investigate for the first time the influence of gut microbiota on aldosterone regulation. To pursue this, we used C57Bl/6 mice (n=20; males) housed in the Johns Hopkins Animal Care Facility and fed a specific pathogen-free diet. Whole blood from submandibular cheek bleeds were collected into K2EDTA-treated tubes at baseline and mice were allowed to recover. Mice were then treated with antibiotics (ABX; 1g/L ampicillin, 1g/L neomycin, and 0.5g/L vancomycin) in their drinking water ad libitum for a total of 7 days to dramatically suppress gut microbes. Whole blood via submandibular cheek bleeds were collected after 24hrs, and at day 7 on ABXs. Mice were sacrificed on day 7 of ABX. Plasma samples from baseline, 24hrs on ABX and 7 days on ABX were used to determine aldosterone concentration and plasma renin activity (PRA) via ELISA. In addition, 24hr urine samples were collected at baseline and at day 7 on ABXs. Statistics were calculated using paired, one-way ANOVA; data below are mean ± SD. Our results demonstrate that plasma aldosterone and PRA, as well as urine aldosterone, were significantly increased from baseline to 7 days on ABX treatment (plasma aldo (pg/mL): 295.1 ± 164.8 vs 634.9 ± 290.4, p= 0.0197; plasma PRA (ng Ang-1/mL/hr): 191.8 ± 137.6 vs 325.9 ± 141.5, p= 0.0108; urine aldo (pg/mL): 2019 ± 322.1 vs 2668 ± 292.0, p = 0.0047). However, there was no significant difference in plasma aldosterone from baseline to 24hrs on ABX treatment (295.1 ± 164.8 vs 434.5 ± 245.3, p= 0.1047). In contrast, we observed a significant increase in angiotensin I from baseline to 24hrs on ABX treatment (191.8 ± 137.6 vs 355.8 ± 139.6, p=0.0041), and this increase was sustained until day 7 (325.9 ± 141.5 vs baseline, p=0.0108). Preliminary data suggest aldosterone is also increased in the plasma of germ-free mice (without gut microbes) compared to conventionalized controls (750.4 ± 265.0, n= 6, vs 314.4 ± 260.3, n=4, p=0.0667), implying that gut microbiota normally act to suppress plasma aldosterone. Taken together, we demonstrate that in the absence of gut microbiota, plasma aldosterone, urine aldosterone, and PRA are significantly increased. Understanding how gut microbes influence aldosterone levels could provide valuable insights into the development and treatment of hypertension. This knowledge may open new avenues for therapeutic interventions, such as probiotics or dietary modifications, to help regulate blood pressure. William Townsend Porter Predoctoral Fellowship from the American Physiological Society (BNM), Johns Hopkins Post-baccalaureate Research Education Program (PREP; R25GM109441 to ADM), the NIDDK Diabetic Complications Consortium (RRID:SCR_001415m www.diacomp.org, grants DK076169 and DK 115255 (subaward to JLP)) and American Heart Association Established Investigator Award (to JLP). 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.

Abstract 1156: Akkermansia (Strain BAA835) And The Microbial ADP-heptose Pathway Are Associated With Reduced Neointimal Hyperplasia After Arterial Injury

Introduction: Despite advances in surgical therapy for atherosclerosis, up to 50% of procedures such as balloon angioplasty, stenting, and bypass fail due to restenosis from neointimal hyperplasia, a cell proliferative process potentiated by inflammation. We hypothesized that neointimal hyperplasia is associated with a distinct gut microbiome profile and can be modulated by fecal transplantation. Methods: We performed fecal transplantation (FT) of C57BL6 mice using donors with differential microbiome profiles and susceptibility to neointimal hyperplasia (Sprague-Dawley [SD] and Lewis [LE] rats, high and low susceptibility, respectively). In separate experiments, germ-free mice underwent FT using C57BL6 donors with and without Akkermansia muciniphila BAA835 ( Amuc ) prior to arterial injury. Microbial communities were characterized using 16S rRNA gene amplicon and shotgun metagenome sequencing. Arterial and immune effects were assessed using frozen arterial sections, multi-parameter flow cytometry of mesenteric lymph nodes, or multiplex cytokine assays after ex vivo stimulation of whole blood with lipopolysaccharide. Results: Paradoxically, neointimal hyperplasia after injury was 67.7% greater in FT recipients of low- compared to high-susceptibility stools (P=0.01). In fecal communities of both FT recipient groups, relative abundance of A. muciniphila (r=-0.66, P<0.001) and of the ADP-heptose biosynthesis pathway (r=-0.7, P<0.001) were negatively associated with neointimal hyperplasia. A. muciniphila was differentially elevated (P<0.001) in SD compared to LE FT recipients and was the main contributing taxa for the enrichment (P<0.001) of the ADP-heptose biosynthesis pathway in the SD recipient group. In mice colonized with communities that differed only by the presence of Amuc , neointimal hyperplasia was 57.8% greater in the Amuc - group compared to Amuc+ (P=0.01). Amuc+ recipients had lower levels of IL-12, MIP-1beta, and MCP-1 in unstimulated blood and higher IP-10 and MCP-1 levels in stimulated blood. Conclusion: A. muciniphila and microbial ADP-heptose biosynthesis pathway are inversely associated with neointimal hyperplasia and are potential targets for novel microbe-based strategies to mitigate restenosis risk.

Multiomics Analysis Reveals Leucine Deprivation Promotes Bile Acid Synthesis by Upregulating Hepatic CYP7A1 and Intestinal Turicibacter sanguinis in Mice

BackgroundLeucine, a branched-chain amino acid, participates in the regulation of lipid metabolism and the composition of the intestinal microbiota. However, the related mechanism remains unclear. ObjectivesHere, we aimed to reveal the potential mechanisms by which hepatic CYP7A1 (a rate-limiting enzyme for bile acid [BA] synthesis) and gut microbiota coregulate BA synthesis under leucine deprivation. MethodsTo this end, 8-wk-old C57BL/6J mice were fed with either regular diets or leucine-free diets for 1 wk. Then, we investigated whether secondary BAs were synthesized by Turicibacter sanguinis in 7-wk-old C57BL/6J germ-free mice gavaged with T. sanguinis for 2 wk by determining BA concentrations in the plasma, liver, and cecum contents using liquid chromatography-tandem mass spectrometry. ResultsThe results showed that leucine deprivation resulted in a significant increase in total BA concentration in the plasma and an increase in the liver, but no difference in total BA was observed in the cecum contents before and after leucine deprivation. Furthermore, leucine deprivation significantly altered BA profiles such as taurocholic acid and ω-muricholic acid in the plasma, liver, and cecum contents. CYP7A1 expression was significantly upregulated in the liver under leucine deprivation. Leucine deprivation also regulated the composition of the gut microbiota; specifically, it significantly upregulated the relative abundance of T. sanguinis, thus enhancing the conversion of primary BAs into secondary BAs by intestinal T. sanguinis in mice. ConclusionsOverall, leucine deprivation regulated BA profiles in enterohepatic circulation by upregulating hepatic CYP7A1 expression and increasing intestinal T. sanguinis abundance. Our findings reveal the contribution of gut microbiota to BA metabolism under dietary leucine deprivation.

Targeting Non-Alcoholic Fatty Liver Disease with Hawthorn Ethanol Extract (HEE): A Comprehensive Examination of Hepatic Lipid Reduction and Gut Microbiota Modulation.

Recent studies have highlighted the lipid-lowering ability of hawthorn ethanol extract (HEE) and the role played by gut flora in the efficacy of HEE. Our study sought to explore the effects of HEE on non-alcoholic fatty liver disease (NAFLD) in normal flora and pseudo germ-free mice. The results showed that HEE effectively diminished hepatic lipid accumulation, ameliorated liver function, reduced inflammatory cytokine levels and blood lipid profiles, and regulated blood glucose levels. HEE facilitated triglyceride breakdown, suppressed fatty acid synthesis, and enhanced intestinal health by modulating the diversity of the gut microbiota and the production of short-chain fatty acids in the gut. In addition, HEE apparently helps to increase the presence of beneficial genera of bacteria, thereby influencing the composition of the gut microbiota, and the absence of gut flora affects the efficacy of HEE. These findings reveal the potential of hawthorn for the prevention and treatment of NAFLD and provide new perspectives on the study of functional plants to improve liver health.

Microbiota metabolism of intestinal amino acids impacts host nutrient homeostasis and physiology

The intestine and liver are thought to metabolize dietary nutrients and regulate host nutrient homeostasis. Here, we find that the gut microbiota also reshapes the host amino acid (aa) landscape via efficiently metabolizing intestinal aa. To identify the responsible microbes/genes, we developed a metabolomics-based assay to screen 104 commensals and identified candidates that efficiently utilize aa. Using genetics, we identified multiple responsible metabolic genes in phylogenetically diverse microbes. By colonizing germ-free mice with the wild-type strain and their isogenic mutant deficient in individual aa-metabolizing genes, we found that these genes regulate the availability of gut and circulatory aa. Notably, microbiota genes for branched-chain amino acids (BCAAs) and tryptophan metabolism indirectly affect host glucose homeostasis via peripheral serotonin. Collectively, at single-gene level, this work characterizes a microbiota-encoded metabolic activity that affects host nutrient homeostasis and provides a roadmap to interrogate microbiota-dependent activity to improve human health.

Single-cell transcriptomics unveiled that early life BDE-99 exposure reprogrammed the gut-liver axis to promote a proinflammatory metabolic signature in male mice at late adulthood.

Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants that bioaccumulate in the environment. The gut microbiome is an important regulator of liver functions including xenobiotic biotransformation and immune regulation. We recently showed that neonatal exposure to polybrominated diphenyl ether-99 (BDE-99), a human breast milk-enriched PBDE congener, up-regulated proinflammation-related and down-regulated drug metabolism-related genes predominantly in males in young adulthood. However, the persistence of this dysregulation into late adulthood, differential impact among hepatic cell types, and the involvement of the gut microbiome from neonatal BDE-99 exposure remain unknown. To address these knowledge gaps, male C57BL/6 mouse pups were orally exposed to corn oil (10 ml/kg) or BDE-99 (57 mg/kg) once daily from postnatal days 2-4. At 15 months of age, neonatal BDE-99 exposure down-regulated xenobiotic and lipid-metabolizing enzymes and up-regulated genes involved in microbial influx in hepatocytes. Neonatal BDE-99 exposure also increased the hepatic proportion of neutrophils and led to a predicted increase of macrophage migration inhibitory factor signaling. This was associated with decreased intestinal tight junction protein (Tjp) transcripts, altered gut environment, and dysregulation of inflammation-related metabolites. ScRNA-seq using germ-free (GF) mice demonstrated the necessity of a normal gut microbiome in maintaining hepatic immune tolerance. Microbiota transplant to GF mice using large intestinal microbiome from adults neonatally exposed to BDE-99 down-regulated Tjp transcripts and up-regulated several cytokines in large intestine. In conclusion, neonatal BDE-99 exposure reprogrammed cell type-specific gene expression and cell-cell communication in liver towards proinflammation, and this may be partly due to the dysregulated gut environment.

Capsaicin modulates Akkermansia muciniphila abundance by enhancing MUCIN2 levels in mice fed with high-fat diets.

Extensive research has been conducted to investigate the impact of capsaicin (CAP) on lipid metabolism, focusing specifically on its interaction with the vanilloid subtype 1 (TRPV1) ion channel. Additionally, studies have illuminated the role of Akkermansia muciniphila (A. muciniphila), a specific strain of intestinal microbiota, in lipid metabolism. In this study, a model utilizing resiniferatoxin (RTX) was employed to deactivate TRPV1 ion channels in germ-free mice, followed by the administration of A. muciniphila via gavage. Following the collection of intestinal tissues for a comprehensive analysis, employing histopathology, qPCR, and ELISA techniques, our findings revealed a significant upregulation of MUC2 and MUC3 expression induced by CAP. This upregulation resulted in the thickening of the colonic mucus layers. Notably, this effect was absent when TRPV1 was selectively inhibited. Moreover, there was no discernible impact on goblet cells. The findings strongly indicate that CAP influences the system by activating the TRPV1 ion channel, thereby enhancing the expression of mucin MUC2 and promoting an augmentation in the thickness of the mucous layer. This activation, in turn, supplies A. muciniphila with an ample source of carbon and nitrogen. This insight potentially clarify the underlying mechanism through which CAP facilitates the increase in A. muciniphila abundance.

A lipidome landscape of aging in mice.

Understanding the molecular mechanisms of aging is crucial for enhancing healthy longevity. We conducted untargeted lipidomics across 13 biological samples from mice at various life stages (2, 12, 19 and 24 months) to explore the potential link between aging and lipid metabolism, considering sex (male or female) and microbiome (specific pathogen-free or germ-free) dependencies. By analyzing 2,704 molecules from 109 lipid subclasses, we characterized common and tissue-specific lipidome alterations associated with aging. For example, the levels of bis(monoacylglycero)phosphate containing polyunsaturated fatty acids increased in various organs during aging, whereas the levels of other phospholipids containing saturated and monounsaturated fatty acids decreased. In addition, we discovered age-dependent sulfonolipid accumulation, absent in germ-free mice, correlating with Alistipes abundance determined by 16S ribosomal RNA gene amplicon sequencing. In the male kidney, glycolipids such as galactosylceramides, galabiosylceramides (Gal2Cer), trihexosylceramides (Hex3Cer), and mono- and digalactosyldiacylglycerols were detected, with two lipid classes-Gal2Cer and Hex3Cer-being significantly enriched in aged mice. Integrated analysis of the kidney transcriptome revealed uridine diphosphate galactosyltransferase 8A (UGT8a), alkylglycerone phosphate synthase and fatty acyl-coenzyme A reductase 1 as potential enzymes responsible for the male-specific glycolipid biosynthesis in vivo, which would be relevant to sex dependency in kidney diseases. Inhibiting UGT8 reduced the levels of these glycolipids and the expression of inflammatory cytokines in the kidney. Our study provides a valuable resource for clarifying potential links between lipid metabolism and aging.

Dermal injury drives a skin to gut axis that disrupts the intestinal microbiome and intestinal immune homeostasis in mice

The composition of the microbial community in the intestine may influence the functions of distant organs such as the brain, lung, and skin. These microbes can promote disease or have beneficial functions, leading to the hypothesis that microbes in the gut explain the co-occurrence of intestinal and skin diseases. Here, we show that the reverse can occur, and that skin directly alters the gut microbiome. Disruption of the dermis by skin wounding or the digestion of dermal hyaluronan results in increased expression in the colon of the host defense genes Reg3 and Muc2, and skin wounding changes the composition and behavior of intestinal bacteria. Enhanced expression Reg3 and Muc2 is induced in vitro by exposure to hyaluronan released by these skin interventions. The change in the colon microbiome after skin wounding is functionally important as these bacteria penetrate the intestinal epithelium and enhance colitis from dextran sodium sulfate (DSS) as seen by the ability to rescue skin associated DSS colitis with oral antibiotics, in germ-free mice, and fecal microbiome transplantation to unwounded mice from mice with skin wounds. These observations provide direct evidence of a skin-gut axis by demonstrating that damage to the skin disrupts homeostasis in intestinal host defense and alters the gut microbiome.

A Gnotobiotic Mouse Model with Divergent Equol-Producing Phenotypes: Potential for Determining Microbial-Driven Health Impacts of Soy Isoflavone Daidzein.

The implications of soy consumption on human health have been a subject of debate, largely due to the mixed evidence regarding its benefits and potential risks. The variability in responses to soy has been partly attributed to differences in the metabolism of soy isoflavones, compounds with structural similarities to estrogen. Approximately one-third of humans possess gut bacteria capable of converting soy isoflavone daidzein into equol, a metabolite produced exclusively by gut microbiota with significant estrogenic potency. In contrast, lab-raised rodents are efficient equol producers, except for those raised germ-free. This discrepancy raises concerns about the applicability of traditional rodent models to humans. Herein, we designed a gnotobiotic mouse model to differentiate between equol producers and non-producers by introducing synthetic bacterial communities with and without the equol-producing capacity into female and male germ-free mice. These gnotobiotic mice display equol-producing phenotypes consistent with the capacity of the gut microbiota received. Our findings confirm the model's efficacy in mimicking human equol production capacity, offering a promising tool for future studies to explore the relationship between endogenous equol production and health outcomes like cardiometabolic health and fertility. This approach aims to refine dietary guidelines by considering individual microbiome differences.

Longitudinal multi-omics analysis uncovers the altered landscape of gut microbiota and plasma metabolome in response to high altitude

BackgroundGut microbiota is significantly influenced by altitude. However, the dynamics of gut microbiota in relation to altitude remains undisclosed.MethodsIn this study, we investigated the microbiome profile of 610 healthy young men from three different places in China, grouped by altitude, duration of residence, and ethnicity. We conducted widely targeted metabolomic profiling and clinical testing to explore metabolic characteristics.ResultsOur findings revealed that as the Han individuals migrated from low altitude to high latitude, the gut microbiota gradually converged towards that of the Tibetan populations but reversed upon returning to lower altitude. Across different cohorts, we identified 51 species specifically enriched during acclimatization and 57 species enriched during deacclimatization to high altitude. Notably, Prevotella copri was found to be the most enriched taxon in both Tibetan and Han populations after ascending to high altitude. Furthermore, significant variations in host plasma metabolome and clinical indices at high altitude could be largely explained by changes in gut microbiota composition. Similar to Tibetans, 41 plasma metabolites, such as lactic acid, sphingosine-1-phosphate, taurine, and inositol, were significantly elevated in Han populations after ascending to high altitude. Germ-free animal experiments demonstrated that certain species, such as Escherichia coli and Klebsiella pneumoniae, which exhibited altitude-dependent variations in human populations, might play crucial roles in host purine metabolism.ConclusionsThis study provides insights into the dynamics of gut microbiota and host plasma metabolome with respect to altitude changes, indicating that their dynamics may have implications for host health at high altitude and contribute to host adaptation.88DwbBo5LkUszxYCpXiUg_Video

Subacute exposure to black phosphorus quantum dots induces cardiac fibrosis and the potential role of gut microbiota

Black phosphorus quantum dots (BPQDs) have been used as the nano-carrier in the field of biomedicine due to their excellent electronic conductivity, optical and thermoelectric properties. However, it is still lack of evaluation of the safety of BPQDs, specifically on the effects and mechanisms of BPQDs on heart. In this study, the specific pathogen-free male mice were orally administered with different doses of BPQDs (0.02, 0.1, 0.5 mg/kg) for 28 days. BPQDs exposure decreased the heart-body ratio and caused cardiac hypertrophy and fibrosis. Additionally, ferroptosis was observed in the cardiac tissue. The recent study has shown BPQDs oral exposure alter gut microbiota. Here, after exposure to 0.1 mg/kg BPQDs for 28 days, the germ-free mice showed neither cardiac injury nor ferroptosis. Taken together, the study demonstrates that BPQDs induce cardiac ferroptosis and fibrosis, possibly mediated by gut microbiota. This research may aid in enhancing understanding of the biosafety of BP nanomaterials and promoting the sustainable development of nanotechnology.

Microbial Indoles: Key Regulators of Organ Growth and Metabolic Function.

Gut microbes supporting body growth are known but the mechanisms are less well documented. Using the microbial tryptophan metabolite indole, known to regulate prokaryotic cell division and metabolic stress conditions, we mono-colonized germ-free (GF) mice with indole-producing wild-type Escherichia coli (E. coli) or tryptophanase-encoding tnaA knockout mutant indole-non-producing E. coli. Indole mutant E. coli mice showed multiorgan growth retardation and lower levels of glycogen, cholesterol, triglycerides, and glucose, resulting in an energy deficiency despite increased food intake. Detailed analysis revealed a malfunctioning intestine, enlarged cecum, and reduced numbers of enterochromaffin cells, correlating with a metabolic phenotype consisting of impaired gut motility, diminished digestion, and lower energy harvest. Furthermore, indole mutant mice displayed reduction in serum levels of tricarboxylic acid (TCA) cycle intermediates and lipids. In stark contrast, a massive increase in serum melatonin was observed-frequently associated with accelerated oxidative stress and mitochondrial dysfunction. This observational report discloses functional roles of microbe-derived indoles regulating multiple organ functions and extends our previous report of indole-linked regulation of adult neurogenesis. Since indoles decline by age, these results imply a correlation with age-linked organ decline and levels of indoles. Interestingly, increased levels of indole-3-acetic acid, a known indole metabolite, have been shown to correlate with younger biological age, further supporting a link between biological age and levels of microbe-derived indole metabolites. The results presented in this resource paper will be useful for the future design of food intervention studies to reduce accelerated age-linked organ decline.

The microbiota and T cells non-genetically modulate inherited phenotypes transgenerationally

The host-microbiota relationship has evolved to shape mammalian physiology, including immunity, metabolism, and development. Germ-free models are widely used to study microbial effects on host processes such as immunity. Here, we find that both germ-free and Tcell-deficient mice exhibit a robust sebum secretion defect persisting across multiple generations despite microbial colonization and Tcell repletion. These phenotypes are inherited by progeny conceived during invitro fertilization using germ-free sperm and eggs, demonstrating that non-genetic information in the gametes is required for microbial-dependent phenotypic transmission. Accordingly, gene expression in early embryos derived from gametes from germ-free or Tcell-deficient mice is strikingly and similarly altered. Our findings demonstrate that microbial- and immune-dependent regulation of non-genetic information in the gametes can transmit inherited phenotypes transgenerationally in mice. This mechanism could rapidly generate phenotypic diversity to enhance host adaptation to environmental perturbations.

Sedanolide alleviates DSS-induced colitis by modulating the intestinal FXR-SMPD3 pathway in mice

IntroductionInflammatory bowel disease (IBD) is a global disease with limited therapy. It is reported that sedanolide exerts anti-oxidative and anti-inflammatory effects as a natural phthalide, but its effects on IBD remain unclear. ObjectivesIn this study, we investigated the impacts of sedanolide on dextran sodium sulfate (DSS)-induced colitis in mice. MethodsThe mice were administered sedanolide or vehicle followed by DSS administration, after which colitis symptoms, inflammation levels, and intestinal barrier function were evaluated. Transcriptome analysis, 16S rRNA sequencing, and targeted metabolomics analysis of bile acids and lipids were performed. ResultsSedanolide protected mice from DSS-induced colitis, suppressed the inflammation, restored the weakened epithelial barrier, and modified the gut microbiota by decreasing bile salt hydrolase (BSH)-expressing bacteria. The downregulation of BSH activity by sedanolide increased the ratio of conjugated/unconjugated bile acids (BAs), thereby inhibiting the intestinal farnesoid X receptor (FXR) pathway. The roles of the FXR pathway and gut microbiota were verified using an intestinal FXR-specific agonist (fexaramine) and germ-free mice, respectively. Furthermore, we identified the key effector ceramide, which is regulated by sphingomyelin phosphodiesterase 3 (SMPD3). The protective effects of ceramide (d18:1/16:0) against inflammation and the gut barrier were demonstrated in vitro using the human cell line Caco-2. ConclusionSedanolide could reshape the intestinal flora and influence BA composition, thus inhibiting the FXR-SMPD3 pathway to stimulate the synthesis of ceramide, which ultimately alleviated DSS-induced colitis in mice. Overall, our research revealed the protective effects of sedanolide against DSS-induced colitis in mice, which indicated that sedanolide may be a clinical treatment for colitis. Additionally, the key lipid ceramide (d18:1/16:0) was shown to mediate the protective effects of sedanolide, providing new insight into the associations between colitis and lipid metabolites.

403 Epithelial hypoxia maintains colonization resistance against Candida albicans

OBJECTIVES/GOALS: Antibiotic treatment sets the stage for intestinal domination by Candida albicanswhich is necessary for development of invasive disease, but the resources driving this bloom remain poorly defined. We sought to determine these factors in order to design novel prophylaxis strategies for reducing gastrointestinal (GI) colonization. METHODS/STUDY POPULATION: We initially developed a generalizable framework, termed metabolic footprinting to determine the metabolites C. albicanspreferentially uses in the mouse GI tract. After identifying the metabolites C. albicansutilizes, we usedin vitro growth assays in the presence and absence of oxygen to validate out metabolomics findings. We next determined if a probiotic E. coli that utilizes oxygen would reduce C. albicanscolonization compared to a mutant E. coli that could not respire oxygen. Finding that oxygen was a necessary resource, we utilized germ-free mice to determine if Clostridiaspp. known to reduce GI oxygen would prevent C. albicanscolonization. Lastly, we sought to see if 5-aminosalicylic acid (5-ASA) could prevent C. albicanscolonization. RESULTS/ANTICIPATED RESULTS: We found that C. albicans preferentially utilizes simple carbohydrates including fructo-oligosaccharides (e.g., 1-kestose), disaccharides (e.g., β-gentiobiose), and alcoholic sugars (e.g., sorbitol) and is able to grow in vitro on minimal media supplemented with either of these nutrients. However, in the hypoxic environment that is found in the “healthy” colon, C. albicans cannot utilize these nutrients. We next found that pre-colonization in a mouse model with a probiotic E. coli significantly reduced C. albicanscolonization, but the mutant E. coli had no effect on colonization. We next showed that Clostridia supplementation restored GI hypoxia and reduced C. albicanscolonization. Remarkably, we found that 5-ASA significantly reduced GI colonization of C. albicans. DISCUSSION/SIGNIFICANCE: We have shown that C. albicans requires oxygen to colonize the GI tract. Importantly, we found that 5-ASA can prevent an antibiotic mediated bloom of C. albicans by restoring GI hypoxia, which warrants additional studies to determine if 5-ASA can be used as an adjunctive prophylactic treatment in high risk patients.

Colitis reduces active social engagement in mice and is ameliorated by supplementation with human microbiota members

Multiple neurological disorders are associated with gastrointestinal (GI) symptoms, including autism spectrum disorder (ASD). However, it is unclear whether GI distress itself can modify aspects of behavior. Here, we show that mice that experience repeated colitis have impaired active social engagement, as measured by interactions with a foreign mouse, even though signs of colitis were no longer present. We then tested the hypothesis that individuals with ASD harbor a microbiota that might differentially influence GI health by performing microbiota transplantation studies into male germfree animals, followed by induction of colitis. Animals that harbor a microbiota from ASD individuals have worsened gut phenotypes when compared to animals colonized with microbiotas from familial neurotypical (NT) controls. We identify the enrichment of Blautia species in all familial NT controls and observe an association between elevated abundance of Bacteroides uniformis and reductions in intestinal injury. Oral treatment with either of these microbes reduces colon injury in mice. Finally, provision of a Blautia isolate from a NT control ameliorates gut injury-associated active social engagement in mice. Collectively, our data demonstrate that past intestinal distress is associated with changes in active social behavior in mice that can be ameliorated by supplementation of members of the human microbiota.

Germ-free status but not subacute polychlorinated biphenyl (PCB) exposure altered hepatic phosphatidylcholine and ether-phosphatidylcholine levels in mice

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that pose a current ecosystem and human health concern. PCB exposure impacts the gut microbiome in animal models, suggesting a mechanistic link between PCB exposure and adverse health outcomes. The presence and absence of the microbiome and exposure to PCBs independently affect the lipid composition in the liver, which in turn affects the PCB disposition in target tissues, such as the liver. Here, we investigated microbiome × subacute PCB effects on the hepatic lipid composition of conventional and germ-free female mice exposed to 0, 6, or 30 mg/kg body weight of an environmental PCB mixture in sterile corn oil once daily for 3 consecutive days. Hepatic triacylglyceride and polar lipid levels were quantified using mass spectrometric methods following the subacute PCB exposure. The lipidomic analysis revealed no PCB effect on the hepatic levels. No microbiome effect was observed on levels of triacylglyceride and most polar lipid classes. The total hepatic levels of phosphatidylcholine (PC) and ether-phosphatidylcholine (ePC) lipids were lower in germ-free mice than the conventional mice from the same exposure group. Moreover, levels of several unsaturated PCs, such as PC(36:5) and PC(42:10), and ePCs, such as ePC(36:2) and ePC(4:2), were lower in germ-free than conventional female mice. Based on a KEGG pathway meta-analysis of RNA sequencing data, the ether lipid metabolism pathway is altered in the germ-free mouse liver. In contrast to the liver, extractable lipid levels, determined gravimetrically, differed in several tissues from naïve conventional vs. germ-free mice. Overall, microbiome × subacute PCB exposure effects on hepatic lipid composition are unlikely to affect PCB distribution into the mouse liver. Further studies are needed to assess how the different extractable lipid levels in other tissues alter PCB distribution in conventional vs. germ-free mice.