Probiotic treatment with viable α-galactosylceramide-producing Bacteroides fragilis reduces diabetes incidence in female nonobese diabetic mice.

BACKGROUND: We aimed to investigate whether alpha-galactosylceramide (α-GalCer)-producing Bacteroides fragilis could induce natural killer T (NKT) cells in nonobese diabetic (NOD) mice and reduce their diabetes incidence. METHODS: Five-week-old female NOD mice were treated orally with B. fragilis, and islet pathology and diabetes onset were monitored. Immune responses were analyzed by flow cytometry and multiplex technology. Effects of ultraviolet (UV)-killed α-GalCer-producing B. fragilis and their culture medium on invariant NKT (iNKT) cells were tested ex vivo on murine splenocytes, and the immunosuppressive capacity of splenocytes from B. fragilis-treated NOD mice were tested by adoptive transfer to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. RESULTS: B. fragilis reduced the diabetes incidence from 69% to 33% and the percent of islets with insulitis from 40% to 7%, which doubled the serum insulin level compared with the vehicle-treated control mice. Furthermore, the early treatment reduced proinflammatory mediators in the serum, whereas the proportion of CD4+ NKT cell population was increased by 33%. B. fragilis growth media stimulated iNKT cells and anti-inflammatory M2 macrophages ex vivo in contrast to UV-killed bacteria, which had no effect, strongly indicating an α-GalCer-mediated effect. Adoptive transfer of splenocytes from B. fragilis-treated NOD mice induced a similar diabetes incidence as splenocytes from untreated NOD mice. CONCLUSIONS: B. fragilis induced iNKT cells and M2 macrophages and reduced type 1 diabetes in NOD mice. The protective effect seemed to be more centered on gut-pancreas interactions rather than a systemic immunosuppression. B. fragilis should be considered for probiotic use in individuals at risk of developing type 1 diabetes.

Inulin Supplementation Modulates the Hepatic Transcriptome, Metabolome, and Ferritin Content in Ovariectomized Rats.

SCOPE: Liver is an important metabolic organ regulating whole-body homeostasis. This study aims to investigate how prebiotic-induced changes in the metabolic activity of the gut microbiome (GM) and dietary calcium depletion modulates the hepatic metabolome and transcriptome. METHODS AND RESULTS: The serum metabolome, liver metabolome, and transcriptome are determined on samples from ovariectomized (OVX) rats fed a control diet (Control, n = 7), a control diet supplemented with 5% w/w inulin (Inulin, n = 7), or a calcium-deficient diet (CaDef, n = 7). Inulin fortification is associated with higher serum concentrations of acetate, 3-hydroxybutyrate, and reduced concentration of dimethyl sulfone, revealing that changes in the metabolic activity of the GM are reflected in circulating metabolites. Metabolomics also reveal that the inulin-fortified diet results in lower concentrations of hepatic glutamate, serine, and hypoxanthine while transcriptomics reveal accompanying effects on the hepatic expression of ferric iron binding-related genes. Inulin fortification also induces effects on the hepatic expression of genes involved in olfactory transduction, suggesting that prebiotics regulate liver function through yet unidentified mechanisms involving olfactory receptors. CONCLUSION: Inulin ingestion impacts hepatic gene expression and is associated with an upregulation of ferritin synthesis-related genes and liver ferritin content.

Yogurt Benefits Bone Mineralization in Ovariectomized Rats with Concomitant Modulation of the Gut Microbiome.

SCOPE: Evidence supports that gut-modulating foods potentially can suppress bone loss in postmenopausal women. This study aims to investigate the effect of milk calcium-enriched milk, yogurt, and yogurt-inulin combination on the gut-bone association. METHODS AND RESULTS: A 6-week intervention study is conducted in ovariectomized rats. Four pastes containing milk calcium-fortified milk (M-Ca), milk calcium-fortified yogurt (Y-Ca), inulin-fortified Y-Ca (Y-I-Ca), or an isoconcentration of calcium carbonate (Ca-N), and a calcium-deficient paste are provided. M-Ca does not influence bone mineral density and content (BMD and BMC), femur mechanical strength, or femoral microstructure compared to Ca-N, but Y-Ca increases spine BMD. The serum metabolome reveals that Y-Ca modulated glycine-related pathways with reduced glycine, serine, and threonine. No additive effects of yogurt and inulin are found on bone parameters. Correlation analysis shows that increased lactobacilli and reduced Clostridiaceae members in Y-Ca is associated with an increased spine BMD. Increases in Bifidobacterium pseudolongum, Turicibacter, Blautia, and Allobaculum and gut short-chain fatty acids in Y-I-Ca are not reflected in bone parameters. CONCLUSION: Yogurt as calcium vehicle contributes to increased spine BMD concomitant with changes in the gut microbiome and glycine-related pathways, while adding inulin to yogurt does not affect bone mineralization in ovariectomized rats.

Effects of Calcium Source, Inulin, and Lactose on Gut-Bone Associations in an Ovarierectomized Rat Model.

SCOPE: Osteoporosis poses a health challenge especially for postmenopausal women. This study aims to explore nutritional strategies to counteract bone demineralization in ovarierectomized (OVX) rats. METHODS AND RESULTS: OVX rats (n = 49) are fed with one of six different diets, where two different calcium sources (dairy calcium or calcium carbonate) are provided alone or in combination with either inulin (5%) or lactose (0.5%). In addition, a calcium-deficient diet is included. Calcium supplementation increases intestinal concentrations of short-chain fatty acids (SCFAs) and the abundance of fecal Acinetobacter and Propionibacterium. Accompanied with these effects, rats fed with calcium-fortified diets have higher bone mineral density, bone mineral content and femur mechanical strength, lower serum levels of bone markers, and lower expression of calcium absorption-related genes (transient receptor potential vanilloid type 6 (TRPV6), calcium-binding protein (CaBP) compared with control. Inulin supplementation results in a markedly increased production of intestinal SCFAs, a decreased intestinal pH, an increased abundance of Allobaculum and Bifidobacterium, and an increased expression of Trpv6. Inulin and lactose show beneficial effects on spine bone. CONCLUSION: Calcium modulates gut microbiome composition and function. A pronounced effect of inulin on metabolic activity in the gastrointestinal tract is evident, and lactose supplementation decreases jejunal pH that might be associated with slightly enhanced bone mineralization.

Gluten-free diet reduces autoimmune diabetes mellitus in mice across multiple generations in a microbiota-independent manner.

Experimental and clinical data suggest that a gluten-free diet attenuates the development of type 1 diabetes. A gluten-free diet changes the gut microbiota composition, and such microbial changes are expected to reduce the autoimmune responses. However, in experiments with laboratory mice, a gluten-free diet changes the gut microbiota differently under varying experimental settings, questioning the specific role of the gut microbes. Here we show that a maternal gluten-free diet until weaning of their pups, delayed type 1 diabetes in both dams (parent generation) and offspring (F1 generation) of untreated non-obese diabetic (NOD) mice and in mice treated with a full cocktail of antibiotics that eradicates most of the existing microbiota. Breeding a second (F2) generation of NOD mice, never exposed to the gluten-free diet or the associated microbial changes, also demonstrated a preventative effect on type 1 diabetes even though their parents (the F1 generation) had only been on a gluten-free diet very early in life. Collectively, the experimental data, thus, points towards microbiota-independent dietary protection. Furthermore, both the perinatal gluten-free diet and antibiotic treatment reduced inflammation in the salivary glands and improved glucose challenged beta cell function in the F1 offspring. However, in contrast to the autoimmune response in the pancreas, those changes appeared to be microbiota dependent, as they were missing in the antibiotic treated mice, and do, therefore, not seem to be related to the preventative effect on type 1 diabetes. Interestingly, adoptive transfer of splenocytes from gluten-free fed mice protected NOD.SCID mice from developing diabetes, demonstrating that the anti-diabetic effect of a gluten-free diet was based on early life changes in the evolving immune system. In particular, genes involved in regulation of lymphocyte activation, proliferation, and cell adhesion were highly expressed in the spleen in gluten-free fed mice at weaning compared to control fed mice of the F1 generation, which suggested that gluten promotes autoimmunity by inhibiting immune regulation, though the involvement of the specific genes needs further investigation. In conclusion, gluten-free diet reduces autoimmune inflammation in salivary glands and pancreas in NOD mice in a microbiota-dependent and -independent manner respectively, and has preventative effect on type 1 diabetes by modulating the systemic immune system.

Dual nuclear magnetic resonance for probing intrinsic bone structure and a potential gut-bone axis in ovariectomized rats.

Currently, the existence of a gut-bone axis receives massive attention, and while sound premises and indirect proofs exist for the gut-bone axis concept, few studies have provided actual data linking the gut and bone physically. This study aimed to exploit the versatile nature of nuclear magnetic resonance (NMR) to link NMR relaxometry data on bone mineralization with NMR spectroscopic profiling of gut metabolites. For this purpose, sample material was obtained from a 6-week intervention study with ovariectomized (OVX) rats (n = 49) fed with seven different diets varying in calcium content (0.2-6.0 mg/kg) and prebiotic fiber content (0-5.0% w/w). This design ensured a span in (i) calcium available for bone mineralization and (ii) metabolic activity in the gut. After termination of the intervention, longitudinal (T1 ), transverse (T2 ) relaxation, and mechanical bone strength were measured on the excised femur bones. A PLS model with high predictability (Q2 = 0.86, R2 = 0.997) was demonstrated between T2 decay curves and femur mechanical strength. Correlations were established between bone T2 populations and gut short-chain fatty acids. In conclusion, the present dual NMR approach showed strong correlation between T2 relaxation and mechanical strength of the bone, and when metabolic activity in the gut was modulated by inulin, the potential existence of a gut-bone axis was demonstrated.

Delayed Gut Colonization Shapes Future Allergic Responses in a Murine Model of Atopic Dermatitis.

Epidemiological studies have long reported that perturbations of the childhood microbiome increase the risk of developing allergies, but a causal relationship with atopic dermatitis remains unclear. Here we colonized germ-free mice at birth or at one or eight week-of-age to investigate the role of prenatal and early postnatal microbial exposure on development of oxozolone-induced dermatitis later in life. We demonstrate that only one week delayed microbial colonization increased IgE levels and the total histological score of the inflamed ear compared to mice colonized throughout life. In parallel, several pro-inflammatory cytokines and chemokines were upregulated in the ear tissue demonstrating an enhanced immunological response following delayed postnatal colonization of the gut. In contrast, sensitivity to oxazolone-induced dermatitis was unaffected by the presence of a maternal microbiota during gestation. Mice colonized at eight week-of-age failed to colonize Rikenellaceae, a group of bacteria previously associated with a high-responding phenotype, and did not develop an immunological response to the same extent as the early colonized mice despite pronounced histopathological manifestations. The study provides proof-of-principle that the first intestinal colonizers of mice pups are crucial for the development of oxazolone-induced dermatitis later in life, and that the status of the maternal microbiota during pregnancy has no influence on the offspring's allergic immune response. This highlights an important window of opportunity following birth for microbiota-mediated interventions to prevent atopic responses later in life. How long such a window is open may vary between mice and humans considering species differences in the ontogeny of the immune system.

Dietary prebiotics promote intestinal Prevotella in association with a low-responding phenotype in a murine oxazolone-induced model of atopic dermatitis.

Atopic dermatitis is a chronic eczema commonly observed among children in Western countries. The gut microbiota is a significant factor in the pathogenesis, and ways to promote intestinal colonizers with anti-inflammatory capabilities are therefore favorable. The present study addressed the effects of a prebiotic, xylooligosaccharide (XOS), on the gut microbiota and ear inflammation in an oxazolone-induced dermatitis model in BALB/c mice. Mice were fed a XOS supplemented or a control diet throughout the experiment. Ear thickness and clinical skin inflammation were scored blindly after three weeks topical challenge with 0.4% oxazolone. The mice were divided into high and low responders to oxazolone-induced dermatitis based on clinical inflammation and histological evaluation of ear biopsies, and significantly fewer high responders were present in the XOS fed group. In addition, XOS fed mice had higher abundance of Prevotella spp. in their gut microbiota compared to the control fed mice. Serum IgE and ear tissue cytokine levels correlated significantly with the clinical scores, and with the abundance of Prevotella spp. The strong association between the low-responding phenotype and high abundance of Prevotella spp., indicates an alleviating effect of this intestinal colonizer in allergic sensitization. Prevotella should be considered as a relevant target for future microbiota-directed treatment strategies in atopic patients.

Betamethasone Treatment for Atopic Dermatitis in Gut Microbiota Transplanted Mice.

Gut microbiota composition correlates strongly with essential disease parameters in the oxazolone-induced mouse model for atopic dermatitis. The phenotype of this model can be transferred to germ-free mice with a gut microbiota transplant to achieve high and low responding mice. Therefore, the production of high responding mice through gut microbiota transplantation may be seen as a tool to reduce group sizes or increase power in intervention studies by increasing effect size. We sought to determine whether high responding mice respond to a common treatment in the same way as low responding mice. We hypothesized that while high responding mice would exhibit a higher clinical score than low responding mice before treatment, the clinical parameters would be similar in both groups after betamethasone treatment. Dermatitis was induced with oxazolone in barrier bred Swiss Webster mice, and a high responding and a low responding donor was selected based upon clinical and pathologic scores, as confirmed by monitoring a range of ear tissue cytokines. Feces from these donors were transplanted to pregnant germ-free Swiss Webster dams, and subsequently to their offspring. Although the overall effect of betamethasone on the clinical dermatitis score and ear thickness was rather small, the high responding recipients had significantly higher clinical dermatitis score and ear thickness than the low responding recipients before treatment, and these differences vanished after betamethasone treatment. We conclude that high responding recipients can be treated to a clinical level comparable with the low responding recipients.

Targeting gut microbiota and barrier function with prebiotics to alleviate autoimmune manifestations in NOD mice.

AIMS/HYPOTHESIS: Adopting a diet containing indigestible fibre compounds such as prebiotics to fuel advantageous bacteria has proven beneficial for alleviating inflammation. The effect of the microbial changes on autoimmunity, however, remains unknown. We studied the effects of prebiotic xylooligosaccharides (XOS) on pancreatic islet and salivary gland inflammation in NOD mice and tested whether these were mediated by the gut microbiota. METHODS: Mother and offspring mice were fed an XOS-supplemented diet until diabetes onset or weaning and were compared with a control-fed group. Diabetes incidence was monitored, insulitis and sialadenitis were scored in histological sections from adult mice, and several metabolic and immune variables were analysed in mice before the development of diabetes. Gut barrier function was assessed using an in vivo FITC-dextran permeability test. The importance of XOS-mediated gut microbial changes were evaluated in antibiotic-treated mice fed either XOS or control diet or given a faecal microbiota transplant from test animals. RESULTS: Diabetes onset was delayed in the XOS-fed mice, which also had fewer cellular infiltrations in their pancreatic islets and salivary glands. Interestingly, insulitis was most reduced in the XOS-fed groups when the mice were also treated with an antibiotic cocktail. There was no difference in sialadenitis between the dietary groups treated with antibiotics; the mice were protected by microbiota depletion regardless of diet. Faecal microbiota transplantation was not able to transfer protection. No major differences in glucose-insulin regulation, glucagon-like peptide-1, or short-chain fatty acid production were related to the XOS diet. The XOS diet did, however, reduce gut permeability markers in the small and large intestine. This was accompanied by a more anti-inflammatory environment locally and systemically, dominated by a shift from M1 to M2 macrophages, a higher abundance of activated regulatory T cells, and lower levels of induction of natural killer T cells and cytotoxic T cells. CONCLUSIONS/INTERPRETATION: Prebiotic XOS have microbiota-dependent effects on salivary gland inflammation and microbiota-independent effects on pancreatic islet pathology that are accompanied by an improved gut barrier that seems able to heighten control of intestinal diabetogenic antigens that have the potential to penetrate the mucosa to activate autoreactive immune responses.