Cell surface polysaccharides of Bifidobacterium bifidum induce the generation of Foxp3+ regulatory T cells.

Dysregulation of intestinal microflora is linked to inflammatory disorders associated with compromised immunosuppressive functions of Foxp3+ T regulatory (Treg) cells. Although mucosa-associated commensal microbiota has been implicated in Treg generation, molecular identities of the "effector" components controlling this process remain largely unknown. Here, we have defined Bifidobacterium bifidum as a potent inducer of Foxp3+ Treg cells with diverse T cell receptor specificity to dietary antigens, commensal bacteria, and B. bifidum itself. Cell surface ß-glucan/galactan (CSGG) polysaccharides of B. bifidum were identified as key components responsible for Treg induction. CSGG efficiently recapitulated the activity of whole bacteria and acted via regulatory dendritic cells through a partially Toll-like receptor 2-mediated mechanism. Treg cells induced by B. bifidum or purified CSGG display stable and robust suppressive capacity toward experimental colitis. By identifying CSGG as a functional component of Treg-inducing bacteria, our studies highlight the immunomodulatory potential of CSGG and CSGG-producing microbes.

Linear α-(1 → 6)-d-glucan from Bifidobacterium bifidum BIM В-733D is low molecular mass biopolymer with unique immunochemical properties.

Role of microorganisms in induction of/protection from autoimmune diseases is proven though molecular mechanisms and bacterial/viral/yeast biopolymers responsible for these effects are in the research stage. Autoantobodies (AAbs) to thyroid peroxidase (anti-TPO) and thyroglobulin (anti-Tg) as well as AAbs to transglutaminase 2 (anti-TG2) and antibodies to gliadins (anti-gliadins) are serological markers of autoimmune thyroid disease and celiac disease, respectively, and players in pathogenesis of these autoimmune diseases. In current study, biopolymer of Bifidobacterium bifidum BIM В-733D that interacts selectively with anti-gliadins (Bb-Ganti-gliadins) was isolated by affinity chromatography with anti-gliadins, purified by size exclusion chromatography on TSK 40 gel and identified by NMR as linear α-(1 → 6)-d-glucan with molecular mass about 5000 Da. It was proven that compounds Bb-Ganti-gliadins and Bb-Ganti-TPO/Bb-Ganti-Tg isolated early from the same strain [Kiseleva, E. P. et al., Benef Microbes.2013, 4, 375 -391] are the same substance designated GBb. Its unique immunochemical property is the ability to interact selectively with anti-TPO, anti-Tg, anti-TG2 and anti-gliadins in presence of no less than 10-fold excess of total immunoglobulins of class G (tIgG), as it was proven by ELISA. Synthesis of GBb-bovine serum albumin (GBb-BSA) conjugate is an example of increasing the reliability and reproducibility of ELISA results by mediated immobilization of a polysaccharide covalently attached to a well-adsorbed protein. Taking into account that there are population of bispecific anti-gliadins (anti-gliadins and anti-TG2 simultaneously) we regard our data as first argument in favor of hypothesis that GBb differentiates between human AAbs per se and other human Ig (e.g. antibodies to antigens of infectious agents) due to its binding with a yet unidentified site which is present in the molecules of all AAbs (independently on their specificity) and absent in other human Igs.

Probiotics modulate gut microbiota and improve insulin sensitivity in DIO mice.

Obesity and type 2 diabetes are characterized by subclinical inflammatory process. Changes in composition or modulation of the gut microbiota may play an important role in the obesity-associated inflammatory process. In the current study, we evaluated the effects of probiotics (Lactobacillus rhamnosus, L. acidophilus and Bifidobacterium bifidumi) on gut microbiota, changes in permeability, and insulin sensitivity and signaling in high-fat diet and control animals. More importantly, we investigated the effects of these gut modulations on hypothalamic control of food intake, and insulin and leptin signaling. Swiss mice were submitted to a high-fat diet (HFD) with probiotics or pair-feeding for 5 weeks. Metagenome analyses were performed on DNA samples from mouse feces. Blood was drawn to determine levels of glucose, insulin, LPS, cytokines and GLP-1. Liver, muscle, ileum and hypothalamus tissue proteins were analyzed by Western blotting and real-time polymerase chain reaction. In addition, liver and adipose tissues were analyzed using histology and immunohistochemistry. The HFD induced huge alterations in gut microbiota accompanied by increased intestinal permeability, LPS translocation and systemic low-grade inflammation, resulting in decreased glucose tolerance and hyperphagic behavior. All these obesity-related features were reversed by changes in the gut microbiota profile induced by probiotics. Probiotics also induced an improvement in hypothalamic insulin and leptin resistance. Our data demonstrate that the intestinal microbiome is a key modulator of inflammatory and metabolic pathways in both peripheral and central tissues. These findings shed light on probiotics as an important tool to prevent and treat patients with obesity and insulin resistance.

The Role of Probiotics in Lipopolysaccharide-Induced Autophagy in Intestinal Epithelial Cells.

BACKGROUND/AIMS: Dysfunction of autophagy has been associated with loss of intestinal homeostasis. Lipopolysaccharide (LPS) from Gram-negative bacteria is known to be a major initiator of intestinal epithelial cell (IEC) autophagy. Although probiotics have been recognized to be involved in many therapeutic properties and participate in host defense responses, the molecular mechanisms by which probiotics exert these positive effects remain unknown. This study assessed the effect of probiotics on LPS-induced physical barrier dysfunction and the underlying mechanism of probiotic action in IECs with a focus on autophagy. METHODS: A LPS-induced autophagic model was established in rat IEC18 cells wherein cells were treated with culture medium supernatants of Bifidobacteria following LPS intervention at indicated times. Autophagosomes in IEC18 cells were visualized by confocal microscopy after transfection with a tandem GFP-mCherry-LC3 construct and also by transmission electron microscopy. Autophagy-associated protein levels were analyzed by western blot and transepithelial electrical resistance (TEER) was measured using an epithelial voltohmmeter. RESULTS: Probiotic treatment could effectively inhibit LPS-induced autophagy, as evidenced by the decreased ratio of microtubule-associated light chain 3 (LC3)-II/LC3-I, fewer autophagic vacuoles, and reduced punctate distribution of GFP-mCherry-LC3. In addition, probiotics prevented chloroquine (CQ) inhibition of autophagic flux and autophagolysosomal fusion as indicated by a failure to recruit LAMP1 and cathepsin D to lysosomes. Interestingly, ATG16L1 knockdown did not inhibit the effect of probiotics on LPS-induced autophagy. Furthermore, the diminished barrier function could be prevented by probiotics. CONCLUSIONS: We provide evidence that autophagy mediation by probiotics may be involved in enteroprotection against LPS-induced intestinal epithelial toxicity, and could serve as a novel mechanism through which probiotics promote and maintain gut homeostasis.

Bifidobacterium bifidum R0071 decreases stress-associated diarrhoea-related symptoms and self-reported stress: a secondary analysis of a randomised trial.

Psychological stress is associated with gastrointestinal (GI) distress. This secondary analysis from a randomised, double-blind, placebo-controlled study examined whether three different probiotics could normalise self-reported stress-associated GI discomfort and reduce overall self-reported stress. Undergraduate students (n=581) received Lactobacillus helveticus R0052, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium bifidum R0071, or placebo. Participants self-reported 2 outcomes for a 6-week period, which included final academic exams: daily level of stress (0=no stress to 10=extremely stressed) and weekly three diarrhoea-related symptoms (DS, 1=no discomfort to 7=severe discomfort) using the GI Symptom Rating Scale. Self-reported stress was positively related to DS (P=0.0068). Mean DS scores were lower with B. bifidum versus placebo at week 2 at the average level of stress and the average body mass index (BMI). DS scores were lower with B. bifidum at week 5 versus week 0 and 1 and with B. infantis R0033 at week 6 versus week 0. DS scores were higher when antibiotics were used in the prior week with placebo (P=0.0092). DS were not different with or without antibiotic use with the probiotics. Only B. bifidum had an effect on self-reported stress scores (P=0.0086). The self-reported stress score was also dependent on hours of sleep per day where it decreased by 0.13 for each additional hour of sleep. During a stressful period, B. bifidum R0071 decreases DS and self-reported stress scores. This trial was registered at clinicaltrials.gov as NCT01709825.

Probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum modulates immunoglobulin levels and cytokines expression in whey proteins sensitised mice.

BACKGROUND: Cow milk allergy is the most common food allergy in children. So far, no effective treatment is available to prevent or cure food allergy. This study investigated whether orally administrated probiotics could suppress sensitisation in whey proteins (WP)-induced allergy mouse model. Two types of probiotic Dahi were prepared by co-culturing Dahi bacteria (Lactococcus lactis ssp. cremoris NCDC-86 and Lactococcus lactis ssp. lactis biovar diacetylactis NCDC-60) along with selected strain of Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3. Mice were fed with probiotic Dahi (La-Dahi and LaBb-Dahi) from 7 days before sensitisation with WP, respectively, in addition to milk protein-free basal diet, and control group received no supplements. RESULTS: Feeding of probiotic Dahi suppressed the elevation of whey proteins-specific IgE and IgG response of WP-sensitised mice. In addition, sIgA levels were significantly (P < 0.001) increased in intestinal fluid collected from mice fed with La-Dahi. Production of T helper (Th)-1 cell-specific cytokines, i.e. interferon-γ (IFN-γ), interleukin (IL)-12, and IL-10 increased, while Th2-specific cytokines, i.e. IL-4 decreased in the supernatant of cultured splenocytes collected from mice fed with probiotic Dahi as compared to the other groups. Moreover, the splenic mRNA levels of IFN-γ, interleukin-10 were found to be significantly increased, while that of IL-4 decreased significantly in La-Dahi groups, as compared to control groups. CONCLUSION: Results of the present study indicate that probiotic Dahi skewed Th2-specific immune response towards Th1-specific response and suppressed IgE in serum. Collectively, this study shows the potential use of probiotics intervention in reducing the allergic response to whey proteins in mice. © 2015 Society of Chemical Industry.