The impact of metabolites derived from the gut microbiota on immune regulation and diseases.

The gut microbiota strongly impacts the physiology and pathology in the host. To understand the complex interactions between host and gut microbiota, an 'integrated omics' approach has been employed, where exhaustive analyses for the different layers of cellular functions, such as epigenomics, transcriptomics and metabolomics, in addition to metagenomics, are combined. With this approach, the mechanisms whereby short-chain fatty acids (SCFAs) regulate host defense and the immune system have been elucidated. In a gnotobiotic mouse model of enterohemorrhagic Escherichia coli infection, Bifidobacterium-derived acetate can protect from infection-mediated death by changing the gene expression profile of colonic epithelial cells. It has also been shown that gut microbiota-derived butyrate enhances colonic regulatory T-cell differentiation through its epigenetic modulatory ability via histone deacetylase inhibition. SCFAs are involved in many other immunomodulatory effects as well as host pathophysiological conditions. Dysbiosis in the gut has been implicated in the pathogenesis of many diseases. Although the causal relationship of gut microbial dysbiosis and/or metabolites with pathogenesis is mostly unknown, mechanistic insights have been elucidated in some cases. Metabolism in the gut microbiota and host liver produces trimethylamine N-oxide, which is known to aggravate atherosclerosis, and a secondary bile acid deoxycholate, which reportedly induces non-alcoholic steatohepatitis-related hepatocellular carcinoma. It has been reported that secondary bile acids could also induce the differentiation of peripherally derived regulatory T cells in the gut. Further studies on the interactions between the host and gut microbiota could lead to the development of new therapeutic strategies as well as in preventive medicine.

Gut microbiota of children with atopic dermatitis: Controlled study in the metropolitan region of São Paulo, Brazil

BACKGROUND: It is possible that imbalances in the composition of the gut microbiota or the relationship of the microbiota with the host may be implicated in the origin of allergy. Therefore, we studied the intestinal microbiota of children with atopic dermatitis (AD). METHODS: Cross-sectional study with 81 children aged 5-11; 23 with AD and 58 controls. Surveys were conducted to obtain demographic, socioeconomic and neonatal data. Diagnosis of AD was made based on the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. Eubacteria, Bacteroidetes, Firmicutes, B. fragilis, E. coli, Lactobacillus spp., S. aureus, E. faecalis, Salmonella spp., M. smithii, Bifidobacterium spp., C. difficile and C. perfringens were quantified using real-time PCR. RESULTS: The analysis showed an association between presence of C. difficile (OR: 5.88; 95 % CI: 1.24; 27.98), greater abundance of bifidobacteria (OR: 11.09; 95 % CI: 2.14; 57.39) and a lower abundance of lactobacilli (OR: 0.07; 95 % CI: 0.01; 0.51) in the gut microbiota of children with AD. Counts of Eubacteria (0,05 × 103 and 8.49 × 103), B. fragilis (0.72 × 109 and 4.5 × 109), Lactobacillus spp. (0.02 × 108 and 0.38 × 108), E. coli (0.13 × 109 and 1.52 × 109) and M. smithii (0.02×108 and 0.31 × 108) were lower in children with AD (P < 0.05). CONCLUSIONS: This study confirmed that children living in the metropolitan area of São Paulo (Brazil) with AD have a different microbiota pattern with higher prevalence of C. difficile, lower abundance of Lactobacillus and greater abundance of bifidobacteria, regardless of socioeconomic status

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Bifidobacterial Dialogue With Its Human Host and Consequent Modulation of the Immune System.

Since bifidobacteria are among the pioneering colonizers of the human infant gut, their interaction with their host is believed to start soon following birth. Several members of the Bifidobacterium genus are purported to exert various health-promoting effects at local and systemic levels, e.g., limiting pathogen colonization/invasion, influencing gut homeostasis, and influencing the immune system through changes in innate and/or adaptive immune responses. This has promoted extensive research efforts to shed light on the precise mechanisms by which bifidobacteria are able to stimulate and interact with the host immune system. These studies uncovered a variety of secreted or surface-associated molecules that act as essential mediators for the establishment of a bifidobacteria-host immune system dialogue, and that allow interactions with mucosa-associated immune cells. Additionally, the by-products generated from bifidobacterial carbohydrate metabolism act as vectors that directly and indirectly trigger the host immune response, the latter by stimulating growth of other commensal microorganisms such as propionate- or butyrate-producing bacteria. This review is aimed to provide a comprehensive overview on the wide variety of strategies employed by bifidobacteria to engage with the host immune system.

Low Interleukin 10 Production at Birth Is a Risk Factor for Atopic Dermatitis in Neonates with Bifidobacterium Colonization.

BACKGROUND: Altered regulatory immune responses to microbial stimuli and intestinal colonization of beneficial bacteria early in life may contribute to the development of allergic diseases (e.g., atopic dermatitis [AD]). However, few reports have investigated these factors simultaneously. The purpose of this study was to analyze neonatal immune responses to microbial stimuli as well as intestinal colonization of beneficial bacteria, in relation to the development of AD in a birth cohort. METHODS: Pregnant women were recruited, and their infants were followed up until 7 months of age. Levels of interleukin (IL)-10 released from cord-blood mononuclear cells (CBMCs) stimulated with heat-killed gram-positive bacteria (Bifidobacterium bifidum and Lactobacillus rhamnosus GG) and Lactobacillus-derived peptidoglycan were measured. Fecal Bifidobacterium counts at 4 days and 1 month were quantified using real-time polymerase chain reaction. The development of AD was determined by means of a questionnaire at 7 months of age. RESULTS: The levels of released IL-10 were significantly lower in infants with AD (n = 17) than in infants without AD (n = 53) for all stimuli. In infants with fecal Bifidobacterium, the incidence of AD was inversely associated with the release of IL-10 from cord blood mononuclear cells. CONCLUSION: Our findings suggest that impaired IL-10 production in response to microbial stimuli at birth may be associated with an increased risk of developing infantile AD, even in infants with early colonization of intestinal bifidobacteria.

Reactivity toward Bifidobacterium longum and Enterococcus hirae demonstrate robust CD8+ T cell response and better prognosis in HBV-related hepatocellular carcinoma.

Recent studies suggest that several bacterial species are involved in tumor immunosurveillance and antitumor immunity. The role of bacteria in immune responses in HBV-related hepatocellular carcinoma (HCC) patients is still unknown. In this study, we examined the bacteria-reactive CD8+ T cell response in patients with HBV-related HCC. We found that circulating CD8+ T cells from healthy individuals demonstrated minimal or zero specificity toward a series of commensals and bacteria previously associated with antitumor effects, including Escherichia coli, Enterococcus faecium, Bifidobacterium longum, Bacteroides fragilis, and Enterococcus hirae. In contrast, the circulating CD8+ T cells from HBV-related HCC patients presented significantly elevated bacteria-reactive responses, albeit with high variations among different HCC individuals. Reactivity toward bacteria was also identified in tumor-infiltrating CD8+ T cells. These bacteria-reactive responses were not primarily induced by TLR ligand, but were dependent on the presence of antigen-presenting monocytes, and were MHC class I-restricted. Interestingly, we observed that the CD8+ T cell-to-Foxp3+ regulatory T cell ratio was positively correlated with the proportions of Bifidobacterium longum-reactive and Enterococcus hirae-reactive CD8+ T cells, while the frequency of PD-1+ CD8+ T cells was negatively correlated with the frequency of Enterococcus hirae-reactive CD8+ T cells. Furthermore, the disease-free survival time of HCC patients after tumor resection was positively correlated with the frequencies of Bifidobacterium longum-reactive and Enterococcus hirae-reactive CD8+ T cells. Together, these results suggested that certain bacterial species might present valuable antitumor effects.

The Surface-Associated Exopolysaccharide of Bifidobacterium longum 35624 Plays an Essential Role in Dampening Host Proinflammatory Responses and Repressing Local TH17 Responses.

The immune-modulating properties of certain bifidobacterial strains, such as Bifidobacterium longum subsp. longum 35624 (B. longum 35624), have been well described, although the strain-specific molecular characteristics associated with such immune-regulatory activity are not well defined. It has previously been demonstrated that B. longum 35624 produces a cell surface exopolysaccharide (sEPS), and in this study, we investigated the role played by this exopolysaccharide in influencing the host immune response. B. longum 35624 induced relatively low levels of cytokine secretion from human dendritic cells, whereas an isogenic exopolysaccharide-negative mutant derivative (termed sEPSneg) induced vastly more cytokines, including interleukin-17 (IL-17), and this response was reversed when exopolysaccharide production was restored in sEPSneg by genetic complementation. Administration of B. longum 35624 to mice of the T cell transfer colitis model prevented disease symptoms, whereas sEPSneg did not protect against the development of colitis, with associated enhanced recruitment of IL-17+ lymphocytes to the gut. Moreover, intranasal administration of sEPSneg also resulted in enhanced recruitment of IL-17+ lymphocytes to the murine lung. These data demonstrate that the particular exopolysaccharide produced by B. longum 35624 plays an essential role in dampening proinflammatory host responses to the strain and that loss of exopolysaccharide production results in the induction of local TH17 responses. IMPORTANCE: Particular gut commensals, such as B. longum 35624, are known to contribute positively to the development of mucosal immune cells, resulting in protection from inflammatory diseases. However, the molecular basis and mechanisms for these commensal-host interactions are poorly described. In this report, an exopolysaccharide was shown to be decisive in influencing the immune response to the bacterium. We generated an isogenic mutant unable to produce exopolysaccharide and observed that this mutation caused a dramatic change in the response of human immune cells in vitro In addition, the use of mouse models confirmed that lack of exopolysaccharide production induces inflammatory responses to the bacterium. These results implicate the surface-associated exopolysaccharide of the B. longum 35624 cell envelope in the prevention of aberrant inflammatory responses.

Impaired responses to gliadin and gut microbes of immune cells from mice with altered stress-related behavior and premature immune senescence.

Stress is associated with impaired communication between the nervous and immune systems leading to immunosenescence and increased disease risk. We investigated whether leukocytes from mice with altered stress-related behavior and premature immunosenescence, as well as from chronologically aged mice differently responded ex vivo to celiac disease (CD) triggers (gliadin) and intestinal bacteria by ELISA and flow cytometry and differed in microbiota composition. We found that altered stress-related behavior and premature immunosenescence led to alterations in T lymphocytes and cytokine release of immune cells basally and in response to peptic fragments of gliadin and commensal and pathogenic bacteria, possibly increasing susceptibility to CD in adulthood.

Probiotic Bifidobacterium breve induces IL-10-producing Tr1 cells in the colon.

Specific intestinal microbiota has been shown to induce Foxp3(+) regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103(+) dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103(+) DCs from Il10(-/-), Tlr2(-/-), and Myd88(-/-) mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103(+) DCs failed to induce IL-10 production from co-cultured Il27ra(-/-) T cells. B. breve treatment of Tlr2(-/-) mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103(+) DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4(+) T cells from wild-type mice, but not Il10(-/-) mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.

Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.

Bifidobacteria comprise a significant proportion of the human gut microbiota. Several bifidobacterial strains are currently used as therapeutic interventions, claiming various health benefits by acting as probiotics. However, the precise mechanisms by which they maintain habitation within their host and consequently provide these benefits are not fully understood. Here we show that Bifidobacterium breve UCC2003 produces a cell surface-associated exopolysaccharide (EPS), the biosynthesis of which is directed by either half of a bidirectional gene cluster, thus leading to production of one of two possible EPSs. Alternate transcription of the two opposing halves of this cluster appears to be the result of promoter reorientation. Surface EPS provided stress tolerance and promoted in vivo persistence, but not initial colonization. Marked differences were observed in host immune response: strains producing surface EPS (EPS(+)) failed to elicit a strong immune response compared with EPS-deficient variants. Specifically, EPS production was shown to be linked to the evasion of adaptive B-cell responses. Furthermore, presence of EPS(+) B. breve reduced colonization levels of the gut pathogen Citrobacter rodentium. Our data thus assigns a pivotal and beneficial role for EPS in modulating various aspects of bifidobacterial-host interaction, including the ability of commensal bacteria to remain immunologically silent and in turn provide pathogen protection. This finding enforces the probiotic concept and provides mechanistic insights into health-promoting benefits for both animal and human hosts.

Differential regulation of Toll-like receptor signalling in spleen and Peyer's patch dendritic cells.

Toll-like receptor (TLR) signalling shapes dendritic cell (DC) responses by inducing co-stimulatory molecule up-regulation and cytokine secretion while TLR regulatory proteins inhibit this process. We aimed to determine if gene expression of TLRs and TLR regulatory proteins underpins the functionally different lipopolysaccharide (LPS) responses of DCs from murine Peyer's patches (PP) and spleen and of murine bacteria-conditioned bone-marrow-derived cells. Isolated spleen and PP DCs were analysed for basal expression of TLRs by flow cytometry and real time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The DCs were stimulated with LPS to determine cytokine secretion by enzyme-linked immunosorbent assay and expression of TLR regulatory proteins by qRT-PCR. In vitro results were confirmed following in vivo intraperitoneal LPS injection. In addition, changes in gene expression of TLR regulatory proteins were assessed in bacteria-conditioned bone-marrow-derived cells. Results indicated that surface expression of TLR2 and TLR4 on PP DCs was decreased compared with spleen DCs. The PP DCs secreted a limited profile of cytokines compared with spleen DCs following LPS stimulation. In vivo LPS exposure up-regulated sigirr, tollip and tmed1 messenger RNA in PP DCs, but not spleen DCs. Similar gene expression changes were observed in bacteria-conditioned bone-marrow-derived cells. Therefore, functionally different LPS responses in PP and spleen DCs reflect their characteristic expression of TLRs and TLR regulatory proteins. Differential regulation of TLR signalling was also evident in bacteria-conditioned bone-marrow-derived cells indicating that bacterial signalling may be a mechanism for inducing altered gene regulation in PP DCs.

Bifidobacterium animalis causes extensive duodenitis and mild colonic inflammation in monoassociated interleukin-10-deficient mice.

BACKGROUND: We recently showed that Bifidobacterium animalis is more prevalent within the colons of interleukin (IL)-10-deficient (-/-) mice than in wildtype (WT) animals colonized with the same specific pathogen-free (SPF) fecal contents. Here we tested the ability of this organism to cause T-cell-mediated intestinal inflammation by introducing it into germ-free (GF) IL-10-/- mice. METHODS: GF IL-10-/- or WT mice were monoassociated with Bifidobacterium animalis subsp. animalis ATCC (American Type Culture Collection, Manassas, VA) 25527(T) or with B. infantis ATCC 15697(T). Inflammation was measured by blinded histologic scores of the duodenum, cecum, and colon and by spontaneous secretion of IL-12/IL-23 p40 from colonic explants. Bacterial antigen-specific CD4(+) mesenteric lymph node (MLN) T-cell recall responses were measured in response to antigen-presenting cells (APC) pulsed with bacterial lysates. RESULTS: B. animalis caused marked duodenal inflammation and mild colitis in monoassociated IL-10-/- mice, whereas the intestinal tracts of WT animals remained free of inflammation. B. infantis colonization resulted in mild inflammation in the duodena of IL-10-/- mice. CD4(+) MLN T cells from B. animalis monoassociated IL-10-/- mice secreted high levels of IFN-gamma and IL-17 in response to B. animalis lysate. B. animalis equally colonized the different intestinal regions of WT and IL-10-/- mice. CONCLUSIONS: B. animalis, a traditional probiotic species that is expanded in experimental colitis in this model, induces marked duodenal and mild colonic inflammation and TH1/TH17 immune responses when introduced alone into GF IL-10-/- mice. This suggests a potential pathogenic role for this commensal bacterial species in a susceptible host.

Dietary nucleic acid and intestinal microbiota synergistically promote a shift in the Th1/Th2 balance toward Th1-skewed immunity.

BACKGROUND: Intestinal microbiota are known to play an important role in the establishment of oral tolerance, thereby protecting the organism from food allergies. Dietary intake of nucleic acid (NA) is also reported to have such an anti-allergic effect; however, one unsolved question is whether or not dietary NA would act through a process of toll-like receptor 9 signaling activated by DNA containing a CpG motif, a well-known sequence leading to immunostimulatory activity. In this study, we focused on the question of whether the addition of dietary NA lacking CpG motifs would allow continued modulation of the Th1/Th2 balance. METHODS: Germ free (GF) and Bifidobacterium-infantis-monoassociated BALB/c mice were maintained on either an NA-free casein diet or on an NA-supplemented casein diet for 4 weeks. Thereafter, both the in vivo anti-casein antibody levels and in vitro splenocyte cytokine secretion pattern were evaluated. RESULTS: Feeding with a casein diet elicited a substantial increase in the serum anti-casein-specific IgG1, IgG2a, and IgE levels of GF mice fed the NA free-diet. The in vitro cytokine production profile showed that enhanced IL-4 production in the GF mice fed the NA free-diet was markedly reduced by the supplementation with dietary NA in both the GF and B.-infantis-monoassociated mice. In addition, IFN-gamma secretion increased in the B.-infantis-reconstituted mice fed the diet containing NA. CONCLUSIONS: These results suggest that dietary intake of NA devoid of CpG motifs may prevent the development of allergies via acceleration of Th1-dominant immunity.

Bifidobacterial species differentially affect expression of cell surface markers and cytokines of dendritic cells harvested from cord blood.

The gut microbiota may be important in the postnatal development of the immune system and hence may influence the prevalence of atopic diseases. Bifidobacteria are the most numerous bacteria in the guts of infants, and the presence or absence of certain species could be important in determining the geographic incidence of atopic diseases. We compared the fecal populations of bifidobacteria from children aged 25 to 35 days in Ghana (which has a low prevalence of atopy), New Zealand, and the United Kingdom (high-prevalence countries). Natal origin influenced the detection of bifidobacterial species in that fecal samples from Ghana almost all contained Bifidobacterium infantis whereas those of the other children did not. Choosing species on the basis of our bacteriological results, we tested bifidobacterial preparations for their effects on cell surface markers and cytokine production by dendritic cells harvested from cord blood. Species-specific effects on the expression of the dendritic-cell activation marker CD83 and the production of interleukin-10 (IL-10) were observed. Whereas CD83 expression was increased and IL-10 production was induced by Bifidobacterium bifidum, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum, B. infantis failed to produce these effects. We concluded that B. infantis does not trigger the activation of dendritic cells to the degree necessary to initiate an immune response but that B. bifidum, B. longum, and B. pseudocatenulatum induce a Th2-driven immune response. A hypothesis is presented to link our observations to the prevalence of atopic diseases in different countries.

Risk factors of development of gut-derived bacterial translocation in thermally injured rats.

AIM: Studies have demonstrated that gut-derived bacterial translocation (BT) might play a role in the occurrence of sepsis and multiple organ dysfunction syndrome (MODS). Yet, no convincing overall analysis of risk factors for BT has been reported. The purpose of this study was to evaluate the related factors for the development of BT in burned rats. METHODS: Wistar rats were subjected to 30% third-degree burns. Then samples were taken on postburn d 1, 3, and 5. Incidence of BT and counts of mucosal bifidobacteria, fungi and E. coli, mucus sIgA, degree of injury to ileal mucosa, and plasma interleukin-6 were observed. Univariate analysis and multivariate logistic regression analysis were performed. RESULTS: The overall BT rate was 53.9% (69 in 128). The result of univariate analysis showed that the levels of plasma endotoxin and interleukin-6, the counts of mucosal fungi and E. coli, and the scores of ileum lesion were markedly increased in animals with BT compared with those without (P=0.000-0.005), while the levels of mucus sIgA and the counts of mucosal bifidobacteria were significantly reduced in animals with translocation compared with those without (P=0.000). There was a significant positive correlation between mucus sIgA and the counts of mucosal bifidobacteria (r=0.74, P=0.001). Moreover, there were strong negative correlations between scores of ileum-lesion and counts of bifidobacteria (r=-0.67, P=0.001). Multivariate logistic regression revealed that ileum lesion score (odds ratio [OR] 45.52, 95% confidence interval [CI] 5.25-394.80), and counts of mucosal bifidobacteria (OR 0.039, 95% CI 0.0032-0.48) were independent predictors of BT secondary to severe burns. CONCLUSION: Ileal lesion score and counts of mucosal bifidobacteria can be chosen as independent prognosis factors of the development of BT. Specific interventions targeting these high-risk factors might be implemented to attenuate BT, including strategies for repair of damaged intestinal mucosae and restoration of the balance of gastrointestinal flora.

Cytokine production in response to endodontic infection in germ-free mice.

This study evaluated the cytokine profiles (type 1 or type 2) that are triggered by and modulate endodontic periapical infections in the root canal system of germ-free mice. Microorganisms isolated from two patients with pulpal necrosis were inoculated into two groups of experimental animals: group I (Gemella morbillorum) and group II (Bifidobacterium adolescentis, Fusobacterium nucleatum and Clostridium butyricum). In vitro, G. morbillorum induced type 1 cytokine synthesis, while the modulation processed in vivo seemed to have the opposite effect, with a reduction in the basal levels of IL-12 and IFN-gamma, IL-4-independent down-modulation. In vitro, microorganisms from group II, in poly-infection, induced a reduction of type 1 cytokine levels from day 10 to day 20, which seemed to be modulated via IL-4. In vivo, however, a predominance of the immune response to one species over the others occurred.