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.     

Gnotobiotic Mouse Immune Response Induced by Bifidobacterium sp. Strains Isolated from Infants

Bifidobacterium, which is a dominant genus in infants’ fecal flora and can be used as a probiotic, has shown beneficial effects in various pathologies, including allergic diseases, but its role in immunity has so far been little known. Numerous studies have shown the crucial role of the initial intestinal colonization in the development of the intestinal immune system, and bifidobacteria could play a major role in this process. For a better understanding of the effect of Bifidobacterium on the immune system, we aimed at determining the impact of Bifidobacterium on the T-helper 1 (T_H1)/T_H2 balance by using gnotobiotic mice. Germfree mice were inoculated with Bifidobacterium longum NCC2705, whose genome is sequenced, and with nine Bifidobacterium strains isolated from infants’ fecal flora. Five days after inoculation, mice were killed. Transforming growth factor β1 (TGF-β1), interleukin-4 (IL-4), IL-10, and gamma interferon (IFN-γ) gene expressions in the ileum and IFN-γ, tumor necrosis factor alpha (TNF-α), IL-10, IL-4, and IL-5 secretions by splenocytes cultivated for 48 h with concanavalin A were quantified. Two Bifidobacterium species had no effect (B. adolescentis) or little effect (B. breve) on the immune system. Bifidobacterium bifidum, Bifidobacterium dentium, and one B. longum strain induced T_H1 and T_H2 cytokines at the systemic and intestinal levels. One B. longum strain induced a T_H2 orientation with high levels of IL-4 and IL-10, both secreted by splenocytes, and of TGF-β gene expression in the ileum. The other two strains induced T_H1 orientations with high levels of IFN-γ and TNF-α splenocyte secretions. Bifidobacterium's capacity to stimulate immunity is species specific, but its influence on the orientation of the immune system is strain specific.     

Preclinical Immunomodulation by the Probiotic Bifidobacterium breve M-16V in Early Life

This study aimed to investigate the effect of supplementation with the probiotic Bifidobacterium breve M-16V on the maturation of the intestinal and circulating immune system during suckling. In order to achieve this purpose, neonatal Lewis rats were supplemented with the probiotic strain from the 6th to the 18th day of life. The animals were weighed during the study, and faecal samples were obtained and evaluated daily. On day 19, rats were euthanized and intestinal wash samples, mesenteric lymph node (MLN) cells, splenocytes and intraepithelial lymphocytes (IEL) were obtained. The probiotic supplementation in early life did not modify the growth curve and did not enhance the systemic immune maturation. However, it increased the proportion of cells bearing TLR4 in the MLN and IEL, and enhanced the percentage of the integrin αEβ7+ and CD62L+ cells in the MLN and that of the integrin αEβ7+ cells in the IEL, suggesting an enhancement of the homing process of naïve T lymphocytes to the MLN, and the retention of activated lymphocytes in the intraepithelial compartment. Interestingly, B. breve M-16V enhanced the intestinal IgA synthesis. In conclusion, supplementation with the probiotic strain B. breve M-16V during suckling improves the development of mucosal immunity in early life.     

Quantitative Real-Time PCR Assays To Identify and Quantify Fecal Bifidobacterium Species in Infants Receiving a Prebiotic Infant Formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5′ nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5′ nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% ± 9.8% to 73.4% ± 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% ± 1.92% and 8.11% ± 4.12%, respectively, versus 0.15% ± 0.11% and 1.38% ± 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

Variation in Consumption of Human Milk Oligosaccharides by Infant Gut-Associated Strains of Bifidobacterium breve

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.     

Lactobacillus rhamnosus GG and Bifidobacterium longum Attenuate Lung Injury and Inflammatory Response in Experimental Sepsis

Introduction

Probiotic use to prevent nosocomial gastrointestinal and potentially respiratory tract infections in critical care has shown great promise in recent clinical trials of adult and pediatric patients. Despite well-documented benefits of probiotic use in intestinal disorders, the potential for probiotic treatment to reduce lung injury following infection and shock has not been well explored.

Objective

Evaluate if Lactobacillus rhamnosus GG (LGG) or Bifidobacterium longum (BL) treatment in a weanling mouse model of cecal ligation and puncture (CLP) peritonitis will protect against lung injury.

Methods

3 week-old FVB/N mice were orally gavaged with 200 µl of either LGG, BL or sterile water (vehicle) immediately prior to CLP. Mice were euthanized at 24 h. Lung injury was evaluated via histology and lung neutrophil infiltration was evaluated by myeloperoxidase (MPO) staining. mRNA levels of IL-6, TNF-α, MyD88, TLR-4, TLR-2, NFΚB (p50/p105) and Cox-2 in the lung analyzed via real-time PCR. TNF-α and IL-6 in lung was analyzed via ELISA.

Results

LGG and BL treatment significantly improved lung injury following experimental infection and sepsis and lung neutrophil infiltration was significantly lower than in untreated septic mice. Lung mRNA and protein levels of IL-6 and TNF-α and gene expression of Cox-2 were also significantly reduced in mice receiving LGG or BL treatment. Gene expression of TLR-2, MyD88 and NFΚB (p50/p105) was significantly increased in septic mice compared to shams and decreased in the lung of mice receiving LGG or BL while TLR-4 levels remained unchanged.

Conclusions

Treatment with LGG and BL can reduce lung injury following experimental infection and sepsis and is associated with reduced lung inflammatory cell infiltrate and decreased markers of lung inflammatory response. Probiotic therapy may be a promising intervention to improve clinical lung injury following systemic infection and sepsis.     

Induction of IL-12p40 and type 1 immunity by Toxoplasma gondii in the absence of the TLR-MyD88 signaling cascade

Toxoplasma gondii is an orally acquired pathogen that induces strong IFN-γ based immunity conferring protection but that can also be the cause of immunopathology. The response in mice is driven in part by well-characterized MyD88-dependent signaling pathways. Here we focus on induction of less well understood immune responses that do not involve this Toll-like receptor (TLR)/IL-1 family receptor adaptor molecule, in particular as they occur in the intestinal mucosa. Using eYFP-IL-12p40 reporter mice on an MyD88^-/- background, we identified dendritic cells, macrophages, and neutrophils as cellular sources of MyD88-independent IL-12 after peroral T. gondii infection. Infection-induced IL-12 was lower in the absence of MyD88, but was still clearly above noninfected levels. Overall, this carried through to the IFN-γ response, which while generally decreased was still remarkably robust in the absence of MyD88. In the latter mice, IL-12 was strictly required to induce type I immunity. Type 1 and type 3 innate lymphoid cells (ILC), CD4⁺ T cells, and CD8⁺ T cells each contributed to the IFN-γ pool. We report that ILC3 were expanded in infected MyD88^-/- mice relative to their MyD88^+/+ counterparts, suggesting a compensatory response triggered by loss of MyD88. Furthermore, bacterial flagellin and Toxoplasma specific CD4⁺ T cell populations in the lamina propria expanded in response to infection in both WT and KO mice. Finally, we show that My88-independent IL-12 and T cell mediated IFN-γ production require the presence of the intestinal microbiota. Our results identify MyD88-independent intestinal immune pathways induced by T. gondii including myeloid cell derived IL-12 production, downstream type I immunity and IFN-γ production by ILC1, ILC3, and T lymphocytes. Collectively, our data reveal an underlying network of immune responses that do not involve signaling through MyD88.     

Isolation of a Bifidogenic Peptide from the Pepsin Hydrolysate of Bovine Lactoferrin

Lactoferrin is an iron-binding glycoprotein found in the milk of most mammals for which various biological functions have been reported, such as antimicrobial activity and bifidogenic activity. In this study, we compared the bifidogenic activity of bovine lactoferrin (bLF) and pepsin hydrolysate of bLF (bLFH), isolated bifidogenic peptide from bLFH, and investigated the bifidogenic spectra of bLF, bLFH, and its active peptide against 42 bifidobacterial strains comprising nine species. Against Bifidobacterium breve ATCC 15700^T, minimal effective concentrations of bLF and bLFH were 300 and 10 μg/ml. Against Bifidobacterium longum subsp. infantis ATCC 15697^T, the minimal effective concentration of bLFH was 30 μg/ml, and bLF did not show bifidogenic activity within 300 μg/ml. As an active peptide, a heterodimer of A¹-W¹⁶ and L⁴³-A⁴⁸ linked by a disulfide bond was isolated. Previously, this peptide was identified as having antibacterial activity. An amino acid mixture with the same composition as this peptide showed no bifidogenic activity. The strains of each species whose growth was highly promoted (>150%) by this peptide at 3.75 μM were as follows: B. breve (7 out of 7 strains [7/7]), B. longum subsp. infantis (5/5), Bifidobacterium bifidum (2/5), B. longum subsp. longum (1/3), Bifidobacterium adolescentis (3/6), Bifidobacterium catenulatum (1/4), Bifidobacterium pseudocatenulatum (0/4), Bifidobacterium dentium (0/5), and Bifidobacterium angulatum (0/3). Growth of none of the strains was highly promoted by bLF at 3.75 μM. We demonstrated that bLFH showed stronger bifidogenic activity than natural bLF, especially against infant-representative species, B. breve and B. longum subsp. infantis; furthermore, we isolated its active peptide. This is the first report about a bifidogenic peptide derived from bLF.     

Commensal Bacteria-induced Interleukin 1β (IL-1β) Secreted by Macrophages Up-regulates Hepcidin Expression in Hepatocytes by Activating the Bone Morphogenetic Protein Signaling Pathway

The liver hormone hepcidin is the central regulator of systemic iron metabolism. Its increased expression in inflammatory states leads to hypoferremia and anemia. Elucidation of the mechanisms that up-regulate hepcidin during inflammation is essential for developing rational therapies for this anemia. Using mouse models of inflammatory bowel disease, we have shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition. Here we investigate how two commensal bacteria, Bifidobacterium longum and Bacteroides fragilis, representative members of the gut microbiota, affect hepcidin expression. We found that supernatants of a human macrophage cell line infected with either of the bacteria up-regulated hepcidin when added to a human hepatocyte cell line. This activity was abrogated by neutralization of IL-1β. Moreover, purified IL-1β increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes and when injected into mice. IL-1β activated the bone morphogenetic protein (BMP) signaling pathway in hepatocytes and in mouse liver, as indicated by increased phosphorylation of small mothers against decapentaplegic proteins. Activation of BMP signaling correlated with IL-1β-induced expression of BMP2 in human hepatocytes and activin B in mouse liver. Treatment of hepatocytes with two different chemical inhibitors of BMP signaling or with a neutralizing antibody to BMP2 prevented IL-1β-induced up-regulation of hepcidin. Our results clarify how commensal bacteria affect hepcidin expression and reveal a novel connection between IL-1β and activation of BMP signaling. They also suggest that there may be differences between mice and humans with respect to the mechanism by which IL-1β up-regulates hepcidin.     

Lysate of probiotic Lactobacillus casei DN-114 001 ameliorates colitis by strengthening the gut barrier function and changing the gut microenvironment

Background

Probiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD.

Methodology/Principal Findings

The preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4⁺FoxP3⁺ regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4⁺FoxP3⁺ regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer''s patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway.

Conclusion/Significance

Our study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment.     

FimH Adhesin of Type 1 Fimbriae Is a Potent Inducer of Innate Antimicrobial Responses Which Requires TLR4 and Type 1 Interferon Signalling

Components of bacteria have been shown to induce innate antiviral immunity via Toll-like receptors (TLRs). We have recently shown that FimH, the adhesin portion of type 1 fimbria, can induce the innate immune system via TLR4. Here we report that FimH induces potent in vitro and in vivo innate antimicrobial responses. FimH induced an innate antiviral state in murine macrophage and primary MEFs which was correlated with IFN-β production. Moreover, FimH induced the innate antiviral responses in cells from wild type, but not from MyD88^−/−, Trif^−/−, IFN−α/βR^−/− or IRF3^−/− mice. Vaginal delivery of FimH, but not LPS, completely protected wild type, but not MyD88^−/−, IFN-α/βR^−/−, IRF3^−/− or TLR4^−/− mice from subsequent genital HSV-2 challenge. The FimH-induced innate antiviral immunity correlated with the production of IFN-β, but not IFN-α or IFN-γ. To examine whether FimH plays a role in innate immune induction in the context of a natural infection, the innate immune responses to wild type uropathogenic E. coli (UPEC) and a FimH null mutant were examined in the urinary tract of C57Bl/6 (B6) mice and TLR4-deficient mice. While UPEC expressing FimH induced a robust polymorphonuclear response in B6, but not TLR4^−/− mice, mutant bacteria lacking FimH did not. In addition, the presence of TLR4 was essential for innate control of and protection against UPEC. Our results demonstrate that FimH is a potent inducer of innate antimicrobial responses and signals differently, from that of LPS, via TLR4 at mucosal surfaces. Our studies suggest that FimH can potentially be used as an innate microbicide against mucosal pathogens.     

Characterization of Two Novel α-Glucosidases from Bifidobacterium breve UCC2003

Two α-glucosidase-encoding genes (agl1 and agl2) from Bifidobacterium breve UCC2003 were identified and characterized. Based on their similarity to characterized carbohydrate hydrolases, the Agl1 and Agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the α-1,6-glucosidases (EC 3.2.1.10). Recombinant Agl1 and Agl2 into which a His₁₂ sequence was incorporated (Agl1_His and Agl2_His, respectively) exhibited hydrolytic activity towards panose, isomaltose, isomaltotriose, and four sucrose isomers—palatinose, trehalulose, turanose, and maltulose—while also degrading trehalose and, to a lesser extent, nigerose. The preferred substrates for both enzymes were panose, isomaltose, and trehalulose. Furthermore, the pH and temperature optima for both enzymes were determined, showing that Agl1_His exhibits higher thermo and pH optima than Agl2_His. The two purified α-1,6-glucosidases were also shown to have transglycosylation activity, synthesizing oligosaccharides from palatinose, trehalulose, trehalose, panose, and isomaltotriose.The gastrointestinal tract is inhabited by a complex community of microorganisms, also referred to as the microbiota, which are believed to play an important role in human health and disease (39). This concept has been driving extensive attempts to positively influence the composition and/or activity of the intestinal microbiota through the use of so-called probiotics and/or prebiotics. A probiotic has been defined as “a preparation or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora (by implantation or colonization) in a compartment of the host and by that exert beneficial health effect in this host” (48). A prebiotic has recently been (re)defined as “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health” (42). Finally, a synbiotic is the combination of a probiotic and a prebiotic (16).One of the dominant bacteria of the intestinal microbiota of humans and animals (51) is bifidobacteria. These are gram-positive, pleomorphic, and anaerobic bacteria that have received increasing scientific attention in recent years due to their perceived probiotic activity (15, 27). The growth of gut-derived bifidobacteria has been shown to be selectively stimulated by various dietary carbohydrates that can thus be considered as prebiotics (30). In this context, it is interesting to note that more than 8% of the identified genes on bifidobacterial genomes are predicted to be involved in sugar metabolism, thus indicative of extensive carbon source-degrading abilities (55, 56).Carbohydrate degradation has been extensively studied in a variety of different Bifidobacterium species (reviewed in reference 53). For example, various α- and β-galactosidases have been characterized in Bifidobacterium breve 203 (60), Bifidobacterium adolescentis DSM20083 (20), Bifidobacterium bifidum NCIMB41171 (18), and Bifidobacterium longum MB219 (43). A number of studies have also shown that Bifidobacterium spp. produce various α- and β-glucosidase activities (reviewed in reference 53), while Bifidobacterium infantis ATCC 15697 (58), Bifidobacterium lactis DSM10140(T) (13), and B. breve UCC2003 (45) have been reported to produce β-fructofuranosidases during growth on fructooligosaccharides. Additionally, starch-, amylopectin-, and pullulan-degrading activities in bifidobacteria have been investigated (36, 44). Several β-glucosidases have been biochemically characterized from a number of strains of bifidobacteria, e.g., B. adolescentis Int-57 (8), B. breve clb (35,) and Bifidobacterium sp. strain SEN (59). To date, only two α-glucosidases (AglA and AglB) have been described from B. adolescentis DSM20083 (54). AglA was shown to preferentially hydrolyze isomaltotriose, while AglB exhibits a high preference to maltose. Both AglA and AglB were also demonstrated to have transglycosylation activity. Aside from this report, little is known about the biochemical characteristics of α-glucosidase enzymes from bifidobacteria, although it is a common activity observed among these bacteria (41).Carbohydrates other than the commercially exploited prebiotics, e.g., fructooligosaccharides (such as inulin) and trans-galactooligosaccharides (42), have received relatively little attention with regard to their possible prebiotic properties. Such potential prebiotics are, for example, honey oligosaccharides, some of which are also interesting because of their noncariogenic properties (14). One of the predominant fractions of noncariogenic sugars in honey is isomaltulose (5, 46), also called palatinose or 6-O-α-d-glucopyranosyl-d-fructose, which is a reducing disaccharide and a functional isomer of sucrose. Palatinose possesses approximately one-third of the sweetness of sucrose and is very resistant to acid and invertase hydrolysis (29, 32). The hydrolysis and adsorption of palatinose in the small intestine thus occurs at a much slower rate than does those of sucrose (17), which results in a reduction of the postprandial plasma glucose and insulin levels (3), which means that most palatinose passes through the small intestine to present a growth substrate for elements of the colonic microbiota.In this study, we describe the identification of two genes, agl1 and agl2, present in the genome of B. breve UCC2003 and responsible for the hydrolysis of α-glycosidic linkages, such as those present in palatinose.     

Modulation of Rat Cecal Microbiota by Administration of Raffinose and Encapsulated Bifidobacterium breve

To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192^T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.     

Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on Hepatic Steatosis in Zucker Rats

We have previously described the safety and immunomodulatory effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 in healthy volunteers. The scope of this work was to evaluate the effects of these probiotic strains on the hepatic steatosis of obese rats. We used the Zucker rat as a genetic model of obesity. Zucker-Lepr^fa/fa rats received one of three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo for 30 days. An additional group of Zucker-lean^+/fa rats received a placebo for 30 days. No alterations in intestinal histology, in the epithelial, lamina propria, muscular layers of the ileal or colonic mucosa, or the submucosae, were observed in any of the experimental groups. Triacylglycerol content decreased in the liver of Zucker-Lepr^fa/fa rats that were fed L. rhamnosus, B. breve, or the mixture of B. breve and L. paracasei. Likewise, the area corresponding to neutral lipids was significantly smaller in the liver of all four groups of Zucker-Lepr^fa/fa rats that received probiotics than in rats fed the placebo. Zucker-Lepr^fa/fa rats exhibited significantly greater serum LPS levels than Zucker-lean^+/fa rats upon administration of placebo for 30 days. In contrast, all four groups of obese Zucker-Lepr^fa/fa rats that received LAB strains exhibited serum LPS concentrations similar to those of Zucker-lean^+/fa rats. Serum TNF-α levels decreased in the Zucker-Lepr^fa/fa rats that received B. breve, L. rhamnosus, or the mixture, whereas L. paracasei feeding decreased IL-6 levels in the serum of Zucker-Lepr^fa/fa rats. In conclusion, the probiotic strains reduced hepatic steatosis in part by lowering serum LPS, and had an anti-inflammatory effect in obese Zucker rats.     

Anti-Inflammatory Effects of Lactobacillus Rahmnosus and Bifidobacterium Breve on Cigarette Smoke Activated Human Macrophages

Background

Chronic obstructive pulmonary disease (COPD) is a major global health problem with cigarette smoke (CS) as the main risk factor for its development. Airway inflammation in COPD involves the increased expression of inflammatory mediators such as CXCL-8 and IL-1β which are important mediators for neutrophil recruitment. Macrophages are an important source of these mediators in COPD. Lactobacillus rhamnosus (L. rhamnosus) and Befidobacterium breve (B. breve) attenuate the development of ‘allergic asthma’ in animals but their effects in COPD are unknown.

Objective

To determine the anti-inflammatory effects of L. rhamnosus and B. breve on CS and Toll-like receptor (TLR) activation.

Design

We stimulated the human macrophage cell line THP-1 with CS extract in the presence and absence of L. rhamnosus and B. breve and measured the expression and release of inflammatory mediators by RT-qPCR and ELISA respectively. An activity assay and Western blotting were used to examine NF-κB activation.

Results

Both L. rhamnosus and B. breve were efficiently phagocytized by human macrophages. L. rhamnosus and B. breve significantly suppressed the ability of CS to induce the expression of IL-1β, IL-6, IL-10, IL-23, TNFα, CXCL-8 and HMGB1 release (all p<0.05) in human THP-1 macrophages. Similar suppression of TLR4- and TLR9-induced CXCL8 expression was also observed (p<0.05). The effect of L. rhamnosus and B. breve on inflammatory mediator release was associated with the suppression of CS-induced NF-κB activation (p<0.05).

Conclusions

This data indicate that these probiotics may be useful anti-inflammatory agents in CS-associated disease such as COPD.     

Lactobacillus rhamnosus GG Protects against Non-Alcoholic Fatty Liver Disease in Mice

Objective

Experimental evidence revealed that obesity-associated non-alcoholic fatty liver disease (NAFLD) is linked to changes in intestinal permeability and translocation of bacterial products to the liver. Hitherto, no reliable therapy is available except for weight reduction. Within this study, we examined the possible effect of the probiotic bacterial strain Lactobacillus rhamnosus GG (LGG) as protective agent against experimental NAFLD in a mouse model.

Methods

Experimental NAFLD was induced by a high-fructose diet over eight weeks in C57BL/J6 mice. Fructose was administered via the drinking water containing 30% fructose with or without LGG at a concentration resulting in approximately 5×10⁷ colony forming units/g body weight. Mice were examined for changes in small intestinal microbiota, gut barrier function, lipopolysaccharide (LPS) concentrations in the portal vein, liver inflammation and fat accumulation in the liver.

Results

LGG increased beneficial bacteria in the distal small intestine. Moreover, LGG reduced duodenal IκB protein levels and restored the duodenal tight junction protein concentration. Portal LPS (P≤0.05) was reduced and tended to attenuate TNF-α, IL-8R and IL-1β mRNA expression in the liver feeding a high-fructose diet supplemented with LGG. Furthermore liver fat accumulation and portal alanine-aminotransferase concentrations (P≤0.05) were attenuated in mice fed the high-fructose diet and LGG.

Conclusions

We show for the first time that LGG protects mice from NAFLD induced by a high-fructose diet. The underlying mechanisms of protection likely involve an increase of beneficial bacteria, restoration of gut barrier function and subsequent attenuation of liver inflammation and steatosis.