Marianna Roselli

Probiotic bacteria Bifidobacterium animalis MB5 and Lactobacillus rhamnosus GG protect intestinal Caco-2 cells from the inflammation-associated response induced by enterotoxigenic Escherichia coli K88

Probiotic bacteria may provide protection against intestinal damage induced by pathogens, but the underlying mechanisms are still largely unknown. We investigated whether Bifidobacterium animalis MB5 and Lactobacillus rhamnosus GG (LGG) protected intestinal Caco-2 cells from the inflammation-associated response induced by enterotoxigenic Escherichia coli (ETEC) K88, by inhibiting pathogen attachment to the cells, which is the first step of ETEC pathogenicity, and regulating neutrophil recruitment, a crucial component of inflammation. A partial reduction of ETEC adhesion was exerted by probiotics and their culture supernatant fractions either undigested or digested with proteases. ETEC viability was unaffected by the presence of B. animalis, LGG or their supernatant fractions in the culture medium, indicating an absence of probiotic bactericidal activity. Probiotics and their supernatant fractions, either undigested or digested with proteases, strongly inhibited the neutrophil transmigration caused by ETEC. Both B. animalis and LGG counteracted the pathogen-induced up regulation of IL-8, growth-related oncogene-alpha and epithelial neutrophil-activating peptide-78 gene expression, which are chemokines essential for neutrophil migration. Moreover, the probiotics prevented the ETEC-induced increased expression of IL-1beta and TNF-alpha and decrease of transforming growth factor-alpha, which are regulators of chemokine expression. These results indicate that B. animalis MB5 and LGG protect intestinal cells from the inflammation-associated response caused by ETEC K88 by partly reducing pathogen adhesion and by counteracting neutrophil migration, probably through the regulation of chemokine and cytokine expression.

Prevention of TNBS-Induced Colitis by Different Lactobacillus and Bifidobacterium Strains Is Associated with an Expansion of γδT and Regulatory T Cells of Intestinal Intraepithelial Lymphocytes

Background: Probiotics may protect against inflammatory bowel disease through regulation of lamina propria lymphocytes (LPLs) function. Data are lacking on possible involvement of intraepithelial lymphocytes (IELs). The aim of this study was to investigate whether different probiotic mixtures prevented gut inflammatory disease and the role of both IELs and LPLs. Methods: BALB/c mice received 2 probiotic mixtures orally for 3 weeks, as Mix1 (Lactobacillus acidophilus and Bifidobacterium longum), or Mix2 (Lactobacillus plantarum, Streptococcus thermophilus, and Bifidobacterium animalis subsp. lactis). Colitis was induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS). Probiotics in stools were analyzed by real‐time polymerase chain reaction (PCR). Colon subpopulations of IELs and LPLs were assayed by flow cytometry. Serum cytokines were measured by cytometric bead array (CBA). Results: All probiotics colonized the intestine. The 2 mixtures prevented the TNBS‐induced intestinal damage, and Mix1 was the most effective. The Mix1 protection was associated with a reduction in CD4+ cells of IELs and LPLs, an increase in &ggr;&dgr;T cells of IELs, and a decrease in &ggr;&dgr;T cells of LPLs. An expansion of T regulatory (Treg) cells of IELs was induced by Mix1 and Mix2. Both probiotic mixtures inhibited tumor necrosis factor (TNF)‐&agr; and monocyte chemotactic protein (MCP)‐1 production and upregulated interleukin (IL)‐10. In addition, Mix1 prevented the TNBS‐induced increase of IL‐12 and interferon (IFN)‐&ggr;. Conclusions: The 2 probiotic mixtures were able to prevent the TNBS‐induced colitis; the L. acidophilus and B. longum mixture was the most effective. Other than an involvement of LPLs, our results report a novel importance of the IELs population in probiotic protection. Inflamm Bowel Dis 2009

Intestinal and Peripheral Immune Response to MON810 Maize Ingestion in Weaning and Old Mice

This study evaluated the gut and peripheral immune response to genetically modified (GM) maize in mice in vulnerable conditions. Weaning and old mice were fed a diet containing MON810 or its parental control maize or a pellet diet containing a GM-free maize for 30 and 90 days. The immunophenotype of intestinal intraepithelial, spleen, and blood lymphocytes of control maize fed mice was similar to that of pellet fed mice. As compared to control maize, MON810 maize induced alterations in the percentage of T and B cells and of CD4(+), CD8(+), gammadeltaT, and alphabetaT subpopulations of weaning and old mice fed for 30 or 90 days, respectively, at the gut and peripheral sites. An increase of serum IL-6, IL-13, IL-12p70, and MIP-1beta after MON810 feeding was also found. These results suggest the importance of the gut and peripheral immune response to GM crop ingestion as well as the age of the consumer in the GMO safety evaluation.

Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKβ, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1β. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans.

Lactobacillus rhamnosus GG and Bifidobacterium animalis MB5 Induce Intestinal but Not Systemic Antigen-Specific Hyporesponsiveness in Ovalbumin-Immunized Rats

Probiotics may modulate the host immune response by mechanisms not yet fully understood. We evaluated the modulation of intestinal and systemic antigen-specific immune response by Lactobacillus rhamnosus GG (LGG) or Bifidobacterium animalis MB5 in tolerized and immunized rats. Three groups of rats received orally LGG, B. animalis, or PBS (control) for 28 d. Each group was divided into two subgroups of tolerized or immunized rats receiving orally ovalbumin (OVA; 7 mg) or PBS on d 7, 9, and 11. All rats were immunized with OVA (300 μg) on d 14 and 21. In tolerized rats, the OVA-induced proliferative response of mesenteric lymph nodes (MLN) and spleen cells did not differ from control, indicating that the two probiotics maintained the tolerance. LGG and B. animalis in immunized rats reduced the OVA-induced proliferative response in MLN (P < 0.01) but not in spleen, whereas the proliferative response to anti-CD3 and concanavalin A of MLN and spleen cells as well as the delayed-type hypersensitivity reaction were not affected by probiotic treatment, indicating OVA-specific hyporesponsiveness restricted to intestinal immunity. This hyporesponsiveness was associated with CD4+CD25+Foxp3+ T cell expansion (P < 0.01) and increased IL-10 and TGFβ after LGG (P < 0.05), and increased apoptosis after B. animalis (P < 0.001) in MLN. In conclusion, we report a novel activity of LGG and B. animalis in inducing OVA-specific hyporesponsiveness in MLN of OVA-immunized rats that can be useful for a therapeutic strategy to prevent undesirable reactions to immunogenic antigens in the gut.

Fecal and urinary NMR-based metabolomics unveil an aging signature in mice

BACKGROUND Aging is characterized by derangements in multiple metabolic pathways that progressively constrict the homeostatic reserve (homeostenosis). The signature of metabolic alterations that accompany aging can be retrieved through the metabolomic profiling of biological fluids. OBJECTIVE To characterize the age-related changes in urinary and fecal metabolic profiles of BALB/c mice through a (1)H nuclear magnetic resonance (NMR)-based metabolomic approach. METHODS Young (n=19) and old (n=13) male BALB/c mice were fed ad libitum standard laboratory chow. Twenty four-hour feces and urine were collected using metabolic cages and analyzed by high-resolution (1)H NMR spectroscopy combined with multivariate statistical analyses. RESULTS An age-related metabolic phenotype was detected both in urine and feces. The metabolic signature of aging consisted of changes in levels of metabolites associated with amino acid metabolism, tricarboxylic acid cycle, tryptophan-nicotinamide adenine dinucleotide pathway, and host-microbiota metabolic axis. CONCLUSIONS Our (1)H NMR-based metabolomic approach was able to characterize the effect of age on urinary and fecal metabotypes. The implementation of this analytical strategy may increase our understanding of the metabolic alterations involved in the aging process and assist in the design of anti-aging interventions.

Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 Induce Different Age-Related Metabolic Profiles Revealed by 1H-NMR Spectroscopy in Urine and Feces of Mice

Age-related dysbioses of intestinal microbiota and decline in the overall metabolic homeostasis are frequently found in the elderly. Probiotic supplementation may represent a way to prevent or reduce the senescence-associated metabolic disorders. The present study evaluated the metabolic impact of Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 supplementation in relation to age by analyzing urine and feces metabolic profiles using (1)H-nuclear magnetic resonance spectroscopy and multivariate analysis. Adult (3 mo old) and aged (16 mo old) mice received an oral supplementation of the 2 probiotics (1 × 10(9) colony-forming units/d each) or phosphate buffered saline (control) daily for 30 d. Urine and feces were collected for 48 h before the end of the study. Partial least squares-discriminant analysis showed that the urinary discriminant metabolites for the probiotic treatment included higher dimethylglycine in adult and aged mice, lower sarcosine and nicotinate in adult mice, higher N-methylnicotinamide in adult mice and lower N-methylnicotinamide in aged mice compared with their controls. These results indicate a probiotic-induced modulation of homocysteine and NAD metabolism pathways, which have important implications because these pathways are involved in essential cellular processes that can be altered in senescence. The probiotic supplementation also modified the fecal metabolic profiles, inducing in both adult and aged mice higher 4-hydroxyphenylacetate and lower xylose in treated mice compared with their control mice, whereas valerate was greater in treated adult mice and lower in treated aged mice compared with their controls. The ANOVA simultaneous component analysis on urinary and fecal metabolic profiling showed an age × treatment interaction (P < 0.05), confirming the age-related modulation of the metabolic response to probiotic supplementation. The results suggest that L. acidophilus and B. lactis may prevent or reduce age-related metabolic dysfunction.

Application of NMR-based metabolomics to the study of gut microbiota in obesity

Lifestyle habits, host gene repertoire, and alterations in the intestinal microbiota concur to the development of obesity. A great deal of research has recently been focused on investigating the role gut microbiota plays in the pathogenesis of metabolic dysfunctions and increased adiposity. Altered microbiota can affect host physiology through several pathways, including enhanced energy harvest, and perturbations in immunity, metabolic signaling, and inflammatory pathways. A broad range of “omics” technologies is now available to help decipher the interactions between the host and the gut microbiota at detailed genetic and functional levels. In particular, metabolomics—the comprehensive analysis of metabolite composition of biological fluids and tissues—could provide breakthrough insights into the links among the gut microbiota, host genetic repertoire, and diet during the development and progression of obesity. Here, we briefly review the most insightful findings on the involvement of gut microbiota in the pathogenesis of obesity. We also discuss how metabolomic approaches based on nuclear magnetic resonance spectroscopy could help understand the activity of gut microbiota in relation to obesity, and assess the effects of gut microbiota modulation in the treatment of this condition.

Regulation of immune response at intestinal and peripheral sites by probiotics

The gut associated lymphoid tissue (GALT) should protect intestinal mucosa against pathogens, but also avoid hypersensitivity reactions to food proteins, normal bacterial flora and other environmental macromolecules. The interaction between epithelial cells and microflora is fundamental to establish gut mucosal barrier and GALT development. The normal colonization of intestine by commensal bacteria is thus crucial for a correct development of mucosal immune system. Probiotic bacteria are normal inhabitants of microflora and may confer health benefits to the host. The modification of the intestinal microflora towards a healthier probiotics enriched microflora may generate beneficial mucosal immunomodulatory effects and may represent a new strategy to cure intestinal and allergic diseases. The health benefits may be specific for different probiotic strains. Ongoing research is providing new insights into the probiotic beneficial effects and related mechanisms. This review represents an update of immunomodulatory activity of different probiotics and of the more accredited mechanisms underlying such activities.

Nanomolar Caffeic Acid Decreases Glucose Uptake and the Effects of High Glucose in Endothelial Cells

Epidemiological studies suggest that moderate and prolonged consumption of coffee is associated with a reduced risk of developing type 2 diabetes but the molecular mechanisms underlying this effect are not known. In this study, we report the effects of physiological concentrations of caffeic acid, easily achievable by normal dietary habits, in endothelial cells cultured in 25 mM of glucose (high glucose, HG). In HG, the presence of 10 nM caffeic acid was associated with a decrease of glucose uptake but not to changes of GLUT-1 membrane localization or mRNA levels. Moreover, caffeic acid countered HG-induced loss of barrier integrity, reducing actin rearrangement and FITC-dextran passage. The decreased flux of glucose associated to caffeic acid affected HG induced apoptosis by down-regulating the expression of initiator (caspase 8 and 9) and effector caspases (caspase 7 and 3) and by increasing the levels of phosphorylated Bcl-2. We also observed that caffeic acid in HG condition was associated to a reduction of p65 subunit nuclear levels with respect to HG alone. NF-κB activation has been shown to lead to apoptosis in HG treated cells and the analysis of the expression of a panel of about 90 genes related to NF-κB signaling pathway revealed that caffeic acid significantly influenced gene expression changes induced by HG. In conclusion, our results suggest that caffeic acid, decreasing the metabolic stress induced by HG, allows the activation of survival mechanisms mediated by a different modulation of NF-κB-related signaling pathways and to the activation of anti-apoptotic proteins.