Elena Mengheri

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.

Altered expression, localization, and phosphorylation of epithelial junctional proteins in celiac disease

We aimed to study the expression and localization of the molecular components of enterocyte junctions in celiac disease together with the level of tyrosine phosphorylation, a phenomenon known to affect their cellular distribution and function, and to explore the influence of proinflammatory cytokines. Duodenal biopsy specimens from patients with celiac disease and control subjects were used for immunoprecipitation, immunoblotting, and immunolocalization by using antioccludin, anti-zonula occludens (ZO)-1, anti-E-cadherin, anti-beta-catenin, and antiphosphotyrosine antibodies. The same procedures were carried out on filter-grown Caco-2 cells incubated in the absence or presence of interferon g and tumor necrosis factor a. In active celiac disease, the absence of a phosphorylated ZO-1 and the extensive phosphorylation of beta-catenin might be responsible for the absence of membranous localization of occludin and E-cadherin, respectively. The in vitro system showed an influence of the cytokines on the assembly of these complexes that proved the opposite to celiac samples as far as tight junctions were concerned because the presence of a phosphorylated ZO-1 enables occludin to localize in the membrane.

Intestinal damage induced by zinc deficiency is associated with enhanced CuZn superoxide dismutase activity in rats: effect of dexamethasone or thyroxine treatment.

Zinc has a wide spectrum of biological activities and its deficiency has been related to various tissue dysfunctions and alterations of normal cell metabolism. Zinc also plays an important role in the antioxidant cellular defenses being a structural element of the non-mitochondrial form of the enzyme superoxide dismutase (CuZnSOD). We have already reported that Zn deficiency induces severe alterations in the rat intestine, that are reverted by treatment with dexamethasone (Dex) or thyroxine (T4). Here we report a paradoxical increase of CuZnSOD activity in rat intestine after 20 and 40 days of zinc deficiency. The increase of CuZnSOD activity is not due to an upregulation of gene expression because both Northern and Western blot analysis indicate that CuZnSOD mRNA and protein levels are not affected by zinc deficiency. A significant increase of lipid peroxidation was also observed in duodenum and jejunum associated with zinc deficiency. Treatment with either Dex or T4 to zinc-deficient rats protects against intestinal oxidative damage and results in SOD activity similar to control rats. Because glutathione peroxidase and catalase activities decreased in zinc deficiency, we speculate that the increase in SOD activity may be associated with an accumulation of hydrogen peroxide that may activate inflammatory molecules, further worsening tissue damage.

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.

Reduction of oxidative stress and cytokine-induced neutrophil chemoattractant (CINC) expression by red wine polyphenols in zinc deficiency induced intestinal damage of rat

Consumption of red wine has been associated with health promotion and disease prevention. We have previously found that the intestine of zinc-deficient (ZD) rats develop oxidative damage associated with inflammation. Here we have used this model to investigate whether red wine polyphenols could protect against intestinal injury and, if so, whether this protection was achieved through antioxidant and anti-inflammatory activity. The intestinal alterations induced by zinc deficiency such as morphological damage, increased TBA-RS level and CuZn-superoxide dismutase activity, and decreased glutathione peroxidase activity, did not develop with the administration to ZD rats of a suspension of dealcoholated red wine (RWS). The same treatment induced in control rats a decrease of TBA-RS level but also of glutathione peroxidase and catalase activity. Treatment with RWS to ZD rats prevented a marked mucosal macrophage and neutrophil infiltration. The expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha and cytokine-induced neutrophil chemoattractant (CINC), was induced by zinc deficiency, whereas that of the anti-inflammatory interleukin-10 was suppressed. Treatment with RWS reduced CINC expression. These results report a novel activity of red wine polyphenols in downregulation of intestinal CINC expression, which likely protects cells against inflammatory processes.

Unique Biochemical Nature of Carp Retinol-binding Protein N-LINKED GLYCOSYLATION AND UNCLEAVABLE NH2-TERMINAL SIGNAL PEPTIDE

Retinol transport and metabolism have been well characterized in mammals; however, very little is known in fish. To study the mechanism by which fish retinol-binding protein (RBP) is able to remain in plasma besides its small molecular size, we isolated RBP cDNA from a carp liver cDNA library. Comparison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp RBP has high homology to the other cloned vertebrate RBPs, but it lacks the COOH-terminal tetrapeptide, RNL(S)L, which is most likely involved in the interaction with transthyretin in mammalian RBPs. In addition, the primary structure of carp RBP contains two consensusN-linked glycosylation sites that represent a unique feature. We have obtained experimental evidence, by in vitro and in vivo expression experiments, that both sites are indeed glycosylated. We have also characterized the protein as a complex type N-linked glycoprotein by lectin binding assay, neuraminidase and endoglycosidase H and F digestion. Inhibition of glycosylation by tunicamycin treatment of transfected cells caused a great reduction of RBP secretion. Since kidney filtration of anionic proteins is less than half that of neutral protein of the same size, this finding strongly suggests that the amount of carp RBP filtration through kidney glomeruli may be reduced by a glycosylation-dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp RBP as secretory protein is the presence of a nonconserved NH2-terminal hydrophobic domain, which functions as an insertion signal but is not cleaved cotranslationally and remains in the secreted RBP.

Cytokine gene expression in intestine of rat during the postnatal developmental period: Increased IL-1 expression at weaning

In the present study we have investigate whether cytokines are constitutively and differently expressed in intestine during the differentiative processes that take place at weaning. We have analyzed the expression of IL-1 beta, IL-2, IL-4 and IFN gamma by polymerase chain reaction in Peyers patches (PP) and in intestine deprived of PP (I-PP) of rats from 16 to 30 days of age. The results showed a constitutive and marked expression of the cytokines already before weaning, with the exception of IL-2 in PP and IFN gamma in I-PP. IL-beta was the only cytokine to show a different expression at various ages with an initial increase at 19 days and a further elevation at 21 days when intestinal epithelium passes through major differentiative stages, suggesting an involvement of this cytokine in intestinal development. We have also tested whether treatment of rats with the immunosuppressor cyclosporin A (CsA) could affect intestinal differentiation. The results showed that only some markers of differentiation were affected (proliferation of staminal crypt cells and length of crypts). This was probably due to a direct effect rather than an immunomediated effect of CsA, since treatment of three intestinal cell lines (Caco-2, HT-29, FRIC) with CsA indicated that this drug can exert a cytostatic activity on intestinal cells.