A Key Role of Dendritic Cells in Probiotic Functionality

BACKGROUND: Disruption of the intestinal homeostasis and tolerance towards the resident microbiota is a major mechanism involved in the development of inflammatory bowel disease. While some bacteria are inducers of disease, others, known as probiotics, are able to reduce inflammation. Because dendritic cells (DCs) play a central role in regulating immune responses and in inducing tolerance, we investigated their role in the anti-inflammatory potential of probiotic lactic acid bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Selected LAB strains, while efficiently taken up by DCs in vitro, induced a partial maturation of the cells. Transfer of probiotic-treated DCs conferred protection against 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Protection was associated with a reduction of inflammatory scores and colonic expression of pro-inflammatory genes, while a high local expression of the immunoregulatory enzyme indolamine 2, 3 dioxgenase (IDO) was observed. The preventive effect of probiotic-pulsed DCs required not only MyD88-, TLR2- and NOD2-dependent signaling but also the induction of CD4+ CD25+ regulatory cells in an IL-10-independent pathway. CONCLUSIONS/SIGNIFICANCE: Altogether, these results suggest that selected probiotics can stimulate DC regulatory functions by targeting specific pattern-recognition receptors and pathways. The results not only emphasize the role of DCs in probiotic immune interactions, but indicate a possible role in immune-intervention therapy for IBD

Proteomics as a quality control tool of pharmaceutical probiotic bacterial lysate products

Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87–89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots

Oral Probiotic Control Skin Inflammation by Acting on Both Effector and Regulatory T Cells

Probiotics are believed to alleviate allergic and inflammatory skin disorders, but their impact on pathogenic effector T cells remains poorly documented. Here we show that oral treatment with the probiotic bacteria L. casei (DN-114 001) alone alleviates antigen-specific skin inflammation mediated by either protein-specific CD4+ T cells or hapten-specific CD8+ T cells. In the model of CD8+ T cell-mediated skin inflammation, which reproduces allergic contact dermatitis in human, inhibition of skin inflammation by L. casei is not due to impaired priming of hapten-specific IFNγ-producing cytolytic CD8+ effector T cells. Alternatively, L. casei treatment reduces the recruitment of CD8+ effector T cells into the skin during the elicitation (i.e. symptomatic) phase of CHS. Inhibition of skin inflammation by L. casei requires MHC class II-restricted CD4+ T cells but not CD1d-restricted NK-T cells. L casei treatment enhanced the frequency of FoxP3+ Treg in the skin and increased the production of IL-10 by CD4+CD25+ regulatory T cells in skin draining lymph nodes of hapten-sensitized mice. These data demonstrate that orally administered L. casei (DN-114 001) efficiently alleviate T cell-mediated skin inflammation without causing immune suppression, via mechanisms that include control of CD8+ effector T cells and involve regulatory CD4+ T cells. L. casei (DN-114 001) may thus represent a probiotic of potential interest for immunomodulation of T cell-mediated allergic skin diseases in human

CARD15/NOD2 Is Required for Peyer's Patches Homeostasis in Mice

BACKGROUND: CARD15/NOD2 mutations are associated with susceptibility to Crohn's Disease (CD) and Graft Versus Host Disease (GVHD). CD and GVHD are suspected to be related with the dysfunction of Peyer's patches (PP) and isolated lymphoid follicles (LFs). Using a new mouse model invalidated for Card15/Nod2 (KO), we thus analysed the impact of the gene in these lymphoid formations together with the development of experimental colitis. METHODOLOGY/PRINCIPAL FINDINGS: At weeks 4, 12 and 52, the numbers of PPs and LFs were higher in KO mice while no difference was observed at birth. At weeks 4 and 12, the size and cellular composition of PPs were analysed by flow cytometry and immunohistochemistry. PPs of KO mice were larger with an increased proportion of M cells and CD4(+) T-cells. KO mice were also characterised by higher concentrations of TNFalpha, IFNgamma, IL12 and IL4 measured by ELISA. In contrast, little differences were found in the PP-free ileum and the spleen of KO mice. By using chamber experiments, we found that this PP phenotype is associated with an increased of both paracellular permeability and yeast/bacterial translocation. Finally, KO mice were more susceptible to the colitis induced by TNBS. CONCLUSIONS: Card15/Nod2 deficiency induces an abnormal development and function of the PPs characterised by an exaggerated immune response and an increased permeability. These observations provide a comprehensive link between the molecular defect and the Human CARD15/NOD2 associated disorders: CD and GVHD

Yersinia pseudotuberculosis Anti-Inflammatory Components Reduce Trinitrobenzene Sulfonic Acid-Induced Colitis in the Mouse

Rectal instillation of trinitrobenzene sulfonic acid (TNBS) induces acute colitis in the mouse. We tested the efficacy of Yersinia pseudotuberculosis anti-inflammatory components in preventing TNBS-triggered colitis. Animals were orally inoculated with virulence-attenuated Yersinia cells (a phoP mutant) prior to TNBS administration. Under these experimental conditions, colonic lesions and tumor necrosis factor alpha mRNA levels were significantly reduced

Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains leads to anti-inflammatory functional food development.

IBD constitute a growing public health concern in western countries. Inflammation is a key parameter involved in many non-communicable chronic diseases. Bacteria with anti-inflammatory properties are lacking in dysbiosis accompanying IBD. Selected strains of probiotic bacteria with anti-inflammatory properties accordingly alleviate symptoms and enhance treatment of ulcerative colitis in clinical trials. Such properties are also found in selected strains of dairy starters, including Propionibacterium freudenreichii and Lactobacillus delbrueckii, which are widely ingested. We investigated the possibility to develop a fermented dairy product, combining both starter and probiotic abilities of both lactic acid and propionic acid bacteria, designed to extend remissions in IBD patients.Methods & ResultsWe developed a two-strain P. freudenreichii and L. delbrueckii-fermented experimental pressed cheese using strains previously selected for their anti-inflammatory properties. This cheese was made in fully mastered conditions and contained only the two selected strains. It was consumed daily by mice during five days before induction of acute colitis by trinitrobenzenesulfonic acid (TNBS). Outcomes of colitis induction were then monitored in mice consuming this cheese or a placebo dairy matrix.For the first time, consumption of a designer P. freudenreichii and Lactobacillus delbrueckii pressed cheese was shown to protect mice against experimental colitis, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages. As a control, the corresponding sterile dairy matrix failed to afford such protection.ConclusionWe eat more bacteria through daily consumption of fermented dairy products then through functional food supplements. This offers a tremendous level to modulate the digestive tract through the intake of desired bacteria. This work reveals the probiotic potential of a mixture of lactic and propionic selected starters. A “two-in-one” ability is revealed for bacteria able both to produce a fermented dairy product and to modulate inflammation. Such an innovation opens new avenues for the reverse engineering development of anti-inflammatory fermented foods designed for target populations with IBD, and has provided evidences leading to an ongoing pilot clinical study in ulcerative colitis patients.This work was financed by the French National Agency for Research

Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains leads to anti-inflammatory functional food development.

IBD constitute a growing public health concern in western countries. Inflammation is a key parameter involved in many non-communicable chronic diseases. Bacteria with anti-inflammatory properties are lacking in dysbiosis accompanying IBD. Selected strains of probiotic bacteria with anti-inflammatory properties accordingly alleviate symptoms and enhance treatment of ulcerative colitis in clinical trials. Such properties are also found in selected strains of dairy starters, including Propionibacterium freudenreichii and Lactobacillus delbrueckii, which are widely ingested. We investigated the possibility to develop a fermented dairy product, combining both starter and probiotic abilities of both lactic acid and propionic acid bacteria, designed to extend remissions in IBD patients.Methods & ResultsWe developed a two-strain P. freudenreichii and L. delbrueckii-fermented experimental pressed cheese using strains previously selected for their anti-inflammatory properties. This cheese was made in fully mastered conditions and contained only the two selected strains. It was consumed daily by mice during five days before induction of acute colitis by trinitrobenzenesulfonic acid (TNBS). Outcomes of colitis induction were then monitored in mice consuming this cheese or a placebo dairy matrix.For the first time, consumption of a designer P. freudenreichii and Lactobacillus delbrueckii pressed cheese was shown to protect mice against experimental colitis, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages. As a control, the corresponding sterile dairy matrix failed to afford such protection.ConclusionWe eat more bacteria through daily consumption of fermented dairy products then through functional food supplements. This offers a tremendous level to modulate the digestive tract through the intake of desired bacteria. This work reveals the probiotic potential of a mixture of lactic and propionic selected starters. A “two-in-one” ability is revealed for bacteria able both to produce a fermented dairy product and to modulate inflammation. Such an innovation opens new avenues for the reverse engineering development of anti-inflammatory fermented foods designed for target populations with IBD, and has provided evidences leading to an ongoing pilot clinical study in ulcerative colitis patients.This work was financed by the French National Agency for Research

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Is cheese a good delivery vehicle for the anti-inflammatory [i]Propionibacterium freudenreichii[/i] probiotic?

Propionibacterium freudenreichii is a beneficial Actinobacterium, (GRAS & QPS status). It is exploited in the food industry for vitamins production and in Swiss type cheeses manufacturing, such as Emmental (1). Dairy propionibacteria have several probiotic features, including strain-dependent anti-inflammatory properties (Interleukine 10 induction by immune cells) (2). Previous studies showed surface protein (S- layer proteins) are involved in these properties (2), particularly the protein SlpB (Data no published).A monoxenic cheese, fermented by P.freudenreichii only, was developed. Consumption of this cheese reduced severity of chemically induced colitis in mice model (3). This shows the probiotic potential of P.freudenreichii for helping of Inflammatory Bowel Diseases (IBD) treatment. We assume cheese may be the best delivery vehicle of anti-inflammatory potential of P.freudenreichii to digestive tract. Indeed, we presume cheese enhances propionibacteria resistance to stress conditions during digestion. The presence of caseins matrix may exert a protective effect, allowing undamaged bacteria and surface proteins to reach the digestive tract.The experimental strategy chosen was to evaluate propionibacteria resistance and slpB protein proteolysis during in vitro digestion, in different delivery vehicles increasingly concentrated in dairy proteins: culture on milk ultrafiltrate (UF), culture on milk and a monoxenic cheese. P.freudenreichii viability and S-layer proteins susceptibility to proteolysis was followed in the different matrix during in vitro digestion. Two anti-inflammatory strains were used for this experiment: CIRM-BIA 129 and CIRM-BIA 118 (4).This will be correlated with in vivo experiments in the perspective to promote dairy products fermented by propionibacteria as probiotics for target populations