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Backgrounds and Aims. Inflammatory Bowel Diseases (IBD), including Ulcerative Colitis (UC), coincide with alterations in the gut microbiota. Consumption of immunomodulatory strains of probiotic bacteria may induce or prolong remission in UC patients. Fermented foods, including cheeses, constitute major vectors for bacteria consumption. New evidences revealed anti-inflammatory effects in selected strains of Propionibacterium freudenreichii. We thus hypothesized that consumption of a functional cheese, fermented by such a strain, may exert a positive effect on IBD. Methods. We investigated the impact of cheese fermented by P. freudenreichii on gut inflammation. We developed an experimental single-strain cheese solely fermented by a selected immunomodulatory strain of P. freudenreichii, CIRM-BIA 129. We moreover produced, in industrial conditions, an Emmental cheese using the same strain, in combination with Lactobacillus delbrueckii CNRZ327 and Streptococcus thermophilus LMD-9, as starters. Consumption of both cheeses was investigated with respect to prevention of Dextran Sodium Sulphate (DSS)-induced colitis in mice. Results. Consumption of the single-strain experimental cheese, or of the industrial Emmental, both fermented by P. freudenreichii CIRM-BIA 129, reduced severity of subsequent DSS-induced colitis, weight loss, disease activity index and histological score. Both treatments, in a preventive way, reduced small bowel Immunoglobulin A (IgA) secretion, restored occludin gene expression and prevented induction of Tumor Necrosis Factor &alpha, (TNF&alpha, ), Interferon &gamma, (IFN&gamma, ) and Interleukin-17 (IL-17). Conclusions. A combination of immunomodulatory strains of starter bacteria can be used to manufacture an anti-inflammatory cheese, as revealed in an animal model of colitis. This opens new perspectives for personalised nutrition in the context of IBD.

Propionibacterium freudenreichii CIRM-BIA 129 (Pf) est un levain fromager et détient plusieurs potentialités probiotiques, dont l’immunomodulation. Cette propriété résulte de la production de divers métabolites bénéfiques et de la présence de protéines de surface S-layer (Slps). Cette propriété « 2-en-1 », c’est-à-dire à la fois fermentaire et probiotique, fait de Pf un bon candidat pour développer des fromages fonctionnels, afin de prévenir les maladies inflammatoires intestinales. L’objectif de cette thèse était d’étudier l’impact de la matrice fromagère sur les propriétés immunomodulatrices de Pf, via ses protéines Slps, par comparaison à une culture sur ultrafiltrat de lait (UF). Les études conduites in vitro suggèrent que la vectorisation par le fromage module la viabilité de Pf lors de la digestion ainsi que dans le côlon. Nous avons montré aussi que la matrice fromagère limite la protéolyse des protéines Slps lors de la digestion gastrique et duodénale. L’étude in vivo a montré que les deux matrices de vectorisation, le fromage et l’UF, présentent des effets anti-inflammatoires différents. Finalement, un emmental a été fabriqué en sélectionnant des souches immunomodulatrices de bactéries lactiques et propioniques. Son ingestion a protégé des souris d’une colite chimiquement induite. Ce travail montre que les propriétés immunomodulatrices de Pf sont matricedépendantes, tout comme elles sont souchedépendantes. Il souligne de plus le potentiel offert par l’utilisation d’un fromage de type emmental probiotique comme un levier pour prévenir les maladies inflammatoires intestinales., Propionibacterium freudenreichii CIRM-BIA 129 (Pf) is a cheese starter and has several probiotic potentialities, including immunomodulation. This property results from the production of various beneficial metabolites, and from the presence of S-layer surface proteins (Slps). The "2-in-1" property, i.e. both fermentative and probiotic properties, makes Pf a suitable candidate to develop functional cheeses, in order to prevent inflammatory bowel disease. The aim of this thesis was to study the impact of the cheese matrix on the immunomodulatory properties of Pf, mediated by its Slps proteins, compared to a culture on milk ultrafiltrate (MUF). In vitro studies suggest that Pf viability is affected by the delivery vehicle, cheese matrix or MUF during digestion as well as in the colon. We also showed that the cheese matrix limits Slps proteins proteolysis during gastric and duodenal digestion. The in vivo study showed that the two delivery vehicles, cheese and MUF, display different antiinflammatory effects. Finally, an industrial-scale Emmental cheese was manufactured using selected immunomodulatory strains of lactic and propionic acid bacteria. It also highlights the potential of using Emmental probiotic cheese as a lever to prevent inflammatory bowel disease.

Probiotic microorganisms may affect human health, partly by modulating the immune response. They are commensals, part of the natural human microbiota, or provided within fermented foods or within food supplements. As an example, some of them colonize the digestive tract at early stages of life and play a key role in the maturation of the immune system. Accordingly, a lack of implantation of these beneficial bacteria correlates with immune and inflammatory disorders, which constitute a growing health concern and are gradually understood, in the light of variations of the gut microbiota structure. Potential applications of probiotic bacteria further involve the treatment of diarrhoea caused by rotavirus, of allergy, of eczema, of irritable bowel syndrome and of inflammatory bowel diseases. The mechanisms underlying interactions between probiotic bacteria and the host thus constitute a hot topic. Surface molecules of probiotic bacteria play a central role in these interactions. Referred to as Microbe-associated Molecular Pattern (MAMP), these key molecules are recognized by corresponding pattern recognition receptors (PRR), present at the surface of the host cells. The role of MAMPs includes adhesion and modulation of the gut associated immune system, prior to that of the systemic immune system, by mediating a host/bacteria cross-talk. MAMPs include surface proteins, glycoproteins, lipoproteins, pilins, flagellins, as well as surface associated polysaccharides. Host PRRs, which can perceive MAMPs, include Toll-like receptors (TLR), as well as the C type lectin DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). They may be present at the surface of epithelial cells, and of dendritic cells. This oral presentation will illustrate the key role of probiotics’ MAMPs through examples of bifidobacteria, lactobacilli, and of propionibacteria. It will present textbook cases spanning from molecular to in vivo studies. Scientific data acquired during the last decade indeed place interactions between MAMP and PRR at the Centre of Probiotic/Host Interaction. The corresponding mechanisms are currently elucidated.

International audience; Propionibacterium freudenreichii is a beneficial bacterium with documented effects on the gut microbiota and on inflammation. Its presence within the animal and human intestinal microbiota was correlated with immunomodulatory effects, mediated by both propionibacterial surface components and by secreted metabolites. It is widely implemented, both in the manufacture of fermented dairy products such as Swiss-type cheeses, and in the production of probiotic food complements, under the form of freeze-dried powders. The bottleneck of this drying process consists in the limited survival of bacteria during drying and storage. Protective pre-treatments have been applied to other bacteria and may, in a strain-dependent manner, confer enhanced resistance. However, very little information was yet published on P. freudenreichii adaptation to freeze-drying. In this report, an immunomodulatory strain of this probiotic bacterium was cultured under hyperosmotic constraint in order to trigger osmoadaptation. This adaptation was then combined with acid or thermal pre-treatment. Such combination led to accumulation of key stress proteins, of intracellular compatible solute glycine betaine, to modulation of the propionibacterial membrane composition, and to enhanced survival upon freeze-drying. This work opens new perspectives for efficient production of live and active probiotic propionibacteria.

International audience; Context and objective: Diplotaxis harra (Forssk.) Boiss. (Brassicaceae) is traditionally used as an antidiabetic, anti-inflammatory or anticancer agent. In these pathologies, the glycogen synthase kinase 3 b (GSK3b) is overactivated and represents an interesting therapeutic target. Several flavonoids can inhibit GSK3b and the purpose of this study was to search for the compounds in Diplotaxis harra which are able to modulate GSK3b. Materials and methods: Methanol extracts from D. harra flowers were prepared and the bio-guided fractionation of their active compounds was performed using inflammatory [protease-activated receptor 2 (PAR2)-stimulated IEC6 cells] and cancer (human Caco-2 cell line) intestinal cells. 50–100 lg/mL of fractions or compounds purified by HPLC were incubated with cells whose inhibited form of GSK3b (Pser9 GSK3b) and survival were analyzed by Western blot at 1 h and colorimetric assay at 24 h, respectively. LC-UV-MS profiles and MS-MS spectra were used for the characterization of extracts and flavonoids-enriched fractions, and the identification of pure flavonoids was achieved by MS and NMR analysis. Results: The methanol extract from D. harra flowers and its flavonoid-enriched fraction inhibit GSK3b in PAR2-stimulated IEC6 cells. GSK3b inhibition by the flavonoid-enriched D. harra fraction was dependent on PKC activation. The flavonoid-enriched D. harra fraction and its purified compound isorhamnetin-3,7- di-O-glucoside induced a 20% decrease of PAR2-stimulated IEC6 and Caco-2 cell survival. Importantly, normal cells (non-stimulated IEC6 cells) were spared by these treatments. Conclusion: This work indicates that flavonoids from D. harra display cytotoxic activity against inflammatory and cancer intestinal cells which could depend on GSK3b inhibition.

International audience; Propionibacterium freudenreichii is a beneficial bacterium, used as a cheese starter, which presents versatile probiotic properties. These properties are strain-dependent. We hypothesized they may also be delivery vehicle-dependent. In this study, we thus explored in healthy piglets how the cheese matrix affects the immunomodulatory properties of P. freudenreichii. During 2 weeks, three groups of weaned piglets consumed, respectively, P. freudenreichii as a liquid culture (PF-culture), P. freudenreichii under the form of a cheese (PF-cheese), or a control sterile cheese matrix (Cheesematrix). The in vivo metabolic activity of P. freudenreichii was assessed by determining short chain fatty acids (SCFA) concentration and bifidobacteria population in feces. Whatever the delivery vehicle, P. freudenreichii was metabolically active in piglets’ colon and enhanced both bifidobacteria and SCFA in feces. P. freudenreichii consumption decreased the secretion of TNFa and of IL-10 by peripheral blood mononuclear cells (PBMC). It did not alter IL-10, IFNg, IL-17, and TNFa secretion in mesenteric lymph node immune cells (MLNC). PF-cheese enhanced significantly Treg phenotype, while PF-culture decreased significantly Th17 phenotype in PBMC and MLNC. Remarkably, only PF-cheese induced an increase of Th2 phenotype in PBMC and MLNC. Ex vivo stimulation of PBMC and MLNC by Lipopolysaccharides and Concanavalin A emphasized the difference in the immunomodulatory responses between PF-culture and PF-cheese group, as well as between PBMC and MLNC. This study shows the importance to consider the delivery vehicle for probiotic administration. It confirms the anti-inflammatory potential of P. freudenreichii. It opens new perspectives for the use propionibacteria-fermented products as preventive agents for inflammatory bowel diseases and intestinal infectious diseases.

Probiotics already in your fridge?. 14.congrès national de la Société Française de Microbiologie (SFM)

INTRODUCTION Propionibacterium freudenreichii is an emergent probiotic bacterium, belonging to dairy propionibacteria, within the group of Actinomycetales, and possessing the GRAS (Generally Recognized As Safe, USA, FDA) and QPS (Qualified Presumption of Safety, EFSA, European Union) status. It is a traditional Swiss-type cheeses starter and an emergent probiotic, exerting several beneficial effects, including anti-inflammatory modulation of gut inflammation. This feature relies on several metabolites and on surface proteins including in particular the surface protein SlpB. METHOD In the present study, we assessed first the impact of proteolytic degradation of SlpB on its immunomodulatory effect on colon epithelial HT-29 cells. Intact P. freudenreichii cells, purified surface layer proteins (SLPs), as well as trypsin-proteolyzed SLPs, were used to stimulate HT-29 cells, in the presence or absence of pro-inflamatory LPS. Cytokines were then quantified to evaluate the inflammatory response. We then investigated the potential of cheese matrix as a delivery vehicle of anti-inflammatory P. freudenreichii MAMP to the colon, by comparing dairy liquids matrices to a solid Swiss-type cheese matrix, using two in vitro digestion models. Integrity of SlpB was monitored using western blotting. RESULTS In this study, we firstly investigated the relevance to avoid SlpB digestive proteolysis, by comparing the effect of i) P. freudenreichii CIRM-BIA 129, ii) its native SLPs, or iii) peptides resulting from SLPs stimulations, with respect to modulation of HT-29 cells response to lipopolysaccharide (LPS) challenge. The anti-inflammatory effect exerted by P. freudenreichii CIRM-BIA 129 and by native surface proteins (SLPs) on HT-29 cells was abolished by digestive proteolysis. This result confirmed the importance to protect immunomodulatory surface proteins from digestive proteolysis in order to allow gut immune system modulation. Thus, we examined the effect of dairy matrices on P. freudenreichii viability and on SlpB integrity during digestion. In comparison with liquid matrices, the cheese matrix provided an enhanced tolerance to digestive stresses and protection of SlpB towards proteolysis, during two in vitro digestion models: static and dynamic. CONCLUSION These in vitro results provide new insights into the matrix effect on P. freudenreichii probiotic functionalities. The cheese matrix offers significant protection against the digestion stresses by enhancing survival, and by protecting surface layer proteins from proteolysis. However, in vivo investigations are necessary to study the matrix effect during digestion, to follow SLPs expression and to evaluate the matrix effect on immunomodulation by P. freudenreichii within the gut. Such data accordingly will allow the development of new functional foods for the delivery of P. freudenreichii to the gastrointestinal tract of humans in the aim to help prevention or treatment of life-style related diseases with an inflammatory component.

Combining selected immunomodulatory strains to develop an anti-inflammatory cheese. WEBINAIRE Aliments Fermentés 24-25 mai 2018

International audience; Propionibacterium freudenreichii is a well-known ripening starter used in the manufacture of Swiss-type cheeses. It exerts several strain-dependent probiotic features, such as the modulation of apoptosis, of immunity and of microbiota, at the gut level. Promoting fermented dairy products as functional foods is a promising manner to reduce risks of specific life-style related diseases, including colitis. In this aim, the selection of starters should be made on both technological and probiotic criteria. Previously, a single-strain cheese fermented exclusively by P. freudenreichii CIRM-BIA 129 revealed a protective effect against induced colitis in mice. Nonetheless, the effect of delivery matrix on immunomodulatory properties of P. freudenreichii is poorly understood. In addition, data on the effect of fermented cheese on healthy subject or animals are scarce. We thus compared three groups of piglets: 1) fed with a sterilized cheese matrix, 2) fed with a P. freudenreichii CIRM-BIA 129-fermented milk ultrafiltrate and 3) fed with a P. freudenreichii CIRM-BIA 129-single-strain cheese. The immune cells from mesenteric lymph nodes (MLN) were extracted and different analysis were carried out: 1) the basal secretion of cytokines TNFα and IFNγ were assessed by ELISA; 2) the expression of transcription factors Tbet and GATA3, marker of lymphocytes Th1 and Th2 respectively were assessed by RT-PCR, 3); then cells were stimulated ex vivo with several stimuli (LPS and Concanavalin A (ConA) and surface proteins of P. freudenreichii (Slps)), TNFα and IFNγ secretion was measured. The ingestion of P. freudenreichii, whatever the delivery vehicle, doesn’t influence the basal secretion of TNFα and IFNγ. However, the ingestion of P. freudenreichii in the form of cheese enhances Th2 response (tolerance response) by increasing GATA3 expression in MLN cells. In addition, MLN cells from different piglet groups don’t respond similarly to ex vivo stimuli. The ingestion of P. freudenreichii in the form of cheese inhibites the TNFα secretion initiated by LPS simulation and enhanced IFNγ response during co-stimulation LPS and Slps or ConA and Slps. Altogether, in healthy piglets, the cheese matrix modulated the immunomodulatory properties of P. freudenreichii in intestinal immunity which lead to a better reaction to exogenous stimulus.

Objectif : Identifier des bactéries qui possèdent à la fois des propriétés techno-fonctionnelles (levain) et des propriétés bio-fonctionnelles (probiotiques) pour orienter les fermentations vers des aliments fermentés probiotiques répondant aux besoins de populations cibles. Contexte . Les bactéries en transit dans notre tractus digestif ont un impact sur des facteurs clés de notre santé : immunité, inflammation, digestion, motilité, sensibilité et perméabilité de l’intestin. . Certains microorganismes sélectionnés probiotiques exercent un effet bénéfique sur l’un de ces paramètres clés, lorsqu’il est perturbé, mais n’ont pas de potentiel techno-fonctionnel reconnu. . Nous consommons beaucoup de bactéries, dans nos aliments fermentés, qui sont sélectionnées pour leurs propriétés techno-fonctionnelles, sans que leur potentiel bio-fonctionnel soit connu. Résultats publiés : . Le laboratoire INRA STLO a mis en évidence des propriétés bio-fonctionnelles, souches-dépendantes, chez la bactérie Propionibacterium freudenreichii utilisée comme levain d’affinage fromager. . Le laboratoire INRA MICALIS a fait de même chez les bactéries Lactobacillus delbrueckii et Streptococcus thermophilus utilisées comme levain des yaourts et de certains fromages. . STLO a identifié les molécules responsables de l’effet anti-inflammatoire de P. freudenreichii, a montré leur production au sein du fromage. . La combinaison de souches sélectionnées de P. freudenreichii et de L. delbrueckii a permis à ces deux équipes de mettre au point un fromage efficace in vivo à prévenir la colite. Perspectives de valorisation pour les entreprises d’une part, et de projets de recherche, d’autre part 9 lignes maximum . L’indentification de souches « 2-en-1 » au sein des espèces bactériennes utilisées en fermentation ouvre la voie à la mise au point d’aliments fermentés fonctionnels. . Bactéries propioniques et lactiques sélectionnées permettent d’envisager une large gamme d’aliments fonctionnels. . Un premier fromage fonctionnel immunomodulateur est en essai clinique dans le cadre d’un projet régional précompétitif . L’indentification des mécanismes immunomodulateurs impliqués offre des perspectives de développement de molécules actives

Development of a anti inflammatory cheese. Cheese symposium - The 2nd Symposium in Future Dairy Manufacturing

Propionibacterium freudenreichii est une bactérie Gram-positive bénéfique, traditionnellement utilisée comme levain d’affinage fromager, qui bénéficie du statut GRAS (Generally Recognized As Safe). P. freudenreichii a révélé un effet immunomodulateur qui a été confirmé in vivo par la capacité à protéger des souris d’une colite aigüe induite. L’effet anti-inflammatoire est cependant hautement souche-dépendant. Il est dû, au moins en partie, à des composés de surface clés qui favorisent ces effets probiotiques. Les bactéries Gram- positives, y compris P. freudenreichii, peuvent être recouvertes d’une couche extérieure protéique, appelée « surface-layer », paracristalline, et formée par l’autoassemblage de protéines dites de S-layer (Slps). Les Slps, dans différentes bactéries, sont impliquées dans plusieurs caractéristiques probiotiques, telles que l’adhésion aux cellules de l’hôte et au mucus, la persistance dans l’intestin, ou encore l’immunomodulation. Le but de cette étude est d’étudier, chez une souche probiotique de P. freudenreichii, la protéine de surface qui joue le principal rôle dans les interactions probiotiques avec l’hôte. La souche P. freudenreichii CIRM-BIA 129, récemment reconnue comme immunomodulatrice prometteuse, possède plusieurs protéines de surface Slps, y compris SlpB. Dans la présente étude, l’inactivation du gène correspondant, dans la souche mutante CB129ΔslpB, a provoqué une baisse drastique de l’adhésion aux cellules intestinales épithéliales HT-29, confirmant le rôle clé des Slps dans l’adhésion aux cellules. P. freudenreichii CIRM-BIA 129. Nous avons étudié la réponse immune de cellules HT-29 stimulées par le sauvage ou par le mutant. Le sauvage induisait principalement l’expression de la cytokine immunomodulatrice IL-10. Le mutant induisait, de façon intéressante, moins d’IL-10, mais plus d’IL-8. Afin d’évaluer the rôle de cette protéines clé SlpB dans les effets antiinflammatoires de P. freudenreichii, nous avons testé ces deux souches dans un modèle murin de mucosite induite par le 5-Fluorouracile. La consommation de la souche sauvage prévenait la perte de poids, préservait l’architecture des villosités, ainsi que la densité des granules de sécrétion au sein des cellules de Paneth, chez les souris atteintes de mucosite. Cependant, le mutant n’avait pas cet effet protecteur. Cette étude a mis en évidence l’implication de la protéine SlpB de P. freudenreichii dans les propriétés anti-inflammatoires de cette bactérie et confirme le rôle versatile joué par les Slps dans les interactions probiotique/hôte., Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese ripening starter, with the GRAS status (Generally Recognized As Safe). P. freudenreichii has revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. The anti- inflammatory effect is however highly strain-dependent and due, at least in part, to key surface compounds favouring probiotic effects. Gram-positive bacteria, including P. freudenreichii, can be covered with an external proteinaceous layer called a surface-layer paracrystalin layer and formed by the self-assembly of surface-layer-proteins (Slps). Slps were shown, in different bacteria, to be involved in several probiotics traits, such as adhesion to host cells and mucus, persistence within the gut, or immunomodulation. The aim of this study is to investigate, in a P. freudenreichii probiotic strain, the surface protein that plays the main role in the probiotic interaction with the host. The P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties and possesses several Slps, including SlpB. In the presented work, inactivation of the corresponding gene, CB129ΔslpB a mutant strain, caused a drastic decrease in adhesion to intestinal epithelial HT-29 cells, further evidencing the key role of Slps in cell adhesion. we investigated immune response of HT-29 cells towards P. freudenreichii CIRM-BIA 129 and CB129ΔslpB. The wild type strain mainly induced expression of the immunomodulatory IL-10 by the cells. Interestingly, the mutant strain induced decreased expression of IL-10, yet increased expression of IL-8. To finally evaluate the role of the key surface protein SlpB in the ant-inflammatory property of P. freudenreichii, we examined both strains in a murine mucositis model induced by 5-Fluorouracil (5-FU). P. freudenreichii wild type strain was able to prevent weight loss, to preserve villous architecture and to preserve secretory granules density inside Paneth cells in mucositis–affected mice. By contrast, the mutant CB129ΔslpB strain failed to exert such protective effect. This study evidenced the involvement of P. freudenreichii SlpB protein in its anti-inflammatory properties and confirmed the versatile role fulfilled by Slps in probiotic host interactions.

Mucositis is a clinically important gastrointestinal inflammatory infirmity, generated by antineoplastic drugs cytotoxic effects. The inflammatory process caused by this disease frequently leads to derangements in the alimentary tract and great malaise for the patient. Novel strategies are necessary for its prevention or treatment, as currently available treatments of mucositis have several limitations in relieving its symptoms. In this context, several research groups have investigated the use of probiotic bacteria, and in particular dairy bacterial strains. Compelling evidences reveal that milk fermented by certain probiotic bacteria has the capacity to ameliorate intestinal inflammatory disorders. In addition, innovative probiotic delivery strategies, based on probiotics incorporation into protective matrices, such as whey proteins, were able to increase the therapeutic effect of probiotic strains by providing extra protection for bacteria against environmental stresses. Therefore, in this study, we evaluated the role of the whey protein isolate (WPI), when added to skim milk fermented by Lactobacillus casei BL23 (L. casei BL23) or by Propionibacterium freudenreichii CIRM-BIA138 (P. freudenreichii 138), as a protective matrix against in vitro stress challenges. In addition, we investigated the therapeutic effect of these fermented beverages in a murine model of mucositis induced by 5-Fluorouracil (5-FU). Our results demonstrated that milk supplementation with 30% (w/v) of WPI increases the survival rate of both strains when challenged with acid, bile salts, high temperature and cold storage stresses, compared to fermented skim milk without the addition of WPI. Moreover, treatment with the probiotic beverages prevented weight loss and intestinal damages in mice receiving 5-FU. We conclude that the presence of WPI maximizes the anti-inflammatory effects of L. casei BL23, but not for P. freudenreichii 138, suggesting that whey protein enhancement of probiotic activity might be strain-dependent.

Dysregulation of intestinal microflora is linked to inflammatory disorders associated with compromised immunosuppressive functions of Foxp3+ T regulatory (Treg) cells. Although mucosa-associated commensal microbiota has been implicated in Treg generation, molecular identities of the “effector” components controlling this process remain largely unknown. Here, we have defined Bifidobacterium bifidum as a potent inducer of Foxp3+ Treg cells with diverse T cell receptor specificity to dietary antigens, commensal bacteria, and B. bifidum itself. Cell surface β-glucan/galactan (CSGG) polysaccharides of B. bifidum were identified as key components responsible for Treg induction. CSGG efficiently recapitulated the activity of whole bacteria and acted via regulatory dendritic cells through a partially Toll-like receptor 2–mediated mechanism. Treg cells induced by B. bifidum or purified CSGG display stable and robust suppressive capacity toward experimental colitis. By identifying CSGG as a functional component of Treg-inducing bacteria, our studies highlight the immunomodulatory potential of CSGG and CSGG-producing microbes.

The human gut microbiota makes fundamental contributions to host metabolism and immune system. Therefore, perturbations in its composition, a process known as dysbiosis, have an important role in the development of several chronicle diseases, mainly intestinal inflammatory disorders. Culture-independent molecular methods are allowing scientific community to uncover substantive findings, thus giving a more detailed description of the human intestinal microbiota. This chapter presents a review on current metagenomic approaches, based on next-generation sequencing technologies, for investigating bacterial taxonomic classification and predictive function associated with the human gut in health and disease. In this context, we describe recent studies that have been trying to elucidate important alterations in microbiome composition across individuals according to delivery mode, aging, diet and medication that might be linked to susceptibility to immune-mediated diseases. A description of the main bacterial taxa and genes acting in dysbiosis during inflammation, focusing on chronic inflammatory bowel diseases and colorectal cancer, is also explored in this chapter.

Aliment fonctionnel : De l'emmental aux propriétés anti-inflammatoire

Some Gram-positive bacteria, including probiotic ones, are covered with an external proteinaceous layer called a surface-layer. Described as a paracrystalline layer and formed by the self-assembly of a surface-layer-protein (Slp), this optional structure is peculiar. The surface layer per se is conserved and encountered in many prokaryotes. However, the sequence of the corresponding Slp protein is highly variable among bacterial species, or even among strains of the same species. Other proteins, including surface layer associated proteins (SLAPs), and other non-covalently surface-bound proteins may also be extracted with this surface structure. They can be involved a various functions. In probiotic Gram-positives, they were shown by different authors and experimental approaches to play a role in key interactions with the host. Depending on the species, and sometime on the strain, they can be involved in stress tolerance, in survival within the host digestive tract, in adhesion to host cells or mucus, or in the modulation of intestinal inflammation. Future trends include the valorization of their properties in the formation of nanoparticles, coating and encapsulation, and in the development of new vaccines.

Introduction:Inflammatory bowel disease (IBD) constitute a growing public health concern in western countries. Bacteria with anti-inflammatory properties are lacking in the 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 such as Propionibacterium freudenreichii and Lactobacillus delbrueckii. We thus 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:We developed a single-strain L. delbrueckii-fermented milk and a two-strain P. freudenreichii and L. delbrueckii-fermented experimental pressed cheese using strains previously selected for their anti-inflammatory properties.Results:Consumption of these experimental fermented dairy products protected mice against TNBS-induced 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.Discussion:This work reveals the probiotic potential of this bacterial mixture, in the context of fermented dairy products. It opens new perspectives 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. Together, the results of this study show the feasibility of the development of fermented milk and cheese with anti-inflammatory properties. The bacteria used are responsible for these anti-inflammatory properties and at the same time assure fermentation, in contrast to probiotic dairy products available today which rely on different bacteria for fermentation and for immune modulation. Accordingly, Lactobacillus casei BL23 was shown to attenuate mice colitis when provided in a fermented milk, yet not in a nutrient-free buffer [43]. Thus, the designed/tailored functional fermented dairy product is more than a food probiotic carrier [44]. Designer fermented milks and cheeses, developed by a reverse engineering strategy using such “2-in-1” strains, open new perspectives for clinical trials on IBD such as ulcerative colitis, in conjunction with usual treatments. This work has provided evidences leading to an ongoing pilot clinical study in IBD patients. This work was part of the “SURFING” project, Starter SURFace against INflammation of the Gut, Grant Number: ANR-2010-ALIA-016, financed by the French National Agency for Research. We thank the CRITT Santé Bretagne for assistance in writing the ANR Grant. We also thank Stéphanie Ferreira, Frédéric Texier, Geoffrey Vauvy and Louise-Eva Vandenborght, from Genoscreen, for carrying out and crucial help in metagenomic data analysis. We thank André Bondance and Mark Hent’hal for fruitful advice.

Dairy propionibacteria are used as cheese ripening starters, as biopreservative and as beneficial additives, in the food industry. The main species, Propionibacterium freudenreichii, is known as GRAS (Generally Recognized As Safe, USA, FDA). In addition to another dairy species, Propionibacterium acidipropionici, they are included in QPS (Qualified Presumption of Safety) list. Additional to their well-known technological application, dairy propionibacteria increasingly attract attention for their promising probiotic properties. The purpose of this review is to summarize the probiotic characteristics of dairy propionibacteria reported by the updated literature. Indeed, they meet the selection criteria for probiotic bacteria, such as the ability to endure digestive stressing conditions and to adhere to intestinal epithelial cells. This is a prerequisite to bacterial persistence within the gut. The reported beneficial effects are ranked according to property’s type: microbiota modulation, immunomodulation, and cancer modulation. The proposed molecular mechanisms are discussed. Dairy propionibacteria are described as producers of nutraceuticals and beneficial metabolites that are responsible for their versatile probiotic attributes include short chain fatty acids (SCFAs), conjugated fatty acids, surface proteins, and 1,4-dihydroxy-2-naphtoic acid (DHNA). These metabolites possess beneficial properties and their production depends on the strain and on the growth medium. The choice of the fermented food matrix may thus determine the probiotic properties of the ingested product. This review approaches dairy propionibacteria, with an interest in both technological abilities and probiotic attributes.

The intestinal microbiota composition has a great impact on physiology and health, since commensal bacteria are crucial to maintain homeostasis and immune regulation of the gut. Consequently, disturbances of this microbiota, a process known as dysbiosis, have severe implications for the host health such as the rise of many gastrointestinal (GI) problems; including inflammatory disorders like the Inflammatory Bowel Diseases (IBD), mucositis, as well as colorectal cancer (CRC). The consumption of probiotics with beneficial effects is a promising tool to help treating such disorders. Indeed, they modulate diverse biological mechanisms involved in GI homeostasis and have been commonly used to reduce such disorders. In this chapter, we present the molecular mechanisms triggered by probiotic bacteria to modulate the gut physiology during gastrointestinal disorder and the importance of the gastrointestinal stresses tolerance as a limiting factors for probiotic application. Moreover, we focus on the emergence of functional probiotic foods, which can act as excellent vehicles, by enhancing stress tolerance and providing a protective matrix towards digestive stresses.

Background and Aims: Regulatory Foxp3(+) CD4(+) T cells [Tregs] have been implicated in the control of colitis in T-cell transfer models, yet their ability to regulate colitis induced by innate immunity and the impact of gut inflammation on their fate and function have been poorly documented.Methods: Colitis was induced by dextran sodium sulphate in DEREG transgenic mice. Tregs ablation and transfer experiments showd that Tregs could limit the severity of colitis in B6 mice.Results: Gut inflammation resulted in increased number of Tregs in mesenteric lymph nodes [MLN] and colon lamina propria [LP], although their frequency decreased due to massive concomitant leukocyte infiltration. This coincided at both sites with a dramatic increase in Ki67(+) Tregs which retained proliferative capacity. Gut inflammation resulted in enhanced suppressive function of Tregs in colon lamina propria and neuropillin-1-[NRP1-] Treg in MLN. Real-time polymerase chain reaction analysis and flow cytometry [using IL10-egfp-reporter mice] showed that compared with NRP1(+) Treg, NRP1-Treg express higher levels of IL-10 transcripts and were enriched in IL10-expressing cells both in the steady state and during colitis. Moreover, Treg conversion in vivo from from naive CD4(+) T cells or Treg precursors was impaired in colitic mice. Finally, gut inflammation caused a decrease in intestinal dendritic cells, affecting both CD103(+) CD11b(+) and CD103(+) CD11b-subsets and affected their Treg conversion capacity.Conclusions: Together, our data indicate that non-specific colon inflammation triggers proliferation and suppressive function of Tregs in the lamina propria and MLN, but impairs their de novo conversion from CD4(+) T cells by intestinal dendritic cells.

International audience; Toxoplasma gondii is a widespread intracellular parasite, which naturally enters the organism via the oral route and crosses the intestinal barrier to disseminate. In addition to neuronal and ocular pathologies, this pathogen also causes gut inflammation in a number of animals. This infection-triggered inflammation has been extensively studied in the C57BL/6 mice, highlighting the importance of the immune cells and their mediators in the development of gut pathology. However, despite their importance in inflammation, the role of protease-activated receptors (PAR) was never reported in the context of T.gondii-mediated small intestine inflammation. Using genetically modified mice, we show that PAR(2) plays a pathogenic role in the development of gut inflammatory lesions. We find that PAR(2) controls the innate inflammatory mediators IL-6, KC/CXCL1, PGE(2) as well as neutrophil infiltration in T. gondii-triggered gut damage. These results bring new knowledge on the mechanisms operating in the gut in response to T. gondii infection.

The EU ban on antimicrobial growth promoters (AGP) has initiated a search for nonantibiotic alternatives. It has been demonstrated that certain antibiotics and non-antibiotic alternatives enhance growth by inhibiting inflammatory cells, i.e. neutrophils and macrophages in the intestine. There is very little information on the effect of anti-inflammatory compounds on intestinal epithelial cells, which are known to play an important role in intestinal inflammatory responses. In order to establish this, a porcine intestinal epithelial cell line (IPEC J2) was incubated with an adherent enterotoxigenic E. coli (ETEC) to stimulate inflammation, using a non-pathogenic non-adherent E. coli (EC) as a control. The influence of the presence of the anti-inflammatory compounds Macleaya cordata extract (MCE) and acetylsalicylic acid (ASA) on inflammatory transcriptional responses was studied. ETEC induced a strong inflammatory response as was most evident from the expression of IL-1β, IL-8 and TNF-α, whereas EC induced IL-1β only. Co-incubation with MCE and ASA significantly reduced the responses of the pro-inflammatory cytokines, similarly for IL-1β and TNF-α, but ASA was more effective than MCE in reducing the IL8 response. The present results suggest that the in vivo anti-inflammatory growth promoting effects of AGP and effective alternatives to AGP such as MCE and ASA are not restricted to inflammatory cells and also involve the more abundant enterocytes. This suggests a major role for epithelial cells in growth promotion livestock, and it further supports the notion that effective alternatives to AGP should have anti-inflammatory activity.

ISI Document Delivery No.: 004QQTimes Cited: 0Cited Reference Count: 46Cited References: Pillon Nicolas J., 2011, CHEMICAL RESEARCH IN TOXICOLOGY, V24, P752Awada, Manar Soulage, Christophe O. Meynier, Anne Debard, Cyrille Plaisancie, Pascale Benoit, Berengere Picard, Gregory Loizon, Emmanuelle Chauvin, Marie-Agnes Estienne, Monique Peretti, Noel Guichardant, Michel Lagarde, Michel Genot, Claude Michalski, Marie-CarolineFrench National Research Agency (ANR)[ANR-08-ALIA-002]; ANRThis work was supported by the French National Research Agency (ANR) Grant ANR-08-ALIA-002, AGECANINOX project.M. Awada acknowledges her PhD grant from ANR. The authors thank Lucie Ribourg and Michele Viau for their work on oil mixtures. Patricia Daira is acknowledged for her help with the 4-hydroxy-2-alkenal assay. The authors thank Jean-Michel Chardigny for useful discussions. Isabelle Jouanin (Plateforme AXIOM-MetaToul, Toulouse) is acknowledged for the synthesis of internal standards to measure 4-hydroxyl-2-alkenals in fat. Yvonne Masson is acknowledged for revising the English-language manuscript.Amer soc biochemistry molecular biology incBethesda[Awada, Manar; Plaisancie, Pascale; Estienne, Monique; Michalski, Marie-Caroline] INRA, CarMeN Lab U1235, F-69621 Villeurbanne, France. [Awada, Manar; Soulage, Christophe O.; Guichardant, Michel; Lagarde, Michel; Michalski, Marie-Caroline] Inst Natl Sci Appl, IMBL, F-69621 Villeurbanne, France. [Meynier, Anne; Genot, Claude] INRA, Biopolymeres Interact Assemblages UR1268, F-44300 Nantes, France. [Debard, Cyrille; Chauvin, Marie-Agnes] INSERM, U1060, CarMeN Lab, F-69921 Oullins, France. [Benoit, Berengere; Picard, Gregory; Loizon, Emmanuelle; Peretti, Noel; Michalski, Marie-Caroline] Univ Lyon 1, F-69622 Villeurbanne, France.marie-caroline.michalski@insa-lyon.fr; International audience; Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P < 0.01) compared with unoxidized diets. Oxidized diets enhanced plasma inflammatory markers and activation of nuclear factor kappaB (NF-κB) in the small intestine along with decreasing Paneth cell number (up to -19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine.

La microbiologie du lait et de sa transformation

Because exacerbation of colitis seems to be associated with stress, we proposed evaluating the influence of stress and the involvement of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) on experimental colitis in rats. Partial restraint stress was applied during 4 consecutive days, before or after intracolonic 2,4,6-trinitrobenzenesulfonic acid (TNB) instillation (15 mg) in rats. Finally, two groups of rats were centrally injected with alpha-helical CRF-(9-41) (5 micrograms) or AVP antagonist (5 micrograms) before each session of stress. Stress was applied before or right after TNB enhanced colitis, with an increase in macroscopic and histological scores and myeloperoxidase activity, alpha-Helical CRF-(9-41) or AVP antagonist had no effect on TNB-induced colitis but enhanced the effects of stress on colitis. These results show that stress may exacerbate experimental colitis in rats and that CRF and AVP are not responsible for this effect.