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International audience; Murine colitis models are crucial tools for understanding intestinal homeostasis and inflammation. However, most current models utilize a highly inbred strain of mice, and often only one sex is employed to limit bias. This targeted approach, which in itself is biased, means that murine genetic diversity and sex-related differences are ignored, making it even more difficult to extend findings to humans, who are highly heterogeneous. Furthermore, most models do not examine the chronic form of colitis, an important fact taking into account the chronic nature of the inflammatory bowel diseases (IBD). Here, we attempted to create a more realistic murine colitis model by addressing these three issues. Using chemically induced chronic colon inflammation in an outbred strain of mice (RjOrl:SWISS [CD-1]), we (i) mimicked the relapsing nature of the disease, (ii) better represented normal genetic variability, and (iii) employed both female and male mice. Colitis was induced by intrarectal administration of dinitrobenzene sulfonic acid (DNBS). After a recovery period and 3 days before the mice were euthanized, colitis was reactivated by a second administration of DNBS. Protocol length was 24 days. Colitis severity was assessed using body mass, macroscopic scores, and histological scores. Myeloperoxidase (MPO) activity, cytokine levels, and lymphocyte populations were also characterized. Our results show that the intrarectal administration of DNBS effectively causes colitis in both female and male CD-1 mice in a dose-dependent manner, as reflected by loss of body mass, macroscopic scores and histological scores. Furthermore, colon cytokine levels and mesenteric lymph node characteristics indicate that this model involves immune system activation. Although some variables were sex-specific, most of the results support including both females and males in the model. Our ultimate goal is to make this model available to researchers for testing candidate anti-inflammatory agents, such as classical or next-generation probiotics; we also aim for the results to be more easily transferrable to human trials.

International audience; The gut barrier plays an important role in human health. When barrier function is impaired, altered permeability and barrier dysfunction can occur, leading to inflammatory bowel diseases, irritable bowel syndrome or obesity. Several bacteria, including pathogens and commensals, have been found to directly or indirectly modulate intestinal barrier function. The use of probiotic strains could be an important landmark in the management of gut dysfunction with a clear impact on the general population. Previously, we found that Lactobacillus rhamnosus CNCM 1-3690 can protect intestinal barrier functions in mice inflammation model. Here, we investigated its mechanism of action. Our results show that CNCM 1-3690 can (i) physically maintain modulated goblet cells and the mucus layer and (ii) counteract changes in local and systemic lymphocytes. Furthermore, mice colonic transcriptome analysis revealed that CNCM 1-3690 enhances the expression of genes related to healthy gut permeability: motility and absorption, cell proliferation; and protective functions by inhibiting endogenous proteases. Finally, Spa FED pili are clearly important effectors since an L. rhamnosus Delta spaF mutant failed to provide the same benefits as the wild type strain. Taken together, our data suggest that CNCM 1-3690 restores impaired intestinal barrier functions via anti-inflammatory and cytoprotective responses.

International audience; Antimicrobial peptides secreted by intestinal immune and epithelial cells are important effectors of innate immunity. They play an essential role in the maintenance of intestinal homeostasis by limiting microbial epithelium interactions and preventing unnecessary microbe‐driven inflammation. Pancreatitis‐associated protein (PAP) belongs to Regenerating islet‐derived III (RegIII) proteins family and is a C‐type (Ca+2 dependent) lectin. PAP protein plays a protective effect presenting anti‐inflammatory properties able to reduce the severity of colitis, preserving gut barrier and epithelial inflammation. Here, we sought to determine whether PAP delivered at intestinal lumen by recombinant Lactococcus lactis strain (LL‐PAP) before and after chemically‐induced colitis is able to reduce the severity in two models of colitis. After construction and characterization of our recombinant strains, we tested their effects in DiNitro‐BenzeneSulfonic‐acid (DNBS) and Dextran Sulfate Sodium (DSS) colitis model. After the DNBS challenge, mice treated with LL‐PAP presented less severe colitis compared to PBS and LL‐empty treated mice groups. After the DSS challenge no protective effects of LL‐PAP could be detected. We determined that after 5 days administration, LL‐PAP increase butyrate producer's bacteria, especially Eubacterium plexicaudatum. Based on our findings, we hypothesize that a treatment with LL‐PAP shifts the microbiota preventing the severity of colon inflammation in DNBS colitis model. These protective roles of LL‐PAP in DNBS colitis model might be through intestinal microbiota modulation.

AGA Abstracts Mo1949; International audience

International audience; The gut microbiota plays a major role in intestinal health, and an imbalance in its composition can lead to chronic gut inflammation and a predisposition to developing colorectal cancer (CRC). Currently, the use of probiotic bacteria represents an emerging alternative to treat and prevent cancer. Moreover, consumption of these beneficial bacteria may also favorably modulate the composition of the gut microbiota, which has been described in several studies to play an important role in CRC carcinogenesis. In this context, the aim of this study was to assess the protective effect of oral treatment with Lactobacillus casei BL23, a probiotic strain well known for its anti-inflammatory and anticancer properties. First, CRC was induced in C57BL6 mice by a single intraperitoneal injection with azoxymethane (8 mg/kg), followed by four courses of dextran sodium sulfate (2.5%) in drinking water that were separated by an adjustable recovery period. At the time of sacrifice (day 46), tumor incidence, histological scores, and epithelial proliferation were determined in colon samples. Our results show that L. casei BL23 significantly protected mice against CRC development; specifically, L. casei BL23 treatment reduced histological scores and proliferative index values. In addition, our analysis revealed that L. casei BL23 had an immunomodulatory effect, mediated through the downregulation of the IL-22 cytokine, and an antiproliferative effect, mediated through the upregulation of caspase-7, caspase-9, and Bik. Finally, L. casei BL23 treatment tended to counterbalance CRC-induced dysbiosis in mice, as demonstrated by an analysis of fecal microbiota. Altogether our results demonstrate the high potential of L. casei BL23 for the development of new, probiotic-based strategies to fight CRC.

Faecalibacterium prausnitzii and its supernatant showed protective effects in different chemically-induced colitis models in mice. Recently, we described 7 peptides found in the F. prausnitzii supernatant, all belonging to a protein called Microbial Anti-inflammatory Molecule (MAM). These peptides were able to inhibit NF-κB pathway in vitro and showed anti-inflammatory properties in vivo in a DiNitroBenzene Sulfate (DNBS)-induced colitis model. In this current proof we tested MAM effect on NF-κB pathway in vivo, using a transgenic model of mice producing luciferase under the control of NF-κB promoter. Moreover, we tested this protein on Dextran Sodium Sulfate (DSS)-induced colitis in mice. To study the effect of MAM we orally administered to the mice a Lactococcus lactis strain carrying a plasmid containing the cDNA of MAM under the control of a eukaryotic promoter. L. lactis delivered plasmids in epithelial cells of the intestinal membrane allowing thus the production of MAM directly by host. We showed that MAM administration inhibits NF-κB pathway in vivo. We confirmed the anti-inflammatory properties of MAM in DNBS-induced colitis but also in DSS model. In DSS model MAM was able to inhibit Th1 and Th17 immune response while in DNBS model MAM reduced Th1, Th2, and Th17 immune response and increased TGFβ production.

Faecalibacterium prausnitzii and its supernatant showed protective effects in different chemically-induced colitis models in mice. Recently, we described 7 peptides found in the F prausnitzii supernatant, all belonging to a protein called Microbial Anti-inflammatory Molecule (MAM). These peptides were able to inhibit NF-kappa B pathway in vitro and showed anti-inflammatory properties in vivo in a DiNitroBenzene Sulfate (DNBS)-induced colitis model. In this current proof we tested MAM effect on NF-kB pathway in vivo, using a transgenic model of mice producing luciferase under the control of NF-kappa B promoter. Moreover, we tested this protein on Dextran Sodium Sulfate (DSS)-induced colitis in mice. To study the effect of MAM we orally administered to the mice a Lactococcus lactis strain carrying a plasmid containing the cDNA of MAM under the control of a eukaryotic promoter. L. lactis delivered plasmids in epithelial cells of the intestinal membrane allowing thus the production of MAM directly by host. We showed that MAM administration inhibits NF-kappa B pathway in vivo. We confirmed the anti-inflammatory properties of MAM in DNBS-induced colitis but also in DSS model. In DSS model MAM was able to inhibit Thl and Th17 immune response while in DNBS model MAM reduced Th1, Th2, and Th17 immune response and increased TGF beta production.

International audience; Faecalibacterium prausnitzii is a commensal bacterium, ubiquitous in the gastrointestinal tracts of animals and humans. This species is a functionally important member of the microbiota and studies suggest it has an impact on the physiology and health of the host. F. prausnitzii is the only identified species in the genus Faecalibacterium, but a recent study clustered strains of this species in two different phylogroups. Here, we propose the existence of distinct species in this genus through the use of comparative genomics. Briefly, we performed analyses of 16S rRNA gene phylogeny, phylogenomics, whole genome Multi-Locus Sequence Typing (wgMLST), Average Nucleotide Identity (ANI), gene synteny, and pangenome to better elucidate the phylogenetic relationships among strains of Faecalibacterium. For this, we used 12 newly sequenced, assembled, and curated genomes of F. prausnitzii, which were isolated from feces of healthy volunteers from France and Australia, and combined these with published data from 5 strains downloaded from public databases. The phylogenetic analysis of the 16S rRNA sequences, together with the wgMLST profiles and a phylogenomic tree based on comparisons of genome similarity, all supported the clustering of Faecalibacterium strains in different genospecies. Additionally, the global analysis of gene synteny among all strains showed a highly fragmented profile, whereas the intra-cluster analyses revealed larger and more conserved collinear blocks. Finally, ANI analysis substantiated the presence of three distinct clusters—A, B, and C—composed of five, four, and four strains, respectively. The pangenome analysis of each cluster corroborated the classification of these clusters into three distinct species, each containing less variability than that found within the global pangenome of all strains. Here, we propose that comparison of pangenome subsets and their associated α values may be used as an alternative approach, together with ANI, in the in silico classification of new species. Altogether, our results provide evidence not only for the reconsideration of the phylogenetic and genomic relatedness among strains currently assigned to F. prausnitzii, but also the need for lineage (strain-based) differentiation of this taxon to better define how specific members might be associated with positive or negative host interactions.

The aim of this review is to summarize the effect in host energy metabolism of the production of B group vitamins and short chain fatty acids (SCFA) by commensal, food-grade and probiotic bacteria, which are also actors of the mammalian nutrition. The mechanisms of how these microbial end products, produced by these bacterial strains, act on energy metabolism will be discussed. We will show that these vitamins and SCFA producing bacteria could be used as tools to recover energy intakes by either optimizing ATP production from foods or by the fermentation of certain fibers in the gastrointestinal tract (GIT). Original data are also presented in this work where SCFA (acetate, butyrate and propionate) and B group vitamins (riboflavin, folate and thiamine) production was determined for selected probiotic bacteria.

Background: Chronic intestinal inflammation alters host physiology and could lead to colorectal cancer (CRC). We have previously reported beneficial effects of the probiotic strain of Lactobacillus casei BL23 in different murine models of intestinal inflammation. In addition, there is an emerging interest on the potential beneficial effects of probiotics to treat CRC. We thus explored whether L. casei BL23 displays protective effects on CRC. Methods: Mice were subcutaneously injected with 1,2-dimethylhydrazine (DMH) weekly during 10 weeks and orally administered with L. casei BL23 in the drinking water until the 10th week. Multiple plaque lesions in the large intestine were observed macroscopically and counted and intestinal tissues were also histologically analyzed. Finally, T-cell populations and cytokine production were evaluated after co-incubation of L. casei BL23 with spleen cells from non-treated mice to determine the immuno-modulatory effects of this bacterium. Results: Our results show that oral treatment with this probiotic bacterium modulates host immune responses and significantly protect mice against DMH-induced CRC. This protection may be associated with the modulation of regulatory T-cells towards a Th17-biased immune response accompanied by the expression of regulatory cytokines (IL-6, IL-17, IL-10 and TGF-β), as demonstrated in L. casei BL23-treated splenocytes, but also with the colonic expression of IL-22 observed in vivo on L. casei BL23-treated mice; suggesting the induction of a fine-tune Th17-biased response. Conclusions: Altogether our results reveal the high potential of L. casei BL23 to treat CRC and opens new frontiers for the study of immunomodulatory functions of probiotics. Fil: Lenoir, Marion. Institut National de la Recherche Agronomique; Francia. AgroParisTech; Francia Fil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Cortes Perez, Naima G.. Institut National de la Recherche Agronomique; Francia Fil: Lozano Ojalvo, Daniel. Institut National de la Recherche Agronomique; Francia. Service de Pharmacologie et d’Immunoanalyse. CEA, IBiTecS; Francia. Consejo Superior de Investigaciones Científicas. Instituto de Investigación en Ciencias de la Alimentación; España Fil: Muñoz Provencio, Diego. Institut National de la Recherche Agronomique; Francia Fil: Chain, Florian. Institut National de la Recherche Agronomique; Francia Fil: Langella, Philippe. Institut National de la Recherche Agronomique; Francia Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Bermudez Humaran, Luis G.. Institut National de la Recherche Agronomique; Francia

Bifidobacterium Animalis SUBSP Lactis CNCM-I2494 Restores Tight Junction Proteins Levels in a Chronic Low-grade Colonic Inflammation Mouse Model. 8. Probiotics, Prebiotics & New Foods for Microbiota and Human Health meeting held

Les mammites bovines représentent un coût économique et sanitaire important pour la production laitière dans le monde. La prévention et le traitement des mammites reposent essentiellement sur les vaccins et l’antibiothérapie, dont l’efficacité est limitée, incitant à explorer de nouvelles stratégies. Ces dernières années, l'utilisation de micro-organismes probiotiques pour lutter contre les infections suscite un intérêt croissant en santé humaine et animale. Le but de cette étude était d'isoler des bactéries lactiques (BL) du microbiote mammaire bovin présentant des propriétés bénéfiques pour la prévention et le traitement des mammites.L'échantillonnage réalisé au niveau du canal du trayon a permis l'isolement de 165 clones appartenant principalement aux genres Enterococcus, Streptococcus, Lactobacillus et Lactococcus. Parmi ces isolats, 10 souches appartenant aux genres Lactobacillus et Lactococcus, et non redondantes d’après leur profil PFGE, ont été retenues pour être caractérisées vis-à-vis de leurs propriétés de surface (hydrophobicité, autoaggrégation), leur capacité à inhiber les trois agents majoritairement impliqués dans les mammites bovines (i.e.Staphylococcus aureus, Escherichia coli et Streptococcus uberis), leurs capacités d'adhésionet d’internalisation aux cellules épithéliales mammaires bovines et leurs propriétés immunomodulatrices.Trois souches, deux Lactobacillus brevis et un Lactobacillus plantarum, présentent de fortes capacités de colonisation et constituent de bons candidats pour limiter la colonisation de la glande mammaire par les pathogènes. Par ailleurs, neuf souches de BL présentent des propriétés anti-inflammatoires : la sécrétion d’IL8 par des cellules épithéliales mammaires bovines stimulées par E. coli est en effet réduite en présence de ces BL. Le séquençage du génome complet de cinq de ces souches candidates a permis de vérifier l’absence de facteurs de risque tels que des gènes d’antibiorésistance et d’identifier des déterminants bactérienspotentiels en lien avec les propriétés probiotiques observées. Ce travail ouvre de nouvelles perspectives sur le développement de solutions alternatives et durables pour le contrôle des mammites bovines.