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Le projet de recherche PROLIFIC (PROduits Laitiers et Ingrédients Fermentés Innovants pour des populations Cibles) va évaluer les bénéfices santé des produits laitiers fermentés à destination des jeunes enfants et des seniors.Le projet est porté par un consortium associant des industriels regroupés au sein de Bba Milk Valley et des équipes de recherche bretonnes et ligériennes.Son coût total est de 14 millions d'euros sur cinq ans, co-financé par les instituts de recherche, les industriels de Bba-Milk Valley et les Régions Bretagne et Pays de la Loire. PROLIFIC se décline en cinq thèses de doctorat, trois post-doctorats et 90 mois de CDD de niveau technicien à ingénieur. Yves Le Loir, directeur de l’UMR STLO, est chargé de coordonner le projet. Son lancement a eu lieu à la rentrée 2020.

International audience; Eukaryotic cells are exposed to environmental and endogenous factors that induce DNA damage, thus affecting genomic integrity. The host cells counteract the consequences of lesions by DNA damage response and checkpoint systems that repair DNA structure or trigger cell death when DNA is irreparably damaged. S. aureus, a highly versatile gram-positive bacterium, can cause a multiplicity of human diseases ranging from mild superficial skin to life-threatening disseminated infections. S. aureus is one of the most prevalent pathogen that cause chronic ruminant mastitis that is very difficult to treat. Epithelial cells are able to sense microbes, creating an early line of defense against pathogens. Chronic S. aureus infection is likely to be associated with the internalization of the pathogen by host cells, where bacteria are protected from host defenses. Host cell cycle alteration is one of the highly sophisticated mechanisms pathogens use to hijack the main (defense) functions of the host cells, thus promoting their invasion and colonization. Recently we have shown S. aureus-induced cell cycle alteration in human and bovine epithelial cells.We aimed to investigate whether S. aureus can compromise host genomic integrity.We found that S. aureus can compromise host genomic integrity as indicated by bacteria-induced histone H2AX phosphorylation, a marker of DNA double strand breaks, in human epithelial HeLa and osteoblast-like MG-63 cells. This DNA damage is mediated by alpha phenol-soluble modulins (PSMα1–4), while a specific class of lipoproteins (Lpls), encoded on a pathogenicity island in S. aureus, dampens the H2AX phosphorylation thus counteracting the DNA damage. We demonstrated that this DNA damage is mediated by ROS (reactive oxygen species). DNA damage is followed by the induction of DNA repair that involves the ATM kinase-signaling pathway. An examination of S. aureus strains, isolated from the same patient during acute initial and recurrent bone and joint infections, showed that recurrent strains produce lower amounts of Lpls, induce stronger DNA-damage and prompt the G2/M transition delay to a greater extent that suggest an involvement of these mechanisms in adaptive processes of bacteria during chronicization. Our findings suggest that S. aureus infection has an impact on the genome and epigenome of host cells, which may exert patho-physiological dysfunctions and indicate that the balance between the levels of PSMα and Lpls expression impacts the persistence of the infection.

Le lait n’est pas un aliment comme les autres. Consommé depuis le Néolithique et la domestication des ruminants, il est décliné en mille et un produits différents, est intégré à mille et une recettes de cuisine, et suscite des passions et des débats agités plus que jamais d’actualité… Aliment exclusif du nourrisson, sa place dans la diète à l’âge adulte dépend des cultures. Inépuisable source d’innovations, c’est un objet d’étude fascinant par sa complexité biochimique et nutritionnelle, optimisée par les mammifères… depuis 310 millions d’années.Par un jeu de questions-réponses, les auteurs donnent les clés de toutes les facettes du lait et des produits laitiers en termes de nutrition, santé, économie, symbolique, histoire, culture…À travers 50 questions des plus insolites — Les hommes peuvent-ils allaiter ? ou Peut-on faire de l’alcool avec du lait ? —, des plus sérieuses — Y a-t-il des utilisations non alimentaires du lait ? Est-ce « durable » de consommer des produits laitiers ? Tous les laits sont-ils fromageables ? — aux plus anecdotiques — C’est quoi les dents de lait ? Le lait de jabot : vrai ou faux lait ? —, vous comprendrez qu’il reste encore beaucoup à découvrir sur les effets du lait (fermenté ou non) sur la santé, sa composition, ses impacts sur l’environnement, sa transformation…De quoi animer vos repas de famille… autour du plateau de fromages !

Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines, mitigates the inflammatory response in vitro and in vivo These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e. modulating NF-κB transcription factor activity and IL-8 release) which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and hence the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical and functional properties of EVs produced from cells cultivated on laboratory Yeast Extract Lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant, smaller in diameter and displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacteria interactions.Importance Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in host-microbe interactions. Recently we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems, and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

The role of Propionibacterium freudenreichii in mitigating inflammation has been a subject of study for many years. Lately, it has been shown that immunomodulation properties are strain- specific and that the major responsible for inflammatory modulation ofimmunomodulation by strain CIRM-BIA129 is the presence of the surface layer protein B (SlpB), which has presented immunomodulation even when expressed in other bacteria. Extracellular vesicles (EVs) are nanosized spherical structures, produced by organisms of all kingdoms, including bacteria. They have been associated with inter-organism communication, pathogenesis, competition, and immunomodulation. Recent studies were aiming to address the role of EVs, in the probiotic effects of bacteria. The properties of P. freudenreichii CIRM-BIA129- derived EVs have been investigated. EVs produced by CIRM-BIA129 cultured in milk ultrafiltrate medium (UF) have been characterized by regarding size and morphology. UF-derived EVs displayed a monodisperse pattern with a modal size of 84.80 ± 2.34 nm. They are composed of a wide variety of proteins, mainly involved in metabolic processes, cellular processes and signaling, and storage and processing of information. A, among these proteins, SlpB was found in high abundance. P. freudenreichii EVs were able to inhibit, in a dose dependent manner, the increase of IL-8 production in HT-29 cells induced with LPS, due to NF-KB pathway inhibition, without causing cell cytotoxicity. EVs produced by a CIRM-BIA129 mutant strain with a knockout for SlpB showed a less efficient reduction in IL-8 production. Results have shown that the environmental conditions are able to modify EVs content and, consequently, their immunomodulatory effects. A change in the growth medium, from UF to YEL (yeast extract-lactate) showed a lower production of EVs, with a slightly larger size. EVs produced in YEL did not perform as well in the inhibition of the NF-KB pathway and had no effect on IL-8 production. Recent results have shown that P. freudenreichii EVs were able to protect Caco-2 cells from inflammation-induced excessive increasing permeability. These Altogether, these results show that Evs produced by beneficial propionibacteria are able to generate immunomodulationtrigger immunomodulation, similar to the parental strain. As for the SlpB role in EVs immunomodulation

SYALSA : Systèmes alimentaires et santé humaineAu cœur des relations entre agriculture, alimentation et environnement, les questions de santé peuvent être un moteur pour transformer les systèmes alimentaires. L’objectif général de SYALSA est d’identifier et d’évaluer les leviers d’action susceptibles de rendre les systèmes alimentaires plus favorables à la santé humaine, à travers l’alimentation, mais aussi à travers leurs effets sur l’environnement, en prenant en compte les co-bénéfices entre santé et environnement. Le métaprogramme vise ainsi à mieux comprendre les divers facteurs et mécanismes d’interaction qui, depuis la production agricole jusqu’à la consommation alimentaire, affectent la santé humaine. Il explore les relations entre les pratiques de production et de transformation, les expositions des populations à des contaminants environnementaux, et les impacts sur la santé. Enfin, il s’attache à caractériser, évaluer et accompagner les changements manifestes ou potentiels (innovations biotechniques, politiques publiques, comportements d’acteurs) susceptibles d’améliorer les bénéfices des systèmes alimentaires sur la santé des individus et des populations. Lancé en janvier 2021, SYALSA mobilise les réflexions de la prospective scientifique interdisciplinaire « Nexus Santé »

Le CBL (Club des Bactéries Lactiques) est une manifestation scientifique qui réunit chercheurs, enseignants-chercheurs et industriels R&D, pour échanger sur les avancées scientifiques et techniques réalisées dans le domaine des bactéries lactiques. Les thèmes abordés sont le reflet des larges potentiels de ces bactéries et de leurs applications : fermentation alimentaire, santé humaine et animale, probiotique, environnement…; International audience; Breastfeeding is recommended by the WHO for the first 6 months of life. It provides health benefits to infants compared to infant formula (IF), including protection against intestinal and respiratory infections in early childhood and a lower risk of metabolic and immune diseases later in life. These differences in health benefits are probably due to the difference in composition between IF and human milk (HM), the latter being more complex and richer in bioactive components. Our hypothesis is that HM health benefits come in part from the HM microbiota -a paucimicrobial ecosystem that presents a great bacterial diversity- through a role on gut immune homeostasis.This study aimed to better understand the immunomodulatory role of HM bacteria. To this end, bacteria were isolated from healthy breast milks and screened for their immunomodulatory potential on two different cellular models in order to select candidates with probiotic interest.The collection of 28 breast milks at the Rennes Sud University Hospital allowed the constitution of a strain library of 1164 isolates with 29 genera and 56 different species. From this library, 88 isolates selected for their prevalence in HM microbiota were first screened on PBMC (blood mononuclear cells) for the production of IL-10 and TNF-alpha cytokines. Among them, 28 isolates exhibiting interesting immunomodulatory properties on PBMC model underwent a second screening on a quadricellular model of intestinal epithelium including enterocytes (Caco2), caliciform cells (HT 29-MTX), M cells (differentiated Caco2) and macrophages (THP-1). This study showed the species- and strain-dependence of HM bacteria immunomodulatory potential and revealed interesting properties of HM bacteria in the context of infant immune system maturation. This study is part of the PROLIFIC project funded by the Régions Bretagne and Pays de la Loire and by the BBA MilkValley industrial consortium.

Additional file 1: Supplementary Figures.

International audience; The roles of bacterial extracellular vesicles (EVs) in cell-to-cell signaling are progressively being unraveled. These membranous spheres released by many living cells carry various macromolecules, some of which influence host-pathogen interactions. Bacterial EVs contain RNA, which may serve in communicating with their infected hosts. Staphylococcus aureus, an opportunistic human and animal pathogen, produces EVs whose RNA content is still poorly characterized. Here, we investigated in depth the RNA content of S. aureus EVs. A high-throughput RNA sequencing approach identified RNAs in EVs produced by the clinical S. aureus strain HG003 under different environmental conditions: early- and late-stationary growth phases, and presence or absence of a sublethal vancomycin concentration. On average, sequences corresponding to 78.0% of the annotated transcripts in HG003 genome were identified in HG003 EVs. However, only ~5% of them were highly covered by reads (≥90% coverage) indicating that a large fraction of EV RNAs, notably mRNAs and sRNAs, were fragmented in EVs. According to growth conditions, from 86 to 273 highly covered RNAs were identified into the EVs. They corresponded to 286 unique RNAs, including 220 mRNAs. They coded for numerous virulence-associated factors (hld encoded by the multifunctional sRNA RNAIII, agrBCD, psmβ1, sbi, spa, and isaB), ribosomal proteins, transcriptional regulators, and metabolic enzymes. Twenty-eight sRNAs were also detected, including bona fide RsaC. The presence of 22 RNAs within HG003 EVs was confirmed by reverse transcription quantitative PCR (RT-qPCR) experiments. Several of these 286 RNAs were shown to belong to the same transcriptional units in S. aureus. Both nature and abundance of the EV RNAs were dramatically affected depending on the growth phase and the presence of vancomycin, whereas much less variations were found in the pool of cellular RNAs of the parent cells. Moreover, the RNA abundance pattern differed between EVs and EV-producing cells according to the growth conditions. Altogether, our findings show that the environment shapes the RNA cargo of the S. aureus EVs. Although the composition of EVs is impacted by the physiological state of the producing cells, our findings suggest a selective packaging of RNAs into EVs, as proposed for EV protein cargo. Our study shedds light to the possible roles of potentially functional RNAs in S. aureus EVs, notably in host-pathogen interactions.

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International audience; Staphylococcus aureus (S. aureus), a versatile Gram-positive bacterium, is the main cause of bone and joint infections, which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1β and IL-18. The role of inflammasomes and caspase-1 in the secretion of mature IL-1β and in the defence of S. aureus-infected non-professional phagocytes, human osteoblast-like MG-63 cells, has not yet been fully investigated. To investigate the role of inflammasomes in S. aureus-infected MG-63 cells, the establishement of CASP1–/–MG-63 cell line was carried out using the CRISPR-Cas9 gene editing system. We and others showed that S. aureus can be internalized and survive within professional phagocytes, such as macrophages as well as within non phagocytic epithelial cells or osteoblasts. To study the capacity of intracellular S. aureus to induce inflammasomes formation in MG-63 cells, we confirmed the presence of intracellular bacteria using transmission electron microscopy We show here that S. aureus-infected MG-63 cells but not caspase-1 knock-out CASP1–/–MG-63 cells activate the inflammasome as monitored by the release of mature IL-1β. The effect was strain-dependent. The quorum-sensing system in S. aureus known as the accessory gene regulator (Agr) regulates the expression of many virulence factors including the expression of most S. aureus toxin genes. Of note, the expression of PSMs encoding genes (PSMα 1 to 4, PSMβ 1 and 2, and δ-toxin sometimes called PSMƔ) is tightly controlled by the Agr system. The use of S. aureus LAC (USA300) wild-type strain, its deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated that PSM toxins are involved in inflammasomes-related IL-1β production by infected MG-63 cells. Furthermore, we found that the lack of caspase-1 in CASP1–/–MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1–/– MG-63 compared to wild-type cells. Our results demonstrate that osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase-1 in bacterial clearance.

International audience; Introduction and aims: Bovine mastitisis an inflammation of the mammary gland generally due to an infection. Itis responsible for considerable economic losses in dairy farms. Preventive and curative strategies that mainly rely on antibiotic therapies are not totally effective and contribute to antibiotic resistance dissemination, prompting the need for alternative or complementary strategies. We previously established a link between teat cistern microbiota composition and bovine mastitisin quarters which had different histories regarding mastitis[1].In thisstudy, to further decipher the relationships between teat cistern microbiota and immune and microbial responses, a switchfrom twice-to once-daily milking (ODM) was used to trigger an udder perturbation. Material and methods: Aswitch to ODM was used in 31initially healthy quarters of dairy cows. Immune and microbial responsesincluding determination of teat microbiotawere monitored just prior to the transition (day 0), and 3 and 14 days following transition to ODM.Results and discussion: Atemporal relationship was reportedbetween initial teat cistern microbiota composition and richness, the immune response to ODM, and mastitis development. Quarters with a low initial microbiota richness and taxonomic markers such as Bacteroidetes and Proteobacteria were associated with a higher rate of mastitis during ODM. Quarters with a higher richness and taxonomic markers such as Firmicutes, Bifidobacteriumand Corynebacteriumdisplayed early inflammation following transition to ODM but without developing mastitis (no infection). Short-term evolution of microbiota indicates that microbiota with a higher initial richness were more strongly altered by transition to ODM, with notably the disappearance of rare OTUs. Microbiota modifications were associated with an early innate immune system stimulation, which, in turn, may have contributed to the prevention of mastitis development[2].These results suggest a role of the bovine teat microbiota composition and richness in the immune response of the mammary gland during perturbations such as transition to ODM or pathogen entrance. They invite us to considerstrategies that can preserve teat microbiota diversityandtaxa associated to a healthy status for the design of next-generation probiotics.

International audience; Introduction 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 of selected strains within the species P.freudenreichii, S.thermophilus and L.delbrueckii. We thus hypothesized that consumption of a functional cheese, fermented by selected strains of these three species, may exert a positive effect on IBD.Methods. Strains of each bacterial species were screened based on the ability to induce regulatory IL-10 in human immune PBMC cells, or to downregulate nuclear factor kappa B (NF-κB) activation in a cultured HT-29 human intestinal epithelial cell line. We then investigated the effect of monostrain experimental cheese. We further investigated the impact two-strains experimental cheese. Finally, we produced in industrial conditions an Emmental cheese using one selected strain of each of the three species, i.e. P. freudenreichii CIRM-BIA 129 in combination with Lactobacillus delbrueckii CNRZ327 and Streptococcus thermophilus LMD-9. Consumption of all cheeses was investigated with respect to prevention of chemically induced colitis in mice.Results. Consumption of the experimental cheeses, or of the industrial Emmental, reduced the severity of subsequent chemically induced colitis, weight loss, disease activity index and histological score, in mice. Furtherrmore, consumption of the Emmental cheese, in a preventive way, reduced small bowel Immunoglobulin A (IgA) secretion, restored occludin gene expression and prevented induction of Tumor Necrosis Factor α (TNFα), Interferon γ (IFNγ) and Interleukin-17 (IL-17).Conclusion. Assembling immunomodulatory strains of both lactic acid and propionic acid starter bacteria leads to an anti-inflammatory Emmental cheese, as revealed in vivo. This opens new perspectives for the development of functional fermented food products for personalised nutrition in the context of IBD.

International audience; Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines and mitigates the inflammatory response in vitro and in vivo. These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e., modulating NF-κB transcription factor activity and interleukin-8 release), which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications, such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here, we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and, hence, the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical, and functional properties of EVs produced from cells cultivated on laboratory yeast extract lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant and smaller in diameter, and they displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacterium interactions. IMPORTANCE Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in hostmicrobe interactions. Recently, we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

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BactInflam IJL focuses on the bacterial components involved in some inflammatory diseases. In particular, in two of them: chronic inflammatory bowel disease (IBD) and mastitis, affecting human health and animal health, respectively.; International audience; Introduction: Bacterial extracellular vesicles (EVs) carry various macromolecules able to affect host-pathogen interactions, such as RNAs. Staphylococcus aureus, an important human and animal pathogen, releases EVs whose RNA content is still unkown. Here, we adress what classes of RNAs compose S. aureus EVs. Methods: S. aureus strain HG003 was cultured in Brain Heart Infusion medium under different in vitro conditions: early- and late-stationary phases, in the presence or absence of a sublethal concentration of vancomycin (0.5 μg/mL). EVswere purified from cell-free culture supernatants using density gradient ultracentrifugation. Bacterial and EV samples were submitted to phenolchloroform RNA extraction, DNAse treatment, and library preparation (Ovation Prokaryotic RNA-Seq, Nugen, rRNA depletion). Sequencing was performed using Illumina, NextSeq500, 75 cycles, single reading, High Output. Results: Particle yields were similar between conditions, however, EVs from late-stationary phases were ∼55% ,larger. On average, 78.0% of HG003 annotated genes were identified in EVs, while only ∼5% presented - 90% read coverage. Highly covered EV RNAs included mRNAs coding for virulence-factors (hld, agrBCD, psmB1, sbi, spa, isaB), ribosomal proteins, transcriptional regulators, and metabolic enzymes. sRNAs were also detected, including the bona fide rsaC. Interestingly, several of these RNAs were shown to belong to the same transcriptional units in S. aureus. Both nature and abundance of the RNAs in EVs were dramatically affected by growth conditions, whereas much less in the parent cells. Finally, the RNA abundance pattern differed between EVs and parent cells. Summary/Conclusion: To our knowledge, this is the first work characterizing the RNA cargo of S. aureus EVs. Our findings show that EV RNAs are shaped by the environment, and suggest the selective packaging of RNAs into EVs. Finally, this study also shedds light to the possible roles of potentially functional RNAs in S. aureus EVs, notably in host-pathogen interactions.

International audience; Introduction Propionibacterium freudenreichii, is a probiotic bacterium, with an established immunomodulatory role. Selected strains indeed induce the release of regulatory IL-10. Extractable surface proteins of the S-layer type (Slp) are involved in such modulation, but the exact mode of interaction with the host remains unknown. Mucositis is a common side effect of cytotoxic chemotherapy characterized by mucosal injury, inflammation, diarrhea, and weight loss. We hypothesized that immunomodulatory P. freudenreichii may mitigate inflammation and that SlpB may be required for such an effect.Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. We hypothesized that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs and thus export immunomodulatory proteins to interact with the host.MethodsP. freudenreichii CIRM-BIA 129, previously selected for its ability to induce IL-10 release by PBMCs, was administered orally to mice, prior to colitis induction using TNBS.The slpB gene was inactivated by insertional inactivation. Both parental wild type and mutant strains were administered orally to mice, prior to induction of mucositis using 5-FU. The SlpB protein was purified and its effect on cultured human intestinal epithelial cells investigated with respect to cytokines expression.EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, prior to physicochemical and functional characterization.ResultsConsumption of P. freudenreichii CIM-BIA129 strain protected mice against TNBS-induced colitis in mice, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages.This strain prevented 5-FU-induced mucositis in mice, regulated key markers, including the expression of Claudin-1 (Cld1) and IL-17a (Il17a), as well as the levels of IL-12 and IL-1β cytokines.The slpB gene was mutated by insertional inactivation and the absence of SlpB protein in the resulting mutant was verified. The mutant strain displayed opposite immunomodulatory effect and failed to mitigate induced mucositis.In HT-29 human intestinal epithelial cells P. freudenreichii reduced expression of IL-8 and TNF-α cytokines in LPS-stimulated cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. In the same cells, purified SlpB was shown to induce expression of IL-10 in a dose-dependent manner.Extracellular vesicles (EVs), they were purified from cell-free culture supernatants of P. freudenreichii. They showed typical shapes and sizes of EVs and contained a broad range of proteins, including SlpB. EVs modulated inflammatory responses, IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells, as well as the NF-κB pathway.DiscussionThis work emphasizes the importance of SlpB in immunomodulatory P. freudenreichii. It constitutes the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. It opens perspectives for the development of functional foods and other bioactive products in order to help preventing and/or treating inflammatory diseases, and to decrease side effects of chemotherapy.