Ligilactobacillus salivarius CNCM I-4866, a potential probiotic candidate, shows anti-inflammatory properties in vitro and in vivo

IntroductionThe aim of this work was to characterize a new strain of Ligilactobacillus salivarius (CNCM I-4866) (CNCM I-4866) to address its potential as probiotic with a special focus on intestinal inflammation. Potential anti-inflammatory abilities of this strain were evaluated through in vivo and in vitro experiments.MethodsFirstly, the strain was tested in a murine acute inflammation colitis model induced by DNBS. In vitro characterization was then performed with diverse tests: modulation capability of intestinal permeability; study of the impact on immunity profile through cytokines dosage; capacity to inhibit pathogens and adhere to intestinal cells lines. Production of metabolites, antibiotic resistance and survival to gastro-intestinal tract conditions were also tested.ResultsIn vitro assay has shown a reduction of colonic damage and markers of inflammation after treatment with CNCM I-4866. Transcriptomic analysis performed on colons showed the capacity of the strain to down-regulate pro-inflammatory cytokines. L. salivarius CNCM I-4866 exerted anti-inflammatory profile by reducing IL-8 production by TNF-α stimulated cell and modulated cytokines profile on peripheral blood mononuclear cells (PBMC). It protected intestinal integrity by increasing trans-epithelial electrical resistance (TEER) on Caco-2 TNF-α inflamed cells. Additionally, L. salivarius CNCM I-4866 displayed inhibition capacity on several intestinal pathogens and adhered to eukaryotic cells. Regarding safety and technical concerns, CNCM I-4866 was highly resistant to 0.3% of bile salts and produced mainly L-lactate. Finally, strain genomic characterization allowed us to confirm safety aspect of our strain, with no antibiotic gene resistance found.DiscussionTaken together, these results indicate that L. salivarius CNCM I-4866 could be a good probiotic candidate for intestinal inflammation, especially with its steady anti-inflammatory profile

Characterization of immunomodulatory and beneficial effects of Christensenella minuta on the host

Le microbiote intestinal, composé de micro-organismes tels que des bactéries ou des virus, assure des fonctions métaboliques, barrières, immunes et protectives permettant le maintien de l’homéostasie générale chez l’hôte. Des études ont montré que la famille des Christensenellaceae étaient considérées comme une signature d’un microbiote intestinal en bonne santé. Elle a également été associée à la longévité du fait de sa présence chez des personnes centenaires et semi-supercentenaires en bonne santé. De plus, la famille des Christensenellaceae est également retrouvée appauvrie chez des patients atteints de maladies inflammatoires chroniques de l’intestin (MICI), comme la maladie de Crohn ou encore la rectocolite hémorragique. Nous nous sommes donc intéressés aux propriétés anti-inflammatoires et bénéfiques liées à la présence de Christensenella minuta DSM 22607, la souche type de la famille des Christensenellaceae. Nous avons i) caractérisé in vitro les propriétés fonctionnelles de notre bactérie d’intérêt ; ii) évalué ses effets sur un modèle d’inflammation pathologique in vivo et iii) déterminé son potentiel bénéfique sur un modèle in vivo d’inflammation physiologique observée lors du vieillissement. Nous avons pu observer que C. minuta DSM 22607 possédait des propriétés anti-inflammatoires in vitro et in vivo et ce dans l’ensemble des modèles étudiés. De plus, l’administration de C. minuta a permis de préserver l’intégrité de la barrière intestinale et de stimuler la production d’acide gras à chaînes courtes, permettant de protéger l’hôte. Ces résultats indiquent que C. minuta joue un rôle clé dans le maintien de l’homéostasie générale et pourrait être utilisée comme biothérapie dans le traitement de l’inflammation et de pathologies associées.The gut microbiota, composed of micro-organisms such as bacteria or viruses, performs metabolic, barrier, immune and protective functions, maintaining the general homeostasis of the host. Studies have shown that the Christensenellaceae family is considered as a signature of a healthy gut microbiota. It has also been associated with longevity due to its presence on healthy centenarians and semi-supercentenarians. Besides, the Christensenellaceae family has been found to be depleted in patients suffering from chronic inflammatory bowel diseases (IBD), such as Crohn's disease or ulcerative colitis. Therefore, we were decided to study the anti-inflammatory and beneficial properties of Christensenella minuta DSM 22607, the type strain of the Christensenellaceae family. We i) characterised the functional properties of our bacterium of interest in vitro; ii) evaluated its effects on an in vivo pathological inflammation model in mice chemically induced and iii) determined its beneficial potential on an in vivo murine physiological inflammation model based on the increase of low-grade inflammation observed during aging. We showed that C. minuta DSM 22607 displayed anti-inflammatory properties in vitro and in vivo in all models studied. In addition, administration of C. minuta preserves the integrity of the intestinal barrier and stimulates the production of short-chain fatty acids, thus protecting the host. These results support the fact that C. minuta plays a key role in gut microbiota homeostasis and could be used as a biotherapy to treat inflammation and/or related-disorders

Rapeseed oil fortified with micronutrients improves cognitive alterations associated with metabolic syndrome

Metabolic syndrome represents a major risk factor for severe comorbidities such as cardiovascular diseases or diabetes. It is also associated with an increased prevalence of emotional and cognitive alterations that in turn aggravate the disease and related outcomes. Identifying therapeutic strategies able to improve those alterations is therefore a major socioeconomical and public health challenge. We previously reported that both hippocampal inflammatory processes and neuronal plasticity contribute to the development of emotional and cognitive alterations in db/db mice, an experimental model of metabolic syndrome that displays most of the classical features of the syndrome. In that context, nutritional interventions with known impact on those neurobiological processes appear as a promising alternative to limit the development of neurobiological comorbidities of metabolic syndrome. We therefore tested here whether n-3 polyunsaturated fatty acids (n-3 PUFAs) associated with a cocktail of antioxidants can protect against the development of behavioral alterations that accompany the metabolic syndrome. Thus, this study aimed: 1) to evaluate if a diet supplemented with the plant-derived n-3 PUFA α-linolenic acid (ALA) and antioxidants (provided by n-3 PUFAs-rich rapeseed oil fortified with a mix of naturally constituting antioxidant micronutrients, including coenzyme Q10, tocopherol, and the phenolic compound canolol) improved behavioral alterations in db/db mice, and 2) to decipher the biological mechanisms underlying this behavioral effect. Although the supplemented diet did not improve anxiety-like behavior and inflammatory abnormalities, it reversed hippocampus-dependent spatial memory deficits displayed by db/db mice in a water maze task. It concomitantly changed subunit composition of glutamatergic AMPA and NMDA receptors in the hippocampus that has been shown to modulate synaptic function related to spatial memory. These data suggest that changes in local neuronal plasticity may underlie cognitive improvements in db/db mice fed the supplemented diet. The current findings might therefore provide valuable data for introducing new nutritional strategies for the treatment of behavioral complications associated with MetS

Lactococcus lactis engineered to deliver hCAP18 cDNA alleviates DNBS-induced colitis in C57BL/6 mice by promoting IL17A and IL10 cytokine expression

International audienceAbstract With its antimicrobial and immunomodulating properties, the cathelicidin (LL37) plays an important role in innate immune system. Here, we attempted to alleviate chemically induced colitis using a lactococci strain that either directly expressed the precursor to LL37, hCAP18 (LL-pSEC:hCAP18), or delivered hCAP18 cDNA to host cells under the control of the cytomegalovirus promoter (LL-Probi-H1:hCAP18). We also investigated whether the alleviation of symptoms could be explained through modification of the gut microbiota by hCAP18. Mice were administered daily doses of LL-pSEC:hCAP18 or LL-Probi-H1:hCAP18. On day 7, colitis was induced by DNBS. During autopsy, we assessed macroscopic tissue damage in the colon and collected tissue samples for the characterization of inflammation markers and histological analysis. Feces were collected at day 7 for 16S DNA sequencing. We also performed a fecal transplant experiment in which mice underwent colon washing and received feces from Lactococcus lactis -treated mice before DNBS-colitis induction. Treatment with LL-Probi-H1:hCAP18 reduced the severity of colitis symptoms. The protective effects were accompanied by increased levels of IL17A and IL10 in mesenteric lymph node cells. L. lactis administration altered the abundance of Lachnospiraceae and Muribaculaceae . However, fecal transplant from L. lactis -treated mice did not improve DNBS-induced symptoms in recipient mice

Evaluation and characterization of a promising anti-ricin antibody against ricin isoforms to treat deadly ricin intoxication

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Effet d'une diète enrichie en acides gras polyinsaturés n-3 et en antioxydants sur les altérations métaboliques associées à l'obésité

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Ricin intoxication: development of monoclonal and recombinant antibodies for diagnostic and therapeutic applications

International audienceIntroduction. Ricin is a protein produced by the plant Ricinus communis, known for its high toxicity. As ricin is readily available in nature, it represents a considerable biothreat. An early diagnosis of ricin intoxication as well as efficient medical countermeasures are therefore urgently needed. In the event of intentional or accidental poisoning, it is essential to treat as quickly as possible. In this context, vaccination, which takes several weeks to several months to achieve the right level of protection, is not appropriate. On the contrary, direct administration of antibodies, provides immediate protection. However, since ricin sequence can differ according to the isoform, developing efficient protective mAbs against ricin is challenging. We have developed and characterized several murine monoclonal antibodies for diagnostic and therapeutic applications. They have enabled the development of a highly sensitive and specific in vitro diagnostic immunoassay (sandwich ELISA) for ricin intoxication, which is now CE-IVD marked and commercially available. Some of these antibodies have been evaluated in in vitro and in vivo models, and have demonstrated excellent protection against ricin poisoning in mice.Materials and Methods. In vitro diagnostic test: monoclonal antibodies were produced by immunizing mice with inactivated ricin. The best antibodies were then selected for their ability to recognize ricin in ELISA. The performance of this test, in its research format, was then evaluated using plasma from ricin-intoxicated animals. An industrial prototype of the test was then designed, and its performance evaluated using human plasma.Medical countermeasures: several generations of ricin antibodies (murine monoclonal and recombinant camelid) were produced and evaluated for their neutralizing capacities in a cellular model. The best ones were then evaluated for their protective activity in an in vivo murine model of intranasal intoxication.Results. In vitro diagnostic test: In its research format, the sandwich ELISA test enables early detection of ricin in the blood of intratracheally poisoned macaques or intranasally poisoned mice.Medical countermeasures: the best antibodies gave intranasally intoxicated mice survival rates of 90% and 50% respectively when administered 6h and 18h post-intoxication.References1. J. Prigent et al., PLoS ONE. 6(5): e20166. (2011)2. M.L. Orsini Delgado et al., Toxins. 13(2):100 (2021

Intraspecific Diversity of Microbial Anti-Inflammatory Molecule (MAM) from Faecalibacterium prausnitzii

International audienceThe commensal bacterium Faecalibacterium prausnitzii has unique anti-inflammatory properties, at least some of which have been attributed to its production of MAM, the Microbial Anti-inflammatory Molecule. Previous phylogenetic studies of F. prausnitzii strains have revealed the existence of various phylogroups. In this work, we address the question of whether MAMs from different phylogroups display distinct anti-inflammatory properties. We first performed wide-scale identification, classification, and phylogenetic analysis of MAM-like proteins encoded in different genomes of F. prausnitzii. When combined with a gene context analysis, this approach distinguished at least 10 distinct clusters of MAMs, providing evidence for functional diversity within this protein. We then selected 11 MAMs from various clusters and evaluated their anti-inflammatory capacities in vitro. A wide range of anti-inflammatory activity was detected. MAM from the M21/2 strain had the highest inhibitory effect (96% inhibition), while MAM from reference strain A2-165 demonstrated only 56% inhibition, and MAM from strain CNCM4541 was almost inactive. These results were confirmed in vivo in murine models of acute and chronic colitis. This study provides insights into the family of MAM proteins and generates clues regarding the choice of F. prausnitzii strains as probiotics for use in targeting chronic inflammatory diseases

From In Vitro to In Vivo: A Rational Flowchart for the Selection and Characterization of Candidate Probiotic Strains in Intestinal Disorders

International audienceExperimental and clinical evidence has demonstrated the potential of probiotic strains in the prevention or treatment of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, there is little data on what the methodology leading to the identification of such strains should be. In this work, we propose a new flowchart to identify strains with probiotic potential for the management of IBS and IBD, which we tested on a collection of 39 lactic acid bacteria and Bifidobacteria strains. This flowchart included in vitro tests of immunomodulatory properties on intestinal and peripheral blood mononuclear cells (PBMCs), assessment of the barrier-strengthening effect by measuring transepithelial electric resistance (TEER) and quantification of short-chain fatty acids (SCFAs) and aryl hydrocarbon receptor (AhR) agonists produced by the strains. The in vitro results were then combined in a principal component analysis (PCA) to identify strains associated with an anti-inflammatory profile. To validate our flowchart, we tested the two most promising strains identified in the PCA in mouse models of post-infectious IBS or chemically induced colitis to mimic IBD. Our results show that this screening strategy allows the identification of strains with potential beneficial effects on colonic inflammation and colonic hypersensitivity