Identification of Nascent Memory CD8 T Cells and Modeling of Their Ontogeny

International audiencePrimary immune responses generate short-term effectors and long-term protective memory cells. The delineation of the genealogy linking naive, effector and memory cells has been complicated by the lack of phenotypes discriminating effector from memory differentiation stages. Using transcriptomics and phenotypic analyses, we identify a novel marker combination that allows us to track nascent memory cells within the effector phase. We then use a formal approach based on mathematical models describing the dynamics of population-size evolutions to test potential progeny links and demonstrate that most cells follow a linear naive-early effector-late effector-memory pathway. Moreover, our mathematical modelallows long-term prediction of memory cell numbers from a few early experimental measurements. Our work thus provides a phenotypic means to identify effector and memory cells, as well as a mathematical framework to investigate the ontology of their generation and to predict the outcome of immunization regimens in terms of memory cell numbers generated

Caecal microbiota compositions from 7-day-old chicks reared in high-performance and low-performance industrial farms and systematic culturomics to select strains with anti-Campylobacter activity.

There is growing interest in exploring the chickens' intestinal microbiota and understanding its interactions with the host. The objective is to optimize this parameter in order to increase the productivity of farm animals. With the goal to isolate candidate probiotic strains, specific culturomic methods were used in our study to culture commensal bacteria from 7-days old chicks raised in two farms presenting long history of high performance. A total of 347 isolates were cultured, corresponding to at least 64 species. Among the isolates affiliated to the Firmicutes, 26 had less than 97% identity of their partial 16S sequence with that of the closest described species, while one presented less than 93% identity, thus revealing a significant potential for new species in this ecosystem. In parallel, and in order to better understand the differences between the microbiota of high-performing and low-performing animals, caecal contents of animals collected from these two farms and from a third farm with long history of low performance were collected and sequenced. This compositional analysis revealed an enrichment of Faecalibacterium-and Campylobacter-related sequences in lower-performing animals whereas there was a higher abundance of enterobacteria-related sequences in high-performing animals. We then investigated antibiosis activity against C. jejuni ATCC 700819 and C. jejuni field isolate as a first phenotypic trait to select probiotic candidates. Antibiosis was found to be limited to a few strains, including several lactic acid bacteria, a strain of Bacillus horneckiae and a strain of Escherichia coli. The antagonist activity depended on test conditions that mimicked the evolution of the intestinal environment of the chicken during its lifetime, i.e. temperature (37°C or 42°C) and oxygen levels (aerobic or anaerobic conditions). This should be taken into account according to the stage of development of the animal at which administration of the active strain is envisaged

A comprehensive evaluation of binning methods to recover human gut microbial species from a non-redundant reference gene catalog

International audienceThe human gut microbiota performs functions that are essential for the maintenance of the host physiology. However, characterizing the functioning of microbial communities in relation to the host remains challenging in reference-based metagenomic analyses. Indeed, as taxonomic and functional analyses are performed independently, the link between genes and species remains unclear. Although a first set of species-level bins was built by clustering coabundant genes, no reference bin set is established on the most used gut microbiota catalog, the Integrated Gene Catalog (IGC). With the aim to identify the best suitable method to group the IGC genes, we benchmarked nine taxonomy-independent binners implementing abundance-based, hybrid and integrative approaches. To this purpose, we designed a simulated non-redundant gene catalog (SGC) and computed adapted assessment metrics. Overall, the best trade-off between the main metrics is reached by an integrative binner. For each approach, we then compared the results of the best-performing binner with our expected community structures and applied the method to the IGC. The three approaches are distinguished by specific advantages, and by inherent or scalability limitations. Hybrid and integrative binners show promising and potentially complementary results but require improvements to be used on the IGC to recover human gut microbial species

An Oral FMT Capsule as Efficient as an Enema for Microbiota Reconstruction Following Disruption by Antibiotics, as Assessed in an In Vitro Human Gut Model

International audienceFecal microbiota transplantation (FMT) is an innovative therapy already used in humans to treat Clostridioides difficile infections associated with massive use of antibiotics. Clinical studies are obviously the gold standard to evaluate FMT efficiency but remain limited by regulatory, ethics, and cost constraints. In the present study, an in vitro model of the human colon reproducing medically relevant perturbation of the colonic ecosystem by antibiotherapy was used to compare the efficiency of traditional FMT enema formulations and a new oral capsule in restoring gut microbiota composition and activity. Loss of microbial diversity, shift in bacterial populations, and sharp decrease in fermentation activities induced in vivo by antibiotherapy were efficiently reproduced in the in vitro model, while capturing inter-individual variability of gut microbiome. Oral capsule was as efficient as enema to decrease the number of disturbed days and bacterial load had no effect on enema performance. This study shows the relevance of human colon models as an alternative approach to in vivo assays during preclinical studies for evaluating FMT efficiency. The potential of this in vitro approach could be extended to FMT testing in the management of many digestive or extra-intestinal pathologies where gut microbial dysbiosis has been evidenced such as inflammatory bowel diseases, obesity or cancers

Lactobacillus paracasei feeding improves immune control of influenza infection in mice.

International audienceRespiratory tract infections such as flu cause severe morbidity and mortality and are among the leading causes of death in children and adults worldwide. Commensal microbiota is critical for orchestrating tissue homeostasis and immunity in the intestine. Probiotics represent an interesting source of immune modulators and several clinical studies have addressed the potential beneficial effects of probiotics against respiratory infections. Therefore, we have investigated the mechanisms of protection conferred by L. paracasei CNCM I-1518 strain in a mouse model of influenza infection. Notably, local myeloid cells accumulation is generated in the lungs after seven days feeding with L. paracasei prior to viral infection. L. paracasei-fed mice showed reduced susceptibility to the influenza infection, associated with less accumulation of inflammatory cells in the lungs, faster viral clearance and general health improvement. Interestingly, Allobaculum was significantly increased in L. paracasei-fed mice 7 days after influenza infection, even if the gut microbiota composition was not altered overall. L. paracasei-purified peptidoglycan partially recapitulated the protective phenotype observed with the entire bacteria. Collectively, our results demonstrate that oral consumption of L. paracasei CNCM I-1518 modulates lung immunity was associated with an improved control of influenza infection. These results further extend the beneficial role for certain lactobacilli to alleviate the burden of respiratory tract infections

Gut Microbiome Integration in Drug Discovery and Development of Small Molecules

International audienceThe ongoing discussion regarding the use of mixed or pure cultures of hydrogenotrophic methanogenic archaea in Power-to-Methane (P2M) bioprocess applications persists, with each option presenting its own advantages and disadvantages. To address this issue, a comparison of methane (CH4) yield between a novel methanogenic archaeon belonging to the species Methanothermobacter marburgensis (strain Clermont) isolated from a biologicalmethanation column, and the community from which it originated, was conducted. This comparison included the type strain M. marburgensis str. Marburg. The evaluation also examined how exposure to oxygen (O2) for up to240 min impacted the CH4 yield across these cultures. While both Methanothermobacter strains exhibit comparable CH4 yield, slightly higher than that of the mixed adapted culture under non-O2-exposed conditions, strainClermont does not display the lag time observed for strain Marburg

Gut microbiome integration in drug discovery and development of small molecules

Human microbiomes, particularly in the gut, could have a major impact on the efficacy and toxicity of drugs. However, gut microbial metabolism is often neglected in the drug discovery and development process. Medicen, a Paris -based human health innovation cluster, has gathered more than 30 international leading experts from pharma, academia, biotech, clinical research organizations, and regulatory science to develop proposals to facilitate the integration of microbiome science into drug discovery and development. Seven subteams were formed to cover the complementary expertise areas of 1) pharma experience and case studies, 2) in silico microbiome-drug interaction, 3) in vitro microbial stability screening, 4) gut fermentation models, 5) animal models, 6) microbiome integration in clinical and regulatory aspects, and 7) microbiome ecosystems and models. Each expert team produced a state-of-the-art report of their respective field highlighting existing microbiome-related tools at every stage of drug discovery and development. The most critical limitations are the growing, but still limited, drug-microbiome interaction data to produce predictive models and the lack of agreed -upon standards despite recent progress. In this paper we will report on and share proposals covering 1) how microbiome tools can support moving a compound from drug discovery to clinical proof -of -concept studies and alert early on potential undesired properties stemming from microbiome-induced drug metabolism and 2) how microbiome data can be generated and integrated in pharmacokinetic models that are predictive of the human situation. Examples of drugs metabolized by the microbiome will be discussed in detail to support recommendations from the working group

Impact on the Gut Microbiota of Intensive and Prolonged Antimicrobial Therapy in Patients With Bone and Joint Infection

International audienceThere is a growing interest in the potentially deleterious impact of antibiotics on gut microbiota. Patients with bone and joint infection (BJI) require prolonged treatment that may impact significantly the gut microbiota. We collected samples from patients with BJI at baseline, end of antibiotics (EOT), and 2 weeks after antibiotic withdrawal (follow-up, FU) in a multicenter prospective cohort in France. Microbiota composition was determined by shotgun metagenomic sequencing. Fecal markers of gut permeability and inflammation as well as multi-drug-resistant bacteria (MDRB) and Clostridioides difficile carriage were assessed at each time point. Sixty-two patients were enrolled: 27 native BJI, 14 osteosynthesis-related BJI, and 21 prosthetic joint infections (PJI). At EOT, there was a significant loss of alpha-diversity that recovered at FU in patients with native BJI and PJI, but not in patients with osteosynthesis-related BJI. At EOT, we observed an increase of Proteobacteria and Bacteroidetes that partially recovered at FU. The principal component analysis (PCoA) of the Bray–Curtis distance showed a significant change of the gut microbiota at the end of treatment compared to baseline that only partially recover at FU. Microbiota composition at FU does not differ significantly at the genus level when comparing patients treated for 6 weeks vs. those treated for 12 weeks. The use of fluoroquinolones was not associated with a lower Shannon index at the end of treatment; however, the PCoA of the Bray–Curtis distance showed a significant change at EOT, compared to baseline, that fully recovered at FU. Levels of fecal neopterin were negatively correlated with the Shannon index along with the follow-up (r2 = 0.17; p < 0.0001). The PCoA analysis of the Bray–Curtis distance shows that patients with an elevated plasma level of C-reactive protein (≥5 mg/L) at EOT had a distinct gut microbial composition compared to others. MDRB and C. difficile acquisition at EOT and FU represented 20% (7/35) and 37.1% (13/35) of all MDRB/C. difficile-free patients at the beginning of the study, respectively. In patients with BJI, antibiotics altered the gut microbiota diversity and composition with only partial recovery, mucosal inflammation, and permeability and acquisition of MDRB carriage. Microbiome interventions should be explored in patients with BJI to address these issues

Nouveaux dispositifs médicaux et nouveau procédé de fabrication pour un transfert de microbiote fécal par voie rectale, sans risque, standardisé et prêt à l’emploi

National audienceFecal microbiota transfer (FMT) consists in the administration of a fecal dilution from a healthy donor into the intestinal tract of a recipient to restore microbial diversity and confer a health benefit. FMT has been demonstrated to successfully treat recurrent Clostridium difficile infection but may also have therapeutic potential for other diseases. In France, a preparation for FMT is considered as a drug and thereby must be prepared under the responsibility of pharmacists. However, the preparation, storage and rectal administration for an efficient FMT is a big challenge for health care professionals, especially hospital pharmacists. MaaT Pharma, a French start-up company has developed patented devices (collection device, storage bag and administration system), and industrialized a manufacturing process developed by The Institut national de la recherche agronomique (Inra), to produce from raw fecal material, an active and stable therapeutic inoculum. Results presented in this study show that solutions developed by MaaT Pharma and Inra help in preserving the viability, diversity and original profile of the microbiota, at all stages, from collection of raw faeces to administration of the FMT. Thereby, MaaT Pharma is able to propose to hospital pharmacists and patients a safe, standardized, ready-to-use FMT, manufactured according to the good manufacturing practices (GMP). The next short-term objective is to propose an oral form product as an alternative to rectal administration for appropriate patients.Le transfert du microbiote fécal (TMF) est l’administration de la dilution des selles d’un donneur sain dans le tractus intestinal d’un receveur afin de restaurer sa diversité microbienne et apporter un bénéfice pour sa santé. Il a été démontré que le TMF peut traiter avec succès une infection récurrente à Clostridium difficile , mais peut également avoir un potentiel thérapeutique pour d’autres maladies. En France, toute préparation pour un TMF est considérée comme un médicament et doit donc être préparée sous la responsabilité des pharmaciens. Cependant, la préparation, le stockage et l’administration rectale d’un TMF efficace représentent un défi important pour les professionnels de santé, en particulier pour les pharmaciens hospitaliers. MaaT Pharma, une start-up franc ̧aise basée à Lyon, a développé des dispositifs médicaux brevetés (dispositif de collecte, poche de stockage et système d’administration), et industrialisé un procédé de fabrication développé par l’Institut national de la recherche agronomique (Inra), pour produire à partir des selles d’un donneur un inoculum thérapeutique actif et stable. Les résultats présentés dans cet article montrent que les solutions développées par MaaT Pharma et l’Inra aident à préserver la viabilité, la diversité et le profil d’origine du microbiote, à toutes les étapes, de la collecte des selles à l’administration du TMF. Ainsi, MaaT Pharma est en mesure de proposer aux pharmaciens hospitaliers et aux patients une solution fabriquée selon les bonnes pratiques de fabrication (BPF), sûre, standardisée et prête à l’emploi pour le TMF. Le prochain objectif à court terme est de proposer un produit pour administration sous forme orale, comme alternative à l’administration rectale pour les patients appropriés

Effect of consumption of <i>L</i>. <i>paracasei</i> (N = 30) on the recruitment of immune cells in the lungs at different days.

<p>A: day 0, B: day 7, C: day 10 after influenza infection, compared with the control group (Mice gavaged with PBS and infected by influenzae virus) (N = 30). Or to mice that were only infected with influenzae virus but with no gavage (N = 5) aM: Alveolar Macrophage, iM: Interstitial Macrophage, DC: Dendritic cells, iM: Inflammatory Monocyte, pM: Patrolling monocytes, Eo: Eosinophils, N: Neutrophils, B: B cells, T: T cells. Results are expressed as mean ± SEM for each group. (*p<0.05, **P<0.01).</p