Enterococcus hirae and Barnesiella intestinihominis Facilitate Cyclophosphamide-Induced Therapeutic Immunomodulatory Effects.

The efficacy of the anti-cancer immunomodulatory agent cyclophosphamide (CTX) relies on intestinal bacteria. How and which relevant bacterial species are involved in tumor immunosurveillance, and their mechanism of action are unclear. Here, we identified two bacterial species, Enterococcus hirae and Barnesiella intestinihominis that are involved during CTX therapy. Whereas E. hirae translocated from the small intestine to secondary lymphoid organs and increased the intratumoral CD8/Treg ratio, B. intestinihominis accumulated in the colon and promoted the infiltration of IFN-γ-producing γδT cells in cancer lesions. The immune sensor, NOD2, limited CTX-induced cancer immunosurveillance and the bioactivity of these microbes. Finally, E. hirae and B. intestinihominis specific-memory Th1 cell immune responses selectively predicted longer progression-free survival in advanced lung and ovarian cancer patients treated with chemo-immunotherapy. Altogether, E. hirae and B. intestinihominis represent valuable "oncomicrobiotics" ameliorating the efficacy of the most common alkylating immunomodulatory compound.

Oral tryptophan activates duodenal aryl hydrocarbon receptor in healthy subjects: a crossover randomized controlled trial.

Tryptophan is an essential amino acid transformed by host and gut microbial enzymes into metabolites that regulate mucosal homeostasis through aryl hydrocarbon receptor (AhR) activation. Alteration of tryptophan metabolism has been associated with chronic inflammation; however, whether tryptophan supplementation affects the metabolite repertoire and AhR activation under physiological conditions in humans is unknown. We performed a randomized, double blind, placebo-controlled, crossover study in 20 healthy volunteers. Subjects on a low tryptophan background diet were randomly assigned to a 3-wk l-tryptophan supplementation (3 g/day) or placebo, and after a 2-wk washout switched to opposite interventions. We assessed gastrointestinal and psychological symptoms by validated questionnaires, AhR activation by cell reporter assay, tryptophan metabolites by liquid chromatography and high-resolution mass spectrometry, cytokine production in isolated monocytes by ELISA, and microbiota profile by 16S rRNA Illumina technique. Oral tryptophan supplementation was well tolerated, with no changes in gastrointestinal or psychological scores. Compared with placebo, tryptophan increased AhR activation capacity by duodenal contents, but not by feces. This was paralleled by higher urinary and plasma kynurenine metabolites and indoles. Tryptophan had a modest impact on fecal microbiome profiles and no significant effect on cytokine production. At the doses used in this study, oral tryptophan supplementation in humans induces microbial indole and host kynurenine metabolic pathways in the small intestine, known to be immunomodulatory. The results should prompt tryptophan intervention strategies in inflammatory conditions of the small intestine where the AhR pathway is impaired.NEW & NOTEWORTHY We demonstrate that in healthy subjects, orally administered tryptophan activates microbial indole and host kynurenine pathways in the small intestine, the primary metabolic site for dietary components, and the richest source of immune cells along the gut. This study provides novel insights in how to optimally activate immunomodulatory AhR pathways and indole metabolism in the small intestine, serving as basis for future therapeutic trials using l-tryptophan supplementation in chronic inflammatory conditions affecting the small intestine.

Counteracting tryptophan metabolism alterations as a new therapeutic strategy for rheumatoid arthritis.

OBJECTIVES: Alterations in tryptophan (Trp) metabolism have been reported in inflammatory diseases, including rheumatoid arthritis (RA). However, understanding whether these alterations participate in RA development and can be considered putative therapeutic targets remains undetermined.In this study, we combined quantitative Trp metabolomics in the serum from patients with RA and corrective administration of a recombinant enzyme in experimental arthritis to address this question. METHODS: Targeted quantitative Trp metabolomics was performed on the serum from 574 previously untreated patients with RA from the ESPOIR (Etude et Suivi des POlyarthrites Indifférenciées Récentes) cohort and 98 healthy subjects. A validation cohort involved 69 established patients with RA. Dosages were also done on the serum of collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) mice and controls. A proof-of-concept study evaluating the therapeutic potency of targeting the kynurenine pathway was performed in the CAIA model. RESULTS: Differential analysis revealed dramatic changes in Trp metabolite levels in patients with RA compared with healthy controls. Decreased levels of kynurenic (KYNA) and xanthurenic (XANA) acids and indole derivatives, as well as an increased level of quinolinic acid (QUIN), were found in the serum of patients with RA. They correlated positively with disease severity (assessed by both circulating biomarkers and disease activity scores) and negatively with quality-of-life scores. Similar profiles of kynurenine pathway metabolites were observed in the CAIA and CIA models. From a mechanistic perspective, we demonstrated that QUIN favours human fibroblast-like synoviocyte proliferation and affected their cellular metabolism, through inducing both mitochondrial respiration and glycolysis. Finally, systemic administration of the recombinant enzyme aminoadipate aminotransferase, responsible for the generation of XANA and KYNA, was protective in the CAIA model. CONCLUSIONS: Altogether, our preclinical and clinical data indicate that alterations in the Trp metabolism play an active role in the pathogenesis of RA and could be considered as a new therapeutic avenue.

Exploring the interaction and impact of probiotic and commensal bacteria on vitamins, minerals and short chain fatty acids metabolism.

There is increasing evidence that probiotic and commensal bacteria play a role in substrate metabolism, energy harvesting and intestinal homeostasis, and may exert immunomodulatory activities on human health. In addition, recent research suggests that these microorganisms interact with vitamins and minerals, promoting intestinal and metabolic well-being while producing vital microbial metabolites such as short-chain fatty acids (SCFAs). In this regard, there is a flourishing field exploring the intricate dynamics between vitamins, minerals, SCFAs, and commensal/probiotic interactions. In this review, we summarize some of the major hypotheses beyond the mechanisms by which commensals/probiotics impact gut health and their additional effects on the absorption and metabolism of vitamins, minerals, and SCFAs. Our analysis includes comprehensive review of existing evidence from preclinical and clinical studies, with particular focus on the potential interaction between commensals/probiotics and micronutrients. Finally, we highlight knowledge gaps and outline directions for future research in this evolving field.

Intestinal Microbiota Increases Cell Proliferation of Colonic Mucosa in Human-Flora-Associated (HFA) Mice.

Intestinal epithelium renewal strictly depends on fine regulation between cell proliferation, differentiation, and apoptosis. While murine intestinal microbiota has been shown to modify some epithelial cell kinetics parameters, less is known about the role of the human intestinal microbiota. Here, we investigated the rate of intestinal cell proliferation in C3H/HeN germ-free mice associated with human flora (HFA, n = 8), and in germ-free (n = 15) and holoxenic mice (n = 16). One hour before sacrifice, all mice were intraperitoneally inoculated with 5-bromodeoxyuridine (BrdU), and the number of BrdU-positive cells/total cells (labelling index, LI), both in the jejunum and the colon, was evaluated by immunohistochemistry. Samples were also observed by scanning electron microscopy (SEM). Moreover, the microbiota composition in the large bowel of the HFA mice was compared to that of of human donor's fecal sample. No differences in LI were found in the small bowels of the HFA, holoxenic, and germ-free mice. Conversely, the LI in the large bowel of the HFA mice was significantly higher than that in the germ-free and holoxenic counterparts (p = 0.017 and p = 0.048, respectively). In the holoxenic and HFA mice, the SEM analysis disclosed different types of bacteria in close contact with the intestinal epithelium. Finally, the colonic microbiota composition of the HFA mice widely overlapped with that of the human donor in terms of dominant populations, although Bifidobacteria and Lactobacilli disappeared. Despite the small sample size analyzed in this study, these preliminary findings suggest that human intestinal microbiota may promote a high proliferation rate of colonic mucosa. In light of the well-known role of uncontrolled proliferation in colorectal carcinogenesis, these results may deserve further investigation in a larger population study.

CARD9 in neutrophils protects from colitis and controls mitochondrial metabolism and cell survival.

OBJECTIVES: Inflammatory bowel disease (IBD) results from a combination of genetic predisposition, dysbiosis of the gut microbiota and environmental factors, leading to alterations in the gastrointestinal immune response and chronic inflammation. Caspase recruitment domain 9 (Card9), one of the IBD susceptibility genes, has been shown to protect against intestinal inflammation and fungal infection. However, the cell types and mechanisms involved in the CARD9 protective role against inflammation remain unknown. DESIGN: We used dextran sulfate sodium (DSS)-induced and adoptive transfer colitis models in total and conditional CARD9 knock-out mice to uncover which cell types play a role in the CARD9 protective phenotype. The impact of Card9 deletion on neutrophil function was assessed by an in vivo model of fungal infection and various functional assays, including endpoint dilution assay, apoptosis assay by flow cytometry, proteomics and real-time bioenergetic profile analysis (Seahorse). RESULTS: Lymphocytes are not intrinsically involved in the CARD9 protective role against colitis. CARD9 expression in neutrophils, but not in epithelial or CD11c+cells, protects against DSS-induced colitis. In the absence of CARD9, mitochondrial dysfunction increases mitochondrial reactive oxygen species production leading to the premature death of neutrophilsthrough apoptosis, especially in oxidative environment. The decreased functional neutrophils in tissues might explain the impaired containment of fungi and increased susceptibility to intestinal inflammation. CONCLUSION: These results provide new insight into the role of CARD9 in neutrophil mitochondrial function and its involvement in intestinal inflammation, paving the way for new therapeutic strategies targeting neutrophils.

Rewiring the altered tryptophan metabolism as a novel therapeutic strategy in inflammatory bowel diseases.

OBJECTIVE: The extent to which tryptophan (Trp) metabolism alterations explain or influence the outcome of inflammatory bowel diseases (IBDs) is still unclear. However, several Trp metabolism end-products are essential to intestinal homeostasis. Here, we investigated the role of metabolites from the kynurenine pathway. DESIGN: Targeted quantitative metabolomics was performed in two large human IBD cohorts (1069 patients with IBD). Dextran sodium sulphate-induced colitis experiments in mice were used to evaluate effects of identified metabolites. In vitro, ex vivo and in vivo experiments were used to decipher mechanisms involved. Effects on energy metabolism were evaluated by different methods including Single Cell mEtabolism by profiling Translation inHibition. RESULTS: In mice and humans, intestinal inflammation severity negatively correlates with the amount of xanthurenic (XANA) and kynurenic (KYNA) acids. Supplementation with XANA or KYNA decreases colitis severity through effects on intestinal epithelial cells and T cells, involving Aryl hydrocarbon Receptor (AhR) activation and the rewiring of cellular energy metabolism. Furthermore, direct modulation of the endogenous tryptophan metabolism, using the recombinant enzyme aminoadipate aminotransferase (AADAT), responsible for the generation of XANA and KYNA, was protective in rodent colitis models. CONCLUSION: Our study identified a new mechanism linking Trp metabolism to intestinal inflammation and IBD. Bringing back XANA and KYNA has protective effects involving AhR and the rewiring of the energy metabolism in intestinal epithelial cells and CD4+ T cells. This study paves the way for new therapeutic strategies aiming at pharmacologically correcting its alterations in IBD by manipulating the endogenous metabolic pathway with AADAT.

The Tolerance of Gut Commensal Faecalibacterium to Oxidative Stress Is Strain Dependent and Relies on Detoxifying Enzymes.

Obligate anaerobic bacteria in genus Faecalibacterium are among the most dominant taxa in the colon of healthy individuals and contribute to intestinal homeostasis. A decline in the abundance of this genus is associated with the occurrence of various gastrointestinal disorders, including inflammatory bowel diseases. In the colon, these diseases are accompanied by an imbalance between the generation and elimination of reactive oxygen species (ROS), and oxidative stress is closely linked to disruptions in anaerobiosis. In this work, we explored the impact of oxidative stress on several strains of faecalibacteria. An in silico analysis of complete genomes of faecalibacteria revealed the presence of genes encoding O2- and/or ROS-detoxifying enzymes, including flavodiiron proteins, rubrerythrins, reverse rubrerythrins, superoxide reductases, and alkyl peroxidase. However, the presence and the number of these detoxification systems varied greatly among faecalibacteria. These results were confirmed by O2 stress survival tests, in which we found that strains differed widely in their sensitivity. We showed the protective role of cysteine, which limited the production of extracellular O2•- and improved the survival of Faecalibacterium longum L2-6 under high O2 tension. In the strain F. longum L2-6, we observed that the expression of genes encoding detoxifying enzymes was upregulated in the response to O2 or H2O2 stress but with different patterns of regulation. Based on these results, we propose a first model of the gene regulatory network involved in the response to oxidative stress in F. longum L2-6. IMPORTANCE Commensal bacteria in the genus Faecalibacterium have been proposed for use as next-generation probiotics, but efforts to cultivate and exploit the potential of these strains have been limited by their sensitivity to O2. More broadly, little is known about how commensal and health-associated bacterial species in the human microbiome respond to the oxidative stress that occurs as a result of inflammation in the colon. In this work, we provide insights regarding the genes that encode potential mechanisms of protection against O2 or ROS stress in faecalibacteria, which may facilitate future advances in work with these important bacteria.

Comprehensive probiogenomics analysis of the commensal Escherichia coli CEC15 as a potential probiotic strain.

BACKGROUND: Probiotics have gained attention for their potential maintaining gut and immune homeostasis. They have been found to confer protection against pathogen colonization, possess immunomodulatory effects, enhance gut barrier functionality, and mitigate inflammation. However, a thorough understanding of the unique mechanisms of effects triggered by individual strains is necessary to optimize their therapeutic efficacy. Probiogenomics, involving high-throughput techniques, can help identify uncharacterized strains and aid in the rational selection of new probiotics. This study evaluates the potential of the Escherichia coli CEC15 strain as a probiotic through in silico, in vitro, and in vivo analyses, comparing it to the well-known probiotic reference E. coli Nissle 1917. Genomic analysis was conducted to identify traits with potential beneficial activity and to assess the safety of each strain (genomic islands, bacteriocin production, antibiotic resistance, production of proteins involved in host homeostasis, and proteins with adhesive properties). In vitro studies assessed survival in gastrointestinal simulated conditions and adhesion to cultured human intestinal cells. Safety was evaluated in BALB/c mice, monitoring the impact of E. coli consumption on clinical signs, intestinal architecture, intestinal permeability, and fecal microbiota. Additionally, the protective effects of both strains were assessed in a murine model of 5-FU-induced mucositis. RESULTS: CEC15 mitigates inflammation, reinforces intestinal barrier, and modulates intestinal microbiota. In silico analysis revealed fewer pathogenicity-related traits in CEC15, when compared to Nissle 1917, with fewer toxin-associated genes and no gene suggesting the production of colibactin (a genotoxic agent). Most predicted antibiotic-resistance genes were neither associated with actual resistance, nor with transposable elements. The genome of CEC15 strain encodes proteins related to stress tolerance and to adhesion, in line with its better survival during digestion and higher adhesion to intestinal cells, when compared to Nissle 1917. Moreover, CEC15 exhibited beneficial effects on mice and their intestinal microbiota, both in healthy animals and against 5FU-induced intestinal mucositis. CONCLUSIONS: These findings suggest that the CEC15 strain holds promise as a probiotic, as it could modulate the intestinal microbiota, providing immunomodulatory and anti-inflammatory effects, and reinforcing the intestinal barrier. These findings may have implications for the treatment of gastrointestinal disorders, particularly some forms of diarrhea.

A Pulmonary Lactobacillus murinus Strain Induces Th17 and RORγt+ Regulatory T Cells and Reduces Lung Inflammation in Tuberculosis.

The lungs harbor multiple resident microbial communities, otherwise known as the microbiota. There is an emerging interest in deciphering whether the pulmonary microbiota modulate local immunity, and whether this knowledge could shed light on mechanisms operating in the response to respiratory pathogens. In this study, we investigate the capacity of a pulmonary Lactobacillus strain to modulate the lung T cell compartment and assess its prophylactic potential upon infection with Mycobacterium tuberculosis, the etiological agent of tuberculosis. In naive mice, we report that a Lactobacillus murinus (Lagilactobacillus murinus) strain (CNCM I-5314) increases the presence of lung Th17 cells and of a regulatory T cell (Treg) subset known as RORγt+ Tregs. In particular, intranasal but not intragastric administration of CNCM I-5314 increases the expansion of these lung leukocytes, suggesting a local rather than systemic effect. Resident Th17 and RORγt+ Tregs display an immunosuppressive phenotype that is accentuated by CNCM I-5314. Despite the well-known ability of M. tuberculosis to modulate lung immunity, the immunomodulatory effect by CNCM I-5314 is dominant, as Th17 and RORγt+ Tregs are still highly increased in the lung at 42-d postinfection. Importantly, CNCM I-5314 administration in M. tuberculosis-infected mice results in reduction of pulmonary inflammation, without increasing M. tuberculosis burden. Collectively, our findings provide evidence for an immunomodulatory capacity of CNCM I-5314 at steady state and in a model of chronic inflammation in which it can display a protective role, suggesting that L. murinus strains found in the lung may shape local T cells in mice and, perhaps, in humans.

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

The 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.

Phosphatidylglycerols are induced by gut dysbiosis and inflammation, and favorably modulate adipose tissue remodeling in obesity.

Lipidomic techniques can improve our understanding of complex lipid interactions that regulate metabolic diseases. Here, a serum phospholipidomics analysis identified associations between phosphatidylglycerols (PGs) and gut microbiota dysbiosis. Compared with the other phospholipids, serum PGs were the most elevated in patients with low microbiota gene richness, which were normalized after a dietary intervention that restored gut microbial diversity. Serum PG levels were positively correlated with metagenomic functional capacities for bacterial LPS synthesis and host markers of low-grade inflammation; transcriptome databases identified PG synthase, the first committed enzyme in PG synthesis, as a potential mediator. Experiments in mice and cultured human-derived macrophages demonstrated that LPS induces PG release. Acute PG treatment in mice altered adipose tissue gene expression toward remodeling and inhibited ex vivo lipolysis in adipose tissue, suggesting that PGs favor lipid storage. Indeed, several PG species were associated with the severity of obesity in mice and humans. Finally, despite enrichment in PGs in bacterial membranes, experiments employing gnotobiotic mice colonized with recombinant PG overproducing Lactococcus lactis showed limited direct contribution of microbial PGs to the host. In summary, PGs are inflammation-responsive lipids indirectly regulated by the gut microbiota via endotoxins and regulate adipose tissue homeostasis in obesity.-Kayser, B. D., Lhomme, M., Prifti, E., Da Cunha, C., Marquet, F., Chain, F., Naas, I., Pelloux, V., Dao, M.-C., Kontush, A., Rizkalla, S. W., Aron-Wisnewsky, J., Bermúdez-Humarán, L. G., Oakley, F., Langella, P., Clément, K., Dugail, I. Phosphatidylglycerols are induced by gut dysbiosis and inflammation, and favorably modulate adipose tissue remodeling in obesity.

The regenerating family member 3 ß instigates IL-17A-mediated neutrophil recruitment downstream of NOD1/2 signalling for controlling colonisation resistance independently of microbiota community structure.

OBJECTIVE: Loss of the Crohn's disease predisposing NOD2 gene results in an intestinal microenvironment conducive for colonisation by attaching-and-effacing enteropathogens. However, it remains elusive whether it relies on the intracellular recruitment of the serine-threonine kinase RIPK2 by NOD2, a step that is required for its activation of the transcription factor NF-κB. DESIGN: Colonisation resistance was evaluated in wild type and mutant mice, as well as in ex-germ-free (ex-GF) mice which were colonised either with faeces from Ripk2-deficient mice or with bacteria with similar preferences for carbohydrates to those acquired by the pathogen. The severity of the mucosal pathology was quantified at several time points postinfection by using a previously established scoring. The community resilience in response to infection was evaluated by 16S ribosomal RNA gene sequence analysis. The control of pathogen virulence was evaluated by monitoring the secretion of Citrobacter-specific antibody response in the faeces. RESULTS: Primary infection was similarly outcompeted in ex-GF Ripk2-deficient and control mice, demonstrating that the susceptibility to infection resulting from RIPK2 deficiency cannot be solely attributed to specific microbiota community structures. In contrast, delayed clearance of Citrobacter rodentium and exacerbated histopathology were preceded by a weakened propensity of intestinal macrophages to afford innate lymphoid cell activation. This tissue protection unexpectedly required the regenerating family member 3ß by instigating interleukin (IL) 17A-mediated neutrophil recruitment to the intestine and subsequent phosphorylation of signal transducer and activator of transcription 3. CONCLUSIONS: These results unveil a previously unrecognised mechanism that efficiently protects from colonisation by diarrhoeagenic bacteria early in infection.

A Putative Type V Pilus Contributes to Bacteroides thetaiotaomicron Biofilm Formation Capacity.

Bacteroides thetaiotaomicron is a prominent anaerobic member of the healthy human gut microbiota. While the majority of functional studies on B. thetaiotaomicron addressed its impact on the immune system and the utilization of diet polysaccharides, B. thetaiotaomicron biofilm capacity and its contribution to intestinal colonization are still poorly characterized. We tested the natural adhesion of 34 B. thetaiotaomicron isolates and showed that although biofilm capacity is widespread among B. thetaiotaomicron strains, this phenotype is masked or repressed in the widely used reference strain VPI 5482. Using transposon mutagenesis followed by a biofilm positive-selection procedure, we identified VPI 5482 mutants with increased biofilm capacity corresponding to an alteration in the C-terminal region of BT3147, encoded by the BT3148-BT3147 locus, which displays homology with Mfa-like type V pili found in many Bacteroidetes We show that BT3147 is exposed on the B. thetaiotaomicron surface and that BT3147-dependent adhesion also requires BT3148, suggesting that BT3148 and BT3147 correspond to the anchor and stalk subunits of a new type V pilus involved in B. thetaiotaomicron adhesion. This study therefore introduces B. thetaiotaomicron as a model to study proteinaceous adhesins and biofilm-related phenotypes in this important intestinal symbiont.IMPORTANCE Although the gut anaerobe Bacteroides thetaiotaomicron is a prominent member of the healthy human gut microbiota, little is known about its capacity to adhere to surfaces and form biofilms. Here, we identify that alteration of a surface-exposed protein corresponding to a type of pili found in many Bacteroidetes increases B. thetaiotaomicron biofilm formation. This study lays the ground for establishing this bacterium as a model organism for in vitro and in vivo studies of biofilm-related phenotypes in gut anaerobes.

Oral delivery of pancreatitis-associated protein by Lactococcus lactis displays protective effects in dinitro-benzenesulfonic-acid-induced colitis model and is able to modulate the composition of the microbiota.

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 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 with 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.

Carriage of λ Latent Virus Is Costly for Its Bacterial Host due to Frequent Reactivation in Monoxenic Mouse Intestine.

Temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. Although these prophages are generally considered to increase their hosts' fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. Here, we investigated the impact of prophage carriage in the gastrointestinal tract of monoxenic mice. Combined with mathematical modelling, these experimental results provided a quantitative estimation of key parameters governing phage-bacteria interactions within this model ecosystem. We used wild-type and mutant strains of the best known host/phage pair, Escherichia coli and phage λ. Unexpectedly, λ prophage caused a significant fitness cost for its carrier, due to an induction rate 50-fold higher than in vitro, with 1 to 2% of the prophage being induced. However, when prophage carriers were in competition with isogenic phage susceptible bacteria, the prophage indirectly benefited its carrier by killing competitors: infection of susceptible bacteria led to phage lytic development in about 80% of cases. The remaining infected bacteria were lysogenized, resulting overall in the rapid lysogenization of the susceptible lineage. Moreover, our setup enabled to demonstrate that rare events of phage gene capture by homologous recombination occurred in the intestine of monoxenic mice. To our knowledge, this study constitutes the first quantitative characterization of temperate phage-bacteria interactions in a simplified gut environment. The high prophage induction rate detected reveals DNA damage-mediated SOS response in monoxenic mouse intestine. We propose that the mammalian gut, the most densely populated bacterial ecosystem on earth, might foster bacterial evolution through high temperate phage activity.

Intestinal dendritic cell licensing through Toll-like receptor 4 is required for oral tolerance in allergic contact dermatitis.

BACKGROUND: Induction of oral tolerance to haptens is an efficient way to prevent allergic contact dermatitis (ACD) in mice. Toll-like receptor (TLR)-mediated sensing of the microbiota contributes to gut homeostasis, yet whether it contributes to induction of oral tolerance has not been documented. OBJECTIVE: We examined whether oral tolerance to the contact sensitizer 2,4-dinitro-fluorobenzene (DNFB) depends on microbiota/TLRs and evaluated the role of TLR4 on the tolerogenic function of intestinal dendritic cells (DCs). METHODS: Oral tolerance was induced by DNFB gavage in germ-free and mice deficient in several TLRs. Tolerance was assessed by means of suppression of contact hypersensitivity and hapten-specific IFN-γ-producing effector T cells. The tolerogenic function of intestinal DCs was tested by adoptive transfer experiments, ex vivo hapten presentation, and forkhead box p3 regulatory T-cell conversion. RESULTS: Oral tolerance induced by DNFB gavage was impaired in germ-free mice and TLR4-deficient mice. Bone marrow chimeras revealed that TLR4 expression on hematopoietic cells was necessary for oral tolerance induction. TLR4 appeared to be essential for the ability of intestinal dendritic cells from DNFB-fed mice to inhibit ACD on adoptive transfer. Indeed, TLR4 conditioned the in vivo mobilization to mesenteric lymph nodes of intestinal migratory CD103+ DCs carrying oral DNFB, especially the CD103+CD11b+ DC subset expressing the vitamin A-converting enzyme retinaldehyde dehydrogenase and specialized in forkhead box p3-positive regulatory T-cell conversion. CONCLUSIONS: Our data demonstrate that TLR4 conditions induction of oral tolerance to DNFB through licensing tolerogenic gut DCs. Oral biotherapy with TLR4 ligands might be useful to potentiate oral tolerance to haptens and alleviate ACD in human subjects.

Card9 mediates susceptibility to intestinal pathogens through microbiota modulation and control of bacterial virulence.

OBJECTIVE: In association with innate and adaptive immunity, the microbiota controls the colonisation resistance against intestinal pathogens. Caspase recruitment domain 9 (CARD9), a key innate immunity gene, is required to shape a normal gut microbiota. Card9-/- mice are more susceptible to the enteric mouse pathogen Citrobacter rodentium that mimics human infections with enteropathogenic and enterohaemorrhagic Escherichia coli. Here, we examined how CARD9 controls C. rodentium infection susceptibility through microbiota-dependent and microbiota-independent mechanisms. DESIGN: C. rodentium infection was assessed in conventional and germ-free (GF) wild-type (WT) and Card9-/- mice. To explore the impact of Card9-/-microbiota in infection susceptibility, GF WT mice were colonised with WT (WT→GF) or Card9-/- (Card9-/- →GF) microbiota before C. rodentium infection. Microbiota composition was determined by 16S rDNA gene sequencing. Inflammation severity was determined by histology score and lipocalin level. Microbiota-host immune system interactions were assessed by quantitative PCR analysis. RESULTS: CARD9 controls pathogen virulence in a microbiota-independent manner by supporting a specific humoral response. Higher susceptibility to C. rodentium-induced colitis was observed in Card9-/- →GF mice. The microbiota of Card9-/- mice failed to outcompete the monosaccharide-consuming C. rodentium, worsening the infection severity. A polysaccharide-enriched diet counteracted the ecological advantage of C. rodentium and the defective pathogen-specific antibody response in Card9-/- mice. CONCLUSIONS: CARD9 modulates the susceptibility to intestinal infection by controlling the pathogen virulence in a microbiota-dependent and microbiota-independent manner. Genetic susceptibility to intestinal pathogens can be overridden by diet intervention that restores humoural immunity and a competing microbiota.

M cell-targeting strategy enhances systemic and mucosal immune responses induced by oral administration of nuclease-producing L. lactis.

Efficient delivery of antigens to the gut-associated lymphoid tissue (GALT) is the most critical step for the induction of mucosal immunity by oral vaccines. As M cells are the main portal for luminal antigens into the GALT, the M cell-targeting of antigens affords a promising strategy toward the development of effective oral vaccines. Lactococcus lactis is a fascinating recombinant host for oral vaccines, as they survive and produce antigens in the gut and have a particularly safe profile for human use. In this study, we developed and evaluated an M cell-targeting oral immunization system using recombinant L. lactis strains. For the purpose, we generated an L. lactis strain that secretes a model antigen fused with the OmpH ß1α1 domain of Yersinia enterocolitica, which has been shown to bind to a complement C5a receptor on the M cell surface. As the model antigen, Staphylococcus aureus nuclease was used for fusion, resulting in L. lactis-expressing Nuc-OmpH (LL/Nuc-OmpH). Ex vivo intestinal loop assays showed that the amount of Nuc-OmpH taken up into Peyer's patches was more than that of the unfused nuclease (Nuc). In addition, oral administration of the recombinant L. lactis strains to mice demonstrated that LL/Nuc-OmpH-induced nuclease-specific fecal IgA and serum IgG titers were significantly higher than those induced by LL/Nuc. These results indicate that OmpH works as an M cell-targeting molecule when fused with antigens secreted from L. lactis and that the M cell-targeting strategy affords a promising platform for L. lactis-based mucosal immunization.

Food-grade TiO2 is trapped by intestinal mucus in vitro but does not impair mucin O-glycosylation and short-chain fatty acid synthesis in vivo: implications for gut barrier protection.

BACKGROUND: Titanium dioxide (TiO2) particles are commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, the risk of gut barrier disruption is an increasing concern because of the presence of a nano-sized fraction. Food-grade E171 may interact with mucus, a gut barrier protagonist still poorly explored in food nanotoxicology. To test this hypothesis, a comprehensive approach was performed to evaluate in vitro and in vivo interactions between TiO2 and intestinal mucus, by comparing food-grade E171 with NM-105 (Aeroxyde P25) OECD reference nanomaterial. RESULTS: We tested E171-trapping properties of mucus in vitro using HT29-MTX intestinal epithelial cells. Time-lapse confocal laser scanning microscopy was performed without labeling to avoid modification of the particle surface. Near-UV irradiation of E171 TiO2 particles at 364 nm resulted in fluorescence emission in the visible range, with a maximum at 510 nm. The penetration of E171 TiO2 into the mucoid area of HT29-MTX cells was visualized in situ. One hour after exposure, TiO2 particles accumulated inside "patchy" regions 20 µm above the substratum. The structure of mucus produced by HT29-MTX cells was characterized by MUC5AC immunofluorescence staining. The mucus layer was thin and organized into regular "islands" located approximately 20 µm above the substratum. The region-specific trapping of food-grade TiO2 particles was attributed to this mucus patchy structure. We compared TiO2-mediated effects in vivo in rats after acute or sub-chronic oral daily administration of food-grade E171 and NM-105 at relevant exposure levels for humans. Cecal short-chain fatty acid profiles and gut mucin O-glycosylation patterns remained unchanged, irrespective of treatment. CONCLUSIONS: Food-grade TiO2 is trapped by intestinal mucus in vitro but does not affect mucin O-glycosylation and short-chain fatty acid synthesis in vivo, suggesting the absence of a mucus barrier impairment under "healthy gut" conditions.