Crosstalk between gut microbiota and the host through L-tryptophan metabolism

Le L-tryptophane (L-trp) est un acide aminé essentiel dont certains métabolites microbiens et humains possèdent des effets immunomodulateurs via leur liaison sur le récepteur aux aryl hydrocarbones (AhR). Placé à l’interface entre l’hôte et le microbiote, AhR présente un fort potentiel thérapeutique dans les maladies inflammatoires. Décoder les mécanismes de son activation et de sa régulation par les métabolites du microbiote est crucial pour comprendre les conséquences des dysbioses qui font le lit de nombreuses pathologies. Nous avons montré une synergie d’activation de AhR en présence de métabolites du L-trp avec les acides gras à chaine courte à la fois sur des lignées cellulaires in vitro ainsi que sur des explants coliques humains. Nos premières investigations ont montré que le butyrate n’agissait pas sur la cascade d’activation de AhR. L’étude des mécanismes indirects d’activation de AhR a montré que le butyrate n’inhibait pas l’activité des cytochromes, n’agissait pas via ses récepteurs et n’agissait pas par l’intermédiaire de son activité inhibitrice du protéasome pour activer AhR. La synergie semblait reposer sur des modifications épigénétiques confirmées par méthode d’immunoprécipitation de la chromatine montrant une augmentation du recrutement de AhR au niveau du promoteur de cyp1a1 lorsque le butyrate était additionné au FICZ et en utilisant l’inhibiteur de HDAC trichostatine A permettant de mimer l’effet du butyrate sur l’activation de AhR médiée par FICZ. En conclusion, ces résultats soutiennent l'existence d'interactions complexes entre de multiples métabolites dérivés de l'intestin qui méritent une exploration approfondie.L-tryptophan (L-trp) is an essential amino acid whose microbial and human metabolites possess immunomodulatory effects through their binding to the aryl hydrocarbon receptor (AhR). At the interface between the host and the microbiota, AhR has a strong therapeutic potential in inflammatory diseases. Deciphering the mechanisms of its activation and regulation by microbiota metabolites is crucial for understanding the consequences of dysbiosis, which is the basis of many pathologies. We have shown a synergistic activation of AhR in the presence of L-trp metabolites with short-chain fatty acids both in in vitro cell lines and in human colonic explants. Our initial investigations showed that butyrate did not affect the AhR activation cascade. The study of indirect mechanisms of AhR activation showed that butyrate did not inhibit cytochrome activity, did not act via its receptors and did not act via its proteasome inhibitory activity to activate AhR. The synergy appeared to be based on epigenetic modifications confirmed by chromatin immunoprecipitation method showing an increase in AhR recruitment at the CYP1A1 promoter when butyrate was added to FICZ and using the HDAC inhibitor trichostatin A to mimic the effect of butyrate on FICZ-mediated AhR activation. In conclusion, these results support the existence of complex interactions between multiple gut-derived metabolites that deserve further exploration

Dialogue entre le microbiote intestinal et l'hôte via le métabolisme du L-tryptophane

L-tryptophan (L-trp) is an essential amino acid whose microbial and human metabolites possess immunomodulatory effects through their binding to the aryl hydrocarbon receptor (AhR). At the interface between the host and the microbiota, AhR has a strong therapeutic potential in inflammatory diseases. Deciphering the mechanisms of its activation and regulation by microbiota metabolites is crucial for understanding the consequences of dysbiosis, which is the basis of many pathologies. We have shown a synergistic activation of AhR in the presence of L-trp metabolites with short-chain fatty acids both in in vitro cell lines and in human colonic explants. Our initial investigations showed that butyrate did not affect the AhR activation cascade. The study of indirect mechanisms of AhR activation showed that butyrate did not inhibit cytochrome activity, did not act via its receptors and did not act via its proteasome inhibitory activity to activate AhR. The synergy appeared to be based on epigenetic modifications confirmed by chromatin immunoprecipitation method showing an increase in AhR recruitment at the CYP1A1 promoter when butyrate was added to FICZ and using the HDAC inhibitor trichostatin A to mimic the effect of butyrate on FICZ-mediated AhR activation. In conclusion, these results support the existence of complex interactions between multiple gut-derived metabolites that deserve further exploration.Le L-tryptophane (L-trp) est un acide aminé essentiel dont certains métabolites microbiens et humains possèdent des effets immunomodulateurs via leur liaison sur le récepteur aux aryl hydrocarbones (AhR). Placé à l’interface entre l’hôte et le microbiote, AhR présente un fort potentiel thérapeutique dans les maladies inflammatoires. Décoder les mécanismes de son activation et de sa régulation par les métabolites du microbiote est crucial pour comprendre les conséquences des dysbioses qui font le lit de nombreuses pathologies. Nous avons montré une synergie d’activation de AhR en présence de métabolites du L-trp avec les acides gras à chaine courte à la fois sur des lignées cellulaires in vitro ainsi que sur des explants coliques humains. Nos premières investigations ont montré que le butyrate n’agissait pas sur la cascade d’activation de AhR. L’étude des mécanismes indirects d’activation de AhR a montré que le butyrate n’inhibait pas l’activité des cytochromes, n’agissait pas via ses récepteurs et n’agissait pas par l’intermédiaire de son activité inhibitrice du protéasome pour activer AhR. La synergie semblait reposer sur des modifications épigénétiques confirmées par méthode d’immunoprécipitation de la chromatine montrant une augmentation du recrutement de AhR au niveau du promoteur de cyp1a1 lorsque le butyrate était additionné au FICZ et en utilisant l’inhibiteur de HDAC trichostatine A permettant de mimer l’effet du butyrate sur l’activation de AhR médiée par FICZ. En conclusion, ces résultats soutiennent l'existence d'interactions complexes entre de multiples métabolites dérivés de l'intestin qui méritent une exploration approfondie

Dialogue entre le microbiote intestinal et l'hôte via le métabolisme du L-tryptophane

L-tryptophan (L-trp) is an essential amino acid whose microbial and human metabolites possess immunomodulatory effects through their binding to the aryl hydrocarbon receptor (AhR). At the interface between the host and the microbiota, AhR has a strong therapeutic potential in inflammatory diseases. Deciphering the mechanisms of its activation and regulation by microbiota metabolites is crucial for understanding the consequences of dysbiosis, which is the basis of many pathologies. We have shown a synergistic activation of AhR in the presence of L-trp metabolites with short-chain fatty acids both in in vitro cell lines and in human colonic explants. Our initial investigations showed that butyrate did not affect the AhR activation cascade. The study of indirect mechanisms of AhR activation showed that butyrate did not inhibit cytochrome activity, did not act via its receptors and did not act via its proteasome inhibitory activity to activate AhR. The synergy appeared to be based on epigenetic modifications confirmed by chromatin immunoprecipitation method showing an increase in AhR recruitment at the CYP1A1 promoter when butyrate was added to FICZ and using the HDAC inhibitor trichostatin A to mimic the effect of butyrate on FICZ-mediated AhR activation. In conclusion, these results support the existence of complex interactions between multiple gut-derived metabolites that deserve further exploration.Le L-tryptophane (L-trp) est un acide aminé essentiel dont certains métabolites microbiens et humains possèdent des effets immunomodulateurs via leur liaison sur le récepteur aux aryl hydrocarbones (AhR). Placé à l’interface entre l’hôte et le microbiote, AhR présente un fort potentiel thérapeutique dans les maladies inflammatoires. Décoder les mécanismes de son activation et de sa régulation par les métabolites du microbiote est crucial pour comprendre les conséquences des dysbioses qui font le lit de nombreuses pathologies. Nous avons montré une synergie d’activation de AhR en présence de métabolites du L-trp avec les acides gras à chaine courte à la fois sur des lignées cellulaires in vitro ainsi que sur des explants coliques humains. Nos premières investigations ont montré que le butyrate n’agissait pas sur la cascade d’activation de AhR. L’étude des mécanismes indirects d’activation de AhR a montré que le butyrate n’inhibait pas l’activité des cytochromes, n’agissait pas via ses récepteurs et n’agissait pas par l’intermédiaire de son activité inhibitrice du protéasome pour activer AhR. La synergie semblait reposer sur des modifications épigénétiques confirmées par méthode d’immunoprécipitation de la chromatine montrant une augmentation du recrutement de AhR au niveau du promoteur de cyp1a1 lorsque le butyrate était additionné au FICZ et en utilisant l’inhibiteur de HDAC trichostatine A permettant de mimer l’effet du butyrate sur l’activation de AhR médiée par FICZ. En conclusion, ces résultats soutiennent l'existence d'interactions complexes entre de multiples métabolites dérivés de l'intestin qui méritent une exploration approfondie

Tryptophan Metabolism as a Pharmacological Target

International audienceL-Tryptophan is an essential amino acid required for protein synthesis. It undergoes an extensive and complex metabolism along several pathways, resulting in many bioactive molecules acting in various organs through different action mechanisms. Enzymes involved in its metabolism, metabolites themselves, or their receptors, represent potential therapeutic targets, which are the subject of dynamic research. Disruptions in L-tryptophan metabolism are reported in several neurological, metabolic, psychiatric, and intestinal disorders, paving the way to develop drugs to target it. This review will briefly describe L-tryptophan metabolism and present and discuss the most recent pharmacological developments targeting it

Butyrate acts through HDAC inhibition to enhance aryl hydrocarbon receptor activation by gut microbiota-derived ligands

Aryl hydrocarbon receptor (AhR) is a critical player in the crosstalk between the gut microbiota and its host. However, factors regulating AhR within the gut, which is a complex metabolomic environment, are poorly understood. This study investigates the effect of a combination of metabolites on the activation mechanism of AhR. AhR activity was evaluated using both a luciferase reporter system and mRNA levels of AhR target genes on human cell lines and human colonic explants. AhR activation was studied by radioligand-binding assay, nuclear translocation of AhR by immuofluorescence and protein co-immunoprecipitation of AhR with ARNT. Indirect activation of AhR was evaluated using several tests and inhibitors. The promoter of the target gene CYP1A1 was studied both by chromatin immunoprecipitation and by using an histone deacetylase HDAC inhibitor (iHDAC). Short-chain fatty acids, and butyrate in particular, enhance AhR activity mediated by endogenous tryptophan metabolites without binding to the receptor. This effect was confirmed in human intestinal explants and did not rely on activation of receptors targeted by SCFAs, inhibition of AhR degradation or clearance of its ligands. Butyrate acted directly on AhR target gene promoter to reshape chromatin through iHDAC activity. Our findings revealed that butyrate is not an AhR ligand but acts as iHDAC leading to an increase recruitment of AhR to the target gene promoter in the presence of tryptophan-derived AhR agonists. These data contribute to a novel understanding of the complex regulation of AhR activation by gut microbiota-derived metabolites

Freeze-dried fecal samples are biologically active after long-lasting storage and suited to fecal microbiota transplantation in a preclinical murine model ofClostridioides difficileinfection

International audienceFecal microbiota transplantation is now recommended for treating recurrent forms ofClostridioides difficileinfection. Recent studies have reported protocols using capsules of either frozen or freeze-dried stool allowing oral administration in in- and out-patient settings. However, a central question remains the viability, engraftment, and efficacy of the microbiome over time during storage life. This study shows that both the freeze-drying and freezing procedures for fecal samples allowed preserving viability, short-chain fatty acids concentration, and anti-Clostridioides difficileproperties of microbiota without significant alteration after storage for 12 months. Fecal transplantation with freeze-dried microbiota allowed engraftment of microbiota leading to clearance ofClostridioides difficileinfection in a preclinical murine model with a survival rate of 70%versus53-60% in mice treated with frozen inocula, and 20% in the untreated group. Moreover, the freeze-dried powder can be used to fill oral hard capsules using a very low amount (0.5%) of glidant excipient, allowing oral formulation. Altogether, this study showed that freeze-dried inocula can be used for the treatment ofClostridioides difficileinfection with long-lasting stability of the fecal microbiota. This formulation facilitates biobanking and allows the use of hard capsules, an essential step to simplify patient access to treatment

AhR/IL-22 pathway as new target for the treatment of post-infectious irritable bowel syndrome symptoms

International audienceAlterations in brain/gut/microbiota axis are linked to Irritable Bowel Syndrome (IBS) physiopathology. Upon gastrointestinal infection, chronic abdominal pain and anxio-depressive comorbidities may persist despite pathogen clearance leading to Post-Infectious IBS (PI-IBS). This study assesses the influence of tryptophan metabolism, and particularly the microbiota-induced AhR expression, on intestinal homeostasis disturbance following gastroenteritis resolution, and evaluates the efficacy of IL-22 cytokine vectorization on PI-IBS symptoms. The Citrobacter rodentium infection model in C57BL6/ J mice was used to mimic Enterobacteria gastroenteritis. Intestinal homeostasis was evaluated as lowgrade inflammation, permeability, mucosa-associated microbiota composition, and colonic sensitivity. Cognitive performances and emotional state of animals were assessed using several tests. Tryptophan metabolism was analyzed by targeted metabolomics. AhR activity was evaluated using a luciferase reporter assay method. One Lactococcus lactis strain carrying an eukaryotic expression plasmid for murine IL-22 (L. lactis IL−22) was used to induce IL-22 production in mouse colonic mucosa. C. rodentiuminfected mice exhibited persistent colonic hypersensitivity and cognitive impairments and anxiety-like behaviors after pathogen clearance. These post-infectious disorders were associated with low-grade inflammation, increased intestinal permeability, decrease of Lactobacillaceae abundance associated with the colonic layer, and increase of short-chain fatty acids (SCFAs). During post-infection period, the indole pathway and AhR activity were decreased due to a reduction of tryptophol production. Treatment with L. lactis IL−22 restored gut permeability and normalized colonic sensitivity, restored cognitive performances and decreased anxiety-like behaviors. Data from the video-tracking system suggested an upgrade of welfare for mice receiving the L.lactis IL−22 strain. Our findings revealed that AhR/IL-22 signaling pathway is altered in a preclinical PI-IBS model. IL-22 delivering alleviate PI-IBS symptoms as colonic hypersensitivity, cognitive impairments, and anxiety-like behaviors by acting on intestinal mucosa integrity. Thus, therapeutic strategies targeting this pathway could be developed to treat IBS patients suffering from chronic abdominal pain and associated well-being disorders

Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome

In pressInternational audienceThe extent to which microbiota alterations define or influence the outcome of metabolic diseases is still unclear, but the byproducts of microbiota metabolism are known to have an important role in mediating the host-microbiota interaction. Here, we identify that in both pre-clinical and clinical settings, metabolic syndrome is associated with the reduced capacity of the microbiota to metabolize tryptophan into derivatives that are able to activate the aryl hydrocarbon receptor. This alteration is not merely an effect of the disease as supplementation with AhR agonist or a Lactobacillus strain, with a high AhR ligand-production capacity, leads to improvement of both dietary- and genetic-induced metabolic impairments, particularly glucose dysmetabolism and liver steatosis, through improvement of intestinal barrier function and secretion of the incretin hormone GLP-1. These results highlight the role of gut microbiota-derived metabolites as a biomarker and as a basis for novel preventative or therapeutic interventions for metabolic disorders