Modulation of the intestinal barrier function and tight junctions by the bacterial quorum sensing molecules N-acly homoserine lactones

L'intestin abrite le principal microbiote du corps humain et exerce donc un rôle de barrière. Les jonctions serrées (JS) contrôlent la perméabilité paracellulaire des cellules épithéliales et sont des acteurs majeurs de la barrière intestinale, dérégulée dans les Maladies Inflammatoires Chroniques de l'Intestin (MICI). Les N-acyl homosérine lactones (AHL), molécules du quorum sensing, un système bactérien de communication, sont également capables d’interagir avec l'hôte. Nous avons récemment identifié l'AHL 3-oxo-C12:2, diminuée dans les MICI. Notre objectif est d’étudier l’impact de cette nouvelle AHL sur la fonction de barrière intestinale et de le comparer à celui de l’AHL 3-oxo-C12 de Pseudomonas aeruginosa, connue pour ses effets délétères sur les barrières épithéliales. Dans la lignée humaine entérocytaire Caco-2/TC7, alors qu'en présence de 3-oxo-C12, la perméabilité paracellulaire est augmentée et le signal de l’occludine et tricelluline est diminuée aux JS bicellulaires et tricellulaires respectivement, la 3-oxo-C12:2 ne modifie ni la perméabilité ni l’organisation des JS. Alors que la 3-oxo-C12 potentialise l’augmentation de la perméabilité induite par l'Interféron-γ et le Tumor Necrosis Factor, la 3-oxo-C12:2 atténue les effets délétères des cytokines sur l'occludine et la tricelluline. De plus, elle préserve la stabilité des complexes jonctionnels à la membrane plasmique en réduisant l’ubiquitination des protéines de JS augmentée par les cytokines. Ainsi, les effets potentiellement protecteurs de la 3-oxo-C12:2 sur la barrière intestinale constitue une nouvelle piste pour mieux comprendre les interactions hôte-microbiote mises en jeu dans les MICI.The intestine is home to the largest microbiota of the human body and therefore has to act as a barrier. Tight junctions (TJ) seal the paracellular space between epithelial cells and thus are major actors of the intestinal barrier function, which is impaired in inflammatory bowel disease (IBD). Dysbiosis is a key feature in IBD. N-acyl homoserine lactones (AHL) are quorum sensing molecules, a bacterial communication network, which also impacts the host. We previously identified 3-oxo-C12:2, a new AHL decreased in IBD. We investigated how 3-oxo-C12:2-AHL impacts the barrier function, in comparison to 3-oxo-C12, a structurally close AHL produced by P. aeruginosa, known to impair TJ. While 3-oxoC12-AHL increased paracellular permeability and decreased Occludin and Tricellulin signal at bicellular and tricellular TJ respectively, 3-oxo-C12:2-AHL modified neither permeability nor TJ integrity in human polarized enterocytes Caco-2/TC7. Whereas 3-oxo-C12-AHL potentiated the increase in permeability induced by Interferon-γ (IFNγ) and Tumor Necrosis Factor-α (TNFα), 3-oxo-C12:2-AHL attenuated the cytokine deleterious effects on Occludin and Tricellulin, as well as on their interaction at the plasma membrane with their cytoplasmic partner ZO-1. We show that the increase in ubiquitination levels of Occludin and Tricellulin caused by cytokines was suppressed in the presence of 3-oxo-C12:2, suggesting that this AHL prevents their endocytosis or degradation. The beneficial role of the 3-oxo-C12:2 AHL, lost in IBD patients, leads to interesting perspectives to better understand host-microbiota interactions in IBD.

Modulation de la fonction de barrière intestinale et des jonctions serrées par les N-acyl homosérine lactones, molécules du quorum sensing bactérien

The intestine is home to the largest microbiota of the human body and therefore has to act as a barrier. Tight junctions (TJ) seal the paracellular space between epithelial cells and thus are major actors of the intestinal barrier function, which is impaired in inflammatory bowel disease (IBD). Dysbiosis is a key feature in IBD. N-acyl homoserine lactones (AHL) are quorum sensing molecules, a bacterial communication network, which also impacts the host. We previously identified 3-oxo-C12:2, a new AHL decreased in IBD. We investigated how 3-oxo-C12:2-AHL impacts the barrier function, in comparison to 3-oxo-C12, a structurally close AHL produced by P. aeruginosa, known to impair TJ. While 3-oxoC12-AHL increased paracellular permeability and decreased Occludin and Tricellulin signal at bicellular and tricellular TJ respectively, 3-oxo-C12:2-AHL modified neither permeability nor TJ integrity in human polarized enterocytes Caco-2/TC7. Whereas 3-oxo-C12-AHL potentiated the increase in permeability induced by Interferon-γ (IFNγ) and Tumor Necrosis Factor-α (TNFα), 3-oxo-C12:2-AHL attenuated the cytokine deleterious effects on Occludin and Tricellulin, as well as on their interaction at the plasma membrane with their cytoplasmic partner ZO-1. We show that the increase in ubiquitination levels of Occludin and Tricellulin caused by cytokines was suppressed in the presence of 3-oxo-C12:2, suggesting that this AHL prevents their endocytosis or degradation. The beneficial role of the 3-oxo-C12:2 AHL, lost in IBD patients, leads to interesting perspectives to better understand host-microbiota interactions in IBD..L'intestin abrite le principal microbiote du corps humain et exerce donc un rôle de barrière. Les jonctions serrées (JS) contrôlent la perméabilité paracellulaire des cellules épithéliales et sont des acteurs majeurs de la barrière intestinale, dérégulée dans les Maladies Inflammatoires Chroniques de l'Intestin (MICI). Les N-acyl homosérine lactones (AHL), molécules du quorum sensing, un système bactérien de communication, sont également capables d’interagir avec l'hôte. Nous avons récemment identifié l'AHL 3-oxo-C12:2, diminuée dans les MICI. Notre objectif est d’étudier l’impact de cette nouvelle AHL sur la fonction de barrière intestinale et de le comparer à celui de l’AHL 3-oxo-C12 de Pseudomonas aeruginosa, connue pour ses effets délétères sur les barrières épithéliales. Dans la lignée humaine entérocytaire Caco-2/TC7, alors qu'en présence de 3-oxo-C12, la perméabilité paracellulaire est augmentée et le signal de l’occludine et tricelluline est diminuée aux JS bicellulaires et tricellulaires respectivement, la 3-oxo-C12:2 ne modifie ni la perméabilité ni l’organisation des JS. Alors que la 3-oxo-C12 potentialise l’augmentation de la perméabilité induite par l'Interféron-γ et le Tumor Necrosis Factor, la 3-oxo-C12:2 atténue les effets délétères des cytokines sur l'occludine et la tricelluline. De plus, elle préserve la stabilité des complexes jonctionnels à la membrane plasmique en réduisant l’ubiquitination des protéines de JS augmentée par les cytokines. Ainsi, les effets potentiellement protecteurs de la 3-oxo-C12:2 sur la barrière intestinale constitue une nouvelle piste pour mieux comprendre les interactions hôte-microbiote mises en jeu dans les MICI

Modeling microbiota-associated human diseases: from minimal models to complex systems

Alterations in the intestinal microbiota are associated with various human diseases of the digestive system, including obesity and its associated metabolic diseases, inflammatory bowel diseases (IBD), and colorectal cancer (CRC). All three diseases are characterized by modifications of the richness, composition, and metabolic functions of the human intestinal microbiota. Despite being multi-factorial diseases, studies in germ-free animal models have unarguably identified the intestinal microbiota as a causal driver of disease pathogenesis. However, for an increased mechanistic understanding of microbial signatures in human diseases, models require detailed refinement to closely mimic the human microbiota and reflect the complexity and range of dysbiosis observed in patients. The transplantation of human fecal microbiota into animal models represents a powerful tool for studying the causal and functional role of the dysbiotic human microbiome in a pathological context. While human microbiota-associated models were initially employed to study obesity, an increasing number of studies have applied this approach in the context of IBD and CRC over the past decade. In this review, we discuss different approaches that allow the functional validation of the bacterial contribution to human diseases, with emphasis on obesity and its associated metabolic diseases, IBD, and CRC. We discuss the utility of simple models, such as in vitro fermentation systems of the human microbiota and ex vivo intestinal organoids, as well as more complex whole organism models. Our focus here lies on human microbiota-associated mouse models in the context of all three diseases, as well as highlighting the advantages and limitations of this approach

Rapid Evaluation of Intestinal Paracellular Permeability Using the Human Enterocytic-Like Caco-2/TC7 Cell Line

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Exploiting the metabolic energy demands of drug efflux pumps provides a strategy to overcome multidrug resistance in cancer

Background: P-glycoprotein (P-gp) is a prevalent resistance mediator and it requires considerable cellular energy to ensure ATP dependent efflux of anticancer drugs. The glycolytic pathway generates the majority of catabolic energy in cancer cells; however, the high rates of P-gp activity places added strain on its inherently limited capacity to generate ATP. This is particularly relevant for compounds such as verapamil that are believed to trap P-gp in a futile transport process that requires continuing ATP consumption. Ultimately, this leads to cell death and the hypersensitivity of resistant cells to verapamil is termed collateral sensitivity. Results: We show that the addition of verapamil to resistant cells produces a prominent reduction in ATP levels that supports the idea of disrupted energy homeostasis. Even in the absence of verapamil, P-gp expressing cells display near maximal rates of glycolysis and oxidative phosphorylation, which prevents an adequate response to the demand for ATP to sustain transport activity. Moreover, the near perpetually maximal rate of oxidative phosphorylation in the presence of verapamil resulted in elevated levels of reactive oxygen species that affect cell survival and underscore collateral sensitivity. Conclusions: Our results demonstrate that the strained metabolic profiles of P-gp expressing resistant cancer cells can be overwhelmed by additional ATP demands. General significance: Consequently, collateral sensitising drugs may overcome the resistant phenotype by exploiting, rather than inhibiting, the energy demanding activity of pumps such as P-gp

Rapid Evaluation of Intestinal Paracellular Permeability Using the Human Enterocytic-Like Caco-2/TC7 Cell Line

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Use of Ussing Chambers to Measure Paracellular Permeability to Macromolecules in Mouse Intestine

International audienceAn increased intestinal permeability has been described in many diseases including inflammatory bowel disease and metabolic disorders, and a better understanding of the contribution of intestinal barrier impairment to pathogenesis is needed. In recent years, attention has been paid to the leak pathway, which is the route of paracellular transport allowing the diffusion of macromolecules through the tight junctions of the intestinal epithelial lining. While the passage of macromolecules by this pathway is very restricted under physiological conditions, its amplification is thought to promote an excessive immune activation in the intestinal mucosa. The Ussing chambers have been widely used to measure both active and passive transepithelial fluxes in intact tissues. In this chapter we present how this simple device can be used to measure paracellular permeability to macromolecules in the mouse intestine. We propose a detailed protocol and describe how to best exploit all the possibilities of this technique, correctly interpret the results, and avoid the main pitfalls

Use of Ussing Chambers to Measure Paracellular Permeability to Macromolecules in Mouse Intestine

International audienceAn increased intestinal permeability has been described in many diseases including inflammatory bowel disease and metabolic disorders, and a better understanding of the contribution of intestinal barrier impairment to pathogenesis is needed. In recent years, attention has been paid to the leak pathway, which is the route of paracellular transport allowing the diffusion of macromolecules through the tight junctions of the intestinal epithelial lining. While the passage of macromolecules by this pathway is very restricted under physiological conditions, its amplification is thought to promote an excessive immune activation in the intestinal mucosa. The Ussing chambers have been widely used to measure both active and passive transepithelial fluxes in intact tissues. In this chapter we present how this simple device can be used to measure paracellular permeability to macromolecules in the mouse intestine. We propose a detailed protocol and describe how to best exploit all the possibilities of this technique, correctly interpret the results, and avoid the main pitfalls

Gossip in the gut: Quorum sensing, a new player in the host-microbiota interactions

International audienceBacteria are known to communicate with each other and regulate their activities in social networks by secreting and sensing signaling molecules called autoinducers, a process known as quorum sensing (QS). This is a growing area of research in which we are expanding our understanding of how bacteria collectively modify their behavior but are also involved in the crosstalk between the host and gut microbiome. This is particularly relevant in the case of pathologies associated with dysbiosis or disorders of the intestinal ecosystem. This review will examine the different QS systems and the evidence for their presence in the intestinal ecosystem. We will also provide clues on the role of QS molecules that may exert, directly or indirectly through their bacterial gossip, an influence on intestinal epithelial barrier function, intestinal inflammation, and intestinal carcinogenesis. This review aims to provide evidence on the role of QS molecules in gut physiology and the potential shared by this new player. Better understanding the impact of intestinal bacterial social networks and ultimately developing new therapeutic strategies to control intestinal disorders remains a challenge that needs to be addressed in the future

P043: 3-oxo-C12:2, a Quorum Sensing molecule from the gut, exerts anti-inflammatory effects through a bitter taste receptor

International audienceBackgroundAcyl-Homoserine Lactones (AHLs) are Quorum Sensing molecules involved in the communication network of bacteria and can also have an impact on the host’s cells. We recently showed, in the human gut ecosystem, the presence of AHLs. Among them, we identified one that has never been described: 3-oxo-C12:2. This molecule was decreased in Inflammatory Bowel Disease (IBD) patients, and its presence was correlated to normobiosis. Interestingly, 3-oxo-C12:2 is structurally close to an AHL well described and synthesized by P. aeruginosa, 3-oxo-C12. We intent to describe 3-oxo-C12:2 effects on gut inflammation and to identified which signalling pathways are involved. Given its analogous structure to 3-oxo-C12, we hypothesized that 3-oxo-C12:2 can interact with the same cellular partners, in particular a bitter taste receptor (BTR), called T2R138, which is a GPCR expressed by immune and epithelial gut cells.MethodsTo test our hypothesis, we used murine macrophages cell line RAW264.7, stimulated by interferon-γ (IFN-γ, 20U/mL) and lipopolysaccharide (LPS, 10ng/mL). Inflammatory response was monitored by measuring cytokine secretion via ELISA We performed a transcriptome analysis to identify inflammatory pathways involved in the effects and analyse pathways by capillary Western blot. Probenecid, a known allosteric inhibitor for T2R138, was used to study T2R138 role in AHL signalling. BTR screening assay was performed to extend search for 3-oxo-C12:2 receptors.ResultsAfter LPS/IFN-γ activation, we observed a decrease of secreted TNFα when cells were exposed to 3-oxo-C12:2, in a dose dependent manner: 15μM (-30%, p<0.05), 25μM (-50%, p<0.001) et 50μM (-65%, p<0.0001) while no change were observed in steady state. This reflects an anti-inflammatory effect, in absence of cytotoxicity. By transcriptomic analysis, we identified the JAK-STAT pathway as differentially down-regulated. Exposing cells to 3-oxo-C12:2 prevented JAK1 and STAT1 protein phosphorylation. In addition, the observed anti-inflammatory effects were lost in presence of Probenecid, a T2R138 inhibitor. In a BTR screening assay, we confirmed that 3-oxo-C12:2 activates T2R38, but also five other BTR (T2R13, T2R8, T2R14, T2R1, T2R10).Conclusion3-oxo-C12:2 exerts a dose dependent anti-inflammatory effect on murine immune cells by preventing the activation of the JAK-STAT pathway. This response is partly mediated by the bitter taste receptor T2R138. This receptor is a potential target of our AHL of interest. Studying the signalling between the receptor and the anti-inflammatory response would allow us to better understand the inter-kingdom dialogue between microbiota involving AHL in IBD