Increased jejunal permeability in human obesity is revealed by a lipid challenge and is linked to inflammation and type 2 diabetes.

Obesity and its metabolic complications are characterized by subclinical systemic and tissue inflammation. In rodent models of obesity, inflammation and metabolic impairments are linked with intestinal barrier damage. However, whether intestinal permeability is altered in human obesity remains to be investigated. In a cohort of 122 severely obese and non-obese patients, we analyzed intestinal barrier function combining in vivo and ex vivo investigations. We found tight junction impairments in the jejunal epithelium of obese patients, evidenced by a reduction of occludin and tricellulin. Serum levels of zonulin and LPS binding protein, two markers usually associated with intestinal barrier alterations, were also increased in obese patients. Intestinal permeability per se was assessed in vivo by quantification of urinary lactitol/mannitol (L/M) and measured directly ex vivo on jejunal samples in Ussing chambers. In the fasting condition, L/M ratio and jejunal permeability were not significantly different between obese and non-obese patients, but high jejunal permeability to small molecules (0.4 kDa) was associated with systemic inflammation within the obese cohort. Altogether, these results suggest that intestinal barrier function is subtly compromised in obese patients. We thus tested whether this barrier impairment could be exacerbated by dietary lipids. To this end, we challenged jejunal samples with lipid micelles and showed that a single exposure increased permeability to macromolecules (4 kDa). Jejunal permeability after the lipid load was two-fold higher in obese patients compared to non-obese controls and correlated with systemic and intestinal inflammation. Moreover, lipid-induced permeability was an explicative variable of type 2 diabetes. In conclusion, intestinal barrier defects are present in human severe obesity and exacerbated by a lipid challenge. This paves the way to the development of novel therapeutic approaches to modulate intestinal barrier function or personalize nutrition therapy to decrease lipid-induced jejunal leakage in metabolic diseases. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mitochondrial perturbation in the intestine causes microbiota-dependent injury and gene signatures discriminative of inflammatory disease.

Mitochondrial dysfunction is associated with inflammatory bowel diseases (IBDs). To understand how microbial-metabolic circuits contribute to intestinal injury, we disrupt mitochondrial function in the epithelium by deleting the mitochondrial chaperone, heat shock protein 60 (Hsp60Δ/ΔIEC). This metabolic perturbation causes self-resolving tissue injury. Regeneration is disrupted in the absence of the aryl hydrocarbon receptor (Hsp60Δ/ΔIEC;AhR-/-) involved in intestinal homeostasis or inflammatory regulator interleukin (IL)-10 (Hsp60Δ/ΔIEC;Il10-/-), causing IBD-like pathology. Injury is absent in the distal colon of germ-free (GF) Hsp60Δ/ΔIEC mice, highlighting bacterial control of metabolic injury. Colonizing GF Hsp60Δ/ΔIEC mice with the synthetic community OMM12 reveals expansion of metabolically flexible Bacteroides, and B. caecimuris mono-colonization recapitulates the injury. Transcriptional profiling of the metabolically impaired epithelium reveals gene signatures involved in oxidative stress (Ido1, Nos2, Duox2). These signatures are observed in samples from Crohn's disease patients, distinguishing active from inactive inflammation. Thus, mitochondrial perturbation of the epithelium causes microbiota-dependent injury with discriminative inflammatory gene profiles relevant for IBD.

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.

3-oxo-C12:2-HSL, quorum sensing molecule from human intestinal microbiota, inhibits pro-inflammatory pathways in immune cells via bitter taste receptors.

In the gut ecosystem, microorganisms regulate group behaviour and interplay with the host via a molecular system called quorum sensing (QS). The QS molecule 3-oxo-C12:2-HSL, first identified in human gut microbiota, exerts anti-inflammatory effects and could play a role in inflammatory bowel diseases where dysbiosis has been described. Our aim was to identify which signalling pathways are involved in this effect. We observed that 3-oxo-C12:2-HSL decreases expression of pro-inflammatory cytokines such as Interleukine-1ß (- 35%) and Tumor Necrosis Factor-α (TNFα) (- 40%) by stimulated immune RAW264.7 cells and decreased TNF secretion by stimulated PBMC in a dose-dependent manner, between 25 to 100 µM. Transcriptomic analysis of RAW264.7 cells exposed to 3-oxo-C12:2-HSL, in a pro-inflammatory context, highlighted JAK-STAT, NF-κB and TFN signalling pathways and we confirmed that 3-oxo-C12:2-HSL inhibited JAK1 and STAT1 phosphorylation. We also showed through a screening assay that 3-oxo-C12:2-HSL interacted with several human bitter taste receptors. Its anti-inflammatory effect involved TAS2R38 as shown by pharmacologic inhibition and led to an increase in intracellular calcium levels. We thus unravelled the involvement of several cellular pathways in the anti-inflammatory effects exerted by the QS molecule 3-oxo-C12:2-HSL.

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.

Paracellular permeability of the intestinal epithelium is a feature of the intestinal barrier, which plays an important role in the physiology of gut and the whole organism. Intestinal paracellular permeability is controlled by complex processes and is involved in the passage of ions and fluids (called pore pathway) and macromolecules (called leak pathway) through tight junctions, which seal the intercellular space. Impairment of intestinal paracellular permeability is associated with several diseases. The identification of a defect in intestinal paracellular permeability may help to understand the implication of gut barrier as a cause or a consequence in human pathology. Here we describe two complementary methods to evaluate alteration of paracellular permeability in cell culture, using the human intestinal cell line Caco-2 and its clone Caco-2/TC7.

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

An 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.

Bacteria 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.

The intestinal quorum sensing 3-oxo-C12:2 Acyl homoserine lactone limits cytokine-induced tight junction disruption.

The intestine is home to the largest microbiota community of the human body and strictly regulates its barrier function. Tight junctions (TJ) are major actors of the intestinal barrier, which is impaired in inflammatory bowel disease (IBD), along with an unbalanced microbiota composition. With the aim to identify new actors involved in host-microbiota interplay in IBD, we studied N-acyl homoserine lactones (AHL), molecules of the bacterial quorum sensing, which also impact the host. We previously identified in the gut a new and prominent AHL, 3-oxo-C12:2, which is lost in IBD. We investigated how 3-oxo-C12:2 impacts the intestinal barrier function, in comparison to 3-oxo-C12, a structurally close AHL produced by the opportunistic pathogen P. aeruginosa. Using Caco-2/TC7 cells as a model of polarized enterocytes, we compared the effects on paracellular permeability and TJ integrity of these two AHL, separately or combined with pro-inflammatory cytokines, Interferon-γ and Tumor Necrosis Factor-α, known to disrupt the barrier function during IBD. While 3-oxo-C12 increased paracellular permeability and decreased occludin and tricellulin signal at bicellular and tricellular TJ, respectively, 3-oxo-C12:2 modified neither permeability nor TJ integrity. Whereas 3-oxo-C12 potentiated the hyperpermeability induced by cytokines, 3-oxo-C12:2 attenuated their deleterious effects on occludin and tricellulin, and maintained their interaction with their partner ZO-1. In addition, 3-oxo-C12:2 limited the cytokine-induced ubiquitination of occludin and tricellulin, suggesting that this AHL prevented their endocytosis. In conclusion, the role of 3-oxo-C12:2 in maintaining TJ integrity under inflammatory conditions identifies this new AHL as a potential beneficial actor of host-microbiota interactions in IBD.

AhR activation defends gut barrier integrity against damage occurring in obesity.

OBJECTIVE: Obesity is characterized by systemic and low-grade tissue inflammation. In the intestine, alteration of the intestinal barrier and accumulation of inflammatory cells in the epithelium are important contributors of gut inflammation. Recent studies demonstrated the role of the aryl hydrocarbon receptor (AhR) in the maintenance of immune cells at mucosal barrier sites. A wide range of ligands of external and local origin can activate this receptor. We studied the causal relationship between AhR activation and gut inflammation in obesity. METHODS: Jejunum samples from subjects with normal weight and severe obesity were phenotyped according to T lymphocyte infiltration in the epithelium from lamina propria and assayed for the mRNA level of AhR target genes. The effect of an AhR agonist was studied in mice and Caco-2/TC7 cells. AhR target gene expression, permeability to small molecules and ions, and location of cell-cell junction proteins were recorded under conditions of altered intestinal permeability. RESULTS: We showed that a low AhR tone correlated with a high inflammatory score in the intestinal epithelium in severe human obesity. Moreover, AhR activation protected junctional complexes in the intestinal epithelium in mice challenged by an oral lipid load. AhR ligands prevented chemically induced damage to barrier integrity and cytokine expression in Caco-2/TC7 cells. The PKC and p38MAPK signaling pathways were involved in this AhR action. CONCLUSIONS: The results of these series of human, mouse, and cell culture experiments demonstrate the protective effect of AhR activation in the intestine targeting particularly tight junctions and cytokine expression. We propose that AhR constitutes a valuable target to protect intestinal functions in metabolic diseases, which can be achieved in the future via food or drug ligands.

Intestinal plasticity in response to nutrition and gastrointestinal surgery.

The plasticity of a material corresponds to its capacity to change its feature under the effect of an external action. Intestinal plasticity could be defined as the ability of the intestine to modify its size or thickness and intestinal cells to modulate their absorption and secretion functions in response to external or internal cues/signals. This review will focus on intestinal adaptation mechanisms in response to diet and nutritional status. These physiological mechanisms allow a fine and rapid adaptation of the gut to promote absorption of ingested food, but they can also lead to obesity in response to overnutrition. This plasticity could thus become a therapeutic target to treat not only undernutrition but also obesity. How the intestine adapts in response to 2 types of surgical remodeling of the digestive tract-extensive bowel resection leading to intestinal failure and surgical treatment of pathological obesity (ie, bariatric surgeries)-will also be reviewed.