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.

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.

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.

Short Term Palmitate Supply Impairs Intestinal Insulin Signaling via Ceramide Production.

The worldwide prevalence of metabolic diseases is increasing, and there are global recommendations to limit consumption of certain nutrients, especially saturated lipids. Insulin resistance, a common trait occurring in obesity and type 2 diabetes, is associated with intestinal lipoprotein overproduction. However, the mechanisms by which the intestine develops insulin resistance in response to lipid overload remain unknown. Here, we show that insulin inhibits triglyceride secretion and intestinal microsomal triglyceride transfer protein expression in vivo in healthy mice force-fed monounsaturated fatty acid-rich olive oil but not in mice force-fed saturated fatty acid-rich palm oil. Moreover, when mouse intestine and human Caco-2/TC7 enterocytes were treated with the saturated fatty acid, palmitic acid, the insulin-signaling pathway was impaired. We show that palmitic acid or palm oil increases ceramide production in intestinal cells and that treatment with a ceramide analogue partially reproduces the effects of palmitic acid on insulin signaling. In Caco-2/TC7 enterocytes, ceramide effects on insulin-dependent AKT phosphorylation are mediated by protein kinase C but not by protein phosphatase 2A. Finally, inhibiting de novo ceramide synthesis improves the response of palmitic acid-treated Caco-2/TC7 enterocytes to insulin. These results demonstrate that a palmitic acid-ceramide pathway accounts for impaired intestinal insulin sensitivity, which occurs within several hours following initial lipid exposure.

Autophagosomes contribute to intracellular lipid distribution in enterocytes.

Enterocytes, the intestinal absorptive cells, have to deal with massive alimentary lipids upon food consumption. They orchestrate complex lipid-trafficking events that lead to the secretion of triglyceride-rich lipoproteins and/or the intracellular transient storage of lipids as lipid droplets (LDs). LDs originate from the endoplasmic reticulum (ER) membrane and are mainly composed of a triglyceride (TG) and cholesterol-ester core surrounded by a phospholipid and cholesterol monolayer and specific coat proteins. The pivotal role of LDs in cellular lipid homeostasis is clearly established, but processes regulating LD dynamics in enterocytes are poorly understood. Here we show that delivery of alimentary lipid micelles to polarized human enterocytes induces an immediate autophagic response, accompanied by phosphatidylinositol-3-phosphate appearance at the ER membrane. We observe a specific and rapid capture of newly synthesized LD at the ER membrane by nascent autophagosomal structures. By combining pharmacological and genetic approaches, we demonstrate that autophagy is a key player in TG targeting to lysosomes. Our results highlight the yet-unraveled role of autophagy in the regulation of TG distribution, trafficking, and turnover in human enterocytes.

Intestinal glucose-dependent expression of glucose-6-phosphatase: involvement of the aryl receptor nuclear translocator transcription factor.

Glucose-6-phosphatase (G6Pase) catalyzes the release of glucose from glucose 6-phosphate. This enzyme was mainly studied in the liver, but while detected in the small intestine little is known about the regulation of its intestinal expression. This study describes the mechanisms of the glucose-dependent regulation of G6Pase expression in intestinal cells. Results obtained in vivo and in Caco-2/TC7 enterocytes showed that glucose increases the G6Pase mRNA level. In Caco-2/TC7 cells, glucose stabilized G6Pase mRNA and activated the transcription of the gene, meaning that glucose-dependent G6Pase expression involved both transcriptional and post-transcriptional mechanisms. Reporter-gene studies showed that, although the -299/+57 region of the human G6Pase promoter was sufficient to trigger the glucose response in the hepatoma cell line HepG2, the -1157/-1133 fragment was required for maximal activation of glucose-6-phosphatase gene transcription in Caco-2/TC7 cells. This fragment binds the aryl receptor nuclear translocator (ARNT), cAMP-responsive element-binding protein, and upstream stimulatory factor transcription factors. The DNA binding activity of these transcription factors was increased in nuclear extracts of differentiated cells from the intestinal villus of mice fed sugar-rich diets as compared with mice fed a no-sugar diet. A direct implication of ARNT in the activation of G6Pase gene transcription by glucose has been observed in Caco-2/TC7 cells using RNA interference experiments. These results support a physiological role for G6Pase in the control of nutrient absorption in the small intestine.

HNF-4-dependent induction of apolipoprotein A-IV gene transcription by an apical supply of lipid micelles in intestinal cells.

Apolipoprotein (apo) A-IV, a component of triglyceride-rich lipoproteins secreted by the small intestine, has been shown to play an important role in the control of lipid homeostasis. Numerous studies have described the induction of apoA-IV gene expression by lipids, but the molecular mechanisms involved in this process remain unknown. In this study, we have demonstrated that a lipid bolus induced transcription of the apoA-IV gene in transgenic mice and that the regulatory region of the apoA-IV gene, composed of the apoC-III enhancer and the apoA-IV promoter (eC3-A4), was responsible for this induction. In enterocyte Caco-2/TC7 cells, a permanent supply of lipids at the basal pole induced expression of the apoA-IV gene both at the transcriptional level and through mRNA stabilization. ApoA-IV gene transcription and protein secretion were further induced by an apical supply of complex lipid micelles mimicking the composition of duodenal micelles, and this effect was not reproduced by apical delivery of different combinations of micelle components. Only induction of the apoA-IV gene by lipid micelles involved the participation of hepatic nuclear factor (HNF)-4, as demonstrated using a dominant negative form of this transcription factor. Accordingly, lipid micelles increased the DNA binding activity of HNF-4 on the eC3-A4 region. These results emphasize the importance of physiological delivery of dietary lipids on apoA-IV gene expression and the implication of HNF-4 in this regulation.

Expression of aminopeptidase B in the developing and adult rat retina.

Aminopeptidase B (Ap-B), a ubiquitous enzyme, catalyses the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. The physiological function of Ap-B still remains an open question, even though its activity suggests that it could be involved in inflammatory processes and proliferation of tumor cells. This study was conducted to determine the expression of Ap-B in the developing and adult retina as a path to envisage physiological roles of Ap-B. RT-PCR and in situ hybridization were used to detect expression of Ap-B mRNA and activity tests, Western blotting and immunofluorescence microscopy were performed to identify and localize the enzyme in the rat retina. These biochemical and morphological methods show that Ap-B is expressed in the retina from embryo to adult. Expression level is restricted to specific layers (pigmented epithelium, outer and inner plexiform layers and ganglion cell layer) and is developmentally regulated. Moreover, a preliminary analysis indicates that Ap-B, the glucose transporter GLUT3 and choline acetyltransferase (ChAT) share a similar expression pattern in retina. Altogether, Ap-B appears predominantly expressed in neuronal cells lying in retinal layers containing neuritic extensions and synaptic junctions. Such expression is up-regulated during ontogenesis allowing to hypothesized that Ap-B participates in processes accompanying retinal neuronal cell differentiation.