Fernand-Pierre Gendron

Intestinal Inflammation Increases the Expression of the P2Y6 Receptor on Epithelial Cells and the Release of CXC Chemokine Ligand 8 by UDP

Epithelial cells participate in the immune response of the intestinal mucosa. Extracellular nucleotides have been recognized as inflammatory molecules. We investigated the role of extracellular nucleotides and their associated P2Y receptors in the secretion of cytokines by epithelial cells. The effect of intestinal inflammation on P2Y6 receptor expression was determined by PCR in the mouse, rat, and human. Localization of the P2Y6 receptor was determined by immunofluorescence microscopy in the colon of normal and dextran sulfate sodium-treated mice. The effect of P2Y6 activation by UDP on cytokine expression and release by epithelial cells was determined using a combination of Western blots, luciferase assays, RT-PCR, cytokine Ab arrays, and ELISA. Inflammation up-regulates P2Y2 as well as P2Y6 receptor expression in the mucosa of the colon of colitic mice. In vitro, we demonstrated that UDP could be released by Caco-2/15 cells. We have confirmed the increased expression of P2Y6 by challenging intestinal epithelial cell-6 and Caco-2/15 cells with TNF-α and IFN-γ and showing that stimulation of epithelial cells by UDP results in an increased expression and release of CXCL8 by an ERK1/2-dependent mechanism. The increase in CXCL8 expression was associated with a transcriptional activation by the P2Y6 receptor. This study is the first report demonstrating the implication of P2Y receptors in the inflammatory response of intestinal epithelial cells. We show for the first time that P2Y6, as well as P2Y2, expression is increased by the stress associated with intestinal inflammation. These results demonstrate the emergence of extracellular nucleotide signaling in the orchestration of intestinal inflammation.

P2X7 nucleotide receptor activation enhances IFNγ‐induced type II nitric oxide synthase activity in BV‐2 microglial cells

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFNγ‐induced NO release in murine BV‐2 microglial cells was investigated. BV‐2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2′,3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP), and 2‐methylthio‐ATP (2‐MeSATP), but not UTP, enhanced IFNγ‐induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal‐regulated kinases ERK1/2, decreased IFNγ‐induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2‐MeSATP at enhancing IFNγ‐induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate‐oxidized ATP, a P2X7 receptor antagonist, and suramin, a non‐specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFNγ‐induced NO production, whereas pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.

Purine signaling and potential new therapeutic approach: possible outcomes of NTPDase inhibition.

Interest for extracellular nucleotides has increased since the pioneer work of Burnstock in the early seventies. Research on cellular functions modulated by purines and pyrimidines has led to the identification and characterization of the different components of purine signaling, namely purinoceptors and ecto-nucleotidases. Receptors for tri- and diphosphonucleosides, known as P2 nucleotide receptors, are designated either P2Y receptors, for those coupled to G-proteins, or P2X for those which are ligand gated-ion channels. Ecto-nucleoside triphosphate diphosphohydrolase (NTPDase; EC 3.6.1.5), previously identified as ecto-ATPase, ecto-ATPDase or CD39, is now considered as the main ecto-nucleotidase responsible for the sequential hydrolysis of beta and gamma phosphates of tri- and diphosphonucleosides. More recently, research has been focused on the development of specific agonists and antagonists to P2 purinoceptors. The need to develop specific inhibitors for NTPDase to understand the role of this enzyme has clearly emerged. This paper covers the development of specific molecules targeting purinergic signaling, more specifically the inhibition of NTPDase and their impact on the different physiological systems.

Loss of Hepatocyte-Nuclear-Factor-4α Affects Colonic Ion Transport and Causes Chronic Inflammation Resembling Inflammatory Bowel Disease in Mice

Background Hnf4α, an epithelial specific transcriptional regulator, is decreased in inflammatory bowel disease and protects against chemically-induced colitis in mice. However, the precise role of this factor in maintaining normal inflammatory homeostasis of the intestine remains unclear. The aim of this study was to evaluate the sole role of epithelial Hnf4α in the maintenance of gut inflammatory homeostasis in mice. Methodology/Principal Findings We show here that specific epithelial deletion of Hnf4α in mice causes spontaneous chronic intestinal inflammation leading to focal areas of crypt dropout, increased cytokines and chemokines secretion, immune cell infiltrates and crypt hyperplasia. A gene profiling analysis in diseased Hnf4α null colon confirms profound genetic changes in cell death and proliferative behaviour related to cancer. Among the genes involved in the immune protection through epithelial barrier function, we identify the ion transporter claudin-15 to be down-modulated early in the colon of Hnf4α mutants. This coincides with a significant decrease of mucosal ion transport but not of barrier permeability in young animals prior to the manifestation of the disease. We confirm that claudin-15 is a direct Hnf4α gene target in the intestinal epithelial context and is down-modulated in mouse experimental colitis and inflammatory bowel disease. Conclusion Our results highlight the critical role of Hnf4α to maintain intestinal inflammatory homeostasis during mouse adult life and uncover a novel function for Hnf4α in the regulation of claudin-15 expression. This establishes Hnf4α as a mediator of ion epithelial transport, an important process for the maintenance of gut inflammatory homeostasis.

P2Y2 Receptor Transcription Is Increased by NF-κB and Stimulates Cyclooxygenase-2 Expression and PGE2 Released by Intestinal Epithelial Cells

Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y2 mRNA receptor expression in the human colon adenocarcinoma cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues of patient suffering from Crohn’s disease and ulcerative colitis. However, the transcriptional events regulating P2Y2 receptor (P2Y2R) expression are not known. We have identified a putative transcription start site in the P2Y2R gene and demonstrated acetylation of Lys14 on histone H3 and Lys8 on histone H4, thus suggesting that the chromatin associated with the P2Y2 promoter is accessible to transcription factors. We also showed that the transcription factor NF-κB p65 regulates P2Y2R transcription under both proinflammatory and basal conditions. A NF-κB-responsive element was identified at −181 to −172 bp in the promoter region of P2Y2. Hence, activation of P2Y2R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE2 secretion by intestinal epithelial cells. These findings demonstrate that P2Y2R expression is regulated during intestinal inflammation through an NF-κB p65-dependent mechanism and could contribute not only to inflammatory bowel disease but also to other inflammatory diseases by regulating PG release.

Hepatocyte Nuclear Factor-4α Promotes Gut Neoplasia in Mice and Protects against the Production of Reactive Oxygen Species

Hepatocyte nuclear factor-4α (Hnf4α) is a transcription factor that controls epithelial cell polarity and morphogenesis. Hnf4α conditional deletion during postnatal development has minor effects on intestinal epithelium integrity but promotes activation of the Wnt/β-catenin pathway without causing tumorigenesis. Here, we show that Hnf4α does not act as a tumor-suppressor gene but is crucial in promoting gut tumorigenesis in mice. Polyp multiplicity in ApcMin mice lacking Hnf4α is suppressed compared with littermate ApcMin controls. Analysis of microarray gene expression profiles from mice lacking Hnf4α in the intestinal epithelium identifies novel functions of this transcription factor in targeting oxidoreductase-related genes involved in the regulation of reactive oxygen species (ROS) levels. This role is supported with the demonstration that HNF4α is functionally involved in the protection against spontaneous and 5-fluorouracil chemotherapy-induced production of ROS in colorectal cancer cell lines. Analysis of a colorectal cancer patient cohort establishes that HNF4α is significantly upregulated compared with adjacent normal epithelial resections. Several genes involved in ROS neutralization are also induced in correlation with HNF4A expression. Altogether, the findings point to the nuclear receptor HNF4α as a potential therapeutic target to eradicate aberrant epithelial cell resistance to ROS production during intestinal tumorigenesis.

P2Y6 receptor contributes to neutrophil recruitment to inflamed intestinal mucosa by increasing CXC chemokine ligand 8 expression in an AP‐1‐dependent manner in epithelial cells

Background: Inflammatory bowel diseases are characterized by the presence of CXCL8 at the site of lesions resulting in neutrophil recruitment and loss of tissue functions. We report that P2Y6 receptor activation stimulates CXCL8 expression and release by intestinal epithelial cells (IECs). In this context, we investigated if uridine 5′‐diphosphate (UDP) enemas stimulate neutrophil recruitment to the mucosa of mice suffering from colitis‐like disease and we characterized the signaling events linking P2Y6 to CXCL8 expression in IEC. Methods: Neutrophil recruitment was monitored by immunofluorescence and FACS analysis. Expression of Cxcl1, a mouse functional homolog of CXCL8, was determined by quantitative real‐time polymerase chain reaction (qPCR). Pharmacological inhibitors and interfering RNAs were used to characterize the signaling pathway. The outcomes of these treatments on protein phosphorylation and on CXCL8 expression were characterized by western blots, qPCR, luciferase, and chromatin immunoprecipitation (ChIP) assays. Results: Mutation of the AP‐1 site in the CXCL8 core promoter abolished the UDP‐stimulating effect. The c‐fos/c‐jun dimer was identified as the AP‐1 complex regulating CXCL8 in response to UDP stimulation. Regulation of CXCL8 expression by P2Y6 required PKC&dgr; activation upstream of the signaling pathway composed of MEK1/2‐ERK1/2 and c‐fos. UDP administration to mice suffering from colitis‐like disease increased the number of neutrophil infiltrating the mucosa, correlating with Cxcl1 increased expression in IEC and the severity of inflammation. Conclusions: This study not only describes the P2Y6 signaling mechanism regulating CXCL8 expression in IEC, but it also illustrates the potential of targeting P2Y6 to reduce intestinal inflammation. (Inflamm Bowel Dis 2012)

Oxidative stress and mitochondrial functions in the intestinal Caco-2/15 Cell Line

Background Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules. Methodology/Principal Findings The objective of the present work was to evaluate how iron-ascorbate (FE/ASC)-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM) (1) increased malondialdehyde levels assessed by HPLC; (2) reduced ATP production noted by luminescence assay; (3) provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4) upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5) affected mitochondrial respiratory chain complexes I, II, III and IV; (6) elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7) lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8) altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2) without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 µM) prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities. Conclusions/Significance Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases.

The P2Y6 Receptor Mediates Clostridium difficile Toxin-Induced CXCL8/IL-8 Production and Intestinal Epithelial Barrier Dysfunction

C. difficile is a Gram-positive spore-forming anaerobic bacterium that is the leading cause of nosocomial diarrhea in the developed world. The pathogenesis of C. difficile infections (CDI) is driven by toxin A (TcdA) and toxin B (TcdB), secreted factors that trigger the release of inflammatory mediators and contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis in CDI. TcdA and TcdB alter cytoskeletal signaling and trigger the release of CXCL8/IL-8, a potent neutrophil chemoattractant, from intestinal epithelial cells; however, little is known about the surface receptor(s) that mediate these events. In the current study, we sought to assess whether toxin-induced CXCL8/IL-8 release and barrier dysfunction are driven by the activation of the P2Y6 receptor following the release of UDP, a danger signal, from intoxicated Caco-2 cells. Caco-2 cells express a functional P2Y6 receptor and release measurable amounts of UDP upon exposure to TcdA/B. Toxin-induced CXCL8/IL-8 production and release were attenuated in the presence of a selective P2Y6 inhibitor (MRS2578). This was associated with inhibition of TcdA/B-induced activation of NFκB. Blockade of the P2Y6 receptor also attenuated toxin-induced barrier dysfunction in polarized Caco-2 cells. Lastly, pretreating mice with the P2Y6 receptor antagonists (MSR2578) attenuated TcdA/B-induced inflammation and intestinal permeability in an intrarectal toxin exposure model. Taken together these data outline a novel role for the P2Y6 receptor in the induction of CXCL8/IL-8 production and barrier dysfunction in response to C. difficile toxin exposure and may provide a new therapeutic target for the treatment of CDI.

The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis

Histone deacetylases (Hdac) remove acetyl groups from proteins, influencing global and specific gene expression. Hdacs control inflammation, as shown by Hdac inhibitor-dependent protection from dextran sulfate sodium (DSS)-induced murine colitis. Although tissue-specific Hdac knockouts show redundant and specific functions, little is known of their intestinal epithelial cell (IEC) role. We have shown previously that dual Hdac1/Hdac2 IEC-specific loss disrupts cell proliferation and determination, with decreased secretory cell numbers and altered barrier function. We thus investigated how compound Hdac1/Hdac2 or Hdac2 IEC-specific deficiency alters the inflammatory response. Floxed Hdac1 and Hdac2 and villin-Cre mice were interbred. Compound Hdac1/Hdac2 IEC-deficient mice showed chronic basal inflammation, with increased basal disease activity index (DAI) and deregulated Reg gene colonic expression. DSS-treated dual Hdac1/Hdac2 IEC-deficient mice displayed increased DAI, histological score, intestinal permeability, and inflammatory gene expression. In contrast to double knockouts, Hdac2 IEC-specific loss did not affect IEC determination and growth, nor result in chronic inflammation. However, Hdac2 disruption protected against DSS colitis, as shown by decreased DAI, intestinal permeability and caspase-3 cleavage. Hdac2 IEC-specific deficient mice displayed increased expression of IEC gene subsets, such as colonic antimicrobial Reg3b and Reg3g mRNAs, and decreased expression of immune cell function-related genes. Our data show that Hdac1 and Hdac2 are essential IEC homeostasis regulators. IEC-specific Hdac1 and Hdac2 may act as epigenetic sensors and transmitters of environmental cues and regulate IEC-mediated mucosal homeostatic and inflammatory responses. Different levels of IEC Hdac activity may lead to positive or negative outcomes on intestinal homeostasis during inflammation.