Up-Regulation of Intestinal Vascular Endothelial Growth Factor by Afa/Dr Diffusely Adhering Escherichia coli

BACKGROUND: Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn's disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC). METHODOLOGY: VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors. PRINCIPAL FINDINGS: C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways. CONCLUSIONS: Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis

cDNA cloning and expression of a hamster α-thrombin receptor coupled to Ca2+ mobilization

AbstractThe serine protease α-thrombin (thrombin) potently stimulates G-protein-coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G-protein-coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C-coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N-terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N-terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion-binding exosite of thrombin

P2RX7B is a new theranostic marker for lung adenocarcinoma patients

International audienc

E. coli adhérentes et invasives et pathogénèse de la maladie de Crohn (rôle du facteur hypoxique HIF-1)

La maladie de Crohn (MC) est une maladie inflammatoire chronique intestinale (MICI). Son incidence et sa prévalence ont augmenté en Europe au cours des dix dernières années (150 pour 100000 habitants) constituant ainsi un problème de santé majeur. L inflammation chronique dans la MC favorise la mise en place d une angiogenèse pathophysiologique. Inflammation et angiogenèse sont deux réponses cellulaires suspectées dans la survenue des cancers coliques associés au MICI. Même si les facteurs favorisant la mise en place de la MC restent non élucidés, la contribution des bactéries exogènes est fortement suspectée. Parmi ces bactéries, les E.coli adhérentes et invasives (AIEC), isolées à partir de la muqueuse iléale de patients porteurs de la MC, sont un bon candidat. Les objectifs de mon projet de thèse étaient de caractériser les mécanismes moléculaires induits par les AIEC et impliqués dans la mise en place des réponses pro inflammatoire et pro angiogénique des cellules intestinales épithéliales. Le facteur de transcription hypoxique (HIF-1) est au cœur de l immunité innée et de l angiogenèse. J ai émis l hypothèse que les AIEC pouvaient moduler le niveau d expression de HIF-1a et ainsi contrôler les réponses pro inflammatoire et pro angiogénique. Dans mon premier article, j ai montré que HIF-1a est maximalement exprimé au niveau de l épithélium iléal des patients porteurs de la MC. Ensuite, j ai montré sur un modèle murin compétent pour l infection par les AIEC, les souris CEABAC10, que les bactéries induisent l augmentation du niveau protéique de HIF-1 a ainsi que l activation de la voie de signalisation du VEGF, le facteur angiogénique le plus puissant.Non communiqué.NICE-Bibliotheque electronique (060889901) / SudocSudocFranceF

Infection bactérienne, inflammation et carcinogenèse (événements moléculaires associés à l'activation d'HIF-1 en réponse à l'infection des cellules épithéliales intestinales par les "Diffusely Adhering Afa/Dr E. coli" (Afa/Dr DAEC))

L angiogenèse participe au processus inflammatoire de type chronique, notamment dans les maladies inflammatoires chroniques intestinales (MICI). L origine de ces maladies est encore mal connue, mais il existe de nombreux arguments incriminant les microorganismes intestinaux. Plusieurs études suggèrent que des E. coli opportunistes présentant des caractéristiques pro-inflammatoires participent à l établissement des MICI. Nous avons émis l hypothèse que ces souches de bactéries pourraient moduler la réponse angiogénique de cellules épithéliales intestinales humaines. L infection de ces cellules par la souche E. coli Afa/Dr DAEC C1845 induit une augmentation de l expression de gènes angiogéniques et inflammatoires dont le Vascular Endothélial Growth Factor et l Interleukine-8. Le VEGF produit est biologiquement actif sur des cellules endothéliales en culture. Le facteur HIF-1 rend compte de la production de VEGF mais aussi de kl IL-8. Contrairement à l hypoxie qui stabilise la protéine HIF-1alpha par inhibition de l activité des prolyl hydroxylases, les bactéries C1845 induisent un macanisme traductionnel conduisant à l activation de la protéine S6 par l intermédiaire des voies MEK/ERK et PI-3K/AKT. La stimulation de HIF 1alpha en réponse aux bactéries requiert la liaison de l adhésine F1845 au récepteur membranaire DAF. Le niveau d expression et l activité du facteur HIF-1 est de plus en plus étudié dans le cadre d inflammations intestinales, dont les MICI, HIF-1 pourraient représenter un élément clé dans la chronicité et l évolution des MICI de part son rôle au cours de l inflammation, de l angiogenèse et dans le maintien de l intégrité de l épithélium intestinal.Angiogenesis is involved in chronic inflammatory processes, including Inflammatory Bowel Disease (IBD). The origin of IBD is not known yet, but there are a lot of arguments incriminating intestinal microorganisms. Some opportunistic E. coli shows inflammatory characteristics in sensitive people. Several studies suggest that bacteria are involved in IBD. We have proposed the hypothesis that bacterial strain might modulate the angiogenic response of human intestinal epithelial cells. The infection of these cells with an inflammatory strain of E. coli Afa/Dr DAEC C1845 induced the expression of inflammatory and angiogenic genes like the Vascular Endothelial Growth Factor (VEGF) and the Interleukin-8 (IL-8). The VEGF product by infected cells is biologically active on endothelial cells in culture. The factor HIF-1 reports the production of VEGF and IL-8. Unlike hypoxia which stabilised HIF-1alpha protein by inhibition of the prolyl hydroxylases activity, C1845 bacteria induced a translational mechanism leading to the activation of the ribosomal protein S6 mediated by the MEK/ERK and PI-3K/AKT pathways. Moreover, the activation of HIF-1alpha in response to the infection requires the binding of the F1845 adhesin to the DAF/CD55 membrane receptor. The level of expression and the activity of HIF-1 are more and more analysed in intestinal inflammations like IBD. HIF-1 could be a key factor in the chronicity if the inflammation and in thhe evolution of IBD thanks to its effect on inflammation, angiogenesis and intestinal epithelium integrity.NICE-BU Sciences (060882101) / SudocSudocFranceF

The Role of IL-18 in P2RX7-Mediated Antitumor Immunity

International audienceCancer is the leading cause of death worldwide despite the variety of treatments that are currently used. This is due to an innate or acquired resistance to therapy that encourages the discovery of novel therapeutic strategies to overcome the resistance. This review will focus on the role of the purinergic receptor P2RX7 in the control of tumor growth, through its ability to modulate antitumor immunity by releasing IL-18. In particular, we describe how the ATP-induced receptor activities (cationic exchange, large pore opening and NLRP3 inflammasome activation) modulate immune cell functions. Furthermore, we recapitulate our current knowledge of the production of IL-18 downstream of P2RX7 activation and how IL-18 controls the fate of tumor growth. Finally, the potential of targeting the P2RX7/IL-18 pathway in combination with classical immunotherapies to fight cancer is discussed

Protocol for evaluating in vivo the activation of the P2RX7 immunomodulator

International audienceBackground: P2RX7 is a purinergic receptor with pleiotropic activities that is activated by high levels of extracellular ATP that are found in inflamed tissues. P2RX7 has immunomodulatory and anti-tumor proprieties and is therefore a therapeutic target for various diseases. Several compounds are developed to either inhibit or enhance its activation. However, studying their effect on P2RX7's activities is limited to in vitro and ex vivo studies that require the use of unphysiological media that could affect its activation. Up to now, the only way to assess the activity of P2RX7 modulators on the receptor in vivo was in an indirect manner. Results: We successfully developed a protocol allowing the detection of P2RX7 activation in vivo in lungs of mice, by taking advantage of its unique macropore formation ability. The protocol is based on intranasal delivery of TO-PRO TM-3, a non-permeant DNA intercalating dye, and fluorescence measurement by flow cytometry. We show that ATP enhances TO-PRO TM-3 fluorescence mainly in lung immune cells of mice in a P2RX7-dependant manner. Conclusions: The described approach has allowed the successful analysis of P2RX7 activity directly in the lungs of WT and transgenic C57BL6 mice. The provided detailed guidelines and recommendations will support the use of this protocol to study the potency of pharmacologic or biologic compounds targeting P2RX7

The Purinergic Landscape of Non-Small Cell Lung Cancer

International audienceLung cancer is the most common cancer worldwide. Despite recent therapeutic advances, including targeted therapies and immune checkpoint inhibitors, the disease progresses in almost all advanced lung cancers and in up to 50% of early-stage cancers. The purpose of this review is to discuss whether purinergic checkpoints (CD39, CD73, P2RX7, and ADORs), which shape the immune response in the tumor microenvironment, may represent novel therapeutic targets to combat progression of non-small cell lung cancer by enhancing the antitumor immune response

To inhibit or to boost the ATP/P2RX7 pathway to fight cancer, that is the question

International audienceDespite new biological insights and recent therapeutic advances, many tumors remain at baseline during treatments. Therefore, there is an urgent need to find new therapeutic strategies to improve the care of patients with solid tumors. P2RX7 receptor (P2XR7), an ATPgated ion channel characterized by its ability to form large pore within the cell membrane, is described by most of the investigators as a "chef d'orchestre" of the antitumor immune response. The purpose of this review is to detail the recent information concerning different cellular mechanisms linking P2RX7 to hallmarks of cancer and to discuss different progresses in elucidating how activation of the ATP/P2RX7/NLRP3/IL-18 pathway is a very promising approach to fight cancer progression by increasing antitumor immune responses