title: Escherichia coli LF82 differentially regulates ROS production and mucin expression in intestinal epithelial T84 cells: implication of NOX1.
abstract: BACKGROUND
  Increased reactive oxygen species (ROS) production is associated with inflamed ileal lesions in Crohn's disease colonized by pathogenic adherent-invasive Escherichia coli LF82. We investigated whether such ileal bacteria can modulate ROS production by epithelial cells, thus impacting on inflammation and mucin expression.
  
  
  METHODS
  Ileal bacteria from patients with Crohn's disease were incubated with cultured epithelial T84 cells, and ROS production was assayed using the luminol-amplified chemiluminescence method. The gentamicin protection assay was used for bacterial invasion of T84 cell. The expression of NADPH oxidase (NOX) subunits, mucin, and IL-8 was analyzed by quantitative real-time PCR and Western blots. Involvement of NOX and ROS was analyzed using diphenyleneiodonium (DPI) and N-acetylcysteine (NAC).
  
  
  RESULTS
  Among different bacteria tested, only LF82 induced an increase of ROS production by T84 cells in a dose-dependent manner. This response was inhibited by DPI and NAC. Heat- or ethanol-attenuated LF82 bacteria and the mutant LF82ΔFimA, which does not express pili type 1 and poorly adheres to epithelial cells, did not induce the oxidative response. The LF82-induced oxidative response coincides with its invasion in T84 cells, and both processes were inhibited by DPI. Also, we observed an increased expression of NOX1 and NOXO1 in response to LF82 bacteria versus the mutant LF82ΔFimA. Furthermore, LF82 inhibited mucin gene expression (MUC2 and MUC5AC) in T84 cells while increasing the chemotactic IL-8 expression, both in a DPI-sensitive manner.
  
  
  CONCLUSIONS
  Adherent-invasive E. coli LF82 induced ROS production by intestinal NADPH oxidase and altered mucin and IL-8 expression, leading to perpetuation of inflammatory lesions in Crohn's disease.
authors.affiliation: *Inserm U1149, CNRS-ERL8252, Centre de Recherche sur l'Inflammation, Paris, France; †Université Paris Diderot, Sorbonne Paris Cité, Laboratoire d'Excellence Inflamex, Faculté de Médecine, Site Xavier Bichat, Paris, France; ‡Inserm U995-Team 1, Faculté de Médecine, Pôle recherche, Laboratoire J&K, Lille, France; §UMR 1071 Inserm/UdA, Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte, Facultés de Médecine et de Pharmacie, Clermont-Ferrand, France; and ‖Aix Marseille Université, INSERM UMR_S 911, Faculté de pharmacie, Marseille, France.
authors.forename: Imen
authors.initials: I
authors.lastname: Elatrech
authors.forename: Viviana
authors.initials: V
authors.lastname: Marzaioli
authors.forename: Hanane
authors.initials: H
authors.lastname: Boukemara
authors.forename: Odile
authors.initials: O
authors.lastname: Bournier
authors.forename: Christel
authors.initials: C
authors.lastname: Neut
authors.forename: Arlette
authors.initials: A
authors.lastname: Darfeuille-Michaud
authors.forename: José
authors.initials: J
authors.lastname: Luis
authors.forename: Laurent
authors.initials: L
authors.lastname: Dubuquoy
authors.forename: Jamel
authors.initials: J
authors.lastname: El-Benna
authors.forename: Pham
authors.initials: P
authors.lastname: My-Chan Dang
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Marie
mesh_terms: D015179:Colorectal Neoplasms / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D004926:Escherichia coli / Q000472:pathogenicity*; D004927:Escherichia coli Infections / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D006801:Humans; D007082:Ileum / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D007124:Immunoenzyme Techniques; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000276:immunology / Q000382:microbiology; D055271:Mucin 5AC / Q000235:genetics / Q000378:metabolism*; D055262:Mucin-2 / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D016209:Interleukin-8; C524625:MUC2 protein, human; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D055262:Mucin-2; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human