title: Targeted CFTR gene disruption with zinc-finger nucleases in human intestinal epithelial cells induces oxidative stress and inflammation.
abstract: Cystic fibrosis (CF) is a multisystemic pathology caused by mutations of the CF transmembrane conductance regulator (CFTR) gene.
  
  
  OBJECTIVE
  As the intestine harbors the greatest number of CFTR transcripts after birth and since CFTR plays a role in glutathione transport, we hypothesized that CFTR deletion might produce oxidative stress (OxS) and inflammation in CF intestinal epithelial cell.
  
  
  METHODS
  CFTR gene was abrogated in Caco-2/15 enterocytes through the zinc-finger nuclease system. Their oxidative and inflammatory characteristics were appreciated under basal conditions and after the treatment with the pro-oxidant iron-ascorbate (Fe/Asc) complex and pro-inflammatory lipopolysaccharide (LPS).
  
  
  RESULTS
  Intestinal epithelial cells with CFTR knockout spontaneously exhibited an increased lipid peroxidation level, reflected by malondialdehyde overproduction and reduced antioxidant defense characterized by low enzymatic activities of glutathione peroxidase and catalase. CFTR silencing also resulted in elevated protein expression of pro-inflammatory tumor necrosis Factor-α, interleukin-6, cyclooxygenase-2, and the transcription factor nuclear factor-κB. Moreover, exaggerated OxS and inflammation processes occurred in CFTR(-/-) cells in response to the addition of Fe/Asc and LPS, respectively.
  
  
  CONCLUSIONS
  Intestinal Caco-2/15 cells with CFTR deletion, display innate oxidative and inflammatory features while being more sensitive to pro-oxidant and pro-inflammatory stimuli. These two pathophysiological processes could be implicated in CF-related intestinal disorders.
authors.affiliation: Research Centre, CHU Sainte-Justine, 3175 Sainte-Catherine Road, Montreal, Quebec H3T 1C4, Canada; Department of Nutrition, Université de Montréal, Montreal, Quebec H3T 1C5, Canada.
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Kleme
authors.affiliation: Research Centre, CHU Sainte-Justine, 3175 Sainte-Catherine Road, Montreal, Quebec H3T 1C4, Canada.
authors.forename: Alain Théophile
authors.initials: AT
authors.lastname: Sané
authors.affiliation: Research Centre, CHU Sainte-Justine, 3175 Sainte-Catherine Road, Montreal, Quebec H3T 1C4, Canada.
authors.forename: Carole
authors.initials: C
authors.lastname: Garofalo
authors.affiliation: Research Centre, CHU Sainte-Justine, 3175 Sainte-Catherine Road, Montreal, Quebec H3T 1C4, Canada; Department of Nutrition, Université de Montréal, Montreal, Quebec H3T 1C5, Canada. Electronic address: emile.levy@recherche-ste-justine.qc.ca.
authors.forename: Emile
authors.initials: E
authors.lastname: Levy
mesh_terms: D018938:Caco-2 Cells; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D003851:Deoxyribonucleases / Q000378:metabolism*; D004847:Epithelial Cells / Q000201:enzymology* / Q000473:pathology; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D006801:Humans; D015151:Immunoblotting; D007249:Inflammation / Q000235:genetics*; D018384:Oxidative Stress / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D016335:Zinc Fingers
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003851:Deoxyribonucleases
keywords: Cystic fibrosis; Inflammation; Oxidative stress; Small intestine