title: Attenuation of Clostridium difficile toxin-induced damage to epithelial barrier by ecto-5'-nucleotidase (CD73) and adenosine receptor signaling.
abstract: BACKGROUND
  Clostridium difficile (Cdf) releases toxins (TcdA and TcdB) that damage the intestinal epithelial barrier. Ecto-5'-nucleotidase (CD73) is expressed on intestinal epithelial cells, and it is hypothesized to protect against toxin-induced epithelial damage through the cleavage of 5'-AMP to adenosine (Ado) and subsequent activation of adenosine receptors (AdoRs). Herein, we sought to assess the potential protective effects of CD73 and AdoR signaling on the injurious effects of Cdf toxins.
  
  
  METHODS
  Barrier function was assessed with T84 colonocytes. Transepithelial electrical resistance (TEER), paracellular fluorescein isothiocyanate (FITC)-dextran flux, and tight junction protein (ZO-1) integrity were monitored. Intrarectal installation of Cdf toxin was used to assess epithelial damage in vivo.
  
  
  RESULTS
  TcdA/B caused reduced TEER and increased paracellular flux in vitro. Concurrent treatment with 5'-AMP attenuated these responses to Cdf toxin; an effect that was blocked with ZM241385 (AdoRA2 antagonist). APCP, a CD73 inhibitor, also suppressed the protective effects of 5'-AMP on paracellular flux. 5'-AMP reduced toxin-induced disruption of ZO-1, an effect that was abolished by APCP and ZM241385. Inhibition of CD73 with APCP during Cdf toxin exposure led to increased intestinal barrier permeability and epithelial damage in vivo. Intrarectal instillation of 5'-AMP had no effect on toxin-induced intestinal injury.
  
  
  CONCLUSIONS
  Our data suggest that CD73 has a protective role against TcdA/B-induced damage. 5'-AMP treatment attenuated the damaging effects of Cdf toxin in vitro, and inhibitors of CD73 (APCP) and AdoRs (ZM241385) revealed that the cleavage of 5'-AMP to Ado was necessary for the protective effects. Inhibition of CD73 in vivo increases colonic tissue damage and epithelial permeability during Cdf toxin exposure.
authors.forename: L P
authors.initials: LP
authors.lastname: Schenck
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, AB, Canada, T2N 4Z6.
authors.forename: L Patrick
authors.initials: LP
authors.lastname: Schenck
authors.forename: S A
authors.initials: SA
authors.lastname: Hirota
authors.forename: C L
authors.initials: CL
authors.lastname: Hirota
authors.forename: P
authors.initials: P
authors.lastname: Boasquevisque
authors.forename: S E
authors.initials: SE
authors.lastname: Tulk
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.forename: A
authors.initials: A
authors.lastname: Wadhwani
authors.forename: C T A
authors.initials: CT
authors.lastname: Doktorchik
authors.forename: W K
authors.initials: WK
authors.lastname: Macnaughton
authors.forename: P L
authors.initials: PL
authors.lastname: Beck
authors.forename: J A
authors.initials: JA
authors.lastname: MacDonald
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism*; D001426:Bacterial Proteins / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004768:Enterotoxins / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism; D010539:Permeability; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D015398:Signal Transduction; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D018047:Receptors, Purinergic P1; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D015720:5'-Nucleotidase