title: Cigarette smoke extract (CSE) delays NOD2 expression and affects NOD2/RIPK2 interactions in intestinal epithelial cells.
abstract: BACKGROUND
  Genetic and environmental factors influence susceptibility to Crohn's disease (CD): NOD2 is the strongest individual genetic determinant and smoking the best-characterised environmental factor. Carriage of NOD2 mutations predispose to small-intestinal, stricturing CD, a phenotype also associated with smoking. We hypothesised that cigarette smoke extract (CSE) altered NOD2 expression and function in intestinal epithelial cells.
  
  
  RESULTS
  Intestinal epithelial cell-lines (SW480, HT29, HCT116) were stimulated with CSE and nicotine (to mimic smoking) ±TNFα (to mimic inflammation). NOD2 expression was measured by qRT-PCR and western blotting; NOD2-RIPK2 interactions by co-immunoprecipitation (CoIP); nuclear NFκB-p65 by ELISA; NFκB activity by luciferase reporter assays and chemokines (CCL20, IL8) in culture supernatants by ELISA. In SW480 and HT29 cells the TNFα-induced NOD2 expression at 4 hours was reduced by CSE (p = 0.0226), a response that was dose-dependent (p = 0.003) and time-dependent (p = 0.0004). Similar effects of CSE on NOD2 expression were seen in cultured ileal biopsies from healthy individuals. In SW480 cells CSE reduced TNFα-induced NFκB-p65 translocation at 15 minutes post-stimulation, upstream of NOD2. Levels of the NOD2-RIPK2 complex were no different at 8 hours post-stimulation with combinations of CSE, nicotine and TNFα, but at 18 hours it was increased in cells stimulated with TNFα+CSE but decreased with TNFα alone (p = 0.0330); CSE reduced TNFα-induced NFκB activity (p = 0.0014) at the same time-point. At 24 hours, basal CCL20 and IL8 (p<0.001 for both) and TNFα-induced CCL20 (p = 0.0330) production were decreased by CSE. CSE also reduced NOD2 expression, CCL20 and IL8 production seen with MDP-stimulation of SW480 cells pre-treated with combinations of TNFα and CSE.
  
  
  CONCLUSIONS
  CSE delayed TNFα-induced NOD2 mRNA expression and was associated with abnormal NOD2/RIPK2 interaction, reduced NFκB activity and decreased chemokine production. These effects may be involved in the pathogenesis of small-intestinal CD and may have wider implications for the effects of smoking in NOD2-mediated responses.
authors.affiliation: Gastrointestinal Unit, Molecular Medicine Centre, Western General Hospital, University of Edinburgh, Edinburgh, Scotland, United Kingdom. maldhous@ed.ac.uk
authors.forename: Marian C
authors.initials: MC
authors.lastname: Aldhous
authors.forename: Kimberley
authors.initials: K
authors.lastname: Soo
authors.forename: Lesley A
authors.initials: LA
authors.lastname: Stark
authors.forename: Agata A
authors.initials: AA
authors.lastname: Ulanicka
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Easterbrook
authors.forename: Malcolm G
authors.initials: MG
authors.lastname: Dunlop
authors.forename: Jack
authors.initials: J
authors.lastname: Satsangi
mesh_terms: D015153:Blotting, Western; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism*; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D007422:Intestines / Q000166:cytology*; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012907:Smoking / Q000009:adverse effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D014409:Tumor Necrosis Factor-alpha; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2