|Title||Cigarette smoke extract (CSE) delays NOD2 expression and affects NOD2/RIPK2 interactions in intestinal epithelial cells.|
|Publication Type||Journal Article|
|Year of Publication||2011|
|Authors||Aldhous MC, Soo K, Stark LA, Ulanicka AA, Easterbrook J, Dunlop MG, Satsangi J|
|Keywords||Blotting, Western, Epithelial Cells, HCT116 Cells, HT29 Cells, Humans, Intestines, Nod2 Signaling Adaptor Protein, Protein Binding, Receptor-Interacting Protein Serine-Threonine Kinase 2, Reverse Transcriptase Polymerase Chain Reaction, Smoking, Tumor Necrosis Factor-alpha|
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.
METHODS AND FINDINGS: 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.
|Alternate Journal||PLoS ONE|
|PubMed Central ID||PMC3171477|
|Grant List||CZB/4/638 / / Chief Scientist Office / United Kingdom |
MC_U127527198 / / Medical Research Council / United Kingdom