Explorer Cigarette Smoke Extract ( CSE ) Delays NOD 2 Expression and Affects NOD 2 / RIPK 2 Interactions in Intestinal Epithelial Cells

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) 6TNFa (to mimic inflammation). NOD2 expression was measured by qRT-PCR and western blotting; NOD2RIPK2 interactions by co-immunoprecipitation (CoIP); nuclear NFkB-p65 by ELISA; NFkB activity by luciferase reporter assays and chemokines (CCL20, IL8) in culture supernatants by ELISA. In SW480 and HT29 cells the TNFa-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 TNFa-induced NFkB-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 TNFa, but at 18 hours it was increased in cells stimulated with TNFa+CSE but decreased with TNFa alone (p = 0.0330); CSE reduced TNFa-induced NFkB activity (p = 0.0014) at the same time-point. At 24 hours, basal CCL20 and IL8 (p,0.001 for both) and TNFa-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 TNFa and CSE. Conclusions: CSE delayed TNFa-induced NOD2 mRNA expression and was associated with abnormal NOD2/RIPK2 interaction, reduced NFkB 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. Citation: Aldhous MC, Soo K, Stark LA, Ulanicka AA, Easterbrook JE, et al. (2011) Cigarette Smoke Extract (CSE) Delays NOD2 Expression and Affects NOD2/RIPK2 Interactions in Intestinal Epithelial Cells. PLoS ONE 6(9): e24715. doi:10.1371/journal.pone. 0024715 Editor: Neeraj Vij, Johns Hopkins School of Medicine, United States of America Received June 9, 2010; Accepted August 19, 2011; Published September 12, 2011 Copyright: 2011 Aldhous et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Chief Scientist’s Office of the Scottish Government, grant numbers CZB/4/68 and CZB/4/638. Website: http://www.cso. scot.nhs. uk/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: maldhous@ed.ac. uk