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

[1]  B. Lambrecht,et al.  Cigarette smoking alters epithelial apoptosis and immune composition in murine GALT , 2011, Laboratory Investigation.

[2]  D. Look,et al.  Cigarette Smoke Inhibition of the NF-κB-Dependent Response to Bacteria in the Airway , 2011 .

[3]  Kaoru Tominaga,et al.  Corrigendum: Activation of innate immune antiviral responses by Nod2 , 2010 .

[4]  E. Montgomery,et al.  Relation between normal rectal methylation, smoking status, and the presence or absence of colorectal adenomas , 2010, Cancer.

[5]  J. Satsangi,et al.  The impact of smoking in Crohn's disease: no smoke without fire , 2010, Frontline Gastroenterology.

[6]  N. Salzman,et al.  Induction and rescue of Nod2-dependent Th1-driven granulomatous inflammation of the ileum , 2010, Proceedings of the National Academy of Sciences.

[7]  G. Kaplan,et al.  Common polymorphisms in the NOD2 gene region are associated with leprosy and its reactive states. , 2010, The Journal of infectious diseases.

[8]  L. Cardell,et al.  The expression and function of Nod‐like receptors in neutrophils , 2010, Immunology.

[9]  B. Whittle,et al.  New light on the anti-colitic actions of therapeutic aminosalicylates: the role of heme oxygenase , 2010, Pharmacological reports : PR.

[10]  M. V. D. van den Brink,et al.  Graft-versus-host disease: regulation by microbe-associated molecules and innate immune receptors. , 2010, Blood.

[11]  Jorge L. Aguilar,et al.  Cigarette Smoke Inhibits Airway Epithelial Cell Innate Immune Responses to Bacteria , 2010, Infection and Immunity.

[12]  A. Hutson,et al.  Inflammatory response of lung macrophages and epithelial cells to tobacco smoke: a literature review of ex vivo investigations , 2010, Immunologic research.

[13]  D. Philpott,et al.  Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry , 2010, Nature Immunology.

[14]  D. Jewell,et al.  NOD2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation , 2010, Nature Medicine.

[15]  S. Fiering,et al.  Exposure to Cigarette Smoke Disrupts CCL20-Mediated Antimicrobial Activity in Respiratory Epithelial Cells. , 2014, The open immunology journal.

[16]  Zhen-hua Hu,et al.  Differential effects of NOD2 polymorphisms on colorectal cancer risk: a meta-analysis , 2010, International Journal of Colorectal Disease.

[17]  Ying Wang,et al.  Genomewide association study of leprosy. , 2009, The New England journal of medicine.

[18]  P. Donnelly,et al.  Genome-wide association study of ulcerative colitis identifies three new susceptibility loci, including the HNF4A region , 2010 .

[19]  Tomas Hrncir,et al.  Nod2 is required for the regulation of commensal microbiota in the intestine , 2009, Proceedings of the National Academy of Sciences.

[20]  J. Satsangi,et al.  The genetics of Crohn's disease. , 2009, Annual review of genomics and human genetics.

[21]  O. Nielsen,et al.  Influence of Smoking on Colonic Gene Expression Profile in Crohn's Disease , 2009, PloS one.

[22]  M. Netea,et al.  A Crohn's disease–associated NOD2 mutation suppresses transcription of human IL10 by inhibiting activity of the nuclear ribonucleoprotein hnRNP-A1 , 2009, Nature Immunology.

[23]  J. Satsangi,et al.  Edinburgh Research Explorer Dysregulation of Human beta-Defensin-2 Protein in Inflammatory Bowel Disease , 2009 .

[24]  Michael A McGuckin,et al.  Intestinal barrier dysfunction in inflammatory bowel diseases. , 2009, Inflammatory bowel diseases.

[25]  S. Youssef,et al.  Differential Regulation of Chemokines by IL-17 in Colonic Epithelial Cells1 , 2008, The Journal of Immunology.

[26]  T. Mussack,et al.  NOD2/CARD15 genotype influences MDP‐induced cytokine release and basal IL‐12p40 levels in primary isolated peripheral blood monocytes , 2008, Inflammatory bowel diseases.

[27]  T. Hibi,et al.  IL23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn’s disease , 2008, Gut.

[28]  D. Artis Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut , 2008, Nature Reviews Immunology.

[29]  J. Rhodes,et al.  Nicotine Enemas for Active Crohn's Colitis: An Open Pilot Study , 2008, Gastroenterology research and practice.

[30]  P. Piirilä,et al.  Decreased Cytokine and Chemokine mRNA Expression in Bronchoalveolar Lavage in Asymptomatic Smoking Subjects , 2008, Respiration.

[31]  I. Rahman,et al.  Differential effects of cigarette smoke on oxidative stress and proinflammatory cytokine release in primary human airway epithelial cells and in a variety of transformed alveolar epithelial cells , 2008, Respiratory Research.

[32]  菊池 英純 Chronic nicotine stimulation modulates the immune response of mucosal T cells to Th1-dominant pattern via nAChR by upregulation of Th1-specific transcriptional factor , 2008 .

[33]  P. Lucas,et al.  A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation. , 2008, The EMBO journal.

[34]  Judy H. Cho,et al.  Chronic stimulation of Nod2 mediates tolerance to bacterial products , 2007, Proceedings of the National Academy of Sciences.

[35]  Hiromu Ito,et al.  Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model , 2007, The Journal of experimental medicine.

[36]  Nathalie Rolhion,et al.  Adherent‐invasive Escherichia coli in inflammatory bowel disease , 2007, Inflammatory bowel diseases.

[37]  R. Xavier,et al.  Unravelling the pathogenesis of inflammatory bowel disease , 2007, Nature.

[38]  S. Catinella,et al.  Alpha,beta-unsaturated aldehydes in cigarette smoke release inflammatory mediators from human macrophages. , 2007, American journal of respiratory cell and molecular biology.

[39]  K. Stringer,et al.  Cigarette smoke extract-induced suppression of caspase-3-like activity impairs human neutrophil phagocytosis. , 2007, American journal of physiology. Lung cellular and molecular physiology.

[40]  K. Fukase,et al.  Various human epithelial cells express functional Toll-like receptors, NOD1 and NOD2 to produce anti-microbial peptides, but not proinflammatory cytokines. , 2007, Molecular immunology.

[41]  D. Slebos,et al.  Cigarette smoke-induced blockade of the mitochondrial respiratory chain switches lung epithelial cell apoptosis into necrosis. , 2007, American journal of physiology. Lung cellular and molecular physiology.

[42]  F. Farina,et al.  Cigarette smoke exposure inhibits extracellular MMP-2 (gelatinase A) activity in human lung fibroblasts , 2007, Respiratory research.

[43]  H. Drummond,et al.  Does Cigarette Smoking Influence the Phenotype of Crohn's Disease? Analysis Using the Montreal Classification , 2007, The American Journal of Gastroenterology.

[44]  H. Drummond,et al.  Smoking Habit and Load Influence Age at Diagnosis and Disease Extent in Ulcerative Colitis , 2007, The American Journal of Gastroenterology.

[45]  S. Brant,et al.  A Population-Based Case-Control Study of CARD15 and Other Risk Factors in Crohn's Disease and Ulcerative Colitis , 2007, The American Journal of Gastroenterology.

[46]  I. Rowland,et al.  Hypothesis about mechanisms through which nicotine might exert its effect on the interdependence of inflammation and gut barrier function in ulcerative colitis , 2007, Inflammatory bowel diseases.

[47]  Hua-Hao Shen,et al.  Effect of cigarette smoke extract on lipopolysaccha-ride-activated mitogen-activated protein kinase signal transduction pathway in cultured cells. , 2007, Chinese medical journal.

[48]  C. Jun,et al.  DA-9601, a standardized extract of Artemisia asiatica, blocks TNF-alpha-induced IL-8 and CCL20 production by inhibiting p38 kinase and NF-kappaB pathways in human gastric epithelial cells. , 2006, World journal of gastroenterology.

[49]  G. Hunninghake,et al.  Cigarette smoke induces cellular senescence. , 2006, American journal of respiratory cell and molecular biology.

[50]  N. Brousse,et al.  Implication of TNF-related apoptosis-inducing ligand in inflammatory intestinal epithelial lesions. , 2006, Gastroenterology.

[51]  William D. Lees,et al.  Defective acute inflammation in Crohn's disease: a clinical investigation , 2006, The Lancet.

[52]  A. Sepulveda,et al.  Carbon monoxide ameliorates chronic murine colitis through a heme oxygenase 1–dependent pathway , 2005, The Journal of experimental medicine.

[53]  J. Ingram,et al.  Mechanisms of Disease: nicotine—a review of its actions in the context of gastrointestinal disease , 2005, Nature Clinical Practice Gastroenterology &Hepatology.

[54]  I. Williams,et al.  Acute induction of human IL-8 production by intestinal epithelium triggers neutrophil infiltration without mucosal injury , 2005, Gut.

[55]  C. Cho,et al.  The diverse actions of nicotine and different extracted fractions from tobacco smoke against hapten-induced colitis in rats. , 2005, Toxicological sciences : an official journal of the Society of Toxicology.

[56]  K. Chung,et al.  Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle , 2005, Respiratory research.

[57]  L. Jacobs,et al.  Nucleotide-Binding Oligomerization Domain-2 Modulates Specific TLR Pathways for the Induction of Cytokine Release 1 , 2005, The Journal of Immunology.

[58]  L. Cantley,et al.  The Crohn's Disease Protein, NOD2, Requires RIP2 in Order to Induce Ubiquitinylation of a Novel Site on NEMO , 2004, Current Biology.

[59]  M Schwab,et al.  NOD2 (CARD15) mutations in Crohn’s disease are associated with diminished mucosal α-defensin expression , 2004, Gut.

[60]  I. Rahman,et al.  Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-kappaB activation and proinflammatory cytokine release in alveolar epithelial cells. , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[61]  W. Wu,et al.  The pharmacological actions of nicotine on the gastrointestinal tract. , 2004, Journal of pharmacological sciences.

[62]  L. Beaugerie,et al.  Gender differences in the response of colitis to smoking. , 2004, Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association.

[63]  A. Hopkins,et al.  Proinflammatory Cytokines Disrupt Epithelial Barrier Function by Apoptosis-Independent Mechanisms 1 , 2003, The Journal of Immunology.

[64]  Judy H. Cho,et al.  Defining Complex Contributions of NOD2/CARD15 Gene Mutations, Age at Onset, and Tobacco Use On Crohn's Disease Phenotypes , 2003, Inflammatory bowel diseases.

[65]  J. Hugot,et al.  Card15 gene overexpression in mononuclear and epithelial cells of the inflamed Crohn’s disease colon , 2003, Gut.

[66]  D. Podolsky,et al.  CARD15/NOD2 functions as an antibacterial factor in human intestinal epithelial cells. , 2003, Gastroenterology.

[67]  M. Chamaillard,et al.  Nod2 Is a General Sensor of Peptidoglycan through Muramyl Dipeptide (MDP) Detection* , 2003, The Journal of Biological Chemistry.

[68]  R. Lazarus,et al.  Is smoking an indirect risk factor for the development of ulcerative colitis? An age‐ and sex‐matched case–control study , 2003, Journal of gastroenterology and hepatology.

[69]  P. Rosenstiel,et al.  TNF-alpha and IFN-gamma regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells. , 2003, Gastroenterology.

[70]  B. Jasani,et al.  Effect of smoking and transdermal nicotine on colonic nicotinic acetylcholine receptors in ulcerative colitis. , 2003, QJM : monthly journal of the Association of Physicians.

[71]  Judy H. Cho,et al.  Crohn's disease-associated NOD2 variants share a signaling defect in response to lipopolysaccharide and peptidoglycan. , 2003, Gastroenterology.

[72]  A. Macpherson,et al.  In siblings with similar genetic susceptibility for inflammatory bowel disease, smokers tend to develop Crohn's disease and non-smokers develop ulcerative colitis , 2002, Gut.

[73]  R. Eliakim,et al.  Chronic nicotine administration differentially alters jejunal and colonic inflammation in interleukin-10 deficient mice , 2002, European journal of gastroenterology & hepatology.

[74]  Alastair Forbes,et al.  The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease. , 2002, Gastroenterology.

[75]  S. Targan,et al.  Mutations in NOD2 are associated with fibrostenosing disease in patients with Crohn's disease. , 2002, Gastroenterology.

[76]  Mourad Sahbatou,et al.  Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease , 2001, Nature.

[77]  Judy H. Cho,et al.  A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease , 2001, Nature.

[78]  F. Carbonnel,et al.  Smoking cessation and the course of Crohn's disease: an intervention study. , 2001, Gastroenterology.

[79]  J. Satsangi,et al.  Review article: the genetics of inflammatory bowel disease. , 2001, Alimentary pharmacology & therapeutics.

[80]  P. J. Kelly,et al.  Inflammatory bowel disease: epidemiology and management in an English general practice population , 2000, Alimentary pharmacology & therapeutics.

[81]  C. Brampton,et al.  An investigation into the effect and mechanisms of action of nicotine in inflammatory bowel disease , 2000, Inflammation Research.

[82]  A. Iwasaki,et al.  Localization of Distinct Peyer's Patch Dendritic Cell Subsets and Their Recruitment by Chemokines Macrophage Inflammatory Protein (Mip)-3α, Mip-3β, and Secondary Lymphoid Organ Chemokine , 2000, The Journal of experimental medicine.

[83]  R. Hamilton,et al.  Effect of acrolein on human alveolar macrophage NF-κB activity. , 1999, American journal of physiology. Lung cellular and molecular physiology.

[84]  M. Hayn,et al.  4-Hydroxynonenal prevents NF-kappaB activation and tumor necrosis factor expression by inhibiting IkappaB phosphorylation and subsequent proteolysis. , 1999, The Journal of biological chemistry.

[85]  D. Thomas,et al.  Identification of lysine residues required for signal-induced ubiquitination and degradation of I kappa B-alpha in vivo. , 1996, Oncogene.

[86]  David E. Irwin,et al.  Nicotine and cotinine in the cervical mucus of smokers, passive smokers, and nonsmokers. , 1992, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.