Dysregulation of Human β-Defensin-2 Protein in Inflammatory Bowel Disease

BACKGROUND:Human beta-defensin-2 (HBD2) is an antimicrobial peptide implicated in the pathogenesis of inflammatory bowel disease (IBD). Low copy number and concomitant low mRNA expression of the HBD2 gene have been implicated in susceptibility to colonic Crohn's Disease (CD). We investigated the colonic distribution of HBD2 mRNA expression, and the contributions of genetic and environmental factors on HBD2 protein production. METHODOLOGY/PRINCIPAL FINDINGS:We examined HBD2 mRNA expression at three colonic locations by microarray analysis of biopsies from 151 patients (53 CD, 67 ulcerative colitis [UC], 31 controls). We investigated environmental and genetic influences on HBD2 protein production using ex vivo cultured sigmoid colon biopsies from 69 patients (22 CD, 26 UC, 21 controls) stimulated with lipopolysaccharide (LPS) and/or nicotine for 24 hours. HBD2 and cytokines were measured in culture supernatants. Using DNA samples from these patients, regions in the HBD2 gene promoter were sequenced for NF-kappaB binding-sites and HBD2 gene copy number was determined. HBD2 mRNA expression was highest in inflamed (vs. uninflamed p = 0.0122) ascending colon in CD and in inflamed (vs. uninflamed p<0.0001) sigmoid colon in UC. HBD2 protein production was increased in inflamed UC biopsies (p = 0.0078). There was no difference in HBD2 protein production from unstimulated biopsies of CD, UC and controls. LPS-induced HBD2 production was significantly increased in CD (p = 0.0375) but not UC (p = 0.2017); this LPS-induced response was augmented by nicotine in UC (p = 0.0308) but not CD (p = 0.6872). Nicotine alone did not affect HBD2 production. HBD2 production correlated with IL8 production in UC (p<0.001) and with IL10 in CD (p<0.05). Variations in the HBD2 promoter and HBD2 gene copy number did not affect HBD2 production. SIGNIFICANCE/CONCLUSIONS:Colonic HBD2 was dysregulated at mRNA and protein level in IBD. Inflammatory status and stimulus but not germline variations influenced these changes

Cigarette Smoke Extract (CSE) Delays NOD2 Expression and Affects NOD2/RIPK2 Interactions in Intestinal Epithelial Cells

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.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.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

Correlations between HBD2 and IL1β, IL8 & IL10 production from unstimulated, LPS- and LPS+nicotine-stimulated biopsies obtained from patients and controls.

<p>Correlations between HBD2 and IL1β, IL8 & IL10 production from unstimulated, LPS- and LPS+nicotine-stimulated biopsies obtained from patients and controls.</p

Expression of HBD2 mRNA with disease (A), inflammation (B) and colonic location of inflamed biopsies (C).

<p>All graphs show the log2 expression values from comparison with a reference marker (of value = 1) for HBD2 mRNA. Horizontal bars denote the median values for each analysis. P values from Mann-Whitney or Kruskall-Wallis tests are given, where significantly different.</p

HBD2 production according to disease and inflammation status.

<p>HBD2 production (mean+sem) from biopsies from CD, UC patients or controls, unstimulated or stimulated with LPS or LPS+nicotine (denoted LN) and grouped according to disease and inflammation. P value given is from a two-way ANOVA compared by disease/inflammation or stimulation.</p

Production of HBD2 protein by LPS or LPS+nicotine in CD and UC patients and controls.

<p>HBD2 protein production (mean+sem) from culture supernatants of unstimulated or LPS- or LPS+nicotine (denoted LN)-stimulated biopsies in organ culture. * denotes P values of<0.05 from the results of Wilcoxon signed rank tests and the groups compared are shown. The actual p values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006285#s3" target="_blank">Results</a>.</p

Patient demographics, disease characteristics and NOD2 genotype.

*<p>Disease location/extent defined according to the Montreal Classification<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006285#pone.0006285-Silverberg1" target="_blank">[35]</a>.</p>†<p>One patient had upper GI disease with L1.</p>‡<p>Surgery was resection for CD, hemi-colectomy for UC.</p>‡‡<p>Disease activity as reported in pathology report.</p>††<p>NOD2 genotyping was carried out as previously described<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006285#pone.0006285-Arnott1" target="_blank">[36]</a>. NA – not applicable.</p