Abstract
BackgroundHuman β-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 FindingsWe 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-κB 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/ConclusionsColonic HBD2 was dysregulated at mRNA and protein level in IBD. Inflammatory status and stimulus but not germline variations influenced these changes.
Highlights
The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) are common causes of chronic disease in the developed world and represent an important public health issue, with a combined prevalence in northern Europe estimated at 1 in 250 [1]
For inflamed biopsies from both Crohn’s Disease (CD) and UC patients we found a differential gradient of Human b-defensin-2 (HBD2) mRNA expression: in CD, the mean and median expression was highest in biopsies obtained from the ascending colon, descending colon with the lowest expression in sigmoid colon; in UC the opposite pattern was seen with the lowest expression in the ascending colon descending colon and highest expression in the sigmoid colon
In CD, this interest has been intensified by the association between NOD2 mutations and decreased adefensin production from Paneth cells [20,21,22], while a-defensins have been found to be dysregulated in UC [23]
Summary
The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) are common causes of chronic disease in the developed world and represent an important public health issue, with a combined prevalence in northern Europe estimated at 1 in 250 [1]. Smoking is the best known environmental factor implicated in the pathogenesis of IBD [7]. Of the.4000 chemicals in cigarette smoke, nicotine has been studied as a constituent potentially affecting intestinal inflammation. Studies of transdermal nicotine therapy suggest that this may be effective in UC patients with active disease [13,14]. The mechanisms whereby nicotine may influence intestinal inflammation are poorly characterised and there have been few studies of the effects of nicotine on the cellular responses of IBD patients, especially in CD. Human b-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
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