Julia Beisner

Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1

Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of −200 mV to –300 mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

Genetic variants of Wnt transcription factor TCF-4 (TCF7L2) putative promoter region are associated with small intestinal Crohn's disease.

Reduced expression of Paneth cell antimicrobial α-defensins, human defensin (HD)-5 and -6, characterizes Crohns disease (CD) of the ileum. TCF-4 (also named TCF7L2), a Wnt signalling pathway transcription factor, orchestrates Paneth cell differentiation, directly regulates the expression of HD-5 and -6, and was previously associated with the decrease of these antimicrobial peptides in a subset of ileal CD. To investigate a potential genetic association of TCF-4 with ileal CD, we sequenced 2.1 kb of the 5′ flanking region of TCF-4 in a small group of ileal CD patients and controls (n = 10 each). We identified eight single nucleotide polymorphisms (SNPs), of which three (rs3814570, rs10885394, rs10885395) were in linkage disequilibrium and found more frequently in patients; one (rs3814570) was thereby located in a predicted regulatory region. We carried out high-throughput analysis of this SNP in three cohorts of inflammatory bowel disease (IBD) patients and controls. Overall 1399 healthy individuals, 785 ulcerative colitis (UC) patients, 225 CD patients with colonic disease only and 784 CD patients with ileal involvement were used to determine frequency distributions. We found an association of rs3814570 with ileal CD but neither with colonic CD or UC, in a combined analysis (allele positivity: OR 1.27, 95% CI 1.07 to 1.52, p = 0.00737), which was the strongest in ileal CD patients with stricturing behaviour (allele frequency: OR 1.32, 95% CI 1.08 to1.62, p = 0.00686) or an additional involvement of the upper GIT (allele frequency: OR 1.38, 95% CI 1.03 to1.84, p = 0.02882). The newly identified genetic association of TCF-4 with ileal CD provides evidence that the decrease in Paneth cell α-defensins is a primary factor in disease pathogenesis.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense.

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl4‐induced ascitic cirrhosis and 2‐day portal vein–ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α‐cryptdins and β‐defensins was determined by real‐time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence‐activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α‐cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver–gut axis including the underlying mechanisms could help us to find new treatment avenues. (HEPATOLOGY 2012)

A Novel Functional Polymorphism in the Transforming Growth Factor-β2 Gene Promoter and Tumor Progression in Breast Cancer

Transforming growth factor-beta (TGF-beta), a multifunctional growth factor, plays an important role in breast cancer. There is increasing evidence that enhanced expression of TGF-beta promotes breast cancer progression contributing to metastasis and invasiveness of the tumor. We identified a functional polymorphism in the TGFB2 promoter, a 4-bp insertion at position -246 relative to the transcriptional start site (-246ins). Transient transfection experiments showed that the -246ins polymorphism significantly increased TGFB2 promoter activity in breast cancer cells. Electrophoretic mobility shift assays revealed binding of the transcription factor Sp1 to the -246ins allele. Overexpression of Sp1 enhanced promoter activity of the -246ins allele, demonstrating that Sp1 mediates transcriptional activation. Furthermore, the -246ins allele was associated with enhanced TGF-beta(2) expression in breast cancer tissue (P = 0.0005). To evaluate the role of the polymorphism in breast cancer, frequency of the -246ins allele was determined in breast cancer patients (n = 78) and healthy female controls (n = 143). No significant differences were found. However, the presence of the -246ins allele was associated with lymph node metastasis (P = 0.003). The -246ins allele was a significant predictor for lymph node metastasis independent of estrogen and progesterone receptor status in a multivariate logistic regression analysis (P = 0.0118, odds ratio, 5.18; 95% confidence interval, 1.44-18.62). We provide evidence that the TGFB2 -246ins polymorphism leads to enhanced TGF-beta(2) expression levels in vivo and might thereby contribute to tumor progression and development of metastases.

Association of a functional variant in the Wnt co-receptor LRP6 with early onset ileal Crohn's disease.

Ileal Crohns Disease (CD), a chronic small intestinal inflammatory disorder, is characterized by reduced levels of the antimicrobial peptides DEFA5 (HD-5) and DEFA6 (HD-6). Both of these α-defensins are exclusively produced in Paneth cells (PCs) at small intestinal crypt bases. Different ileal CD–associated genes including NOD2, ATG16L1, and recently the β-catenin–dependant Wnt transcription factor TCF7L2 have been linked to impaired PC antimicrobial function. The Wnt pathway influences gut mucosal homeostasis and PC maturation, besides directly controlling HD-5/6 gene expression. The herein reported candidate gene study focuses on another crucial Wnt factor, the co-receptor low density lipoprotein receptor-related protein 6 (LRP6). We analysed exonic single nucleotide polymorphisms (SNPs) in a large cohort (Oxford: n = 1,893) and prospectively tested 2 additional European sample sets (Leuven: n = 688, Vienna: n = 1,628). We revealed an association of a non-synonymous SNP (rs2302685; Ile1062Val) with early onset ileal CD (OR 1.8; p = 0.00034; for homozygous carriers: OR 4.1; p = 0.00004) and additionally with penetrating ileal CD behaviour (OR 1.3; p = 0.00917). In contrast, it was not linked to adult onset ileal CD, colonic CD, or ulcerative colitis. Since the rare variant is known to impair LRP6 activity, we investigated its role in patient mucosa. Overall, LRP6 mRNA was diminished in patients independently from the genotype. Analysing the mRNA levels of PC product in biopsies from genotyped individuals (15 controls, 32 ileal, and 12 exclusively colonic CD), we found particularly low defensin levels in ileal CD patients who were carrying the variant. In addition, we confirmed a direct relationship between LRP6 activity and the transcriptional expression of HD-5 using transient transfection. Taken together, we identified LRP6 as a new candidate gene in ileal CD. Impairments in Wnt signalling and Paneth cell biology seem to represent pathophysiological hallmarks in small intestinal inflammation and should therefore be considered as interesting targets for new therapeutic approaches.

Influence of standard treatment on ileal and colonic antimicrobial defensin expression in active Crohn’s disease

Background  Crohn’s Disease (CD), a chronic intestinal inflammation, is currently treated primarily by therapeutics which are directed against inflammatory responses. Recent findings though suggest a central role of the innate immune barrier in the pathophysiology. Important factors providing this barrier are antimicrobial peptides like the α‐ and β‐defensins. Little is known about in vivo effects of common drugs on their expression.

Innate antimicrobial immunity in inflammatory bowel diseases

Inflammatory bowel diseases are characterized by chronic intestinal inflammation at different sites. Data from animal models as well as human patients including gene-association studies suggest that different components of the innate barrier function are primarily defective. These recent advances support the evolving hypothesis that intestinal bacteria induce inflammation predominantly as a result of a weakened innate mucosal barrier in genetically predisposed individuals. This article discusses our current understanding of the primary events of disease. Together, these findings should result in new therapeutic avenues aimed at restoring antimicrobial barrier function to prevent a bacterial-triggered inflammatory response.

Gastric Antimicrobial Peptides Fail to Eradicate Helicobacter pylori Infection Due to Selective Induction and Resistance

Background Although antimicrobial peptides protect mucus and mucosa from bacteria, Helicobacter pylori is able to colonize the gastric mucus. To clarify in which extend Helicobacter escapes the antimicrobial defense, we systematically assessed susceptibility and expression levels of different antimicrobial host factors in gastric mucosa with and without H. pylori infection. Materials and Methods We investigated the expression levels of HBD1 (gene name DEFB1), HBD2 (DEFB4A), HBD3 (DEFB103A), HBD4 (DEFB104A), LL37 (CAMP) and elafin (PI3) by real time PCR in gastric biopsy samples in a total of 20 controls versus 12 patients colonized with H. pylori. Immunostaining was performed for HBD2 and HBD3. We assessed antimicrobial susceptibility by flow cytometry, growth on blood agar, radial diffusion assay and electron microscopy. Results H. pylori infection was associated with increased gastric levels of the inducible defensin HBD2 and of the antiprotease elafin, whereas the expression levels of the constitutive defensin HBD1, inducible HBD3 and LL37 remained unchanged. HBD4 was not expressed in significant levels in gastric mucosa. H. pylori strains were resistant to the defensins HBD1 as well as to elafin, and strain specific minimally susceptible to HBD2, whereas HBD3 and LL37 killed all H. pylori strains effectively. We demonstrated the binding of HBD2 and LL37 on the surface of H. pylori cells. Comparing the antibacterial activity of extracts from H. pylori negative and positive biopsies, we found only a minimal killing against H. pylori that was not increased by the induction of HBD2 in H. pylori positive samples. Conclusion These data support the hypothesis that gastric H. pylori evades the host defense shield to allow colonization.

Paneth cell function - Implications in pediatric Crohn disease

Defects in the intestinal barrier play a central role in disease pathogenesis. Recently we have demonstrated that children with ileal Crohn´s disease show a reduced expression of small intestinal HD-5 at the age of onset suggesting that a compromised mucosal barrier function might be a key factor in the early disease pathogenesis. We also identified a disturbance of the Wnt signaling transcription factor TCF-4 as a major mechanism for this deficiency in children which might result in a compromised innate immune function of small intestinal Paneth cells via defensin secretion. Here we provide a summary on our recent findings and discuss the data in more detail especially focusing on the role of Paneth cell differentiation and function in the pathogenesis of pediatric ileal Crohn’s disease.