Wnt signaling regulates small intestinal stem cell maintenance and Paneth cell differentiation. In patients with ileal Crohn's disease (CD), a decrease of Paneth cell α-defensins has been observed that is partially caused by impaired TCF-4 and LRP6 function. Here we show reduced expression of the Wnt signaling effector TCF-1 (also known as TCF-7) in patients with ileal CD. Reporter gene assays and in vitro promoter binding analysis revealed that TCF-1 activates α-defensin HD-5 and HD-6 transcription in cooperation with β-catenin and that activation is mediated by three distinct TCF binding sites. EMSA analysis showed binding of TCF-1 to the respective motifs. In ileal CD patients, TCF-1 mRNA expression levels were significantly reduced. Moreover, we found specifically reduced expression of active TCF-1 mRNA isoforms. Tcf-1 knockout mice exhibited reduced cryptdin expression in the jejunum, which was not consistently seen at other small intestinal locations. Our data provide evidence that TCF-1-mediated Wnt signaling is disturbed in small intestinal CD, which might contribute to the observed barrier dysfunction in the disease.

Human β-defensin 1 (hBD-1) is an antimicrobial peptide expressed by epithelia and hematopoietic cells. We demonstrated recently that hBD-1 shows activity against enteric commensals and Candida species only after its disulfide bonds have been reduced by thioredoxin (TRX) or a reducing environment. Here we show that besides TRX, glutaredoxin (GRX) is also able to reduce hBD-1, although with far less efficacy. Moreover, living intestinal and lymphoid cells can effectively catalyze reduction of extracellular hBD-1. By chemical inhibition of the TRX system or specific knockdown of TRX, we demonstrate that cell-mediated reduction is largely dependent on TRX. Quantitative PCR in intestinal tissues of healthy controls and inflammatory bowel disease patients revealed altered expression of some, although not all, redox enzymes, especially in ulcerative colitis. Reduced hBD-1 and TRX localize to extracellular colonic mucus, suggesting that secreted or membrane-bound TRX converts hBD-1 to a potent antimicrobial peptide in vivo.

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.

Background: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell.Methods: Expression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/- flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted.Results: Expression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice.Conclusions: Intestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch.

BACKGROUND AND AIMS:To prevent bacterial adherence and translocation, the colonic mucosa is covered by a protecting mucus layer and the epithelium synthesizes antimicrobial peptides. The present qualitative study investigated the contents and interaction of these peptides in and with rectal mucus.METHODS:Rectal mucus extracts were analyzed for antimicrobial activity and screened with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Dot blot and immunohistochemistry for antimicrobial peptides. In addition, binding of AMPs to mucins was investigated by Western blot and enzyme-linked lectin assays.RESULTS:In functional tests the mucus layer exhibited a strong antimicrobial activity. We detected 11 antimicrobial peptides in mucus extracts from healthy persons including the defensins HBD-1 and -3, the cathelicidin LL-37, ubiquitin, lysozyme, histones, high mobility group nucleosome-binding domain-containing protein 2, ubiquicidin and other ribosomal proteins. AMPs were bound by mucins but this was demonstrated to be reversible and inhibition of antibacterial activity was limited.CONCLUSION:These findings indicate that epithelial antimicrobial peptides are retained in the intestinal mucus layer without losing their efficacy. Thus, the mucus layer and its composition provide an attractive drug target to restore antimicrobial barrier function in intestinal diseases.

Ileal Crohn's 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.

fibrosis deposition were: presence of a stricture (p=0.005), and changes in pattern of enhancement over time (p=0,000).There were a significant correlation between the degree of fibrosis and relative contrast enhancement SI at 70 seconds and 7 minutes (R=0.7,P=0.000).The persistence of homogeneous enhancement over time, presence of blurring, presence of comb sign and presence of ulcers were associated with a major presence of vessels and edema on pathology specimens, markers of inflammatory.Furthermore the presence of blurring and the presence of comb sign were inversely correlated with Masson-trichrome staining.The MRI variable specifically associated with the inflammatory score was the presence of ulcers (p=0.05).Conclusions: The fibrotic component of CD lesions can be determined by MRI based on the pattern of enhancement over time, the relative early and late contrast enhancement and presence of strictures.By contrast an homogeneous enhancement over time and the presence of comb sign, blurring and ulcers are markers of the inflammatory component.

Einleitung: Im Gegensatz zu M. Crohn (MC) ist bei Colitis ulcerosa (CU) die schützende Mukusschicht im Vergleich zu Kontrollen defekt. Gleichzeitig findet sich eine verminderte Zahl an Becherzellen, dies könnte auf einer defekten Differenzierung beruhen. Die hierfür relevanten Transkriptionsfaktoren sind Hath1 und Klf4, während Hes1 die Differenzierung absorptiver Zellen stimuliert.

Background: Olfactomedin-4 (OLFM4) is a glycoprotein characteristic of intestinal stem cells and apparently involved in mucosal defense of the stomach and colon. Here we studied its expression, regulation and function in IBD. Methods: The expression of OLFM4, mucins Muc1 and Muc2, the goblet cell differentiation factor Hath1 and the proinflammatory cytokine IL-8 was measured in inflamed or noninflamed colon in IBD patients and controls. OLFM4 protein was located by immunohistochemistry, quantified by Dot Blot and its binding capacity to defensins HBD1-3 was investigated. The influence of bacteria with or without the Notch blocker dibenzazepine (DBZ) and of several cytokines on OLFM4 expression was determined in LS174T cells. Results: OLFM4 mRNA and protein were significantly upregulated in inflamed CD (4.3 and 1.7-fold) and even more pronounced in UC (24.8 and 3.7-fold). OLFM4 expression was correlated to IL-8 but not to Hath1. In controls immunostaining was restricted to the lower crypts but in inflamed IBD it expanded up to the epithelial surface including the mucus. OLFM4 bound to HBD1-3 without profoundly inactivating these defensins. In LS174T-cells OLFM4 mRNA was significantly augmented after incubation with Escherichia coli K12, Escherichia coli Nissle and Bacteroides vulgatus. DBZ downregulated OLFM4 expression and blocked bacterial induction whereas IL-22 but not TNF-α was stimulatory. Conclusions: OLFM4 is overexpressed in active IBD and secreted into mucus. The induction is triggered by bacteria through the Notch pathway and also by the cytokine IL-22. OLFM4 seems to be of functional relevance in IBD as a mucus component, possibly by binding defensins.

The pathogenesis of both entities of inflammatory bowel disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC), is still complex and under investigation. The importance of the microbial flora in developing IBD is beyond debate. In the last few years, the focus has changed from adaptive towards innate immunity. Crohn's ileitis is associated with a deficiency of the antimicrobial shield, as shown by a reduced expression and secretion of the Paneth cell defensin HD5 and HD6, which is related to a Paneth cell differentiation defect mediated by a diminished expression of the Wnt transcription factor TCF4. In UC, the protective mucus layer, acting as a physical and chemical barrier between the gut epithelium and the luminal microbes, is thinner and in part denuded as compared to controls. This could be caused by a missing induction of the goblet cell differentiation factors Hath1 and KLF4 leading to immature goblet cells. This defective Paneth and goblet cell differentiation in Crohn's ileitis and UC may enable the luminal microbes to invade the mucosa and trigger the inflammation. The exact molecular mechanisms behind ileal CD and also UC must be further clarified, but these observations could give rise to new therapeutic strategies based on a stimulation of the protective innate immune system.

Einleitung: Olfactomedin4 (OLFM4) wird als Marker für humane intestinale Stammzellen angesehen. Zudem ist OLFM4 bei der aktiven vs. inaktiven Colitis ulcerosa (CU) signifikant induziert.

For nearly fifty years, azathioprine has been a reliable therapy for IBD.However, the mechanism by which this purine antimetabolite controls intestinal inflammation remains obscure.FOXP3+ regulatory T cells (Tregs) are critical for limiting intestinal inflammation in mice and humans, so we evaluated the effect of azathioprine on intramucosal FOXP3+ Treg frequency.A blinded, cross-sectional, retrospective analysis of archived colon biopsies from IBD patients was performed.Using immunohistochemical (IHC) quantification of FOXP3+ and CD4+ T cells, we found that a significantly higher percent of CD4+ T cells were FOXP3+ in the mucosa of patients taking azathioprine at the time of biopsy.This increase was primarily due to a decrease in the relative number of CD4+, but not FOXP3+, T cells in the mucosa of azathioprine recipients.To determine the effect of azathioprine on FOXP3+ versus FOXP3-T cells In Vitro, we cultured T cells from healthy donor blood in the presence or absence of 6-thioguanine nucleotide (6-TGN)-the active metabolite of azathioprine.We found that the proliferation of both naïve and effector/memory CD4+ T cells was inhibited by similar concentrations of 6-TGN.Likewise, proliferation of both FOXP3+ and FOXP3-T cells was inhibited by similar concentrations of 6-TGN, in the presence or absence of IL-2.However, de novo activation-induced expression of FOXP3 in sorted CD25-T cells was much less sensitive to inhibition by 6-TGN.Thus, 6-TGN was able to dissociate FOXP3 induction from T cell proliferation.We hypothesize that azathioprine selectively enriches FOXP3+ T cells in the inflamed bowel by inhibiting T cell proliferation without inhibiting induced expression of FOXP3.Such local enrichment of induced Tregs ("iTregs") may be a major mechanism by which azathioprine controls intestinal inflammation in IBD.

Einleitung: hBD-1 (humanes ß-defensin-1) erlangt nach Reduktion seiner charakteristischen Disulfid-Brücken eine neue Funktion. Das reduzierte Peptid besitzt antibakterielle Aktivität gegen Bifidobakterien, Lactobazillen und gegen C. albicans. In vitro kann hBD-1 durch die Oxidoreduktase Thioredoxin (Trx) in reduziertes hBD-1 (redhBD-1) umgewandelt werden (Schroeder et al., Nature 2011). Sollte aufgrund einer verminderten Expression von redox-aktiven Faktoren die Aktivierung von hBD-1 unterbleiben, könnte dies die mukosale Abwehr beeinträchtigen.

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 alpha- and beta-defensins. Little is known about in vivo effects of common drugs on their expression.AIM:To analyse the influence of corticosteroids, azathioprine and aminosalicylate treatment on ileal and colonic antimicrobial peptides in active CD and also assess the role of inflammation.METHODS:We measured the expression of antimicrobial peptides and pro-inflammatory cytokines in 75 patients with active CD.RESULTS:Ileal and colonic alpha- and beta-defensins as well as LL37 remained unaffected by corticosteroids, azathioprine or aminosalicylate treatment. Additionally, we did not observe a negative coherency between Paneth cell alpha-defensins and any measured cytokines. HBD2 and LL37 unlike HBD1 levels were linked to inflammatory cytokines and increased in highly inflamed samples.CONCLUSIONS:Current oral drug treatment seems to have no major effect on the expression of antimicrobial peptides. In contrast to HBD2 and LL37, ileal levels of HD5 and HD6 and colonic HBD1 level are independent of current inflammation. Innovative drugs should aim to strengthen protective innate immunity.

Crohn’s d isease may pr in ic ipa l ly invo lve the whole gastrointestinal tract. Most commonly, the inflammation occurs in the small intestine and/or in the colon with stable disease location over the years. The pathogenesis of both disease phenotypes is complex, the likely primary defect lies in the innate rather than adaptive immunity, particularly in the chemical antimicrobial barrier of the mucosa. Crohn’s ileitis is associated with a reduced expression of the Wnt signalling pathway transcription factor T-cell factor 4 (TCF4), which is regulating Paneth cell differentiation. As a result, the alpha-defensins and principal Paneth cell products HD5 and HD6 are deficiently expressed in ileal disease, independent of current inflammation. In contrast, Crohn’s colitis is typically associated with an impaired induction of the beta-defensins HBD2 and HBD3 caused by fewer gene copy numbers in the gene locus of the beta-defensins on chromosome 8. This ileal and colonic defect in innate defence mediated by a deficiency of the protective alphaand betadefensins may enable the luminal microbes to invade the mucosa and trigger the inflammation. A better understanding of the exact molecular mechanisms behind ileal and colonic Crohn’s disease may give rise to new therapeutic strategies based on a stimulation of the protective innate immune system. © 2008 The WJG Press. All rights reserved.

Goblet cells are mucin-secreting intestinal cells forming the mucus layer that protects the mucosal surface. Ulcerative colitis (UC) has been associated with a defective colonic mucus layer and a reduced number of goblet cells. In experimental animals, colonic goblet cell differentiation is regulated by interacting transcription factors Hath1, KLF4 and the Notch, as well as Wnt pathways, whereas data in humans are limited. We investigated goblet cell differentiation factors and mucins in controls and in inflammatory bowel diseases (IBDs). We performed real-time PCR for Hath1, KLF4, several ligands, receptors and target genes of the Notch and Wnt pathways, as well as several mucins in biopsies from the sigmoid colon of controls (n=21), Crohn's disease (CD, n=48) and UC (n=40). In addition, Hath1 protein was quantitated with Western blot and localized with immunohistochemistry. Notably, the degree of inflammation as measured by IL-8 and histology was similar in both disease entities. The proportion of goblet cells was lowered in both IBDs, but specifically diminished in the upper third of the crypt in UC. Comparable levels of inflammation induced both Hath1 (2.0-fold, p<0.001) and KLF4 (1.8-fold for KLF4, p=0.031) mRNA expression in CD but not in UC (0.8–0.9-fold, ns). The differential induction was confirmed for Hath1 protein using Western blot. Hath1 immunostaining was found mostly in the lower half of the colonic crypts. Hath1, KLF4 and the Notch target gene Hes1 were significantly (p<0.001) and positively correlated. Moreover, both Hath1 and KLF4 were correlated (p<0.001) with MUC1, MUC2 as well as MUC4 in all control and IBD cohorts. The results indicate that both transcription factors are key regulators of goblet cell differentiation and mucin formation in the human colon. Conspicuously, inflammation is associated with an enhanced goblet cell differentiation in CD but not in UC, a defect possibly of pathogenic importance.