Regulation of antimicrobial peptides in the gastrointestinal tract

The intestinal epithelium has an enormously large surface, which is constantly exposed to high amounts of pathogens and commensal bacteria. In order to prevent pathogens or other microorganisms from overcoming this barrier, good protection is needed. In the small intestine, human α-defensin 5 (HD-5) and 6 (HD-6) are important components of this barrier. Paneth cells, which are secretory epithelial cells, produce these antimicrobial peptides. The cells are located at the base of the crypt in the small intestine, where they play a central role in the microbial defense and regulating the composition of the intestinal microbiota. In ileal Crohn's disease, which belongs to the inflammatory bowel diseases, HD-5 and HD-6 levels are reduced, which leads to a changed composition of the intestinal flora and a poorer barrier function of the intestinal mucosa. Underlying mechanisms are genetically but also non-genetically influencing the Paneth cell function on different levels. The Wnt signaling pathway plays a key role in maintaining intestinal homeostasis. On the one hand it keeps the intestinal epithelial cells in a proliferating state, on the other hand it regulates the differentiation and maturation of Paneth cells. It is also instrumental in the regulation of HD-5 and -6. Former results from our group revealed a link between the decreased expression of HD-5 and -6 and the Wnt transcription factor TCF-4. Independent of inflammation, patients with ileal Crohn's disease show decreased TCF-4 expression levels. But not only TCF-4 is affected, our group could also show that the receptor LRP6, which is essential for the stabilization of β-catenin in the cytoplasm is affected. Studies showed a decreased expression in ileal Crohn's disease. The results of genetic investigations are also exciting; In the LRP6 gene, a polymorphism was identified that occurs significantly more frequently in a subgroup (patients> 18) than in healthy subjects. In the present work the consequences of a disturbed Paneth cell function, but also potential causes were further investigated. Emphasis was placed on the patient side, but the microbial side of the balance was also examined using animal models. It could be shown that LRP6 directly regulates the expression of HD-5 and -6 whereas the expression of LRP5, as a co-expressed receptor of LRP6, is barely altered in patients and also in vitro there is no evidence for a direct regulation of HD-5 and -6. It was possible to show the high heterogeneity of LRP5 in several populations studied and no genetic association in all subgroups of Crohn's disease was found. Studying TCF-1 expression revealed decreased levels in patients with ileal Crohn's disease. Further results from a knockout mouse model showed that a TCF-1 knockout leads to a lower expression of cryptdines (mouse α-defensins). Furthermore, the role of antimicrobial peptides in the intestine could be further clarified in a rat liver cirrhosis model, since the transfer of commensal bacteria occurring here through the intestinal epithelium could be explained by a reduced expression of some antimicrobial peptides. Although many factors which play a role in the development of ileal Crohn's disease are still unknown, the present work has provided new insights into the mechanisms that lead to a well-balanced homeostasis at the epithelial site in the small intestine. Furthermore, the important role of the Wnt signaling pathway for the development of ileal Crohn's disease was underlined. Hopefully, these additional insights into the complex pathogenesis of the disease will one day provide new approaches to therapy.Das Epithel des Darmes besitzt eine enorm große Oberfläche, welche ständig erheblichen Mengen von Pathogenen und kommensalen Bakterien ausgesetzt ist. Um zu verhindern, dass Pathogene oder auch andere Mikroorganismen diese Barriere überwinden, ist ein guter Schutz nötig. Im Dünndarm sind die humanen α- Defensine 5 (HD-5) und 6 (HD-6) wichtige Komponenten dieser Barriere. Diese antimikrobiellen Peptide werden von Panethzellen produziert. Panethzellen sind sekretorischen Epithelzellen und befinden sich an der Basis der Krypte im Dünndarm. Dort spielen sie eine zentrale Rolle bei der mikrobiellen Abwehr und der Regulation der bakteriellen Zusammensetzung der Darmflora. Bei Morbus Crohn des Dünndarms, diese Erkrankung zählt zu den chronisch entzündlichen Darmerkrankungen, kommt es zu einer verminderten Expression von HD-5 und HD-6 und dadurch zu einer veränderten Zusammensetzung der Darmflora und zu einer schlechteren Barrierefunktion der Darmmukosa. Grundlage hierfür sind verschiedene genetische aber auch nicht- genetische Defekte, welche die Panethzellfunktion auf verschiedenen Ebenen beeinflussen. Der Wnt Signalweg spielt eine zentrale Rolle, wenn es darum geht die Homöostase im Darm aufrecht zu erhalten. Er hält die intestinalen Epithelzellen einerseits in einem proliferierenden Zustand, andererseits reguliert er die Differenzierung und das Ausreifen der Panethzellen. Ebenso ist er an der Regulation von HD-5 und -6 maßgeblich beteiligt. Unsere Arbeitsgruppe konnte bereits zeigen, dass es eine Verbindung zwischen der verminderten Expression von HD-5 und -6 und dem Wnt Transkriptionsfaktor TCF-4 gibt. Entzündungsunabhängig zeigen Patienten mit Morbus Crohn des Dünndarms eine verminderte TCF-4 Expression. Aber nicht nur TCF-4 ist betroffen, es konnte auch gezeigt werden, dass der Rezeptor LRP6 betroffen ist, welcher für die Stabilisierung von ß- Catenin im Cytoplasma essentiell ist. Untersuchungen zeigten eine verminderte Expression bei Morbus Crohn Patienten. Spannend sind auch die Ergebnisse genetischer Untersuchungen; im LRP6 Gen wurde ein Polymorphismus identifiziert, der in einer Untergruppe (Patienten >18) deutlich häufiger auftritt als in Gesunden. In der hier vorgestellten Arbeit wurde an diese Ergebnisse angeknüpft und die Folgen einer gestörten Panethzellfunktion, aber auch die Faktoren die potentiell dazu führen, weiter untersucht. Hierbei wurde ein Schwerpunkt auf die Patientenseite gelegt, aber auch die mikrobielle Seite wurde mithilfe von Modellen untersucht. Es konnte gezeigt werden, dass LRP6 die Expression von HD-5 und -6 direkt reguliert während die Expression von LRP5, als ein Co-Rezeptor von LRP6, in Patienten kaum verändert ist und sich auch in vitro keine Hinweise auf eine direkte Regulation von HD-5 und -6 finden lassen. Ebenso zeigte sich, dass LRP5 sehr heterogen in mehreren untersuchten Populationen vorliegt und keine genetische Assoziation in allen untersuchten Subgruppen von Morbus Crohn vorliegt. Die Untersuchungen von TCF-1 zeigten eine erniedrigte Expression in Patienten mit Morbus Crohn des Dünndarms. Ergebnisse aus einem Knockout Mausmodell zeigten, dass ein TCF-1 Knockout zu einer niedrigeren Expression von Cryptdinen führt. Weiter konnte in einem Leberzirrhose- Tiermodel die Rolle antimikrobieller Substanzen weiter geklärt werden, da der hier auftretende Übertritt von kommensalen Bakterien durch das Darmepithel mit einer verminderten Expression einiger antimikrobieller Peptide erklärt werden konnte. Obwohl immer noch viele Faktoren, die in der Entstehung von Morbus Crohn des Dünndarms eine Rolle spielen unbekannt sind, konnte die hier vorliegende Arbeit neue Einblicke in die Mechanismen liefern, die das Aufrechterhalten der Balance zwischen Bakterien und Mensch im Dünndarm ermöglichen. Weiter wurde die wichtige Rolle des Wnt Signalwegs für die Entstehung von Morbus Crohn des Dünndarms unterstrichen. Hoffentlich ermöglichen diese weiteren Einblicke in die komplexe Pathogenese der Erkrankung eines Tages neue Ansätze für eine Therapie

Association of a Functional Variant in the Wnt Co-Receptor LRP6 with Early Onset Ileal Crohn's Disease

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

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, CCl(4)-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

Increased activity of β-catenin–dependent Wnt via overexpression of LRP6 transactivates human α-defensin 5 promoter activity in HEK-293 cells.

<p>As demonstrated with immunohistochemistry staining A), the Wnt co-receptor LRP6 is widely expressed in the cells of the small intestinal epithelium and also found at the base of the crypts where Paneth and stem cells are located. Additionally, LRP6 is sporadically found in infiltrating immune cells. As previously demonstrated, overexpression of LRP6 in HEK293 cells leads to an activation of β-catenin dependent Wnt signalling B) as monitored by TopFlash activity. The transcriptional activity of a luciferase reporter under the control of an 1 kb HD-5 promoter was also increased after LRP6 mediated activation of Wnt C). Both effects were not seen using a non-functional version of the LRP6 Co-receptor which is lacking cytoplasmatic parts that are necessary for signal transduction. Values are the average of triple determinations with the SEM indicated by error bars.</p

Age of included individuals.

<p>Provided are the mean age ± standard deviation. For controls we listed the age at time of DNA sampling, for CD patients we provide the mean age at onset.</p

Risk for rs2302685 C-allele carriers in the different disease subgroups and the influence of detailed phenotyping.

<p>A) Odds ratios and confidence intervals for the different comparisons are shown. The frequency distribution of the minor rs2302685 allele was analysed in different cohorts and combined samples: odds ratios and 95% confidence interval for the allele frequency are shown for patients with specific disease subtypes of ileal CD (classified as either L1 (solely ileal) or L3 (ileal and colonic involvement)) compared to healthy control individuals. The early onset as well as the penetrating behaviour subgroup displays an significantly increased Minor allele frequencies (MAF). B) An especially high risk for early onset ileal CD is found for homozygous minor C - allele carriers. This becomes apparent in the high odds ratios for such individuals in the different cohorts and combined samples (left panel) and an highly increased CC genotype frequency compared to the control samples and other disease groups (right panel). C) The MAF distribution was analysed in different cohorts and combined samples: odds ratios and 95% confidence interval for the MAF are shown for patients with ulcerative colitis (UC) (left panel) and CD patients with solely colonic involvement (right panel) compared to healthy control individuals. The lack of an increase in the odds ratios for the different comparisons allows excluding major effects of the variant in the colonic IBD subgroups.</p

Known exonic SNPs in LRP6.

<p>(A) LRP6 gene card with locations of exonic SNPs published in the NCBI SNPdb. Variants which lead to an amino acid exchange are marked in red. A deletion mutation causing a different protein chain is marked in blue, silent variants are marked in green. (B) Linkage disequilibria (LD) of the exonic SNPs in CD patients of the Oxford cohort. 7 of the genotyped SNPs were not present. We conclude that there is no significant LD between the sole coding variant (rs2302685) and any of the synonymous SNPs.</p