Miriam Schlee

Publications of Miriam Schlee

• Antimicrobial host defense in the upper gastrointestinal tract.

  Authors: Yoshio Hosaka, Maureen Koslowski, Sabine Nuding, Guoxing Wang, Miriam Schlee, Christian Schäfer, Katunori Saigenji, Eduard F Stange, Jan Wehkamp

  European journal of gastroenterology & hepatology. 01/2009; 20(12):1151-8.

  BACKGROUND: With the exception of fungi, microbial infections are rare in the oesophagus. Herein, we aimed to systematically assess the distribution and quantity of different antimicrobial hostBACKGROUND: With the exception of fungi, microbial infections are rare in the oesophagus. Herein, we aimed to systematically assess the distribution and quantity of different antimicrobial host factors as well as, for the first time, functional mucosal antimicrobial activity in the upper gastrointestinal tract. METHODS: We investigated biopsies from the healthy oesophagus, three different locations in the stomach and the duodenum in a total of 12 individuals. Using real-time PCR with external standards, we compared absolute expression of mRNA encoding antimicrobial peptides including defensins, cathelicidin, bactericidal/permeability-increasing protein, psoriasin, and elafin. In addition, we performed immunostaining for human-beta-defensin-1 (HBD1), elafin, and psoriasin. To test functional relevance, we assessed antimicrobial as well as antifungal activity of cationic extracts from biopsies against E. coli ATCC 25922 and a clinical isolate of Candida albicans. RESULTS: In contrast to HBD1 which was similarly expressed in all tissues, inducible beta-defensins in the healthy oesophagus were much higher compared with the stomach and duodenum (for HBD2-4: P<0.01). In addition, the antiproteases elafin and psoriasin were also predominantly expressed in the oesophagus (P<0.005). In contrast, LL-37 and bactericidal/permeability-increasing protein were only marginally expressed. Cationic tissue extracts from both the oesophagus as well as the stomach showed potent antibacterial activity against E. coli. Consistent with susceptibility to Candida infection, the esophageal extracts exhibited a weaker activity against C. albicans (P=0.026). CONCLUSION: Despite dominant expression of antimicrobial host peptides, oesophageal tissue shows a weakened potency to kill C. albicans. These data suggest an important role of yet unknown antimicrobial molecules.

• Induction of human beta-defensin 2 by the probiotic Escherichia coli Nissle 1917 is mediated through flagellin.

  Authors: Miriam Schlee, Jan Wehkamp, Artur Altenhoefer, Tobias A Oelschlaeger, Eduard F Stange, Klaus Fellermann

  Infection and immunity. 06/2007; 75(5):2399-407.

  Human beta-defensin 2 (hBD-2) is an inducible antimicrobial peptide synthesized by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certainHuman beta-defensin 2 (hBD-2) is an inducible antimicrobial peptide synthesized by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certain bacterial strains, have been identified as potent endogenous inducers. Recently, we have found that hBD-2 induction by probiotic Escherichia coli Nissle 1917 was mediated through NF-kappaB- and AP-1-dependent pathways. The aim of the present study was to identify the responsible bacterial factor. E. coli Nissle 1917 culture supernatant was found to be more potent than the pellet, indicating a soluble or shed factor. Chemical analysis demonstrated the factor to be heat resistant and proteinase digestible. Several E. coli Nissle 1917 deletion mutants were constructed and tested for their ability to induce hBD-2 expression in Caco-2 cells. Deletion mutants for flagellin specifically exhibited an impaired immunostimulatory capacity. Reinsertion of the flagellin gene restored the induction capacity to normal levels. Isolated flagellin from E. coli Nissle 1917 and from Salmonella enterica serovar Enteritidis induced hBD-2 mRNA significantly in contrast to the flagellin of the apathogenic E. coli strain ATCC 25922. H1 flagellin antiserum abrogated hBD-2 expression induced by flagellin as well as E. coli Nissle 1917 supernatant, confirming that flagellin is the major stimulatory factor of E. coli Nissle 1917.

• NF-kappaB- and AP-1-mediated induction of human beta defensin-2 in intestinal epithelial cells by Escherichia coli Nissle 1917: a novel effect of a probiotic bacterium.

  Authors: Jan Wehkamp, Jürgen Harder, Kai Wehkamp, Birte Wehkamp-von Meissner, Miriam Schlee, Corinne Enders, Ulrich Sonnenborn, Sabine Nuding, Stig Bengmark, Klaus Fellermann, Jens-Michael Schröder, Eduard F Stange

  Infection and immunity. 11/2004; 72(10):5750-8.

  Little is known about the defensive mechanisms induced in epithelial cells by pathogenic versus probiotic bacteria. The aim of our study was to compare probiotic bacterial strains such as EscherichiaLittle is known about the defensive mechanisms induced in epithelial cells by pathogenic versus probiotic bacteria. The aim of our study was to compare probiotic bacterial strains such as Escherichia coli Nissle 1917 with nonprobiotic, pathogenic and nonpathogenic bacteria with respect to innate defense mechanisms in the intestinal mucosal cell. Here we report that E. coli strain Nissle 1917 and a variety of other probiotic bacteria, including lactobacilli--in contrast to more than 40 different E. coli strains tested--strongly induce the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) in Caco-2 intestinal epithelial cells in a time- and dose-dependent manner. Induction of hBD-2 through E. coli Nissle 1917 was further confirmed by activation of the hBD-2 promoter and detection of the hBD-2 peptide in the culture supernatants of E. coli Nissle 1917-treated Caco-2 cells. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-kappaB and AP-1 in the hBD-2 promoter are required for induction of hBD-2 through E. coli Nissle 1917. Treatment with the NF-kappaB inhibitor Helenalin, as well as with SP600125, a selective inhibitor of c-Jun N-terminal kinase, blocked hBD-2 induction by E. coli Nissle 1917 in Caco-2 cells. SB 202190, a specific p38 mitogen-activated protein kinase inhibitor, and PD 98059, a selective inhibitor of extracellular signal-regulated kinase 1/2, were ineffective. This report demonstrates that probiotic bacteria may stimulate the intestinal innate defense through the upregulation of inducible antimicrobial peptides such as hBD-2. The induction of hBD-2 may contribute to an enhanced mucosal barrier to the luminal bacteria.

• Probiotic bacteria enhance the antibacterial barrier of enterocytes: insights into their mechanism of action

  Authors: Miriam Schlee

  In the healthy intestine there is a stable balance of luminal bacteria and host factors to prevent infections or inflammatory bowel diseases (IBD). A complex network of environmental, genetic, andIn the healthy intestine there is a stable balance of luminal bacteria and host factors to prevent infections or inflammatory bowel diseases (IBD). A complex network of environmental, genetic, and immunoregulatory factors may precipitate the onset of ulcerative colitis (UC) and Crohn's disease (CD), the primary manifestations of inflammatory bowel disease (IBD). It is currently believed that IBD results from an aberrant immune response of the intestinal mucosa towards the normal commensal bacterial flora. Alternatively, a primary defect in the mucosal barrier might permit bacterial invasion and trigger inflammation. In our research group the hypothesis was proposed that the defective barrier in Crohn´s disease may be due to a lack of defensins which form a chemical barrier against luminal bacteria. A major gut defensin is the human beta defensin-2 (hBD-2) which is an inducible antimicrobial peptide synthesized and secreted by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certain bacterial strains, have been identified as potent endogenous inducers. In recent studies, Fellermann et al demonstrated that the defective expression of hBD-2 which was measured in the gut mucosa of patients with Crohn´s disease was due to a reduced copy number of the hBD-2 gene. In patients with ulcerative colitis beta-defensin expression is low in the colon during remission, but readily inducible during inflammation. Probiotic bacteria might act beneficially in the human gut by inducing the expression of defensins and thereby reinforcing the mucosal barrier. Recently, our group has been the first to describe hBD-2 induction by the probiotic strain E. coli Nissle (Mutaflor®) which is an effective treatment for ulcerative colitis during remission. The aim of the present work was to determine the underlying molecular mechanisms. We determined a time- and dose-dependent expression pattern of hBD-2 in Caco-2 cells upon stimulation with IL-1 beta;, E. coli Nissle culture supernatant and diverse other probiotic strains. We further investigated the transcriptional regulation of hBD-2 expression mediated by probiotics. The hBD-2 promoter contains several elements known to be involved in transcriptional upregulation such as the NF-kappa B element, which is believed to be one of the main regulators of the hBD-2 gene expression. However, for certain signals, the expression of the hBD-2 gene has been reported to depend on the activation of a second transcription factor, such as AP-1. Most importantly, E. coli Nissle was shown to shed or secrete factors, contained in the bacterial supernatant, which were sufficient to trigger activation of NF-kappa B and AP-1 and to induce hBD-2. Our results indicated further that the supernatant-induced activation of the MAP kinase pathways ERK½, JNK, and p38 may be directly responsible for the probiotic supernatant-induced activation of the transcription factors AP-1 and NF-kappa B and subsequent synthesis of hBD-2. A further aim of the present study was to identify and isolate the bacterial components which are responsible for E. coli Nissle mediated hBD-2 induction. As E. coli Nissle culture supernatant was found to be a more potent stimulant than the bacterial pellet, we investigated the characteristics of the unknown soluble or shed molecules in the bacterial culture media. The first analysis revealed the factor as a heat resistant and proteinase sensitive molecule. Both, E. coli Nissle specific lipopolysaccharide (LPS) and bacterial DNA, which might contain immunostimulatory DNA motifs, failed to trigger hBD-2 expression. Based on the knowledge of the surface composition several E. coli Nissle deletion mutants were constructed and tested for their ability to induce hBD-2 expression in Caco-2 cells. Deletion mutants for flagellin, the flagella filament protein, specifically exhibited an impaired immunostimulatory capacity. Reinsertion of the flagellin gene restored the induction capacity to normal levels. Next, we isolated flagellins from different bacteria strains (Salmonella enterica serovar Enteritidis, E. coli ATCC 25922, E. coli Nissle and the uropathogenic E. coli strain CFT073 Delta hly, whose genome structure resembles closely that of E. coli Nissle). In the Western blot anti-H1 flagellin displayed immunoreactivity against the different types of flagellins, due to the highly conserved central region of the flagellin filament structure. Incubation of Caco-2 cells with isolated E. coli Nissle flagellin (molecular size 60.81 kDa) induced hBD-2 promoter activation in a dose-dependent manner. The induction of hBD-2 expression by flagellin was confirmed with a positive control (Salmonella flagellin). Interestingly, the serotype-identical CFT073 Delta hly flagellin expressed only moderate hBD-2 inducing ability compared to E. coli Nissle flagellin. Thus, differences in extracellular matrix e.g. the glycosilation degree might underlie the differentially modulated hBD-2 response of Caco-2 cells by the two flagellins. H1 flagellin antiserum abrogated hBD-2 expression induced by flagellin as well as E. coli Nissle supernatant, confirming that flagellin is the major stimulatory factor of E. coli Nissle. In conclusion, flagellin of E. coli Nissle provides reinforcement of mucosal antimicrobial function, apparently without inducing inflammation. This might explain the beneficial effects of E. coli Nissle on remission maintenance in ulcerative colitis. In patients with Crohn´s disease there is evidence against a therapeutic effect of probiotics and this may be explained by a defective defensin system. Future investigations about strain-specific beneficial functions might contribute to the therapeutic application of science-based probiotic products. Im gesunden Darm herrscht ein stabiles Gleichgewicht zwischen der luminalen Flora und körpereigenen Abwehrkräften, das Infektionen oder entzündlichen Erkrankungen im Darm vorbeugt. Ein komplexes Netzwerk aus Umwelteinflüssen, genetischen und immunregulatorischen Faktoren kann den Ausbruch von Colitis ulcerosa oder Morbus Crohn, den Haupterscheinungsformen der chronisch entzündlichen Darmerkrankungen (CED) herbeiführen. Eine überschießende Immunantwort der intestinalen Epithelzellen gegenüber der normalen Darmflora wird als auslösender Faktor für die intestinale Entzündung postuliert. Alternativ, könnte auch ein Primärdefekt der mukosalen Barriere verantwortlich für eine bakterielle Invasion und die darauffolgende Entzündung sein. In unserer Arbeitsgruppe wurde vor kurzem die Hypothese entwickelt, dass die gestörte Darmbarrierefunktion bei Morbus Crohn in einem Mangel an Defensinen begründet sein könnte, welche eine Art chemische Barriere gegen luminale Bakterien darstellen. Eines der wichtigsten Defensine des Darms ist das humane beta Defensin-2 (hBD-2) welches ein, durch proinflammatorische Zytokine sowie spezielle Bakterienstämme induzierbares, antimikrobiell wirksames Peptid ist. Es wird von Epithelzellen synthetisiert und sezerniert zur Abwehr einer bakteriellen Adhärenz und Invasion. Fellermann et al wies vor kurzem nach, dass die in der Darmschleimhaut des Kolons von Morbus Crohn Patienten gemessene defekte hBD-2 Expression durch eine verringerte Genkopienzahl des humanen beta-Defensins-2 bedingt ist. Bei Patienten mit Colitis ulcerosa ist die Beta-Defensin Expression im Kolon in der beschwerdefreien Phase (=Remission) niedrig, in der Entzündung wird diese jedoch umgehend induziert. Probiotika könnten durch eine Induktion der Defensinexpression im Darm einen günstigen Einfluss ausüben und somit die Darmbarriere stärken. Unsere Arbeitsgruppe hat vor kurzem erstmals eine hBD-2 Induktion durch den probiotischen Keim E. coli Nissle (Mutaflor®) nachgewiesen, der mit gutem Behandlungserfolg in der Remission der Colitis ulcerosa Einsatz findet. Das Ziel der vorliegenden Arbeit war, die zugrunde liegenden molekularen Mechanismen zu untersuchen. Wir bestätigten ein zeit- und dosisabhängiges hBD-2 Expressionsmuster in Caco-2 Zellen durch Stimulation mit IL-1 beta, E. coli Nissle Kulturüberstand und diversen anderen probiotischen Keimen. Des Weiteren untersuchten wir die transkriptionelle Regulation der probiotika-vermittelten hBD-2 Expression. Der hBD-2 Promotor enthält verschiedene, an der Aktivierung der Transkription beteiligte Elemente, wie z.B. dem nukleären Faktor NF-kappa B, welches ein Hauptregulator der hBD-2 Expression zu sein scheint. Es gibt jedoch Hinweise darauf, dass die Expression des hBD-2 Gens durch gewisse Stimulanzien noch von einem weiteren Transkriptionsfaktor abhängt, wie z.B. AP-1. Sezernierte oder abgelöste Faktoren des E. coli Nissle, welche sich im Kulturüberstand befinden, waren ausreichend in der Lage hBD-2 durch die Aktivierung von NF-kappa B und AP-1 zu induzieren. Die überstandsinduzierte Aktivierung der MAP-Kinase-Signalwege über ERK½, JNK und p38 scheint hierbei direkt verantwortlich zu sein für die über AP-1 und NF-kappa B aktivierte Synthese von hBD-2. Ein weiteres Ziel dieser Arbeit war die Identifizierung und Isolation der bakteriellen Be-standteile des E. coli Nissle welche für die hBD-2 Induktion verantwortlich sind. Da der Bakterienkulturüberstand ein stärkerer Stimulus als das Bakterienpellet war, untersuchten wir die Charakteristiken der unbekannten löslichen oder abgegebenen Moleküle im bakteriellen Kulturmedium. Erste Untersuchungen ergaben, dass der gesuchte Faktor eine hitzeresistente, durch Proteinase verdaubare Komponente des Bakteriums darstellt. Das für E. coli Nissle spezifische Lipopolysaccharid (LPS) sowie dessen bakterielle DNA, welche immunstimulatorische DNA-Motive enthalten könnte, hatten keinen hBD-2 induzierenden Einfluss. Auf der Grundlage bekannter oberflächlich exprimierter Moleküle des E. coli Nissle wurden mehrere Deletionsmutanten konstruiert und auf ihre Fähigkeit, die hBD-2 Expression in Caco-2 Zellen auszulösen, untersucht. Flagellin-Deletionsmutanten wiesen eine stark verminderte immunstimulatorische Kapazität auf. Das erneute Einfügen des deletierten Flagellingens regenerierte die Fähigkeit zur hBD-2 Induktion vollständig. Des Weiteren isolierten wir Flagellen aus verschiedenen Bakterienstämmen (Salmonella enterica serovar Enteritidis, E. coli ATCC 25922, E. coli Nissle und dem uropathogenen E. coli Stamm CFT073 Delta hly, dessen Genom dem des E. coli Nissle sehr ähnlich ist). Im Western Blot wurde eine Immunreaktivität von Anti-H1 Flagellin gegen die verschiedenen Flagellintypen nachgewiesen, was sich durch die hoch konservierte Zentralregion der Flagellinfilamentstruktur erklären lässt. Die Inkubation von Caco-2 Zellen mit dem isolierten E. coli Nissle Flagellin (Molekulargewicht: 60.81 kDa) induzierte dosisabhängig die hBD-2 Promotoraktivierung. Die Induktion der hBD-2 Expression durch Flagellin wurde mit einer Positivkontrolle (Salmonella Flagellin) bestätigt. Interessanterweise war die Induzierbarkeit durch das serotypidentische CFT073 Delta hly Flagellin, verglichen mit der des E. coli Nissle Flagellins reduziert. Unterschiede in der Extrazellulärmatrix, z.B. dem Glykosilierungsgrad, könnten die verschieden ausgeprägte hBD-2 Antwort der Caco-2 Zellen auf die beiden Flagelline erklären. Zusätzlich wurde die Flagellin- und E. coli Nissle Überstand-induzierte hBD-2 Expression durch H1-Flagellin Antiserum gehemmt. Diese Befunde lassen schließen, dass Flagellin der hauptverantwortliche Faktor für die Stimulation der hBD-2 Expression durch E. coli Nissle ist. Das Flagellin des E. coli Nissle stärkt somit die mukosale antimikrobielle Funktion des Darmepithels ohne eine Entzündung auszulösen. Dies könnte die positive Wirkung des E. coli Nissle auf die Remissionserhaltung der Colitis ulcerosa erklären. Bei Patienten mit Morbus Crohn gibt es Hinweise gegen einen therapeutischen Nutzen von Probiotika, was durch ein defektes Defensinsystem erklärt werden könnte. Die Charakterisierung positiver Eigenschaften relevanter Keime könnte einen Beitrag zur evidenz-basierten therapeutischen Verwendung probiotischer Produkte liefern.