the intestinal barrier
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1 intestinal defensins/antimicrobial peptides the intestinal barrier Jan Wehkamp, Stuttgart, tt t Germany Dr. Margarete Fischer Bosch Institute of Clinical Pharmacology, Stuttgart, Germany Medicine I Robert Bosch Hospital, Stuttgart, Germany
2
3 CCF 2002 Mammalian Mucosal Surfaces Continual contact with microbes Low incidence of infection Infrequent inflammatory response Effective Mucosal Defense Mechanisms
4 Normal Low counts of microbes Steril Intestinal barrier intestinal lumen loads of bacteria outer mucuslayer (700 um) antimicrobial peptides (defensins, cathelicidine, others) inner mucusslayer (100 um) adhaerent, antibakt. peptides colon-crypts (200 um) Secretion of mucus and antibakt. peptides epithelialbarrier M. Crohn A. Swidsinski
5 Normal mucosa is protected by epithelial antibacterial peptides: Constitutive HBD-1 Permanent war at host interface Harder et al α- (Ileum) and β-defensins Cthliidi Cathelicidins Elafin / Antiproteases BPI and Lysozyme others Mucins mucus! Mucus seems to be sterile! A. Swidsinski
6 part of the innate immune system exist in all multicellular organisms Antimicrobial peptides (AMPs) expressed mainly, but not exclusively, by epithelia characterized by their capacity to kill a wide variety of microorganisms i four major classes of human AMPs: defensins, cathelicidins, S100 proteins and ribonucleases Defensins α-defensin consensus β -defensin consensus C C C C CC C C C C CC
7 Every eucarytic organism is provided by an arsenal of antimicrobial peptides
8
9 Relationship between humans and microbiota the human gut harbours trillion microorganisms between 1 2 kg of weight outnumber the total number of human cells by factor 10 composed of at least different bacterial species most of them have beneficial symbiotic relationships with host separation between microbiota and intestinal tissue by a single epithelial layer that is only ~20 mm thick, but which encompasses about 200 m 2 of surface area
10 Bacterial Species Colonization Birth : gut is sterile Environment & Host Factors acquisition of an «adult» microbiota (2 years) Biodiversity Stability Functions (protection)
11 70% of bacterial species remain not cultivable cultivated fraction Tannock % Suau % Needs : culture independent technics
12 No bacteria - no inflammation Microbiota are essential in inflammatory process Diversion of the fecal stream is effective in ameliorating CD distally, luminal contents trigger inflammation Harper et. al. Gut 1983, Rutgeerts et.al., Lancet 1991 D Haens et al., Gastroenterology 1998 T-cell response against the autologous bacterial flora in Crohn s disease Duchmann et al., Gut 1999 Serological response to bacteria/fungal antigens (ANCA, ASCA, anti-flagellin) Mow et al. Gastroenterology 2004) Lunardi et al. J Intern Med 2008) Adherent-invasive E. Coli.. and others... Darfeuille-Michaud et al., Gastro 2004 Swidsinski et al.,gastro
13 Bacteria in the center of disease t ll d b? controlled by.?
14 Enteric defensins are essential regulators of intestinal microbiology Luminal Flora in transgenic HD5 mice What else? Wehkamp, Salzman et al. PNAS 2005 Salzman et al. Nature Immunology 2009
15 Different antimicrobial peptides at different sites along the gastrointestinaltract Colon Dünndarm Panethzell- epithelzellα-defensins: β-defensins: HD-5 / HD-6 HBD1 (constitutive) (constitutive) LL3 HBD 2,3 and 4 (inducible) Broadspectrum antibiotics- controling the host environment!
16 Figure 1: Overview of Defensins in the GI tract Wehkamp J et al. (2005) Mechanisms of Disease: defensins in gastrointestinal diseases Nat Clin Pract Gastroenterol Hepatol 2:
17 Distribution of bacterial species along the digestive tract > sequences ADNr 16S Stomach Colon Streptococcus Bacteroides Eubacterium Small bowell (ileum) Clostridium Lactobacillus Ruminococcus Streptococcus Bifidobacterium E. coli Streptococcus Clostridium Lactobacillus Bacteroides E. coli Eubacterium non-e. colicoliformes Veillonella bacteria (gut) cells (human being) microbial census exceeds the total body number of our own human cells by ~10 fold Eckburg et al. Science 2005
18 Summary 1 Different Defensins are expressed in the intestinal tract Constitutive expression and expression on demand d (inducible) Control environment: microbial flora
19 Inflammatory bowel ldiseases Wehkamp et al. Nature Clin Pract, 2006
20 Regulation and function
21 Current work. Epithelial stem cell regulation, Wnt and notch pathway
22 The impact of human beta defensine 1 (hbd1) hbd1 ubiquitously it expressed by human epithelia (not only intestine!) ti but: only minor antibiotic killing activity! Wehkamp et al. Nature clin. Practice 2005
23 Constitutive HBD-1 Permanent WAR at host interface
24 PNAS May 2010
25 Human β-defensin 1 (hbd-1) identified as first human beta defensin in plasma different N-terminal truncated peptides, ranging from 36 to 47 aa constitutively i expressed by all human epithelia regulated by PPRy Pazgier et al., JBC 2006 Why although does constitutively an organism expressed by produce all human epithelia, high only amounts weak antimicrobial of an ineffective activity comparison defense with other molecule? AMPs
26 Life in the intestine the gut is strongly challenged by intestinal microbes majority of intestinal microbes are facultative or strict anaerobes absence of oxygen and bacterial fermentation produce reducing environment when analyzing antimicrobial defence in the intestine, these factors should be considered What about the interaction between antimicrobial BUT: Intestinal antimicrobial peptides have been tested only under oxidizing conditions! peptides and anaerobic bacteria from the normal flora under reducing conditions? 26
27
28 0 mm DTT 1mM DTT 2 mm DTT hbd-1: reduced to action! Modified radial diffusion assay measuring antimicrobial activity against Gram-positive anaerobe Bifidobacterium adolescentis under reducing conditions 0 mm DTT 1mM DTT 2 mm DTT Schroeder et al., Nature 2011
29 How does reduced hbd-1 affect bacteria growth? flow cytometric killing assay electron microscopy depolarized bacterial membrane clearly bactericidal! Schroeder et al., Nature 2011
30 The thioredoxin (TRX) system thioredoxin reductase hbd-1(ox) hbd-1(red) not active active Colon: oxygen partial pressure is low TRX a multifunctional oxidoreductase ubiquitously expressed (also by mucosal surfaces)
31 Role of redox biology: Thioredoxin (TRX) catalyses reduction of oxidized hbd-1 1andcolocalizes co-localizes with redhbd-1 in vivo. Inflammation hbd-1 incubated distal with to TRX proximal? plus NADPH and TRX-Reductase Role of oxydative pressure hbd-1 ox Controling innate hbd-1 immunity? ox hbd-1 red hbd-1 ox hbd-1 red hbd-1 red Schroeder et al.; Nature 469
32 current (and preliminary) work. Mucus?!? Mucus?!? needs special conservation Work in progress, experiments by M. Gersemann and S. Nuding
33 hbd-1 is produced by all human surfaces Summary 2 it is much more active after reduction of disulphide-bonds reduced hbd-1 differs structurally from oxidized hbd-1 free cysteines in the C-terminus seem important for the antimicrobial activity reduction can be performed either by the oxido-reductase thioredoxin or by a reducing environment reduced hbd-1 might shield the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi No organism produces high amounts of an ineffective defense molecule!! l
34 Current work and summary: Translation of findings in therapy!! treatments which strengthen barrier function Thank you! We need new concepts (beyond anti-inflammation)
35 IKP Stuttgart Jan Wehkamp Bjoern Schroeder Michelle Katajew Heidi Loeffler Dr. Sabine Nuding Dr. Julia Beisner Jutta Bader Kathleen Siegel Robert Kuechler Svetlana Becker Zora Teltschik Marion Schiffmann Maureen Koslowski Lioba Courth Dr. Sabine Nuding Acknowledgement Robert-Bosch-Hospital Stuttgart UC Davis, CA Charles L. Bevins Eduard d F. Stange Simon Jaeger Michael Gersemann Ardeypharm GmbH Ulrich Sonnenborn MPI Evolutionary Biologie, Tübingen Sandra Groscurth University Hospital Kiel Jens-M. Schroeder Zhihong Wu Ulf Meyer-Hoffert TU München Johannes Buchner Moritz Marcinowski University Tübingen Martin Schaller
36 Open questions: Interaction of different arms of immunity: innate and adaptive e.g. Novel concept: Th17-cell/AMP-circuit circuit Antimicrobial peptides/proteins Anti- Il 17 is worsening Crohn s disease, ECCO 2011 IL-17 and IL-22 Th17 Recruitment of neutrophils Bacteria APC:DC
37 Therapeutic targets?! yet unknown components of barrier e.g.?????? function
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