They all do it! What?

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1 They all do it! What?

2 Defensins: the endogenous antibiotics of body surfaces Wehkamp J et al. (2005) Mechanisms of Disease: defensins in gastrointestinal diseases Nat Clin Pract Gastroenterol Hepatol 2: Are defensins important?

3 Normal mice die after S. typhimurium HD-5 transgenics are resistant Salzman et al. Nature Apr 3;422(6931)

4 Defensins: Mechanism of Action control S. aureus HBD-3, 30 min. Defensins are endogenous antimicrobial peptides that kill bacteria by pore formation in the bacterial membrane J. Harder and J. Schröder JBC 2003 HBD-3, 120 min HBD-3, 120 min

5 Adherent intestinal bacteria in IBD: Key to pathogenesis Bacteria sit there because T-cells are dysregulated? Macrophages are angry at them? Granulocytes are weaklings? Swidsinski et al., 2002

6 The defensin deficiency hypothesis in Crohn s disease TLR s NOD s Microbe Antimicrobial Peptides Microbe elimination Defective Response Antimicrobial Peptides Epithelium Inflammation Acquired Immunity Fellermann et al. Eur J Gastro Hepatol 2003

7 Defensin Expression Permanent protection Protection on demand Paneth cell defensins in the small intestine in very high concentrations Induction of β-defensins and cathelicidins by normal epithelial cells Human β-defensin 1 expression throughout the intestinal tract (about 1000 fold lower compared to Paneth cell defensin expression in the small intestine) Expression of Paneth cell defensins by metaplastic Paneth cells

8 Crohn s disease In 1/3 patients disease is selectively in the ileum because Only ileal T-cells are dysregulated? Only ileal macrophages are angry? Only ileal granulocytes are weaklings?

9 Ileal Paneth Cells Express antimicrobials, including α-defensins, lysozyme, and spla2 Release their granules in response to bacterial products (LPS and MDP) into the lumen Express NOD2, the susceptibility gene for ileal Crohn s disease References Porter et al. Cell Mol Life Sci 59, 156 (2002) Ayabe et al. Nature Immunol. 1, 113 (2000) Ghosh et al. Nature Immunol. 3, 583 (2002) Lala et al. Gastroenterology 125:47 (2003) Ogura et al. Gut 52:1591 (2003)

10 Are Paneth cell defensins decreased in Crohn's disease? Biopsies from 45 Crohn's disease patients (24 with NOD-2 mutations) compared with 12 controls Wehkamp et al. Gut 53: 1658 (2004)

11 Paneth cell defensins in controls and ileal Crohn s disease US patients from the Cleveland Clinic foundation Ileal tissue Ileal tissue HD5 mrna Transcripts per μg RNA p= HD6 mrna Transcripts per μg RNA p= Controls (11) CD Ileitis (34) Controls (11) CD Ileitis (34) HD-5 and HD-6 are diminished in Crohn s disease Wehkamp et al. (2005) Proc. Natl. Acad. Sci. USA 102,

12 Paneth cell products in ileal Crohn s disease: NOD2 wildtype versus NOD2 mutation HD5 mrna Transcripts per μg RNA n.s HD5 mrna Transcripts per μg RNA n.s. p= Ileal tissue NOD2: wt (11) snp8 (10) snp12 (3) snp13 (10) HD6 mrna Transcripts per mg RNA p= Ileal tissue Ileal tissue p=0.03 n.s. n.s NOD2: wt (11) snp8 (10) snp12 (3) snp13 (10) CD Ileitis Controls (11) CD Ileitis (34) CD Ileitis HD-5 and HD-6 are further diminished in Ileal disease associated with NOD2 SNP13 mutation

13 At the protein level? Coomassie Western Other Paneth cell products

14 Functional consequences? Is the antimicrobial activity diminished in ileal Crohn s disease biopsies? E. coli Percentage Killing S. aureus Percentage Killing Controls (9) CD Ileitis (14) 0 Controls (9) CD Ileitis (14) Antimicrobial activity is diminished in mucosal biopsies of ileal Crohn s disease patients

15 Can inflammation alone account for the decreased expression of Paneth cell defensins? Pouchitis as a model

16 Paneth cell defensins are not diminished in pouchitis IL-8 HD5 p<0.01 p: n.s.

17 Colonic Crohn s disease

18 Induction of colonic HBD2 and 3 is diminished in Crohn s HBD-2 HBD-3 Cathelicidin LL 37 A cathelicidin expression ** *** n.s. * Wehkamp Eur J Gastro Hepatol 2002 Wehkamp IBD 2003 Fahlgren Clin Exp Immunol 2003 Fahlgren Clin Exp Immunol 2004 Schauber Eur J Gastro Hepat 2006 controls UC non-inflamed UC inflamed CD non-inflamed CD inflamed

19 Conclusion Crohn s ileitis is associated with a decrease of Paneth cell defensins Wehkamp et al., Gut 2004 Wehkamp et al., PNAS 2005 Ayabe et al., abstract, DDW 2005 Crohn s colitis is associated with a lack of induction of beta defensins and a cathelicidin Wehkamp et al., Europ J Gastroenterol Hepatol, 2002 Fahlgren et al. Clin Exp Immunol Wehkamp et al. Innfamm. Bowel. Dis., 2003 Fahlgren et al. Clin Exp Immunol Aldhaus et Satsangi, DDW 2004 Schauber et al. 2006

20 Induction of defensins by administration of stimulators Probiotic bacteria Probiotic bacteria stimulate defensin expression

21 Induction of defensins by administration of stimulators Probiotic bacteria mimickry as bad guys

22 Morning Renewal Crème Ingredients: Dl water, Aloe Vera gel, Emu oil, Raffermine, Cetyl alcohol, Glycerin, Reductine, Sodium Hyaluronate, Antiglyskin, Liftline, Defensine, Polysorbate 20, Sorbitol 70%, Elastin, DMAE, Description: Nourish your skin with emu oil plus a blend of nutrients and antioxidants including Furfuryladenine, Chlorelline, Defensine, DMAE, Alpha Lipoic Acid, Collagen, Elastin, Vitamin E, CoQ10 and many more for an age defying start to your day.

23 The Teams at Bosch and UC Davis Funded by Robert Bosch Foundation and NIH

24 Thank you!

25 Adherent intestinal bacteria in IBD: due to a loss of epithelial antibacterial defense? HBD-2 Schauber er al Wehkamp et al Cathelicidine LL 37

26 Can inflammation alone account for the decreased expression of Paneth cell defensins? HD5 not related to severity of inflammation

27 CD non-inflamed CD inflamed control B A Cathelicidin expression in IBD UC, CD: Colon ** *** n.s. * controls UC non-inflamed UC inflamed CD non-inflamed CD inflamed n.s Colonic CD4+ in CD * C 0 CD non-inflamed CD inflamed cathelicidin expression CD4 expression cathelicidinexpression CD: Ileum

28 Das Konzept eines kausalen Defensindefektes bei M. Crohn ist attraktiv

29 Thank you!!! Davis, California The Bevins Lab Charles L. Bevins Ryan Feathers Hiu Chu Mark Underwood Robert Kays Tsang Lau Jenny Karlsson Bjoern Schroeder The Computer Department David Curtis Blaire Everybody in the Department of Microbiology and Immunology Satya Dandekar Kathy Blaisdell The Department of Pathology Maggy Laser Capture Core Facility Christian Leutenegger Marko Estrada

30 Barrier problem

31 Possible Mechanism- Variation in β-defensin HBD2-DNA in Controls: DNA copy numbers? HBD2 DNA in ileal Crohn s disease HBD2-DNA in colonic Crohn s disease Manuscript in preperation

32 Summary 2 Attenuated induction of inducible β-defensins in colonic Crohn s disease (but still work to do)

33 Human Paneth Cell Antimicrobials Small intestine Colon Mouth Rectum HD5 mrna copies per 10ng RNA Paneth Cells Wehkamp, Chu, Feathers and Bevins, manuscript in preparation

34 Why are defensins candidate molecules for pathophysiology in Crohn's disease? Defensins are major mucosal antibiotics Defensins are active against lumenal microbes, both flora and ingested pathogens Paneth cells are located in small intestine, abundant in ileum Express defensins AND Crohn's susceptibility gene, NOD2 So, ileal Crohn's disease may be "a defensin deficiency syndrome" Fellerman, Wehkamp & Stange 2003

35 Antimicrobial peptides in IBD: HBD-2 expression in colon Attenuated induction of human beta defensin 2 in colonic Crohn s disease Wehkamp et al., IBD 2003

36 Fig. 1. PC {alpha}-defensins in controls and IBD patients Wehkamp, Jan et al. (2005) Proc. Natl. Acad. Sci. USA 102, Copyright 2005 by the National Academy of Sciences

37 Fig. 3. Quantitative analysis of antimicrobial activity and PC mrna transcript copy numbers in ileal mucosa Wehkamp, Jan et al. (2005) Proc. Natl. Acad. Sci. USA 102, Copyright 2005 by the National Academy of Sciences

38 Fig. 4. Bacterial microbiota in wild-type, heterozygous, and homozygous HD5 TG mice Wehkamp, Jan et al. (2005) Proc. Natl. Acad. Sci. USA 102, Copyright 2005 by the National Academy of Sciences

39 Fig. 2. Mucosal inflammation and expression of PC defensin HD5 Wehkamp, Jan et al. (2005) Proc. Natl. Acad. Sci. USA 102, Copyright 2005 by the National Academy of Sciences

40 Hypothesis Are Paneth cell defensins altered in Crohn s disease patients (especially those with ileal disease)? Are NOD2 mutations in Crohn s disease associated with an altered expression of Paneth cell defensins?

41 Paneth Cell Expression in Ileal Crohn s Disease Surgical specimens from the Cleveland Clinic foundation N: N: 11 34

42 Do small changes in HD5 expression levels impact on the luminal flora? Small Bacilli Predominate Larger Fusiform Bacteria Predominate

43

44 Wehkamp, Fellermann, Herrlinger, Bevins and Stange, Nature Clinical Practice, September 2005

45 Primary genetic defect? Mapping studies Gene copies Alternative ideas? Host factors impact on PC antimicrobials NOD2 Microbes impact on PC antimicrobials Salzman et al I&I 71:1109 (2003)

46 Speculation Data may provide rationale for alternative therapies

47 Administration of defensins experiment, preclinical stage * The difference is statistical significant (p= ) and representative for 5 independent experiments 100% (2 out of 11 dead *) Survival rate 75% 50% experiment, clinical stage (5 out of 8 dead *) 25% HD5 tg wt HD5 protects against colonic inflammation (in a Mouse DSS model) 9 12 Days of 2.5% DSS Studies in the works

48 Acknowledgements 1

49 Thank you!!! Davis, California The Bevins Lab Charles L. Bevins Ryan Feathers Hiu Chu Mark Underwood Robert Kays Tsang Lau Jenny Karlsson Bjoern Schroeder The Computer Department David Curtis Blaire Everybody in the Department of Microbiology and Immunology Satya Dandekar Kathy Blaisdell The Department of Pathology Maggy Laser Capture Core Facility Christian Leutenegger Marko Estrada

50 Acknowledgements 2 Davis, California Charles L. Bevins Ryan Feathers Hiu Chu Los Angeles Tom Ganz Edith Porter Rose Linzmeier Herdecke Corinne Enders Milwaukee Nita Salzman Stuttgart Ed Stange Klaus Fellermann Elke Schaeffeler Mathias Schwab Miriam Schlee Kathleen Siegel Cleveland Bo Shen Robert Petras Kiel Jens Schroeder Juergen Harder Michael Weichenthal Funded by Robert Bosch Foundation (Germany) Funded by Natianal Institute of Health. (USA) Supported by Ardeypharm, Inc. (Manufactor of Mutaflor (E. coli Nissle 1917))

51 Dysregulation

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