Molecular mechanisms of bacteria-host interactions at the epithelial cell level

Transcription

1 Technical University of Munich Prof. Dr. Dirk Haller Molecular mechanisms of bacteria-host interactions at the epithelial cell level Chair for Biofunctionality Department of Food and Nutrition Science Department of Medicine (

2 Lessons from the past: germ theory vs. organ pathology Rudolf Virchow ( ) Theory about the cellular pathology of the diseased organ (1858) Disease due to organ failure Robert Koch ( ) Theory about germs that cause infectious disease (1876) Disease due to infectious microbes

3 Etiology of chronic inflammatory bowel diseases (IBD): a paradigm for bacteria-host interactions Microbial defense Immune function Epithelial function CARD15, NCF4, ATG16L1 TNFSF15, PTPN2, IL-23R ABCB1, PTGER4, SLC22A Ulcerative colitis Genetic predisposition Normal gut Environmental triggers Microorganisms in the gut (Dysbiosis) Infections, Smoking, Diet, NSAIDs Crohn s disease

4 Enteric bacteria are not all equal in their capability to trigger chronic intestinal inflammation Enteric Bacteroides bacteria vulgatus (SPF) Enteric Enterococcus bacteria faecalis (SPF) Enteric Enterococcus bacteria faecalis (SPF) Enteric Bacteroides bacteria vulgatus (SPF) Wild IL-10 type -/- Wild HLA-B27 type IL-10 -/- HLA-B27 Normal Gut Chronic Inflammation Kim et al Inflam. Bowel Dis. and 2005 Gastroenterology

5 Failure of the epithelial cell homeostasis facilitates the loss of barrier function and chronic inflammation CpG DNA p40/70 Protective Defensines and mucus Colitogenic Flagellin GelE P RelA U UU P Normal gut SIgA (Tr1) IL-10 (Th3) TGF-β IL-11 IL-15 Epithelial cell integrity Chronic inflammation IL-12 - IFN-γ (Th1) IL-23 - IL-17 (Th17) IL-13 (Th2) Smad7

6 Interface function of intestinal epithelial cells: from the proteome analysis to molecular mechanisms Proteome analysis Disease mechanisms Cell isolation Cellular extract and fractions Microbial and nutritional signals Ligand Receptor Protein Signal transduction 2D-PAGE and MALDI-TOF MS Host signals Gene- and proteinexpression Clavel and Haller 2007 Inflam. Bowel. Dis. Werner and Haller 2007 Mutation Res.

7 What are the molecular mechanisms of bacteria-host interactions Microbial determinants for pro-and antiinflammatory effects at the epithelial cell level Inflammation meets metabolism: cellular stress in the pathogenesis of chronic inflammation

8 Germ-free (sterile) mouse models for chronic intestinal inflammation: modeling bacteria-host cross-talk Germ-free Germ-free Wild type Protective effects Colitogenic effects IL-10-/- Signal integration Wild type E. faecalis IL-10 - / - E. faecalis

9 Intestinal epithelial cell activation at early and late stages of bacterial colonization in wild type and IL-10-/- mice IP-10 mrna expression (fold increase) Wild type IL-10 -/- * * * E. faecalis OG1RF GF 1 14 GF 1 14 time (weeks) Histopathology (Score ± SD) 0.2 (0.1) 0.6 (0.1) 0.5 (0.1) 0.3 (0.1) 0.8 (0.1) 3.6 (0.2) Distal colon Ruiz et al J. Immunol.

10 Host-derived anti-inflammatory feed-back mechanisms in epithelial cells: signals from a complex environment TGF-β Enterococcus faecalis TGF-βR1 TGF-βR2 TLR2 2 IKK-β P-Smad2/3 15-deoxy-Δ 12,14 - prostaglandin J 2 PP2A IκB/ NF-κB Smad4 1 P-Ac-Histone CBP/ p300 3 NF-κB IP-10 Haller et al J.BC Ruiz et al J. Immunol. Ruiz et al JBC Haller et al JBC

11 Microbial-host cross-talk: from physiological inflammation to chronic inflammation Healing Inflammation Enteric bacteria Chronic inflammation Inflammation Chronic inflammation Homeostasis Time

12 Reconstituted lactobacilli-free (RLF) mice as a model of bacteria-host interaction in the presence of a microbiota time post inoculation 2d 6d 21d Histoscore (±std) L.reuteri count (cfu) 1 x 10 8 per gram Ileum Jejunum Ileum RLF-mice 0,7 (±0,24) - - RLF-mice + 0,5 (±0,0) 3,9 5,4 RLF-mice 0,7 (±0,24) - - RLF-mice + 1,1 (±0,2)* 6,5 7,8 RLF-mice 0,38 (±0,13) - - RLF-mice + 0,55 (±0,24) 6,7 7,9 J. Nutr. 2008

13 Reconstituted lactobacilli-free (RLF) mice as a model of bacteria-host interaction in the presence of a microbiota IL-1α IL-6 IL-1β MIP-2 A20 ** Mass kda 100 CTRL days after inoculation with L.reuteri 30 ** fold change * * days 6 days 21 days 1 2 days 6 days 21 days 2 2 days 6 days 21 days 3 2 days 6 days 21 days L.reuteri L.reuteri L.reuteri L.reuteri grp58 I-FABP β-actin L. reuteri days after inoculation 15 ph 3 ph 10 Number of regulated / identified proteins ph 3 ph inoculation days

14 What are the molecular mechanisms of bacteria-host interactions Microbial determinants for pro-and antiinflammatory effects at the epithelial cell level Inflammation meets metabolism: cellular stress in the pathogenesis of chronic inflammation

15 Quantification and identification of protein expression changes in complex samples: from health to disease Mass spectrometry (MS) x10 4 MALDI-TOF ESI Proteolytic digestion Spot intensity Gel-based protein separation Intens. [a.u.] m/z m/z 2D-SDS-PAGE Intensity

16 Induction of the ER stress response protein (Grp)-78 in the epithelium: from animal models to human IBD A Proteome analysis C Validation in IBD patients WT Grp-78 3,6 NI I IL-10 -/- B Western Blot Western Blot Grp-78 4,6 Grp-78 β-actin β-actin WT IL-10 -/- NI I Shkoda et al Gastroenterology

17 Endoplasmic reticulum (ER) stress contributes to the pathogenesis of chronic degenerative diseases Ribosome S-S + ADP Grp-78 ATF-6 IRE-1α/β PERK Adaptation Endoplasmic reticulum stress Apoptosis Chronic inflammation

18 Epithelial cells are susceptible for ER-associated stress mechanisms in IBD: what is the evidence? Bertolotti et al J. Clin. Invest. ER-stress increased sensitivity to DSS colitis in IRE1β-deficient mice Shkoda et al Gastroenterology ATF-6-mediated ER-stress in the epithelium of IL-10-deficient mice and IBD patients: protective role for IL-10 Heazlewood et al PLOS Medicine Abberent mucin assemly causes ER stress-associated colitis and goblet cell loss in the epithelium Kaser et al Cell XBP-1 variants are associated with ER-stress in Paneth cells and human IBD

19 Cellular mechanisms for the regulation of stress responses: inflammation meets metabolism IL-10 Bacteria TNF IL-10 TLR IKKα/β/γ TNFR Cpn60 Cpn60 ROS P U U U P RelA Grp-78 Cpn60 NF-κB ATF6 IRE-1 UPR P P P P RelA Inflammation PERK ATF6 XBP1s AP-1 CHOP Stress response mtupr UPRE Nucleus

20 Differential protein expression profile in primary IEC from colon and ileum of IBD patients: a pilot study CRC UC UC CD N=6 N=6 N=6 N=2 17/9* 8/4* 38/38* 3/3* Shkoda et al J. Proteome Res.

21 Comparison of inflamed versus non-inflamed tissue regions: the better-matched controls? Up Down UC Patient (No 1) UC Patient (No 2) Non-inflamed (NI) Inflamed (I)

22 Highly interrelated protein networks indicate metabolic changes in primary IEC under chronic inflammation Protektive signals Inflammatory signals Bibliometric co-citation analysis with 66 proteins Homeostasis Increase in glycolosyis Increase in β-oxidation Increase in apoptotic mechanisms Increase in anti-oxidative responses Increase in cell stress responses Decrease in mitochondrial function Tissue pathology Shkoda et al J. Proteome Res. Werner et al J. Proteome Res.

23 Inflammation meet metabolism: common disease mechanisms for chronic degenerative pathologies? Inflammatory Bowel Diseases and Colorectal Cancer Inflammatory stress response Neurodegenerative Diseases Autoimmunity and Allergy Metabolic stress response Obesity, insulin resistance and Type 2 Diabetes

24 Lessons from the past may be valid in the future Rudolf Virchow Theory about the cellular pathology of the diseased organ (1858) Disease due to organ failure Elias Metchnikoff Gut bacteria contribute to disease development (1907) Disease due to microbial dysbalance

25 Acknowledgements UNC, Chapel Hill, USA Balfour R. Sartor Sandra C. Kim Charité Berlin Ulf Göbel Stefan Bereswill Rowett Institute Denise Kelly Caroline Reiff DIfE Michael Blaut Gunnar Loh University of Zürich Gerhard Rogler Funding DFG, BMBF, Danone Stiftung Industry, EU, NuGo Biofunctionality, TUM Dirk Haller Ingrid Schmöller Sigrid Kiesling Thomas Clavel Susan Chang Micha Hoffmann Anja Messlik Tanja Werner Eva Rath Gabriele Hörmannsperger Natalie Steck Emanuel Berger Marie Anne von Schillde Stephan Wagner Nico Gebhardt Benjamin Tiemann