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1 Dept. of Internal Medicine/Infectious and Respiratory Diseases Stefan Hippenstiel Epigenetics as regulator of inflammation
2 Host cell activation LPS TLR NOD2 MDP TRAF IKK NF-κB IL-x, TNFα,...
3 Chromatin organisation Heterochromatin transcriptionally inactive coiled Euchromatin gene-rich transcriptionally active uncoiled DAPI
4 Chromatin organisation
5 Nucleosome core particel N-terminal Histone octamer (H2A, H2B, H3, H4) bp DNA wrapped around octamer in 1.65 turns Covalent modification of N-terminal histone tails regulates DNAhistone binding
6 Nucleosome core particel histone modifications Acetylation usually turns a gene on Methylation (mono,di,tri) usually turns a gene off Phosphorylation we re not sure what that does Ubiquitinylation? ADP-ribosylation? Sumoylation?
7 Histone acetylation Lysine O N C C C α β γ C C δ ε C N+ O - O P O O P DNA backbone binding Acetyl-CoA HAT (Histone Acetyl-Transferase) CoA reversible reactions Histone Deacetylase ε-n-acetyl-lysine O N C C C C C ε C N C O C no DNA binding Hypoacetylation (Blue): Strong internucleosomal interactions: histone tails constrain wrapping of DNA on nucleosome surface Hyperacetylation (Yellow): Weak internucleosomal interactions: histone tails do not constrain DNA, which is accessible to transcription factors
8 HATs & HDACs HAT (histone acetyltransferase) CBP/p300, Esa1, Gcn5, Hat1, p160 family, P/CAF, TAFII250, Tip60, HDAC (histone deacetylases) Classical HDACs Class I HDAC ( almost exclusively nuclear ) 1, 2, 3, 8 Class II HDAC (shuttle between nucleus and cytosol) HDAC 11 4, 5, 6, 7, 9a/b, HDRP SIR2-family (NAD + -dependent HDACs)
9 Prepairing the target for the bullet NcoR No transcription Transcription
10 Chromatin immunoprecipitation (ChIP) Cross-link chromatin Sonication to shear chromatin Immunoprecipitate Anti-histone Anti-transciption factor Anti-accessory proteins Reverse crosslinking Purify DNA Detection (Quantitative) PCR Promotor microarray (Chip-on-ChIP)
11 Model cytokines Interleukin-8 (IL-8) Pro-inflammatory CXC-chemokine Strong long-lasting chemoattractant for leucocytes Regulation by e.g. NF-κB and AP-1 Interferon-γ (IFN-γ) Type-II IFN Anti-viral and immunoregulatory protein Potentiate type-i IFN effects
12 Listeria monocytogenes Gram-positive facultative intracellular pathogen Causes food-borne septicaemia and meningitis in immunocompromised hosts, pregnant women, neonates Multiple virulence factors (e.g. listeriolysin, phospholipases, ) Efficient infection of and replication in human endothelial cells Well-known model organism
13 Intracellular Listeria induced IL-8 and IFNγ expression in HUVEC C control Dapi EGD INN F-actin overlay Cytosol Extracell. Cytosol Extracell.
14 Listeria induced histone modifications in endothelial cells Schmeck et al. J. Immunol 2005
15 Kinase inhibition reduced IL-8, but not IFNγ expression A B - - +SB +U L.m.
16 Acetylation - difference between IL-8 and IFNγ Schmeck et al. J. Immunol 2005
17 MAPK-inhibition reduced CBP recruitment to the il8 promotor
18 Summary (I) EGD Nod1 Rac1 ERK p38 NF-κB A P CBP Pol II NF-κB IL-8
19 Summary (II) EGD EGD Nod1 Rac1 Nod1 Rac1 ERK p38 NF-κB ERK p38 NF-κB CBP A P CBP Pol II NF-κB HDAC1 NF-κB Pol II IL-8 IL-8
20 Legionella pneumophila Gram-negative facultative intracellular pathogen Causes severe pneumonia (Legionnaires` disease) Multiple virulence factors (e.g. phospholipases, glycosyltransferases, RalF, ) Infects lung mononuclear cells and alveolar epithelium Very limited information about molecular mechanisms of Legionella-host interaction
21 Legionella induced histone modifications in human lung epithelium Schmeck et al. submitted
22 HDAC-inhibition increased Legionalla-related gene transcription
23 Legionella increased HDAC5 expression in lung epithelium
24 No effect of HDAC5 silencing on Legionella-related IL-8 expression 20 IL-8 [ng/ml]
25 Summary (III) Leg x y MAPK PKC NF-κB? HDAC1 A P CBP NF-κB Pol II IL-8
26 Summary (IV) Leg Leg x y x y MAPK PKCα NF-κB MAPK PKC NF-κB? HDAC1 CBP Pol II P CBP A NF-κB Pol II HDAC5 HDAC1 NF-κB IL-8 IL-8
27 Moraxella catarrhalis Gram-negative facultative intracellular pathogen Major cause of infectious exacerbations of COPD (4. often cause of death worldwide) Adheres to and infects tracheobronchial epithelial cells Very limited information about molecular mechanisms of Moraxella-host interaction Role in COPD-disease progression unclear
28 Moraxella induced IL-8 expression
29 M. catarrhalis related histone modifications C Slevogt H et al. Am. J. Physiol. 2006
30 Moraxella decreased HDAC2 expression and HDAC activity in lung cells A C B
31 Summary (V) Mor x y MAPK NF-κB HDAC2? HDAC-activity A P NF-κB Pol II chronic stimulation?? IL-8
32 Histone modifications Contribute to gene regulation in infectious diseases, including lung infections Open questions: Which pathogenic factors involved? Which PRRs and signaling pathways involved? Role in infection resolution, chronic inflammation, and tissue repair? Role in differentiation of (immune) cells (heritable changes!?)
33 Dept. of Internal Medicine/Infectious and Respiratory Diseases Stefan Hippenstiel T H A N K Y O U! Berlin: All the members in the lab!!!! Collaborators: Michael Kracht Antje Flieger Trinad Chakraborty
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