EPIGENETIC MODIFICATION FOR THE FUTURE TREATMENT OF INFLAMMATORY DISEASE

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1 EPIGENETIC MODIFICATION FOR THE FUTURE TREATMENT OF INFLAMMATORY DISEASE Peter J Barnes FRS, FMedSci National Heart & Lung Institute Imperial College, London, UK Inflammation 21: RSC Meeting November 21 Imperial College Royal Brompton Hospital THERAPEUTIC POTENTIAL OF EPIGENETICS EPIGENETICS: non genetic changes in chromatin structure resulting in changes in gene expression DNA methylation- long-term changes, developmental Histone modification DNA methylation DNA methyltransferase inhibitors (e.g. azacytidine): reverse silencing of good genes Stimulate methylation: silence bad genes Applicable to lung cancer, inflammation? Problems of specificity and targeting Histone modification Involved in cancer, fibrosis, inflammation Small molecule modifiers now identified (including existing therapies) 1

2 INFLAMMATORY GENE TRANSCRIPTION Multiple transcription factors CREB AP-1 NF- B HAT = histone acetyltransferase CBP STATs mrna Core histones Histone acetylation HAT RNA polymerase II Repressive chromatin Decreased transcription Inflammatory gene repression tive chromatin Increased transcription Inflammatory gene expression CHROMATIN STRUCTURE H4 H4 DNA * =etylation sites: Lysine residues 2

3 HISTONE ACETYLATION H3 H3 H4 H2A H2B DNA Nucleosome Histone octamer N terminal SGRGKGG GGKGLGKGGAKRHRK Histone 4 -CH 2 -CH 2 -CH 2 -CH 2 -NH Lysine NH 3 + HAT HDAC -CH 2 -CH 2 -CH 2 -CH 2 -NH-CO-CHCH 3 -acetyl-lysine HISTONE ACETYLATION AND GENE TRANSCRIPTION Gene repression Gene transcription DNA Nucleosome (histone octamers) RNA polymerase II Transcription factor Histone deacetylation HDAC COREPRESSORS etylation of Lys Histone acetylation HATs: CBP, p3, pcaf etc COACTIVATORS 3

4 HISTONE ACETYLATION Anti-acetylated histone H4 A549 cells Histone acetyltransferase + IL-1ß HAT activity (dpm/µg protein) * nucleus Ito K et al: Mol Cell Biol IL-1ß (ng/ml) HISTONE ACETYLATION AND GENE EXPRESSION Inflammatory stimuli e.g. IL-1ß, TNF- IKK2 NF-κB B regulated genes Chemokines: p65cxcl1, NF- B I B CXCL8, CCL2, CCL5, CCL11 Cytokines: p5 GM-CSF, activation TNF-α,, IL-1β,, IL-6 Inflammatory protein Enzymes: inos, cpla 2, COX-2, MMP-9 (e.g. GM-CSF) Receptors: NK 1, NK 2, bradykinin B 1, B 2 Peptides: p65 endothelin-1 CBP Co-activators Adhesion p5mols: ICAM-1 HAT etylation Inflammatory Gene transcription Gene activation Gene repression 4

5 ACTIVATION OF INFLAMMATORY GENES INFLAMMATION Transcription factors e.g. NF- B INFLAMMATORY GENE REGULATION Coactivators e.g. CBP HAT Core histones - INFLAMMATORY PROTEINs e.g. GM-CSF, IL-8 HAT Histone acetylation - - Corticosteroids Pol HDAC HDAC2 mrna Histone deacetylation INFLAMMATION mrna Repressed chromatin CLOSED - - tivated chromatin OPEN Repressed chromatin CLOSED 5

6 EFFECT OF CORTICOSTEROID ON HDAC A549 cells p65 (NF- B) immunoprecipitates HDAC activity (dpm) HDAC activity IL-1ß Dex * Anti-HDAC2 Anti-p65 HDAC2 protein - - IL-1ß Dex (1-1 M) Ito K et al: Mol Cell Biol 2 CHROMATIN IMMUNOPRECIPITATION (ChIP)) ASSAY GM-CSF promoter (-7-+32bp) K8 IP NS IL-1 Dex (-log M) etylation of lysine 8 on histone H4 6

7 CORTICOSTEROID SUPPRESSION OF INFLAMMATORY GENES Inflammatory stimuli e.g. IL-1ß,, TNF- Corticosteroids NF- B p65 tivated GR: highly specific for GR p5 activated inflammatory gene complex Inflammatory protein (recognition Co-activators of histone acetylation signature) (e.g. GM-CSF) Recruitment CBP p65 p5 etylation HAT GR HDAC2 Co-repressors Deacetylation Inflammatory Gene transcription Gene activation Gene repression Inflammatory gene transcription EFFECT OF STEROID ON INFLAMMATORY GENES 7

8 HDAC activity (dpm/µg protein) HDAC EXPRESSION IN COPD MACROPHAGES reverse histone acetylation Alveolar macrophages switch off gene transcription 1.5 IgG 15 HDAC2 HDAC switches activity off inflammatory control genes HDAC activated inflammatory genes: Non-smoker smoker P< Histone deacetylases (HDAC1-11): 11): r=.92, p=<.1 HDAC2 recruited by glucocorticoid receptors to Non-sm Smoker Ito K et al: FASEB J 21 P<.1 COPD TNF- production (vs. TSA inducible TNF- prod). Smoker HDAC TNFα HDAC2 mediates suppression of inflammation by steroids non-smoker Total HDAC activity (dpm) Inhibitory effect of Dex on TNF- (%) CORRELATION OF HDAC TO STEROID RESPONSE Alveolar macrophage: normal smokers and non-smokers 1 5 TNF- inhibition r =.88 p = HDAC activity dpm/mg protein Ito K et al: FASEB J 21 Inhibitory effect of Dex on TNF- (%) 1 5 IL-8 8 inhibition r =.65 p = HDAC activity dpm/mg protein 8

9 HDAC2 HDAC2 KNOCK-DOWN: RNAi Alveolar/sputum macrophages H1 KD H2 KD Sc H2 KD NT GM-CSF(ng/ml) * * Non-treated Scrambled HDAC2 KD Non-treated LPS LPS + Dex (1-6 M) Ito K et al:: J Exp Med 26 HDAC2 expression (ratio vs histone-1) Ito K et al: N Engl J Med 25 HDAC2 IN COPD LUNG Peripheral lung (surgical resection) HDAC2 Histone acetylation of * IL-8 8 gene correlated with HDAC2.5 Neutrophilic inflammation * IL-8 mrna Normal smokers COPD. H4 acetylation of κb binding site on IL-8 promoter (ChIP) 1. Nonsmokers Nonsmokers Normal smokers COPD IL-8 mrna (RT-PCR) IL-8 promoter acetylation (x1-3 M) 9

10 HDAC activity (ΔAFU) HDAC2 AND STEROID RESPONSIVENESS IN COPD Alveolar macrophages HDAC activity COPD macrophages Plasmid vector with HDAC2 5. Restores n=6 HDAC2 to normal GM-CSF secretion GM-CSF (ng/ml) 2.5 HDAC2 non-smoker NT Em H2 COPD NT Em H2 normal smoker COPD Ito K et al: J Exp Med 26 Control Empty HDAC2 HDAC1 vector vector vector LPS LPS + dexamethasone (1μM) HDAC2 immunoprecipitates Anti-NT Anti-HDAC2 NITRATION AND HDAC2 ACTIVITY O NO. superoxide nitric anions oxide Ito K et al: BBRC 24 C Sm COPD Nitro-Tyr/HDAC2 ratio HDAC2 activity (dpm/hdac2).75.5 ONOO - peroxynitrite.5 4 Peroxynitrite (nm) Nitrotyrosine-HDAC2 Exhaled Peroxynitrite 2 1 Normal Smoker COPD 3-nitrotyrosine HDAC2 activity stable N p<.1 COPD Osoata G et al: Chest 29 Altered function? 1

11 PEROXYNITRITE INDUCES STEROID RESISTANCE Human airway epithelial cells GM-CSF (% of control) C [Dexamethasone (-log M)] SIN-1: peroxynitrite generator IL-1ß + SIN-1 (5µM) IL-1ß Ito K et al: BBRC 24 CORTICOSTEROID RESISTANCE IN COPD Cigarette smoke ANTIOXIDANTS THEOPHYLLINE HDAC activator Osoata G et al: BBRC 29. O 2 - HDAC2 Inflammation NO Peroxynitrite NO Tyr146 inos Tyr253 NO Barnes PJ: Ann Rev Physiol 29 COPD inos INHIBITORS Peroxynitrite scavengers Ub Destruction by Ub Ub 28S proteasome UbUb Proteasome Ub inhibitors Inflammatory Ub E3 ligase inhibitors genes Inflammatory genes Response to to steroids 11

12 CORTICOSTEROID RESISTANCE Oxidative stress Peroxynitrite Nitrative stress PI3K-δ Cell membrane Ub Ub Ub Ub Akt P P HDAC2 NO Tyr Ub UbUbUb HDAC2 Steroid resistance Oxidative stress PI3K Akt (PKB) HDAC2 PI3K-Akt PATHWAY 11α 11β 11γ 11δ P P STEROID RESISTANCE To Y et al: Am J Respir Crit Care Med 21 PI3K- )/GNB2L1 pakt / Akt Normal PI3K- mrna Normal Peripheral lung PI3K activation * COPD COPD 12

13 THEOPHYLLINE AS HDAC ACTIVATOR HDAC activity (AFU/1µg) 2 COPD macrophages: nuclear lysates Theophylline in low 15therapeutic concentrations: activates HDAC- esp when reduced 1 via a novel mechanism (not PDE/adenosine antag) 5 markedly potentiates steroid effects reverses steroid resistance Ito K et al: PNAS 22, Cosio B et al: B/L J Exp Med Theo 24 (1-6 M) Cosio B et al: J Exp Med 24 THEOPHYLLINE RESTORES STEROID RESPONSE 7.5 Alveolar macrophages: smokers HDAC inhibitor C IL-8 (ng/ml) *. Cntrl Cosio B et al: J Exp Med 24 LPS Theo (1μM) Dex (1nM) Theo +Dex TSA 13

14 THEOPHYLLINE EFFECT ON ChIP ANALYSIS Histone acetylation of NF-κB B site of IL-8 8 promoter H4 acetylation at κb site TNF-α Cntl TNF-α Theo Dex Marwick J et al: BBRC 28 ChIP analysis Dex+ Theo TNF-α + smoke TNF-α + CSM Theo Dex * Dex+ Theo HDAC activity (mg of standard) EFFECT OF THEOPHYLLINE IN SMOKING MICE 2 1 Theophylline 3 mg/kg p.o. (plasma concentration 1.5 mg/l) Lung HDAC activity Lung Inflammation * 1 Smoking - + Fox JC et al: ATS 27 Alveolar Macrophages (% non-treated) Similar results with inhaled Reversed theophylline NS by HDAC NS inhibitor No detectable plasma levels (TSA) Daily cigarette x 11 days 5 Neutrophils Macrophages Steroid-resistant resistant inflammation + Theo NT Dex Theo Dex+ Theo 14

15 REVERSAL OF SMOKE-INDUCED INFLAMMATION A/J Mice Drugs Cigarette smoke (4%, 3 min) days Neutrophils (x1 4 cells/ml).5. BAL Neutrophils 1. NS Air To Y et al: AJRCCM 21 Smoke Dex NS BAL Theophylline 1mg/kg orally (plasma conc 4.±.9.9mg/L) Theo Dex+Theo Neutrophils (%) COPD PATIENTS: CORTICOSTEROIDS + THEOPHYLLINE Fluticasone F+T combination Induced sputum Placebo Theophylline Plasma theophylline~8mg/l n=3 4 8 wk Sputum neutrophils Sputum neutrophil elastase 1 HDAC activity 15 1 p <.1 PBMCs 75 No 5 difference in fluticasone 5 or theophylline alone treatment 25 Total HDAC activity [relative light units] Baseline Ford P et al: Chest p<.1 FP+T HNE (μg/ml) No difference in fluticasone or theophylline alone treatment 25 FP 1 3 FP + theo Baseline p<.1 FP&T 15

16 STEROID RESISTANCE IN SMOKING ASTHMATICS NON-SMOKING ASTHMA SMOKING ASTHMA Inflammatory stimuli Corticosteroids Cigarette smoke Oxidative stress NF- B GR Peroxynitrite NF- B Histone acetylation GM-CSF IL-8 eotaxin HDAC2 Steroid response Histone acetylation HDAC2 Steroid resistance Histone acetylation GM-CSF GM IL-8 eotaxin GM-CSF eotaxin THEOPHYLLINE + ICS IN SMOKING ASTHMATICS Change in PEF (L/min) Spears M et al: ERJ 29 P=.6 Serum theophylline 5 mg/l Duration (days) P=.8 Theophylline + inhaled BDP (n=22) Theophylline (n=23) Inhaled BDP (n=23) 16

17 HDAC activity ( g of standard) HOW DOES THEOPHYLLINE RESTORE HDAC? U937 cells * Nontreated Nontreated Theo (1μM) H 2 O 2 [2 μm] Enzyme activity (NT-1) LY Oxidative stress Immunoprecipitated PI3K-δ A549 cells PI3K Black Box HDAC2 activity H 2 O 2 stimulated (IC 5 =2.1µM) 9 Steroid [Theophylline sensitivity (-log 1 M)] LY: LY 2942, non-selective PI3K inhibitor 8 Intact (IC 5 =134µM) Theophylline 4 3 A/J Mice PI3K-δ INHIBITION IN VIVO Drugs Cigarette smoke (4%, 3 min) days Neutrophils (x1 4 cells/ml) Air * NS Dex BAL IC87114: PI3K-δ inhibitor LY2942: pan PI3K inhibitor NS Smoke IC Dex+IC Dex+LY 17

18 PI3K-δ NULL MICE BAL Neutrophils/ml x 1 3 ) Sham Smoke Smoke + budesonide Steroid-resistant resistant Wild type (balb/c) Marwick J et al: AJRCCM 29 * PI3K-δ null Steroid- Steroid-resistant resistant responsive PI3K-γ null UNEXPECTED SYNERGY Borisy AA et al: PNAS 23 Cells x High Brown throughput Norway screening: rats: inhaled synergy ovalbumin challenge Targeting multiple interacting pathways Inhaled administration * B/L Vehicle Bud NT Bud+NT (CRx-17) 18

19 HDAC activity (fold change) NORTRIPTYLENE AND HDAC REVERSAL 6 1. % Inhibition Effect of nortriptylene hydrochloride HDAC activity p<.5 PI3Kδ inhibition Imminoprecipitated enzyme IC 5 =.82μM (No effect on PI3Kα, PI3Kγ) Control [Nortiptylene H 2 O 2 (μm)] H 2 O 2 + NH (1μM) U937 cells pakt/akt 4 p< PI3K activity Control H 2 O 2 H 2 O 2 + NH (1μM) REVERSAL OF CORTICOSTEROID RESISTANCE Oxidative stress PI3K-δ P Akt-1 P HDAC2 HDAC2 Reversal Steroid of steroid resistance resistance Antioxidants Nrf2 activators (sulforaphane) Cell membrane THEOPHYLLINE Nortriptyline PI3K-δ inhibitors Akt inhibitors HDAC2 activators? Macrolides (non-antibiotic) 19

20 Relative luminescence MACROLIDES Macrolides REVERSE prevent decrease HDAC2 in promoter TRANSCRIPTION activity 1 HDAC2 promoter activity Erythromycin Non-antibiotic macrolide Normoxia Hypoxia EM EM73 EPIGENETIC MODIFICATION OF HISTONES Phosphorylation Kinases Phosphatases Kinase inhibitors P Ser etylation HAT HDAC2 HAT inhibitors HDAC - Lys activators Nitration Denitrases NO Tyr HISTONES (H3, H4) Ubiquitination Ub E3 ligases Proteasome Deubiquitinases inhibitors Ub SUMOylation Lys Su Methylation HMT Demethylases Methyltransferase Lys inhibitors Arg Me Inflammatory genes 2

21 METHYLATION AND STEROID ACTION 5-aza-dC: 5-aza-2 -deoxycytidine: Methytransferase inhibitor GM-CSF release (ng/ml) IL-1ß Mometasone Kagoshima M et al: Eur J Pharmacol 21 Mometasone + 5-aza-dC EFFECT OF STEROID ON HISTONE METHYLATION TGF-β1 1 promoter Enrichment of IP DNA Anti-diMeH3K9 ChIP Contr IL-1β IL-1β + FP GR HMT associates with GR Contr FP (1-8 M) IP: SUV39H1 (HMT) IB: GR FP: fluticasone propionate 21

22 CORTICOSTEROID INHIBITION OF TGF-β1 IL-1 TGF-β1 CBP p65 RNA Pol2 Steroid GR HDAC2 SUV H4 MeH3K9 MeH3K4 H3 TGF- 1 1 mrna TGF- 1 1 Promoter Lee K et al: J Immunol 26 SUV = histone methyltransferase CONCLUSIONS Multiple histone modifications regulated by enzymes involved in regulation of inflammatory genes acetylation, methylation, phosphorylation, nitration, ubiquitination, itination, sumoylation HDAC2 recruitment mediates antiinfl effects of corticosteroids in COPD: - due to oxidative/nitrative stress HDAC2 activity restored by gene transfer, theophylline reverse corticosteroid resistance in COPD cells Theophylline in HDAC2 mediated by PI3Kδ inhibition Histone methylation (H3K9) by corticosteroids HMT (SUV39H1) recruited by corticosteroids New therapeutic approaches targeting epigenetic changes now possible 22

23 NHLI Imperial College Royal Brompton Hospital ACKNOWLEDGEMENTS Ian Adcock Caterina Brindicci Borja Cosio Gaetano Caramori Fan Chung Louise Donnelly Paul Ford Mark Hew Kaz Ito Ellen Jazrawi Masa Kagoshima Vicki Katsaounou Vera Keatings John Marwick Grace Osoata Yasuo To Loukia Tsaprouni Satoshi Yamamura Jim Hogg (UBC, Vancouver) Mary FitzGerald (Argenta) Yasuo Kizawa (Nihon University) Neil Thomson (Glasgow University) FUNDED BY: Wellcome Trust MRC Asthma UK GSK Mitsubishi-Tanabe Novartis Pfizer 23