EPIGENETIC MODIFICATION FOR
THE FUTURE TREATMENT
OF INFLAMMATORY DISEASE
Peter J Barnes FRS, FMedSci
National Heart & Lung Institute
Imperial College, London, UK
Inflammation 2010: RSC Meeting November 2010
Imperial College
Royal Brompton Hospital
THERAPEUTIC POTENTIAL OF EPIGENETICS
EPIGENETICS: non genetic changes in chromatin structure
resulting in changes in gene expression
• DNA methylationmethylation- longlong-term changes, developmental
• Histone modification
DNA methylation
• DNA methyltransferase inhibitors (e.g. azacytidine):
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
INFLAMMATORY GENE TRANSCRIPTION
HAT = histone acetyltransferase
Multiple
transcription
factors
NFNF-B STATs
mRNA
APAP-1
CBP
CREB
HAT
Core
histones
RNA
polymerase II
Histone
acetylation
Ac
Ac
Ac
Ac
Ac
Ac
Repressive chromatin
Decreased transcription
Inflammatory gene repression
Ac
Ac
Ac
Ac
Ac
Ac
Active chromatin
Increased transcription
Inflammatory gene expression
CHROMATIN STRUCTURE
H4
H4
DNA
* =Acetylation sites:
Lysine residues
2
HISTONE ACETYLATION
DNA
Nucleosome
H4
H3
H3
Histone octamer
H2A H2B
N terminal
SGRGKGGKGLGKGGAKRHRK
5
8
12
-CH2-CH2-CH2-CH2-NH3+
Lysine
16
HAT
HDAC
Histone 4
-CH2-CH2-CH2-CH2-NHNH-COCO-CH3
-acetyl-Lysine
HISTONE ACETYLATION AND GENE TRANSCRIPTION
Gene repression
Gene transcription
DNA
Nucleosome
(histone octamers) RNA
polymerase II
Transcription factor
Acetylation of Lys
Histone deacetylation
HDAC 11-11
COREPRESSORS
Histone acetylation
HATs: CBP, p300, pCAF etc
COACTIVATORS
3
HISTONE ACETYLATION
A549 cells
HAT activity
(dpm/µg protein)
AntiAnti-acetylated
histone H4
+ ILIL-1ß
Histone
acetyltransferase
**
150
100
*
50
0
nucleus
0
0.01
0.1
1
IL-1ß (ng/mL)
Ito K et al: Mol Cell Biol 2000
HISTONE ACETYLATION AND GENE EXPRESSION
Inflammatory stimuli
e.g. IL-1ß, TNF-
IKK2
NF-κB regulated
genes
p65CXCL1,
NF-B
• Chemokines:
CXCL1
CXCL8, CCL2,
CCL2, CCL5,
CCL5, CCL11
IB , CXCL8,
•
Cytokines: p50
GMTNFGM-CSF, activation
TNF-α, ILIL-1β, ILIL-6
•
Enzymes:
iNOS,
cPLA
,
COX2,
MMPMMP-9
Inflammatory protein
2 COX
(e.g. GM-CSF)
• Receptors: NK1, NK2, bradykinin B1, B2
• Peptides:p65 endothelin-1
endothelin
CBP
Co-activators
• Adhesion
mols:
:
ICAM1
ICAM
mols
p50
HAT
Acetylation
 Inflammatory Gene
transcription
Gene
activation
Gene
repression
4
ACTIVATION OF INFLAMMATORY GENES
INFLAMMATORY GENE REGULATION
INFLAMMATION
INFLAMMATORY
PROTEINs
 INFLAMMATION
Corticosteroids
e.g. GMGM-CSF, ILIL-8
Coactivators
e.g. CBP
↓ HAT
↑HDAC
HDAC2
HAT
Transcription
factors
e.g. NFNF-B
Pol2
Histone
acetylation
AcAc-
Core
histones
Repressed chromatin
CLOSED
Ac-
mRNA
Histone
deacetylation
Ac-
mRNA
AcAcAcAc- Activated chromatin
OPEN
Repressed chromatin
CLOSED
5
EFFECT OF CORTICOSTEROID ON HDAC
A549 cells
HDAC activity
75
HDAC activity (dpm)
p65 (NF-B) immunoprecipitates
HDAC2 protein
**
**
50
-
*
-
IL-1ß
Dex
(10-10M)
Anti-HDAC2
25
Anti-p65
0
IL-1ß
-
+
+
+
+
+
Dex
-
-
12
10
8
6
Ito K et al: Mol Cell Biol 2000
CHROMATIN IMMUNOPRECIPITATION (ChIP) ASSAY
GM-CSF promoter (-70-+32bp)
AcK8 IP
NS IL-1
Dex 10
8
6
6 (-log M)
Acetylation of lysine 8 on histone H4
6
CORTICOSTEROID SUPPRESSION OF INFLAMMATORY GENES
Inflammatory stimuli
Corticosteroids
e.g. ILIL-1ß, TNFTNF-
NFNF-B
Activated GR: highly specific for
p65
GR
p50
activated inflammatory gene complex
(recognition
of histone acetylation signature)
Inflammatory protein
CoCo-activators
(e.g. GMGM-CSF)
Recruitment
p65
p50
CBP
HAT
Acetylation
 Inflammatory Gene
transcription
Gene
activation
GR HDAC2
CoCo-repressors
Deacetylation
Gene
repression
 Inflammatory
gene transcription
EFFECT OF STEROID ON INFLAMMATORY GENES
7
↓HDAC EXPRESSION IN COPD MACROPHAGES
Histone deacetylases (HDAC1-11):
(vs. TSA inducible TNF-  prod)
• reverse
histone acetylation
Alveolar macrophages
• switch off gene transcription ↓HDAC→↑ TNFα
HDAC
activity
• HDAC2
switches
off inflammatory genes
r=0.92, p=<0.001
• HDAC2 recruited by glucocorticoid
receptors to
(dpm/µg protein)
HDAC activity
TNF- production
1.5
150
IgG control
HDAC1
1.0
HDAC2
100
activated
inflammatory genes:
smoker
NonNon-smoker
P<0.01
mediates suppression
of inflammation
by steroids
0.5
50
0
P<0.001
nonnon-smoker
Smoker
0.0
0
50
NonNon-sm Smoker COPD
100
150
200
Total HDAC activity (dpm)
Ito K et al: FASEB J 2001
CORRELATION OF HDAC TO STEROID RESPONSE
Alveolar macrophage: normal smokers and nonnon-smokers
ILIL-8 inhibition
r = 0.88
p =0.0001
100
Inhibitory effect of
Dex on TNF- (%)
Inhibitory effect of
Dex on TNF- (%)
TNFTNF- inhibition
50
0
r = 0.65
p =0.024
100
50
0
0
50
100
150
200
HDAC activity dpm/mg protein
0
50
100
150
200
HDAC activity dpm/mg protein
Ito K et al: FASEB J 2001
8
HDAC2 KNOCK-DOWN: RNAi
Alveolar/sputum macrophages
H1
KD
H2
KD
H2
KD
Sc
NT
GM-CSF(ng/ml)
HDAC2
3.0
2.0
1.0
0
*
*
Non-treated
Ito K et al:: J Exp Med 2006
Scrambled
Non-treated
LPS
HDAC2 KD
LPS + Dex (10-6M)
 HDAC2 IN COPD LUNG
Peripheral lung (surgical resection)
1.0
**
3
**
2
1
↑ Histone acetylation of *IL-8 gene
correlated with ↓ HDAC2
0.5
→ Neutrophilic
inflammation
1
***
0
NonNormal COPD
smokers smokers
Ito K et al: N Engl J Med 2005
2
IL-8 promoter
acetylation (x10-3M)
HDAC2
IL-8 mRNA
HDAC2 expression
(ratio vs histone-1)
3
H4 acetylation of κB binding
site on IL-8 promoter (ChIP)
0
0.0
NonNormal
smokers smokers COPD
IL-8 mRNA (RT-PCR)
9
HDAC2 AND STEROID RESPONSIVENESS IN COPD
Alveolar macrophages
HDAC2
COPD
NT Em H2
HDAC activity
COPD macrophages
Plasmid vector with HDAC2
5.0
GMGM-CSF secretion
Restores
n=6 HDAC2 to normal
200
**
100
GM-CSF (ng/ml)
HDAC activity (ΔAFU)
300
non-smoker
NT Em H2
0
**
0
normal smoker COPD
Ito K et al: J Exp Med 2006
2.5
Control
Empty
vector
LPS
HDAC2
vector
HDAC1
vector
LPS + dexamethasone (1μ
(1μM)
Sm COPD
AntiAnti-NT
O2.- +
NO.
superoxide nitric
anions
oxide
Ito K et al: BBRC 2004
HDAC2 activity
AntiAnti-HDAC2
ONOO-
(dpm/HDAC2)
C
0.75
NitrotyrosineNitrotyrosine-HDAC2
Exhaled Peroxynitrite
0.50 400
Peroxynitrite (nM)
HDAC2 immunoprecipitates
Nitro--Tyr/HDAC2 ratio
Nitro
NITRATION AND HDAC2 ACTIVITY
p<0.001
0.25 300
0
200
Normal Smoker COPD
0.50 100
peroxynitrite
0.25
3-nitrotyrosine
HDAC2 activity
stable
0
N
COPD
Osoata G et al: Chest 2009
Altered function?
10
PEROXYNITRITE INDUCES STEROID RESISTANCE
GM-CSF (% of control)
Human airway epithelial cells
100
SINSIN-1: peroxynitrite generator
75
IL-1ß + SIN-1 (500µM)
50
25
IL-1ß
0
C
-12 -11 -10
-9
-8
-7
-6
[Dexamethasone (-log M)]
Ito K et al: BBRC 2004
CORTICOSTEROID RESISTANCE IN COPD
Barnes PJ: Ann Rev Physiol 2009
Cigarette smoke
ANTIOXIDANTS
.O 2
NO
iNOS
Peroxynitrite
THEOPHYLLINE
HDAC activator
COPD
Inflammation
NO
Tyr146
HDAC2
iNOS INHIBITORS
Peroxynitrite scavengers
Tyr253 NO
Ub
Destruction by
Ub
28S proteasome
Ub
Ub
Proteasome
inhibitors
Ub
Ub E3 ligase inhibitors
Inflammatory
genes
Osoata G et al: BBRC 2009
 Inflammatory
Inflammatory genes
genes
Response
Responsetotosteroids
steroids
11
CORTICOSTEROID RESISTANCE
Nitrative stress
Oxidative stress
Peroxynitrite
Cell membrane
↑PI3K-δ
P
Ub
Akt
Ub
Ub Ub
NO
Ub
Tyr UbUbUb
P
↓↓HDAC2
HDAC2
↓HDAC2
Steroid resistance
PI3K-Akt PATHWAY
Oxidative
stress
Akt (PKB)
HDAC2
P
P
STEROID RESISTANCE
To Y et al: Am J Respir Crit Care Med 2010
PI3K activation
**
0.09
0.06
0.03
0.00
PI3K-)/GNB2L1
110α
110α
110β
PI3K 110β
110γ
110γ
110δ
110δ
pAkt / Akt
0.12
Peripheral lung
0.4
Normal
COPD
PI3KPI3K- mRNA
*
0.3
0.2
0.1
0.0
Normal
COPD
12
HDAC activity (AFU/10µg)
THEOPHYLLINE AS HDAC ACTIVATOR
20000
COPD macrophages:
nuclear lysates
**
15000 therapeutic concentrations:
Theophylline in low
• activates HDAC- esp when reduced
• via a novel mechanism (not PDE/adenosine antag)
antag)
• markedly potentiates steroid effects
• reverses steroid resistance
10000
5000
0
B/L
Ito K et al: PNAS 2002, Cosio B et al:
J Exp Med
2004
Theo
(10-6M)
Cosio B et al: J Exp Med 2004
THEOPHYLLINE RESTORES STEROID RESPONSE
C
IL-8 (ng/ml)
7.5
Alveolar macrophages: smokers
HDAC
inhibitor
5.0
*
2.5
0.0
Cntrl
LPS
Cosio B et al: J Exp Med 2004
Theo
Dex
(1μ
(1μM)
(1nM)
Theo
+Dex
TSA
13
THEOPHYLLINE EFFECT ON ChIP ANALYSIS
H4 acetylation at κB site
Histone acetylation of NFNF-κB site of ILIL-8 promoter
0.6
ChIP analysis
+ TNFTNF-α
TNFTNF-α +
smoke
0.4
**
0.2
**
***
0
Cntl TNF-α
Theo Dex
Marwick J et al: BBRC 2008
Dex+
Theo
TNF-α + CSM
Theo Dex
Dex+
Theo
EFFECT OF THEOPHYLLINE IN SMOKING MICE
Theophylline 3 mg/kg p.o.
p.o.
(plasma concentration 1.5 mg/L)
Alveolar Macrophages
(% non-treated)
Lung HDAC activity
*
**
HDAC activity
(mg of standard)
20
10
100
Similar results with inhaled
Reversed
theophylline
by HDAC
NS
NS inhibitor
No detectable plasma levels
(TSA)
TSA)
Daily cigarette x 11
days
50
↑ Neutrophils
↑ Macrophages
SteroidSteroid-resistant inflammation
0
Smoking
Lung Inflammation
-
+
Fox JC et al: ATS 2007
+
Theo
**
0
NT
Dex Theo Dex+
Theo
14
REVERSAL OF SMOKESMOKE-INDUCED INFLAMMATION
A/J Mice
Drugs
Cigarette smoke (4%, 30 min)
Neutrophils (x104 cells/ml)
1 2 3 4
5 6 7 8 9 10 11 12 13 14 days
BAL
BAL Neutrophils
NS
NS
1.0
Theophylline 10mg/kg orally
(plasma conc 4.0±
4.0±0.9mg/L)
0.9mg/L)
**
0.5
**
0.0
Smoke
Dex
Theo
Air
To Y et al: AJRCCM 2010
Dex+Theo
COPD PATIENTS: CORTICOSTEROIDS + THEOPHYLLINE
Fluticasone
F+T combination
Theophylline
Placebo
n=30
4
75
8 wk
Sputum neutrophil elastase
Sputum neutrophils
150
HDAC activity
1000
p < 0.01
PBMCs
750
HNE (μg/mL)
100
Total HDAC activity
[relative light units]
Neutrophils (%)
0
Induced sputum
Plasma theophylline~8mg/L
10
3
500
No 50
difference in fluticasone
or theophylline alone treatment
25
0
Baseline
250
p<0.01
0
FP
FP+T
FP + theo
0
Baseline
p<0.01
FP&T
Ford P et al: Chest 2010
15
STEROID RESISTANCE IN SMOKING ASTHMATICS
NONNON-SMOKING ASTHMA
SMOKING ASTHMA
Cigarette smoke
Inflammatory stimuli
Corticosteroids
Oxidative stress
Peroxynitrite
GR
NFNF-B
NFNF-B
 HDAC2
 GMGM-CSF
 ILIL-8
 eotaxin
 HDAC2
Histone
Histone
acetylation
Steroid acetylation
resistance
Steroid
response
GMGMCSF
GM--CSF
GM
ILIL8
IL--8
IL
eotaxin
eotaxin
 Histone acetylation
THEOPHYLLINE + ICS IN SMOKING ASTHMATICS
Serum theophylline 5 mg/l
Change in PEF (L/min)
40
P=0.06
P=0.008
Theophylline +
inhaled BDP (n=22)
30
20
10
0
Theophylline (n=23)
1
-10
14
Duration (days)
28
Inhaled BDP (n=23)
-20
Spears M et al: ERJ 2009
16
HOW DOES THEOPHYLLINE RESTORE HDAC?
**
125
**
15
10
5
0
Oxidative stress
Immunoprecipitated PI3KPI3K-δ
A549 cells
*
Enzyme activity (NT-100)
HDAC activity (g of standard)
U937 cells
NonNontreated treated
Theo
(1μM)
LY
Intact (IC50=134µ
=134µM)
Black Box
PI3K

100
Theophylline
75
50
HDAC2 activity
25
H2O2 stimulated (IC50=2.1µ
2.1µM)
0
9
8
7
6
5
4
3
Steroid
sensitivity
[Theophylline
(-log10M)]
H2O2 [200 μM]
LY: LY 294002, non-selective PI3K inhibitor
PI3K-δ INHIBITION IN VIVO
A/J Mice
Drugs
Cigarette smoke (4%, 30 min)
Neutrophils (x104 cells/ml)
1 2 3 4
5 6 7 8 9 10 11 12 13 14 days
***
2.0
BAL
NS
IC87114: PI3KPI3K-δ inhibitor
LY294002: pan PI3K inhibitor
NS
1.0
**
0
Air
Dex
Smoke
Dex+IC
IC
**
Dex+LY
17
BAL Neutrophils/ml x 103)
PI3K-δ NULL MICE
250
200
Sham
Smoke + budesonide
Smoke
SteroidSteroid-resistant
SteroidSteroidSteroidSteroid-resistant
responsive
150
*
100
50
0
Wild type
(balb/c)
PI3K-γ null
PI3K-δ null
Marwick J et al: AJRCCM 2009
UNEXPECTED SYNERGY
Borisy AA et al: PNAS 2003
Cells x106
5
4
Brown
Norwayscreening:
rats: inhaled
ovalbumin challenge
High
throughput
synergy
Targeting multiple interacting pathways
Inhaled administration
3
2
***
1
0
B/L
Vehicle
Bud
NT
Bud+NT
(CRx-170)
18
NORTRIPTYLENE AND HDAC REVERSAL
Effect of nortriptylene hydrochloride
HDAC activity
U937 cells
1.0
0.5
40
20
0
0
PI3Kδ
PI3Kδ inhibition
4
Imminoprecipitated
enzyme
3
pAkt/Akt
60
% Inhibition
HDAC activity (fold change)
p<0.05
IC50=0.82μ
=0.82μM
(No effect on
PI3Kα, PI3Kγ)
0.001
0.01
PI3K activity
p<0.05
2
1
0.1
1
Control
H2O2
H2O2 +
[Nortiptylene
(μM)]
NH (1μM)
0
Control
H2O2
H2O2 +
NH (1μM)
REVERSAL OF CORTICOSTEROID RESISTANCE
Oxidative stress
Antioxidants
Nrf2 activators
(sulforaphane)
Cell membrane
↑PI3K-δ
↓ PI3KPI3K-δ
P
Akt
↓ AktAkt-1
P
HDAC2
↑↓HDAC2
Reversal
Steroid of
steroid
resistance
resistance
THEOPHYLLINE
Nortriptyline
PI3KPI3K-δ inhibitors
Akt inhibitors
HDAC2 activators?
Macrolides
(non(non-antibiotic)
19
MACROLIDES
↓HDAC2
TRANSCRIPTION
MacrolidesREVERSE
prevent decrease
in promoter
activity
Relative luminescence
HDAC2 promoter activity
1
Non-antibiotic
macrolide
Erythromycin
0.8
0.6
0.4
0.2
0
Normoxia
Hypoxia
EM
EM703
EPIGENETIC MODIFICATION OF HISTONES
Phosphorylation
Kinases Phosphatases
Kinase inhibitors
P
Ser
Acetylation
HAT
HDAC2
HAT inhibitors
Lys
AcAc- activators
HDAC
Nitration
Denitrases
NO
Tyr
Ubiquitination
Ub E3 ligases
Proteasome
Deubiquitinases
inhibitors
Ub
SUMOylation
HISTONES
(H3, H4)
Lys Su
Methylation
HMT Demethylases
Methyltransferase
Lys
inhibitors
Arg Me
Inflammatory genes
20
METHYLATION AND STEROID ACTION
GM-CSF release (ng/ml)
5-aza-dC: 5-aza-2’-deoxycytidine: Methytransferase inhibitor
**
1.4
1.2
1.0
**
0.8
0.6
0.4
0.2
0
IL-1ß
Mometasone
Kagoshima M et al: Eur J Pharmacol 2001
Mometasone
+ 5-aza-dC
EFFECT OF STEROID ON HISTONE METHYLATION
TGFTGF-β1 promoter
AntiAnti-diMeH3K9 ChIP
**
1.25
Enrichment of IP DNA
HMT associates with GR
Contr
1.00
GR
**
0.75
0.50
IP: SUV39H1 (HMT)
IB: GR
0.25
0.00
FP (10-8M)
Contr
IL-1β
IL-1β
+ FP
FP: fluticasone propionate
21
CORTICOSTEROID INHIBITION OF TGF-β1
IL-1
TGF-β1
CBP
p65
GR
Steroid
HDAC2
RNA Pol2
MeH3K9
AcH4 MeH3K9
MeH3K4
AcH3
TGFTGF-1 mRNA
SUV
TGF-1 Promoter
SUV = histone methyltransferase
Lee K et al: J Immunol 2006
•
•
•
CONCLUSIONS
Multiple histone modifications regulated by enzymes
involved in regulation of inflammatory genes
acetylation, methylation, phosphorylation, nitration, ubiquitination,
ubiquitination, 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
PI3Kδ inhibition
• Histone methylation (H3K9) ↑ by corticosteroids
HMT (SUV39H1) recruited by corticosteroids
• New therapeutic approaches targeting epigenetic
changes now possible
22
ACKNOWLEDGEMENTS
NHLI
Imperial College
Royal Brompton
Hospital
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)
Argenta)
Yasuo Kizawa
(Nihon University)
Neil Thomson
(Glasgow University)
FUNDED BY:
Wellcome Trust
MRC
Asthma UK
GSK
MitsubishiMitsubishi-Tanabe
Novartis
Pfizer
23