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NEW MECHANISMS AND NEW MECHANISMS AND TREATMENT TREATMENT OF COPDOF COPD
Peter BarnesNational Heart & Lung InstituteImperial College, London, UK
Imperial College Royal Brompton Hospital
Luxembourg December 2008Luxembourg December 2008
A DEFINITION OF COPDA DEFINITION OF COPD
GGlobal Initiative for Chronic lobal Initiative for Chronic OObstructive bstructive LLung ung DDisease (isease (GOLDGOLD) 2006) 2006
“A preventable and treatable disease with some A preventable and treatable disease with some significant extrapulmonary effects that may significant extrapulmonary effects that may contribute to the severity in individual patients. Its contribute to the severity in individual patients. Its pulmonary component is characterized by airflow pulmonary component is characterized by airflow limitation that is not fully reversible. The airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated limitation is usually progressive and associated with an abnormal inflammatory response of the lung with an abnormal inflammatory response of the lung to noxious particles or gases”to noxious particles or gases”
NormalNormal COPDCOPD
PATHOLOGY OF COPDPATHOLOGY OF COPD
Peripheral lungPeripheral lung
Dr Manuel Cosio
BronchioleBronchiole
AlveolarAlveolarwallwall
Loss ofLoss ofattachmentsattachments
EMPHYSEMAEMPHYSEMA
FibrosisFibrosisInflammationInflammation
Chronic obstructive bronchitisChronic obstructive bronchitis
Small airway inflammationSmall airway inflammation• Correlated with diseased severityCorrelated with diseased severity
• Inflammatory cell numbersInflammatory cell numbers
• Inflammatory cell exudate in lumenInflammatory cell exudate in lumen
• Peribronchiolar fibrosisPeribronchiolar fibrosisHogg JC et al: NEJM 2004
Inflammation
NormalNormal
Inspiration
Expiration
AIR TRAPPING IN COPDAIR TRAPPING IN COPD
alveolar attachments
small small airwayairway
COPD COPD
loss of alveolar attachments loss of elasticity (emphysema)
airway closure
thickened airway
• Hyperinflation: air trappingHyperinflation: air trapping• ↑ ↑ TLCTLC• ↑ ↑ Residual volumeResidual volume• ↑ ↑ FRCFRC• ↓ ↓ Inspiratory capacityInspiratory capacity
↑ ExertionalExertional dyspnoeadyspnoea
↓ ↓ Exercise Exercise tolerancetolerance
BRONCHODILATORSBRONCHODILATORSLAMA: tiotropium bromide, …..LAMA: tiotropium bromide, …..LABA: salmeterol, formoterol, ….LABA: salmeterol, formoterol, ….
NormalNormalHyperinflationHyperinflation
KEY QUESTIONS ABOUT INFLAMMATION IN COPDKEY QUESTIONS ABOUT INFLAMMATION IN COPD
• Why is there a specific pattern of inflammation?Why is there a specific pattern of inflammation? macrophages, neutrophils, CD8macrophages, neutrophils, CD8++/CD4/CD4++cells, B lymphocytescells, B lymphocytes
• Why is inflammation in COPD patients amplified?Why is inflammation in COPD patients amplified? compared to matched “normal” smokerscompared to matched “normal” smokers susceptibility genes? lack of inhibitory mechanisms?susceptibility genes? lack of inhibitory mechanisms?
• Why does inflammation persist in ex-smokers?Why does inflammation persist in ex-smokers? memory cells? structural changes?memory cells? structural changes?
• Why is inflammation in COPD resistant to steroids?Why is inflammation in COPD resistant to steroids?
• How does inflammation affect clinical features:How does inflammation affect clinical features: progression, symptoms, exacerbations, mortalityprogression, symptoms, exacerbations, mortality
NeutrophilsNeutrophils MacrophagesMacrophages CytokinesCytokines MediatorsMediators ProteasesProteases
Non-smokers Normal Non-smokers Normal smokerssmokers
Infl
amm
atio
n
0
+
++++
AMPLIFICATION OF INFLAMMATION IN COPDAMPLIFICATION OF INFLAMMATION IN COPD
ExacerbationExacerbation MildMildCOPDCOPD
SevereSevere COPDCOPD
++
+++
What are the molecularWhat are the molecularmechanisms of amplification?mechanisms of amplification?Is it genetically determined?Is it genetically determined?
Cigarette smoke (and other irritants)
Macrophage
Epithelial cells
Alveolar wall destruction(Emphysema)
PROTEASES PROTEASES Neutrophil elastaseMMP-9
Mucus hypersecretion
TGF-β
Fibrosis(Small airways)
Fibroblast
CELLULAR MECHANISMS OF COPDCELLULAR MECHANISMS OF COPD
Neutrophil
Tc1 cell
Monocyte
Th1 cell
CXCL1CXCL8
CXCR3CXCR3 CXCR2CXCR2 CCR2CCR2
CCL2 CXCL9,10,11
Eosinophil
CCL5(IL-4, IL-5)
CCR3
SPUTUM CYTOKINES IN COPDSPUTUM CYTOKINES IN COPD
COPD patients: 62.5 ±3.2y; FEV1 = 34.6±4 % predicted
L
[ T
NF
- (
nm
ol/l
)]
Controls(n=16)
Smokers(n=12)
COPD(n=14)
Asthma(n=22)
*
*
TNF-
Keatings V et al: AJRCCM 1996
0
2
4
6
8 **
L [I
L-8
(n
mo
l/l)]
Controls(n=16)
Smokers(n=12)
COPD(n=14)
Asthma(n=22)
*
**
IL-8 (CXCL8)
0
1
2
3
4
Cigarette smoke
Alveolar macrophage
TNF-TNF- and IL-8 in COPD and IL-8 in COPD
4 ))TNF-
Epithelialcells
TNF-
IL-8
NF-B
IL-8 gene
Neutrophils
IL-8
IL-8
p65 antibodyp65 antibody (NF-(NF-b)b)
anti-CD68anti-CD68(macrophage)(macrophage)
Lung parenchymaLung parenchyma
Anti-p65 antibodyAlveolar macrophages
Stable Exacerbation12.4% 44.2%
DiStefano A et al: ERJ 2002Caramori G et al: Thorax 2003
NF-NF-B IN COPD MACROPHAGESB IN COPD MACROPHAGES
Induced sputum
Nuclear stainingNuclear staining
NF-NF-B IN COPDB IN COPD
Inflammatory genes
NF-B
IKK2IB
B
Oxidative stress
Cigarette smokeIrritants
TNF-IL-1ß
mRNA
CytokinesTNF-, IL-1ß
GM-CSF
ChemokinesIL-8, GRO-α
MIP-1, MCP-1
Adhesionmolecules
ICAM-1
EnzymesMMP-9
Stimulus
Inflammation
IL-8 gene IL-6 geneTNF- gene
IL-8, ENA-78TNF- IL-6
Macrophage Neutrophil T-lymphocyte
Oxidative stress
Amplification loops
COPD EXACERBATIONS COPD EXACERBATIONS
TNF-
NF-B
BacteriaBacteriaVirusesViruses
0
10
20
30
40
50
+ve
p65
nu
clei
(%
)
Stable ExacerbationCaramori G et al: Thorax 2003
↑ ↑ NF-NF-κκB activationB activationIn COPD exacerbationIn COPD exacerbation
Sputummacrophages
0 24 48 72
0
1
2
3
4
5
To
tal E
last
in D
egra
ded
(µ
g)
Time (hours)
Healthy smokersNon-smokers
Ex-smokers
COPD
**
**
**3H-elastin released
BAL macrophages
ELASTASE ACTIVITY OF MACROPHAGES IN COPDELASTASE ACTIVITY OF MACROPHAGES IN COPD
Russell R et al: AJP Lung 2002
Mainly MMP-9Mainly MMP-9
Chemotactic peptides
NeutrophilsNeutrophils
NeutrophilNeutrophilelastaseelastase
Macrophage
EFFECTS OF MMP-9EFFECTS OF MMP-9
Elastolysis
1-AT
EmphysemaEmphysema
MMP-9MMP-9Pro-MMP-9
Latent TGF-ßLatent TGF-ß Active TGF-ß Active TGF-ß
Small airway fibrosisSmall airway fibrosis(chronic obstructive bronchiolitis)(chronic obstructive bronchiolitis)
COPDCOPD
CXC CHEMOKINE RECEPTORSCXC CHEMOKINE RECEPTORS
CXC chemokines
CXCR1
Adhesion
Activation
O2- MPO Enzymes
(low affinity)
GRO-GRO-ßGRO-ENA 78GCP2PF4CTAP IIIßTGNAP-2
CXCR2
Chemotaxis
(high affinity)
IL-8NeutrophilNeutrophil
Placebo AZD8309300mg b.d. x 3d
Ne
utr
op
hils
x1
06/g
p<0.05
0.0
5.0
10.0
15.0
-79%
O’Connor BJ et al: ERS 2007
Inhaled LPS challenge (30 Inhaled LPS challenge (30 μμg)g): normal subjects (n=16): normal subjects (n=16)
ADZ8309: CXCR1/2 antagonist
CXCR1/2 ANTAGONISTS ON LPS CHALLENGECXCR1/2 ANTAGONISTS ON LPS CHALLENGE
↓ ↓ Macrophages (47%)Macrophages (47%)↓ ↓ IL-8 (52%)IL-8 (52%)↓ ↓ GRO-GRO-αα (25%) (25%)↓↓ LTBLTB4 4 (39%)(39%)
↓ ↓ neutrophil elastaseneutrophil elastase
+6 hours+6 hours
INCREASED GRO-INCREASED GRO- IN COPD IN COPD
0
20
40
60
80
100
HealthyNormals
n=17
COPDPatients
n=16
HealthySmokers
n=12
****
GR
O-
(n
g/m
L)
Induced sputumGrowth-related oncogene-
GRO-
CXCR2
Neutrophil Monocyte
Traves S et al: Thorax 2002
MØ
Nu
mb
er (
x108 )
ControlControl(52 pk.yr)(52 pk.yr)
EmphysemaEmphysema(87 pk.yr)(87 pk.yr)
*100
0
50
Retamales et al: AJRCCM 2001
Alveolar TissueAlveolar Tissue
0
1000
2000
3000
4000
5000
*
MØ
Nu
mb
er (
x108 )
ControlControl(52 pk.yr)(52 pk.yr)
EmphysemaEmphysema(87 pk.yr)(87 pk.yr)
Alveolar spaceAlveolar space
MACROPHAGES IN COPDMACROPHAGES IN COPD
bloodbloodmonocytemonocyte
alveolaralveolarmacrophagemacrophage
ChemotaxisChemotaxisDifferentiationDifferentiation(Proliferation)(Proliferation)
0
20
40
60
80
100
0.1 1 5 10 50 1000
GRO- (ng/mL)
Cel
ls P
er F
ield
Healthy smokers (n=8)Healthy normal (n=8)
MONOCYTE CHEMOTACTIC RESPONSE TO GRO-MONOCYTE CHEMOTACTIC RESPONSE TO GRO-
*
*** COPD (n=11)
Increased response to GRO- in COPD
Traves S et al: J Leuk Biol 2004
Not seen with IL-8 or ENA-78 Not seen with IL-8 or ENA-78 Mimicked by NAP2Mimicked by NAP2Not due to Not due to ↑ CXCR2↑ CXCR2Due to ↑ recycling of CXCR2Due to ↑ recycling of CXCR2
Cytotoxic T cell (CD8+: Tc1 cell)
IP-10, Mig, I-TAC IP-10, Mig, I-TAC
CXCR3CXCR3
PerforinsPerforinsGranzyme BGranzyme B
EmphysemaEmphysema(apoptosis of type I (apoptosis of type I pneumocytes)pneumocytes)
CD8 CELLS IN COPDCD8 CELLS IN COPD
Macrophage Bronchiolarepithelial cells
IFN-IFN-
CD8CD8++ cells in small airways cells in small airways and lung parenchyma and lung parenchyma Correlate with disease severityCorrelate with disease severity
IP-10 (CXCL10)
MIG CXCL9)
I-TAC (CXCL11)
Basal
0
2.5
5.0
7.5
10.0
1 10 100
[IFN-γ (ng/ml)]
[Cy
tok
ine
] n
g/m
l
Epithelial cells Epithelial cells in vitroin vitro
CXCR3 CHEMOKINESCXCR3 CHEMOKINES
Costa C et al: Chest 2008
CXCL9CXCL9
CXCL10CXCL10
CXCL11CXCL11
Sputum CXCR3 chemokinesSputum CXCR3 chemokines
Phagocytosis
CigaretteCigarette smokesmokeWood smokeWood smoke
ElastolysisElastolysis MMP-9, MMP-12MMP-9, MMP-12 Cathepsins B,L,KCathepsins B,L,K
ALVEOLAR MACROPHAGES IN COPDALVEOLAR MACROPHAGES IN COPD
Numbers (25X)Numbers (25X)SecretionSecretion Steroid resistanceSteroid resistance
NeutrophilsNeutrophils
LTBLTB44
IL-8IL-8GRO-GRO-
CXCR2
MonocytesMonocytes
MCP-1MCP-1GRO- GRO-
CCR2CXCR2
CD8CD8++ cells cells EmphysemaEmphysema
IP-10IP-10 MigMigI-TACI-TAC
CXCR3
NONOROSROS
HDACHDAC Steroid Steroid responseresponse
LONG-TERM INHALED STEROIDS IN COPDLONG-TERM INHALED STEROIDS IN COPD
TRIAL n DURATION SEVERITYTRIAL n DURATION SEVERITY
Copenhagen City 290Copenhagen City 290 3 yr mild 3 yr mild
EUROSCOP 1277 3 yr mildEUROSCOP 1277 3 yr mild
ISOLDE 751 3 yr moderateISOLDE 751 3 yr moderate
Lung Health 2 1116 3.5 yr moderateLung Health 2 1116 3.5 yr moderate11oo outcome = decline in lung function outcome = decline in lung function
OUTCOMEOUTCOME
no effectno effect
no effectno effect
no effectno effect
no effectno effect
Cochrane Database Systematic Review: >13,000 COPD patientsCochrane Database Systematic Review: >13,000 COPD patientsno no ↓ FEV↓ FEV1 1 decline decline (Yang IM et al 2007)(Yang IM et al 2007)
No ↓ mortality No ↓ mortality (TORCH: Calverley PMA et al NEJM 2007)(TORCH: Calverley PMA et al NEJM 2007)
10
8
6
4
2
[TN
F-
(n
mo
l/mL
)]
0
TNF-TNF-
PlaceboPlacebo Budesonide (800 µg b.d.x 2 wk)Budesonide (800 µg b.d.x 2 wk)BaselineBaseline
COPD patients (n=14): age 65 1.1 yr; FEV1 = 35 1.3%
CYTOKINES IN INDUCED SPUTUM IN COPD:CYTOKINES IN INDUCED SPUTUM IN COPD:LACK OF EFFECT OF INHALED CORTICOSTEROIDLACK OF EFFECT OF INHALED CORTICOSTEROID
[IL
-8 (
nm
ol/m
L)]
0
2
4
6
8
IL-8IL-8
Keatings V et al: Am J Respir Crit Care Med 1997
N.SN.S.N.SN.S.
IS THERE AN ACTIVE STEROID IS THERE AN ACTIVE STEROID RESISTANCE MECHANISM IN COPD?RESISTANCE MECHANISM IN COPD?
00
100100
200200
MIP
-1M
IP-1
(
ng
/ml)
(n
g/m
l)
1010-10-10 10 10-8-8 10 10-6-6
Dexamethasone (M)Dexamethasone (M)
LPSLPSNSNS
Non-smokerNon-smoker
NS LPSNS LPS1010-8-8MM
DexDex
COPDCOPD
Bronchoalveolar lavage macrophagesBronchoalveolar lavage macrophagesREDUCED EFFECT OF CORTICOSTEROIDS IN COPDREDUCED EFFECT OF CORTICOSTEROIDS IN COPD
ALVEOLAR MACROPHAGES AREALVEOLAR MACROPHAGES ARESTEROID-RESISTANT IN COPDSTEROID-RESISTANT IN COPD
(SIMILAR RESULTS WITH IL-8, MMP-9)(SIMILAR RESULTS WITH IL-8, MMP-9)
Culpitt SV et al: Am J Respir Crit Care Med 2002
Cigarette smokeCigarette smoke
Oxidative stressOxidative stress
AMPLIFICATION AND STEROID RESISTANCEAMPLIFICATION AND STEROID RESISTANCE
NF-NF-κκBBGlucocorticoid Glucocorticoid receptorreceptor
HDAC2HDAC2
CorticosteroidsCorticosteroids
HistoneHistoneacetylationacetylation
InflammationInflammation
Inflammatory Inflammatory genes genes e.g. IL-8, MMP-9e.g. IL-8, MMP-9
Cigarette smokeCigarette smoke
Oxidative stressOxidative stress
AMPLIFICATION AND STEROID RESISTANCEAMPLIFICATION AND STEROID RESISTANCE
NF-NF-κκBB
HistoneHistoneacetylationacetylation
Inflammatory Inflammatory genes genes e.g. IL-8, MMP-9e.g. IL-8, MMP-9
HDAC2HDAC2
↑ ↑ InflammationInflammation
SteroidSteroidresistanceresistance
↓↓HDAC EXPRESSION IN COPD MACROPHAGESHDAC EXPRESSION IN COPD MACROPHAGES
IgG controlIgG control
HDAC2HDAC2HDAC1HDAC1
SmokerSmoker
Non-smokerNon-smoker
HD
AC
act
ivit
y(d
pm
/µg
pro
tein
)
HDAC activity
P<0.01
Non-smNon-sm
Ito K et al: FASEB J 2001
SmokerSmoker0
50
100
150
COPDCOPD
Alveolar macrophagesAlveolar macrophages
P<0.001
Histone deacetylases (HDAC1-11): Histone deacetylases (HDAC1-11): • reverse histone acetylationreverse histone acetylation• switch off gene transcriptionswitch off gene transcription• HDAC2 switches off inflammatory genesHDAC2 switches off inflammatory genes• HDAC2 recruited by glucocorticoid receptors toHDAC2 recruited by glucocorticoid receptors to activated inflammatory genes: activated inflammatory genes: mediates suppression of inflammation by steroidsmediates suppression of inflammation by steroids
Total HDAC activity (dpm)0 50 100 150 200
0.0
0.5
1.0
1.5
TN
F-
pro
du
ctio
n
(vs.
TS
A i
nd
uci
ble
TN
F-
pro
d)
r=0.92, p=<0.001r=0.92, p=<0.001
non-smokernon-smoker
smokersmoker
↓HDAC→↑ TNFα
HDAC activity dpm/mg protein
r = 0.88 r = 0.88 p =0.0001p =0.0001
Inh
ibit
ory
eff
ect
of
D
ex o
n T
NF
- (
%)
0 50 100 150 200
100
50
0
TNF-TNF- inhibition inhibition
p =0.024p =0.024r = 0.65 r = 0.65
HDAC activity dpm/mg protein
Inh
ibit
ory
eff
ect
of
D
ex o
n T
NF
- (
%)
0 50 100 150 200
100
50
0
IL-8 inhibitionIL-8 inhibition
Alveolar macrophage: normal smokers and non-smokersAlveolar macrophage: normal smokers and non-smokers
CORRELATION OF HDAC TO STEROID RESPONSECORRELATION OF HDAC TO STEROID RESPONSE
Ito K et al: FASEB J 2001
Peripheral lung (surgical resection)Peripheral lung (surgical resection)
HDAC2 IN COPD LUNGHDAC2 IN COPD LUNG
Ito K et al: N Engl J Med 2005
HDAC2HDAC2
HD
AC
2 e
xp
res
sio
n(r
ati
o v
s h
ist o
ne-
1)
0
1
2
3
Non-smokers
***
Normalsmokers
COPD
0
1
2
3
IL-8
pro
mo
ter
acet
ylat
ion
(x1
0-3M
)**
H4 acetylation of κB binding site on IL-8 promoter (ChIP)
IL-8
mR
NA
0.0
0.5
1.0 **
*
Non-smokers
Normalsmokers COPD
IL-8 mRNA (RT-PCR)
↑ ↑ Histone acetylation of IL-8 geneHistone acetylation of IL-8 genecorrelated with ↓ HDAC2correlated with ↓ HDAC2→ → Neutrophilic inflammationNeutrophilic inflammation
normal smoker COPD0
100
200
300
HD
AC
ac
tiv
ity
(Δ
AF
U)
****
n=6
Alveolar macrophagesAlveolar macrophages
HDAC activityHDAC activity
HDAC2 AND STEROID RESPONSIVENESS IN COPDHDAC2 AND STEROID RESPONSIVENESS IN COPD
HDAC1HDAC1 vectorvector
HDAC2HDAC2vectorvector
****
ControlControl LPSLPS LPS + dexamethasone (1LPS + dexamethasone (1μμM)M)
0
2.5
5.0
GM
-CS
F (
ng
/ml)
GM-CSF secretionGM-CSF secretion
EmptyEmptyvectorvector
COPD macrophagesCOPD macrophagesPlasmid vector with HDAC2Plasmid vector with HDAC2Restores HDAC2 to normalRestores HDAC2 to normal
Ito K et al: J Exp Med 2006
Em H2 non-smoker COPD
NTEm H2NTHDAC2
-12 -11 -10 -9 -8 -7 -6 C
0
20
40
60
80
100
120
ControlControl
[Dexamethasone (log M)]
IL-8
(%
of
co
ntr
ol)
LPSLPS
U937 macrophage–like cell lineU937 macrophage–like cell line
CIGARETTE SMOKE EFFECTS ON STEROID RESPONSE CIGARETTE SMOKE EFFECTS ON STEROID RESPONSE
CSMCSM
Effect of CSMEffect of CSM
CSM: cigarette smoke-conditioned medium: dilution = 0.15CSM: cigarette smoke-conditioned medium: dilution = 0.15
IL-8
(p
g/m
l)
ControlControl CSMCSM0
1000
2000
3000
CSM +CSM +NAC (10 mM)NAC (10 mM)
Effect of antioxidantEffect of antioxidantN-acetylcysteine
Inflammatory genesInflammatory genes Response to steroidsResponse to steroids Inflammatory genesInflammatory genes Response to steroidsResponse to steroids
HDAC2HDAC2Inflammatory genesInflammatory genes
CORTICOSTEROID RESISTANCE IN COPDCORTICOSTEROID RESISTANCE IN COPD
Cigarette smokeCigarette smoke InflammationInflammation
..OO22-- NONO
iNOSiNOSCOPDCOPD
Barnes PJ et al: Lancet 2004
UbUb
UbUbDestruction byDestruction byproteasomeproteasome
Tyr253 Tyr253 NONO
Tyr146Tyr146NONO
PeroxynitritePeroxynitrite
Osoata G et al: ATS 2008
CORTICOSTEROID RESISTANCE IN COPDCORTICOSTEROID RESISTANCE IN COPD
Cell membraneCell membrane
Steroid resistanceSteroid resistance
↑PI3K-δ
AktAkt
↓↓HDAC2HDAC2
PP
PP
Nitrative stressNitrative stress
PeroxynitritePeroxynitrite
↓↓HDAC2HDAC2
NONOTyrTyr Ub
Oxidative stressOxidative stress
Ub
HDAC2HDAC2
Akt (PKB)Akt (PKB)
Oxidative Oxidative stressstress
P
P
PI3K-Akt PATHWAYPI3K-Akt PATHWAY
110110αα110110ββ110110γγ110110δδ
0.0
0.1
0.2
0.3
0.4
PI3
K-
)/G
NB
2L1
*PI3K-PI3K- mRNA mRNA
Normal COPD
0.00
0.03
0.06
0.09
0.12
pA
kt /
Akt
**
Normal COPD
PI3K activationPI3K activation
Peripheral lungPeripheral lung
PI3KPI3K
Theophylline in therapeutic concentrations:Theophylline in therapeutic concentrations:
• activates HDAC (restores to normal in COPD) activates HDAC (restores to normal in COPD)
• via a novel mechanism via a novel mechanism (not PDE/adenosine antag)(not PDE/adenosine antag)
• markedly potentiates steroid effectsmarkedly potentiates steroid effects
• reverses steroid resistance reverses steroid resistance in vitroin vitroIto K et al: PNAS 2002, Cosio B et al: J Exp Med 2004Ito K et al: PNAS 2002, Cosio B et al: J Exp Med 2004
THEOPHYLLINE AS HDAC ACTIVATORTHEOPHYLLINE AS HDAC ACTIVATOR
0
5000
10000
15000
20000
HD
AC
ac
tiv
ity
(A
FU
/10µ
g)
****
B/L Theo (10-6M)
COPD macrophages: COPD macrophages: nuclear lysatesnuclear lysates
Cosio B et al: J Exp Med 2004
C
0.0
2.5
5.0
7.5
IL-8
(n
g/m
l)
Cntrl LPSCntrl LPS TheoTheo (1(1μμM)M)
DexDex (1nM)(1nM)
*
TheoTheo +Dex+Dex
Alveolar macrophages: smokersAlveolar macrophages: smokers
Cosio B et al: J Exp Med 2004
TSATSA
HDACHDACinhibitorinhibitor
THEOPHYLLINE RESTORES STEROID RESPONSETHEOPHYLLINE RESTORES STEROID RESPONSE
Dex+Theo
**
Dex+Theo
Alv
eola
r M
acro
ph
ages
(
% n
on
-tre
ated
)
NT
50
100
Theo0
NS NS
Dex
Lung InflammationLung Inflammation
Theophylline 3 mg/kg p.o.Theophylline 3 mg/kg p.o.(plasma concentration 1.5 mg/L)(plasma concentration 1.5 mg/L)
TheoTheo
**
+
EFFECT OF THEOPHYLLINE IN SMOKING MICEEFFECT OF THEOPHYLLINE IN SMOKING MICE
Daily cigarette x 11 daysDaily cigarette x 11 days↑ ↑ NeutrophilsNeutrophils↑ ↑ MacrophagesMacrophagesSteroid-resistant inflammationSteroid-resistant inflammation
0
10
20
HD
AC
ac
tiv
ity
(m
g o
f st
and
ard
)
*
Smoking +-
Lung HDAC activityLung HDAC activity
Fox JC et al: ATS 2007
Similar results with inhaled theophyllineSimilar results with inhaled theophyllineNo detectable plasma levelsNo detectable plasma levels
Reversed by HDAC inhibitorReversed by HDAC inhibitor(sodium valproate)(sodium valproate)
**
Dex+Theo
BALBAL
Cigarette smoke (4%, 30 min)Cigarette smoke (4%, 30 min)
DrugsDrugs
days1 2 3 4 5 6 7 8 9 10 11 12 13 14
REVERSAL OF SMOKE-INDUCED INFLAMMATIONREVERSAL OF SMOKE-INDUCED INFLAMMATION
Air
BAL NeutrophilsBAL Neutrophils
0.0
0.5
1.0 NSNS
Neu
tro
ph
ils (
x104
cells
/ml)
Smoke Dex Theo
**
Theophylline 10mg/kg orallyTheophylline 10mg/kg orally (plasma conc 4.0(plasma conc 4.0±0.9±0.9mg/L)mg/L)
A/J MiceA/J Mice
Placebo
Fluticasone
Theophylline
F+T combination
0 4 8 wk
COPD PATIENTS: CORTICOSTEROIDS + THEOPHYLLINECOPD PATIENTS: CORTICOSTEROIDS + THEOPHYLLINE
Plasma theophylline~8mg/L
0
25
50
75
100
Neu
tro
ph
ils
(%)
p<0.01
Baseline F+T
Sputum neutrophilsSputum neutrophils
Baseline F&T0
310
150
HN
E (
μg
/mL
)
Sputum neutrophil elastase
p<0.01
n=30
No difference in fluticasone or theophylline alone treatmentNo difference in fluticasone or theophylline alone treatment
0
250
500
750
1000 p < 0.01 T
ota
l HD
AC
ac
tivi
ty [
rela
tiv
e li
gh
t u
nit
s]PBMCs
FP FP + theo
HDAC activityHDAC activity
Placebo
Fluticasone
Theophylline
F+T combination
0 4 8 wk
COPD PATIENTS: CORTICOSTEROIDS + THEOPHYLLINECOPD PATIENTS: CORTICOSTEROIDS + THEOPHYLLINE
Plasma theophylline~8mg/L
FE
F 2
5-7
5 (
L.s
-1)
0
0.5
1.00
Baseline F+T
Airway functionAirway function
p<0.05
n=30
0
1
2
3
4
5
6
7
Dy
sp
no
ea
Sc
ore
Baseline F+T
p<0.05
SymptomsSymptoms
TheophyllineTheophylline
Oxidative stressOxidative stress
HDAC2 activityHDAC2 activity
Steroid sensitivitySteroid sensitivity
HD
AC
act
ivit
y (
g o
f st
and
ard
)
15
10
5
0Non-treated
H2O2 [200 μM]
#
Theo 1μM
*
Non-treated
Black BoxPI3KPI3K
LY: LY 294002, non-selective PI3K inhibitor
LY
**
HOW DOES THEOPHYLLINE RESTORE HDAC?HOW DOES THEOPHYLLINE RESTORE HDAC?
U937 cellsU937 cells
0
25
50
75
100
125
En
zym
e a
ctiv
ity
(NT
-10
0)
Theophylline (-log10 M)
3456789
Immunoprecipitated Immunoprecipitated PI3K-PI3K-δδ
ICIC5050=2.1=2.1µMµM
A549 cells
PI3K-PI3K-δδKD AND STEROID SENSITIVITYKD AND STEROID SENSITIVITY
U937: TNF-U937: TNF-ααstim, IL-8 ELISA stim, IL-8 ELISA
H2O2 +-Scr. OligoScr. Oligo
IC50
-Dex
(n
M)
10
100
1
1000
0.1
21
P<0.05
213
Ste
roid
Inse
nsi
tive
siRNA knock downsiRNA knock down
+-PI3K-PI3K-δδ-KD-KD
28
NS
39
δδ
+-PI3K-PI3K-γγ-KD-KD
26
P<0.05
272γγ
0
1.0
2.0
Air
Ne
utr
op
hils
(x
104
ce
lls/m
l)
NS
BALBAL
Cigarette smoke (4%, 30 min)Cigarette smoke (4%, 30 min)
DrugsDrugs
days1 2 3 4 5 6 7 8 9 10 11 12 13 14
***
Dex
NS
****
Smoke
IC87114: PI3K-IC87114: PI3K-δδ inhibitor inhibitorLY294002: pan PI3K inhibitorLY294002: pan PI3K inhibitor
IC Dex+IC Dex+LY
PI3K-PI3K-δδ INHIBITION INHIBITION IN VIVOIN VIVOA/J MiceA/J Mice
0
50
100
150
200
250
BA
L N
eutr
op
hil
s/m
l x
103)
Wild type (balb/c)
*
PI3K-δ null PI3K-γ null
Sham Smoke Smoke + budesonide
Marwick J et al: AJRCCM 2008
PI3K-PI3K-δδ KNOCK-OUT MICE KNOCK-OUT MICE
Budesonide also Budesonide also ↓ KC (IL-8)↓ KC (IL-8)and IL-6 in -and IL-6 in -δδ but not - but not -γγ K.O. K.O.
Cell membraneCell membrane
Oxidative stressOxidative stress
REVERSAL OF CORTICOSTEROID RESISTANCEREVERSAL OF CORTICOSTEROID RESISTANCE
PP
SteroidSteroidresistanceresistance
PP
↑PI3K-δ
AktAkt
↓↓HDAC2HDAC2
Reversal of Reversal of steroid resistancesteroid resistance
↓ ↓ PI3K-PI3K-δδ THEOPHYLLINETHEOPHYLLINE
↓ ↓ Akt-1Akt-1
↑↑HDAC2HDAC2
PI3K-PI3K-δδ inhibitors inhibitors
AntioxidantsAntioxidants
Akt inhibitorsAkt inhibitors
HDAC2 activators?HDAC2 activators?
MacrolidesMacrolides(non-antibiotic)(non-antibiotic)
IMPLICATIONS FOR NEW COPD TREATMENTSIMPLICATIONS FOR NEW COPD TREATMENTS
New anti-inflammatory treatmentsNew anti-inflammatory treatments
• PDE4 inhibitors PDE4 inhibitors • p38 MAP kinase inhibitorsp38 MAP kinase inhibitors• IKK-2 (NF-IKK-2 (NF-κκB) inhibitorsB) inhibitors• PI3-kinase-PI3-kinase-γγ inhibitors inhibitors
Alternative approaches: Restore steroid sensitivityAlternative approaches: Restore steroid sensitivity!!
• Low dose theophylline: cheap, combination with steroidLow dose theophylline: cheap, combination with steroid
• Effective antioxidants: e.g. EC-SODEffective antioxidants: e.g. EC-SOD
• PI3-kinase-PI3-kinase-δδ inhibitors inhibitors
• Other HDAC2-selective activators (macrolides?)Other HDAC2-selective activators (macrolides?)
High risk of side effectsHigh risk of side effectsInhaled deliveryInhaled delivery
CONCLUSIONSCONCLUSIONS
• Amplification of inflammatory response to irritantsAmplification of inflammatory response to irritants• LTBLTB44, IL-8, GRO-, IL-8, GRO-, TNF-, TNF-, CXCR3 agonists, MMP-9, CXCR3 agonists, MMP-9
• ↑ ↑ Chemotactic response to GRO-Chemotactic response to GRO-αα (↑ macrophage/neutrophil) (↑ macrophage/neutrophil)
• Resistance to anti-inflammatory effects of corticosteroidsResistance to anti-inflammatory effects of corticosteroids
• HDAC2 in macrophages and peripheral lungHDAC2 in macrophages and peripheral lung amplification of inflammation, steroid resistance, amplification of inflammation, steroid resistance, due to oxidative stress (due to oxidative stress (→→PI3KPI3Kδδ activation) activation)• ↓ ↓ HDAC2 rHDAC2 reversed by low dose theophylline eversed by low dose theophylline - reverses steroid resistance in COPD cells, smoking mice- reverses steroid resistance in COPD cells, smoking mice - mediated via direct PI3K-- mediated via direct PI3K-δδ inhibition inhibition
• New therapeutic approaches now possible New therapeutic approaches now possible (reversal of steroid resistance)(reversal of steroid resistance)
ACKNOWLEDGEMENTSACKNOWLEDGEMENTSIan AdcockIan AdcockBorja CosioBorja Cosio
Gaetano CaramoriGaetano CaramoriFan ChungFan Chung
Sarah CulpittSarah CulpittLouise DonnellyLouise Donnelly
Paul FordPaul FordKaz ItoKaz Ito
Ellen JazwrawiEllen JazwrawiMasa KagoshimaMasa KagoshimaVicki KatsaounouVicki Katsaounou
Vera KeatingsVera KeatingsJohn MarwickJohn MarwickGrace OsoataGrace Osoata
Richard RussellRichard RussellYasuo ToYasuo To
Loukia TsaprouniLoukia TsaprouniSatoshi YamamuraSatoshi Yamamura
NHLIImperial College
Royal Brompton Hospital
Jim HoggJim Hogg (UBC, Vancouver)(UBC, Vancouver)Mary FitzGeraldMary FitzGerald (Argenta)(Argenta)Yasuo KizawaYasuo Kizawa (Nihon University)(Nihon University)Neil ThomsonNeil Thomson (Glasgow University)(Glasgow University)
FUNDED BY:FUNDED BY:Wellcome TrustWellcome TrustMRCMRCAsthma UKAsthma UK