NEW MECHANISMS AND NEW MECHANISMS AND TREATMENT TREATMENT OF COPDOF COPD

Peter BarnesNational Heart & Lung InstituteImperial College, London, UK

Imperial College Royal Brompton Hospital

p.j.barnes@imperial.ac.uk

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