NEW MECHANISMS ANDNEW MECHANISMS ANDTHERAPEUTIC TARGETS IN COPDTHERAPEUTIC TARGETS IN COPD

Peter Barnes FRS, FMedSciNational Heart & Lung InstituteImperial College, London, UK

Imperial College Royal Brompton Hospital

Turkish Thoracic Society: April 2014Turkish Thoracic Society: April 2014

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?What are the genetic/epigeneticWhat are the genetic/epigeneticmechanisms?mechanisms?

Cigarette smoke (and other irritants)

MacrophageEpithelial cells

Fibrosis(Small airways)

TGF-β CTGF

Fibroblast

PROTEASES PROTEASES

Alveolar wall destruction(Emphysema) Mucus hypersecretion

Neutrophil elastaseMMP-9

Neutrophil

CXCL1CXCL8

CXCR2CXCR2

Monocyte

CCR2CCR2

CCL2

INFLAMMATION IN COPDINFLAMMATION IN COPDBarnes PJ: JCI 2008

Tc1Th1

CXCR3CXCR3

CXCL9 CXCL10CXCL11

Th17Tc1

IL-23COPD inflammationCOPD inflammationis corticosteroid-resistantis corticosteroid-resistant

(in contrast to asthma)(in contrast to asthma)

WHY?WHY?

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

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

HDAC2HDAC2

Peripheral lung

Ito K et al: N Engl J Med 2005

CORTICOSTEROID RESISTANCE IN COPDCORTICOSTEROID RESISTANCE IN COPD

Cell membraneCell membrane

Steroid resistanceSteroid resistance

Nitrative stressNitrative stress

↓↓HDAC2HDAC2

NONOTyrTyr

Oxidative stressOxidative stress

PeroxynitritePeroxynitrite

↓↓HDAC2HDAC2UbUbUb

Ub

AktAkt

↓↓HDAC2HDAC2

PP

PP

↑ ↑PI3KPI3Kδδ

↓↓HDAC2HDAC2

Ub

UbUb

Ub

Exhaled PeroxynitriteExhaled Peroxynitrite

0

100

200

300

Pe

roxy

nit

rite

(n

M)

N COPD

400

p<0.001

Osoata G et al: Chest 20090

5000

10000

15000

20000

HD

AC

ac

tiv

ity

(A

FU

/10µ

g)

****

B/L Theophylline (10-6M)

COPD macrophages COPD macrophages

Cosio B et al: J Exp Med 20 04

HDACHDAC

↑PI3Kδ

Cell membraneCell membrane

Oxidative stressOxidative stress

REVERSAL OF CORTICOSTEROID RESISTANCEREVERSAL OF CORTICOSTEROID RESISTANCE

PP

SteroidSteroidresistanceresistance

PP

AktAkt

↓↓HDAC2HDAC2

THEOPHYLLINETHEOPHYLLINE

Reversal of Reversal of steroid resistancesteroid resistance

↓ ↓ PI3KPI3Kδδ

↓ ↓ Akt-1Akt-1

↑↑HDAC2HDAC2

MacrolidesMacrolides

PI3KPI3Kδδ inhibitors inhibitors

NortriptylineNortriptyline

AntioxidantsAntioxidantsNrf2 activatorsNrf2 activators

Akt inhibitorsAkt inhibitors

HDAC2 activators?HDAC2 activators?

GSK3GSK3ββ activators activators

Nrf2 AND ANTIOXIDANT GENE REGULATIONNrf2 AND ANTIOXIDANT GENE REGULATION

Antioxidant genesAntioxidant genes(GPX, HO-1, catalase etc)(GPX, HO-1, catalase etc)

NucleusNucleus

Nrf2Nrf2

ARE

mRNAmRNA

AntioxidantsAntioxidantsKeap-1Keap-1

OXIDATIVE STRESSOXIDATIVE STRESS

UbUb

BZip transcription factor

Nrf2(-/-): ↑ emphysema in smoking miceRangasamy T et al: :JCI 2004;Ishii et al: J Immunol 2005

Nrf2 activity in lungNrf2 activity in lung↑ ↑ in normal smokersin normal smokers↓ ↓ in COPD patientsin COPD patientsMalhotra D et al: AJRCCM 2008

No ↑ with ox stress in COPDNo ↑ with ox stress in COPDDue to Nrf2 acetylationDue to Nrf2 acetylationfrom ↓ HDAC2from ↓ HDAC2Mercado N et al: BBRC 2010

Nrf2-ARE bindingNrf2-ARE bindingR

elat

ive

adso

rban

ce

0

0.25

0.50

Healthy Smoker

*

Mercado N et al: BBRC 2011

2.0

Ace

tyla

ted

:to

tal N

rf2

0.0

0.5

1.0

1.5

C TSA

Nrf2 acetylationNrf2 acetylation*

Lung+H2O2

**

Nrf2 REGULATION IN COPDNrf2 REGULATION IN COPD

COPD

Oxidative stressOxidative stress

Nrf2Nrf2

ARE gene transcriptionARE gene transcription

AcAc

↓↓HDAC2HDAC2

↓↓AntioxidantsAntioxidants

↑↑Oxidative stressOxidative stress

SULFORAPHANE ↑ HDAC2 VIA Nrf2 ACTIVATIONSULFORAPHANE ↑ HDAC2 VIA Nrf2 ACTIVATION

Nrf2-/-

0

10

20

30

HD

AC

act

ivit

y (μ

M/μ

g p

rote

in)

Contr Sulfo

Nrf2+/+

HDAC activity in vivoHDAC activity in vivo

**Mice

BroccoliBroccoliBroccoli sproutsBroccoli sproutsWasabiWasabi

Sulforaphane (isothiocyanate) Sulforaphane (isothiocyanate) Destabilises KEAP-1→↑Nrf2Destabilises KEAP-1→↑Nrf2

HDAC2 activityHDAC2 activity (COPD macrophages)(COPD macrophages)

HD

AC

2 (μ

M/μ

g p

rote

in)

0

2

4

6

8

10

Contr Sulfo

**

Malhotra D et al: J Clin Invest 2011

Nrf2 activators as therapeuticsNrf2 activators as therapeutics• Sulforaphane: clinical trial in COPDSulforaphane: clinical trial in COPD• Triterpenoids: bardoxelone methyl (CDDO)Triterpenoids: bardoxelone methyl (CDDO) high toxicityhigh toxicity• Olipraz (dithiolethione)Olipraz (dithiolethione)• Dimethyl fumarate (BG12): now approved in MSDimethyl fumarate (BG12): now approved in MS• Novel Nrf2 activators Novel Nrf2 activators

IMPLICATIONS FOR NEW TREATMENTSIMPLICATIONS FOR NEW 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• pan-JAKpan-JAK inhibitorsinhibitors

Alternative approach: Alternative approach: Restore steroid sensitivityRestore steroid sensitivity!!

Repurposing existing therapies:Repurposing existing therapies:

• Low dose oral theophyllineLow dose oral theophylline

• Oral nortriptyline, macrolidesOral nortriptyline, macrolidesDevelopment of new therapies:Development of new therapies:

• Inhaled PI3KInhaled PI3Kδδ, PI3K, PI3Kγγ//δδ inhibitors (RespiVert/J&J) inhibitors (RespiVert/J&J)

• Non-antibiotic macrolidesNon-antibiotic macrolides

High risk of side effectsHigh risk of side effectsInhaled delivery neededInhaled delivery needed

Theophylline is cheap, safe in low doses, oral (small airways)Theophylline is cheap, safe in low doses, oral (small airways)suitable for oral combinationssuitable for oral combinationsLong-term controlled trials (1 yr) with theophylline in COPDLong-term controlled trials (1 yr) with theophylline in COPD• Low dose oral theophylline (plasma conc ~5mg/L)Low dose oral theophylline (plasma conc ~5mg/L) + low dose + low dose inhaledinhaled steroid: UK NHS study (TWICS) steroid: UK NHS study (TWICS)• Low dose oral theophylline Low dose oral theophylline + low dose + low dose oraloral steroid (prednisone 5mg): China (TASCS) steroid (prednisone 5mg): China (TASCS)

Lu

ng

fu

nct

ion

(%

max

)

Age (yr)

Anti-ageing molecules?Anti-ageing molecules?

16060-70 80-90 25

100

50

0

COPDCOPD

Oxidative stressOxidative stress

Normal ageingNormal ageing

SenileSenileemphysemaemphysema

ACCELERATED AGEING IN COPDACCELERATED AGEING IN COPD

Ito K, Barnes PJ: Chest 2009

GeroprotectorsGeroprotectors

AntioxidantsAntioxidants

AGEINGAGEING

PROGRAMMEDPROGRAMMED

Telomere shorteningTelomere shorteningReplicative senescenceReplicative senescence

NON-PROGRAMMEDNON-PROGRAMMED

Oxidative stressOxidative stress

UV irradiationUV irradiation

DNA damageDNA damage

Counteracted by anti-ageing molecules Counteracted by anti-ageing molecules including: sirtuins SIRT1, SIRT6including: sirtuins SIRT1, SIRT6 HDAC2HDAC2 FOXO3aFOXO3a

Nrf2Nrf2 Klotho, SMP30Klotho, SMP30

Sirtuin SIRT1: anti-ageing and repair moleculeSirtuin SIRT1: anti-ageing and repair molecule protein deacetylase (non-histone proteins)protein deacetylase (non-histone proteins)

↓ ↓ SIRT1 IN COPDSIRT1 IN COPD

0 25 50 75 1000.0

0.1

0.2

FEV1/FVC (%)

SIR

T1/

GN

B2L

1

p<0.001r=0.658

Good correlation with FEV1 (r=0.72)non-smoker smoker

0

2

4

6

COPD

SIR

T1/

lam

ine

A/C p<0.001

SIRT1 protein

Nakamuru Y et al: FASEB J 2009

↓ ↓ SIRT1 expression in COPD lung and PBMCSIRT1 expression in COPD lung and PBMC Also ↓SIRT6; SIRT2,3,4,5,7 normalAlso ↓SIRT6; SIRT2,3,4,5,7 normal

0

1000

2000

3000

4000

Cntrl H2O2

(200μM)

AF

U

**

SIRT1 activitySIRT1 activity

H2O20 4 24 h

SIRT1

lamin

(200μM)

SIRT1 mRNASIRT1 mRNA

OXIDATIVE STRESS OXIDATIVE STRESS ↓ SIRT1↓ SIRT1

U937 cellsU937 cells

Nakamuru Y et al: FASEB J 2009

MM

P-9

mR

NA

(% G

AP

DH

)

0

5

10

15 *

Ac

tivi

ty-O

D

0

0.5

1.0

1.5 *

B/L PMA PMA + Splitomycin (100 μM)

↓ ↓ SIRT1 ↑MMP9SIRT1 ↑MMP9U937 cells

MMP-9 mRNAMMP-9 mRNA

MMP-9 activityMMP-9 activity

SIRT inhibitorSIRT inhibitor

*

0

0.5

1.0

1.5

MM

P-9

/GA

PD

H

mR

NA

1

2

3

4

5

0

*

Ac

tivi

ty-O

DB/L PMA Scr SIRT1-

MMP-9 mRNAMMP-9 mRNA

MMP-9 activityMMP-9 activity

SIRT1 siRNASIRT1 siRNA

Nakamuru Y et al: FASEB J 20099

↓↓SIRT1SIRT1

↑ ↑ Oxidative stressOxidative stress

↓↓Ku70Ku70

↓ ↓ DNA RepairDNA Repair

AcAc

↓↓FoxO3aFoxO3a↑ ↑ p53p53

AcAc AcAc

↓↓Nrf2Nrf2

↑ ↑ Oxidative stressOxidative stress

AcAc

↑↑NF-NF-κκBB

↑ ↑ InflammationInflammation↑ ↑ AgingAging

AcAc

↑ ↑ IL-8IL-8↑ ↑ MMP-9MMP-9

EFFECTS OF EFFECTS OF ↓ SIRT1↓ SIRT1

Cell membraneCell membrane

RESTORING SIRT1 RESTORING SIRT1

↑ ↑ LUNG AGEINGLUNG AGEING

↑↑PI3KPI3Kδδ

↑↑AktAkt

↑ ↑ mTORmTOR

AMPKAMPK MetforminMetformin

RapamycinRapamycin

TheophyllineTheophyllineNortriptylineNortriptylinePI3KPI3Kδδ inhibitors inhibitors

↓↓SIRT1SIRT1

AntioxidantsAntioxidantsNrf2 activatorsNrf2 activators

Sirtuin activatorsSirtuin activatorsResveratrolResveratrol

Oxidative stressOxidative stress

0 1 10 1000

500

1000

SIR

T1

acti

vity

(%

bas

al)

Resveratrol (μM)

10x↑

SIRT1 activitySIRT1 activity

0.1 1 10 1000

20

40

60

80

100

120

MM

P-9

(%

co

ntr

ol)

IC50=9μM

PMA

Resveratrol (μM)

PMA+RV

MMP-9

MMP-9 activityMMP-9 activity

ZymographyZymography

RESVERATROL ON SIRT1 AND MMP-9RESVERATROL ON SIRT1 AND MMP-9

U937 cells

SIRTUIN ACTIVATORS (STACs)SIRTUIN ACTIVATORS (STACs)Plant polyphenols:Plant polyphenols:• ResveratrolResveratrol• QuercitinQuercitin• PiceatannolPiceatannol Poor oral bioavailabilityPoor oral bioavailability More potent synthetic SIRT1 activators (e.g. SRT2172)More potent synthetic SIRT1 activators (e.g. SRT2172) Inhaled resveratrol?Inhaled resveratrol?

SIRT1 activationSIRT1 activation

Concentration (-log,M)

% A

ctiv

atio

n

80

120

160

200

240

280

8 7 6 59

SRT-2172(Sirtris)

Resveratrol

% o

f c

on

tro

l0 9 8 7 6 5

0

20

40

60

80

100

120

Concentration (-log,M)4

SRT-2172

MMP-9 activityMMP-9 activity

U937 cells exposed to H2O2

SIRT ACTIVATORSIRT ACTIVATOR

Nakamuru Y et al: FASEB J 2009

0

1

2

3

4

5

MM

P-9

/GA

PD

H

** ** **

SRTSalSal Dex

MMP-9 expressionMMP-9 expression

AirSmoke

SIRT ACTIVATOR IN SMOKING MICESIRT ACTIVATOR IN SMOKING MICES

IRT

1 ac

tivi

ty

(un

it/u

g p

rote

in)

0

5

10

15

20

AirSalSal

Smoke

SIRT1 activitySIRT1 activity

**

SRT Dex

**** **

SRT-2171

Nakamuru Y et al: FASEB J 2009

↑↑PI3KPI3Kδδ

↓↓SIRT1SIRT1

Oxidative stressOxidative stress

↑ ↑ LUNG AGINGLUNG AGING

↑ ↑ CARDIOVASCULAR AGING CARDIOVASCULAR AGING

↓↓eNOSeNOS ↑↑MMP9MMP9

↑ ↑ Arterial stiffnessArterial stiffness

AtherosclerosisAtherosclerosis HypertensionHypertension

EmphysemaEmphysema

↓↓LV functionLV function

Cardiac failureCardiac failure

↓ ↓ SIRT1 AND ACCELERATED AGINGSIRT1 AND ACCELERATED AGING

DiabetesDiabetesMetabolic syndromeMetabolic syndrome

OsteoporosisOsteoporosisSkin Skin

wrinklingwrinkling

Increased skin-wrinklingIn COPD patientsPatel BD et al: Thorax 2006

Skin wrinkling associated with ↑ dermal MMP-9

Endothelial colony forming cellsEndothelial colony forming cells

VWFVWF++/CD45/CD45--

7-22 days7-22 days

ENDOTHELIAL PROGENITOR CELLSENDOTHELIAL PROGENITOR CELLS

Paschalaki K et al: Stem Cells 2013Paschalaki K et al: Stem Cells 2013

Cel

l n

um

ber

/wel

l x

10C

ell

nu

mb

er/w

ell

x 10

3300

11

22

33

Normal COPD Normal COPD

ECFCECFC

MatrigelMatrigel

COPD COPD vsvs controls controls• Proliferation +/- VEGF: unchangedProliferation +/- VEGF: unchanged• Migration: unchangedMigration: unchanged• ↓ ↓ AngiogenesisAngiogenesis• ↑ ↑ ApoptosisApoptosis• Failure to repair endothelial injuryFailure to repair endothelial injury

Normal (72yr)Normal (72yr)

COPD (69 yr)COPD (69 yr)

↑ ↑ CELLULAR SENESCENCE IN EPC FROM COPDCELLULAR SENESCENCE IN EPC FROM COPD

SA-SA-ββ-GAL-GAL SA

-β-G

AL

+ /

tota

l ce

lls

0

0.1

0.2

0.3

Normal COPDNormal COPD (n=9) (n=11)(n=9) (n=11)

PP<0.01<0.01

Senescence-associated Senescence-associated ββ-galactosidase-galactosidase

0

100

200

S

IRT

1 ac

tivi

ty

(OD

/μg

pro

tein

)

Normal COPDNormal COPD

PP<0.05<0.05

Correlated with Correlated with ↑ SA-↑ SA-ββ-GAL-GAL

EPC SIRT1 activityEPC SIRT1 activity

Paschalaki K et al: Stem Cells 2013Paschalaki K et al: Stem Cells 2013

eNO

S p

rote

in

Normal COPD

*

EPC eNOSEPC eNOS

MonocytesMonocytes

CCL2CCL2CXCL1CXCL1

CCR2CXCR2

CD8CD8++ cells cells

CXCL9CXCL9CXCL10CXCL10CXCL11CXCL11

CXCR3

NeutrophilsNeutrophils

LTBLTB44

CXCL1CXCL1CXCL8CXCL8

CXCR2

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

EmphysemaEmphysema

NONOROSROS

HDACHDAC Steroid Steroid responseresponse

• Numbers (25X)Numbers (25X)• ↑ ↑ Mediator secretionMediator secretion• Steroid resistanceSteroid resistance• ““M2-like”M2-like”Chana K et al: JACI 2013

MDMMDM

+10% FCS+10% FCS+GM-CSF x 12d+GM-CSF x 12d

SmSmNon-smNon-sm COPDCOPD00

22

44

66

**

Ph

ago

cyto

sed

P

hag

ocy

tose

d E

co

liE

co

li ( (μμ

g)

g)

nsns

MonocytesMonocytes

Ph

ago

cyto

sed

P

hag

ocy

tose

d E

co

liE

co

li ( (μμ

g)

g)

SmSmNon-smNon-sm00

22

44

66

COPDCOPD

nsnsnsns

↓↓ MACROPHAGE PHAGOCYTOSIS IN COPDMACROPHAGE PHAGOCYTOSIS IN COPD

SmSmNon-smNon-sm00

22

44

66

Ph

ago

cyto

sed

P

hag

ocy

tose

d E

co

liE

co

li ( (μμ

g)

g)

COPDCOPD

**

nsns

Taylor AE et al: Eur Resp J 2010

Alveolar MacrophagesAlveolar Macrophages

Fluorescent Fluorescent E coli uptakeE coli uptake

0

0.01

0.02

0.03

0.04

0.05

Ph

ago

cyto

sis

**

Haemophilus influenzaeHaemophilus influenzae

Normal Smoker COPD

Monocyte-derived macrophages

↓↓ MACROPHAGE PHAGOCYTOSIS IN COPDMACROPHAGE PHAGOCYTOSIS IN COPD

Taylor AE et al: Eur Resp J 2010

0.00

0.02

0.04

0.06

0.08

0.10

Ph

ago

cyt

os

is

Strep. Strep. pneumoniaepneumoniae

Normal Smoker COPD

**

↓↓Phagocytosis of bacteria (Gram +ve and -ve)Phagocytosis of bacteria (Gram +ve and -ve)NormalNormal phagocytosis of inert beads phagocytosis of inert beadsAbnormality in scavenger receptors?Abnormality in scavenger receptors?

Alveolar macrophage

Bacteria

Sterilisation ofSterilisation ofRespiratory tractRespiratory tract

Apoptoticneutrophils

Resolution ofResolution ofinflammationinflammation

• Non-smoker• Normal smoker• COPD

Chronic colonisationChronic colonisationPersistent inflammationPersistent inflammation

MACROPHAGE PHAGOCYTOSISMACROPHAGE PHAGOCYTOSIS

DefectiveDefectivephagocytosisphagocytosis

↑ ↑ InflammatoryInflammatorymediatorsmediators

Scavenger receptorsMARCO, CD36, CD163,Mannose receptor, PS receptor

≤1 exac/yr ≥3 exac/y0

5

10

15

20

**

RF

U (

x103

)

Infrequent Frequent

H. influenzaeH. influenzae

Catherine Thomas

Bacterial phagocytosis by MDMsBacterial phagocytosis by MDMs

FREQUENT FREQUENT vsvs INFREQUENT EXACERBATORS INFREQUENT EXACERBATORS

Strep. pneumoniaeStrep. pneumoniae

≤1 exac/yr ≥3 exac/y0

5

10

15

RF

U (

x103

)

Infrequent Frequent

* New treatments to restore bacterial phagocytosisNew treatments to restore bacterial phagocytosisMicrotubular stabilisers (epothilone)Microtubular stabilisers (epothilone)New signalling pathways identified (e.g. S1P pathway)New signalling pathways identified (e.g. S1P pathway)

CONCLUSIONSCONCLUSIONS

• Corticosteroid resistance in COPD - via oxidative stress: ↓HDAC2, ↑PI3Kδ - reversed by theophylline, macrolides, PI3Kδ inhibitors, Nrf2 activators• Accelerated aging in COPD: cellular senescence Anti-aging molecules: SRT activators, PI3K inhibs, metformin

• New approaches include restoring defective phagocytosis e.g. microtubular stabilisersUnderstanding COPD mechanismsUnderstanding COPD mechanisms

Identifies new treatment targetsIdentifies new treatment targets More effective therapy in the future toMore effective therapy in the future to ↓ ↓ disease progression, ↓ mortalitydisease progression, ↓ mortality ↓ ↓ exacerbatons, ↓ comorbiditiesexacerbatons, ↓ comorbidities

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

Ian AdcockIan AdcockKylie BelchamberKylie Belchamber

Borja CosioBorja CosioGaetano CaramoriGaetano Caramori

Kiran ChanaKiran ChanaFan ChungFan Chung

Louise DonnellyLouise DonnellyPaul FordPaul Ford

Kaz ItoKaz ItoEllen JazrawiEllen Jazrawi

Masa KagoshimaMasa KagoshimaYoshi KobayashiYoshi Kobayashi

John MarwickJohn MarwickNico MercadoNico MercadoGrace OsoataGrace Osoata

Koralia PaschalakiKoralia PaschalakiAnna RandiAnna RandiRicha SinghRicha Singh

Abigail TaylorAbigail TaylorCatherine ThomasCatherine Thomas

Yasuo ToYasuo ToWisia WedzichaWisia Wedzicha

NHLIImperial College

Royal Brompton Hospital

Jim HoggJim Hogg (Vancouver, Canada)(Vancouver, Canada)Yasuo KizawaYasuo Kizawa (Tokyo, Japan)(Tokyo, Japan)Shyam BiswalShyam Biswal (Baltimore, USA)(Baltimore, USA)Sundeep SalviSundeep Salvi (Pune, India)(Pune, India)

FUNDED BY:FUNDED BY:Wellcome TrustWellcome TrustMRCMRCAstraZenecaAstraZenecaCempraCempraGSKGSKMitsubishi-TanabeMitsubishi-TanabeNovartisNovartisPfizerPfizer