Common links between Corticosteroid Resistance and Inflammatory Lung Diseases

Common links between Corticosteroid Resistance and Inflammatory Lung

Diseases

Ian Adcock

National Heart and Lung Institute,Imperial College London

Overview• Steroid resistance occurs in airways

disease• Inflammatory signalling pathways

modulate GR function– MAPK, JAK/STAT, NF-kB

• Oxidative stress from cigarette smoke affects GR function– Affects GR nuclear translocation

• INFLAMMATORY PATHWAYS REDUCE THE ABILITY OF ACTIVATED GR TO ENTER THE NUCLEUS

Worldwide prevalence of inflammatory lung diseases

Donnelly & Rogers 2008

Reduced steroid responsiveness seen in severe asthma, COPD, CF and IPF – all chronic inflammatory diseases – associated with oxidative stress and – activation of intracellular transcription pathways

NF-kB is a key mediator of inflammation

p65

p50

IL-1

TNF

LPS

haematopoieticcytokines

Oxidativestress

UV light

TLRs

Cigarette Smoke

Cytokines (IL-6, IL-8, etc)

Adhesion molecules(e.g. ICAM)

inflammatory enzymesiNOS, COX-2

Receptors

NF-kB is central to inflammatory processes

But other TFs also important in cell/stimulus context

0

20

40

60

80

Nucl

ear

stain

ing (

%)

NormalAsthmatic

AsthmaNormal

Normal

COPD

TNFaIL-1 , b TLRs

AP-1

Ras

MEKK-1

JNKK

JNK

P

P

FOS

JUN

NF-AT

CaN

NF-ATc

NF-ATc

P

AP-1

Antigen

STAT

JAK

STAT

P P

Cytokines

NF-kB

TNFa

IkB Kinase

Ub-Proteasome

NF-kB

IkBP

Stimuli

Cytoplasm

Nucleus

NF-kB and other transcription factors

Skin Blanching Response to Topical Budesonide

SRSS

Severe asthma macrophages have less steroid-suppression of cytokine release

GM-C

SF IN

F-IL

-10

IL-1 IL

-6IL

-8

MCP-1

TNF-

RANTES

MIP

-1

0

25

50

75

100

Dexamethasone 10-6 M

**

* ** **

*

Severe asthma

non-severe asthma

non asthmatic

% c

yto

kin

e re

leas

e re

lati

ve t

oL

PS

-sti

mu

lati

on

alo

ne

Bhavsar et al , Thorax 2008

Co-repressor

Mechanisms of steroid action

IkBa, GILZAP-1/NF-kB

X

O

OH

CH2OH

C=OOHCH3

CH3 CH3

F

Cell membrane

OOH

CH2OHC=O

OHCH3

CH3 CH3

F

O

OH

CH2OHC=O

OHCH3

CH3 CH3

FO

OH

CH2OHC=O

OHCH3

CH3 CH3

F

O

OH

CH2OHC=O

OHCH3

CH3 CH3

F

Ligand binding

Nuclear translocation

DNA binding/co-activator function Co-repressor recruitment/activity

0

1

2

3

4

5

0

1

2

3

4

5

Baselin

e t

ota

l G

R/

-acti

n

(fo

ld s

tan

da

rd p

rote

in)

0

1

2

3

4

5 **

Non-asthmatics

(n=10)

Asthmatics(n=14)

Baselin

e t

ota

l G

R/

-acti

n

(fo

ld s

tan

dard

pro

tein

)

US 30 min 1 h 2 h 4 h0

1

2

3

4

Dexamethasone 10-7 M

Non-asthmatics (n=5)Non-severe asthmatics (n=8)Severe asthmatics (n=7)

*##

#

Nu

cle

ar

GR

/TB

P v

s U

S

Reduced GR expression and nuclear translocation in ASM cells from severe

asthma

Chang et al., 2012

MAPKs

0

100

200

300

+---

-

- -

-++

++ +

+

+

IL-2, IL-4PMA/ PHADex

GM

-CS

F p

g/m

l

+--

NS

*

* ** ** *

IL-2/IL-4 induces dex-insensitivity in PBMCs

Irusen et al., 2002

GR nuclear translocation is reduced in IL-2/IL-4 treated U937 cells: role of p38

MAPKNuclear extract

GR

Lamin A

Dex (10-7M)- + - +

IL-2/IL-4 -- + +

Yasuo To/Kaz Ito

IL-2 + IL-4

SB 205380 (1mM)

IP- GR in PBMCs

+

+

+

- +

++

----

-

Therefore postulated that p38 MAPK drives relative steroid resistance in severe asthma

Targets reduced GR nuclear import

Enhanced p38 MAPK activity in PBMC and ASMC of severe asthma

Non-Severe Severe0.0

0.5

1.0

1.5*

p-p3

8 M

APK

/tota

l p38

MAP

K (F

old

US

)

LPS 10 ng/ml

Khorasani et al, 2012 Chang et al, 2012

p38 MAPK sensitivity in BAL macrophages from patients with severe asthma

Æp38 Dex

& Dex

Æp38

Æp38 Dex

& Dex

Æp38

Æp38 Dex

& Dex

Æp38

0

25

50

75

100 * **

IL-1b IL-6 MIP-1 a

% c

ytok

ine

rele

ase

com

pare

d to

LPS

alo

ne

Bhavsar et al., 2008

GW856553 enhances CS mediated suppression of LPS-induced CXCL8 release in severe asthma

0

20

40

60

80

100

+GW 10-9M

+ GW 10-10M

-9 -8 -7 -6

***

***

***

***

Dex [Log M]

% I

L-8

Su

pp

res

sio

n v

s L

PS

Bhavsar et al, 2010

Impaired steroid responsiveness of ASM cells in severe asthma

Po-Jui Chang

GW856553 enhances dex-mediated suppression of TNFα-induced CCL11 release in severe asthmatics

Chang et al, 2012

ASM cells

Targeting p38 MAPK restores steroid sensitivity in COPD macrophages

Kent et al., JPET 2009

MAPK activation in airway disease affects GR nuclear import and function

Opportunity for new combination drugs

Other signalling pathways

JAK-STAT signalling pathwayJAK is an associated tyrosine kinase.

Upon receptor activation, it phosphorylatesseveral tyrosine residues on the receptor.

An inactive STAT binds the phosphorylated receptor. It is then phosphorylated byJAK and dissociates.

Cytokine receptors

0

25

50

75

100

125

-12 -10 -8 -6 -4IFNlog [Drug (M)]

CX

CL

10 (

% C

on

tro

l)

0

25

50

75

100

125

-12 -10 -8 -6 -4IFNlog [Drug (M)]

CX

CL

10 (

% C

on

tro

l)

Airway epithelial cells (BEAS2B, primary cells)

• JAK inhibitor more effective with IFNγ+TNFα (X-talk with NF-κB signalling)

• No effect on IL-1-induced IL-6 (steroid responsive)

• IFNγ-induced CXCR3 chemokines (CXCL9, CXCL10, CXCL11) (relatively steroid ineffective)

JAK inhibitor

Dexa

IFNγ-induced CXCL10 release

STAT6 activation in severe asthma

Mullings et al., J Allergy Clin Immunol. 2001

Normal

Severe asthma Mild asthma

Mild asthma

STAT6 STAT6

STAT6 control

STAT4 activation in COPD

Di Stefano et al., ERJ 2004

Viral infection activates STAT signalling

Kong et al., BBRC 2003

Human Epithelial cells

Liu et al., JBC 2008

EGFR mutant (K721R) kinase inactive defective STAT activation

RV16 infection attenuates steroid function

RV16 infection reduces GR nuclear import:

reversed by NF-kB and JNK inhibitors

LY reverses Bud-insensitivity induced by IL-17

Smoking and oxidative stress

-5

0

5

10

15

20

25

30

Chan

ge in

mor

ning

PE

F (%

)

Placebo

Fluticasone (1mg/day)

Non-smokers (n=21)

Sputum Eos 0 -1.8*% change

**

Chalmers GW et al: Thorax 2002

Smoking and steroid responses in asthma

Smokers (n=17)

0 0

Cigarette smoking inhibits the inflammatory response to corticosteroids in asthma

NAC (-)NAC (+)

0

1000

2000

3000

IL-1bH2O2

- - + +

- + - +

IL-8

(pg/m

l)

*

#

†

IL-8

(pg/m

)

GM

-CSF

(pg/m

)

0

1000

2000

3000IL-8GM-CSF

0

200

400

600

A-549 cells

- - + + IL-1b- + - + H2O2

**

#

**

# #

Oxidative stress enhances IL-1b-stimulated inflammatory gene expression

BUD FP

Oxidative stress reduces corticosteroid activity

-12 -10 -8 -60

20

40

60

80

IL-1IL-1 plus H2O2

Log[BUD], M

CX

CL

8 s

tim

ula

tio

n (

% c

on

tro

l)

-14 -12 -10 -8 -60

20

40

60

80

IL-1IL-1 plus H2O2

Log[FP], MC

XC

L8 s

tim

ula

tio

n (

% c

on

tro

l)

Amir Hakim 2012

? Importance in COPD, smoking asthma and severe asthma

Oxidatives stress reduces GR nuclear translocation

TATA BP

Nuclear GR

* p < 0.05 vs baseline # p < 0.05 vs. corticosteroid

#

*

#

*

BUD FP

Due to effects on Importin 7/Ran GTP systemAmir Hakim 2012

Summary• Chronic inflammation is associated with relative

corticosteroid insensitivity• Linked to activation of key transcription pathways

• NF-kB• p38 MAPK• JAK/STAT activation

– Viral infection and exacerbation• Oxidative stress

• Affects NF-kB-dependent transcription• ROS stress also affects GR function

• GR nuclear import/expression, HDAC2

• The current drivers for excessive pathway activation are unknown in disease – not affect therapeutic strategies

Acknowledgements

Funded by:

Fan Chung Peter J. BarnesKazuhiro ItoPank BhavsarMark HewJohn MatthewsElvis Irusen John MarwickKarina Enesa Paul EvansAmir Hakim Elen Jazrawi

Nadia KhorasaniPo-Jui ChangLouise Donnelly

Imperial College Trust

NIH-RO1 grant HL-69155

Alberto PapiGaetano CaramoriAntonino Di Stefano

Is There Anything Common between Corticosteroid Resistance and Inflammatory Lung Diseases?

30 mins

Increased oxidative stress in COPD

Smoker

COPD0 25 50 75 100 125 150

0

1

2

3

FEV1 (% pred.)

r= -0.76p<0.05

4

4-HNE staining

Irfan Rahman

Kinnula et al., ERJ 2007

Brindicci et al., Chest 2007

Altered glutathione homeostasis in children with severe asthma: ELF

measurements

Fitzpatrick et al., JACI 2009

31 6 31 25

Sputum 8-isoprostane in severe asthma

Wood et al., AJRCCM 2005

Macrophage

IL-6IL-8GM-CSFTNFPDGFPGE2

Proteases

Epitheium

IL-1IL-6IL-8 GM-CSFEicosanoids

Migration

TNF-INF-PDGF

EosinophilIL-3IL-5IL-6GM-CSF

MigrationAdhesion

TNF-TGF-/-

T-lymphocyteIL-2INF-GM-CSF

Migration

NeutrophilIL-3IL-4IL-5IL-6

IL-1IL-8GM-CSF

Adhesion

Endothelium

Cytokines, Cigarette smoke, infectious agents

Inflammatory mediators in respiratory disease

Mast Cell

IL-3IL-4IL-5IL-6

TNF-

Clinical phenotypes of asthma

Bel EH, Curr Opin Pulm Med 2004;10:44-50Wenzel S, Lancet 2006;386:804-813

Eosinophilicsteroid-responsive

ExacerbationproneSevere

Fixedobstruction

Allergic

Reduced steroid responsiveness in severe asthma, COPD, CF and IPF – all chronic inflammatory diseases – associated with oxidative stress and – activation of intracellular transcription pathways

PNAS 2012

Dura

tion o

f p6

5-

dsR

ed n

ucl

ear

loca

lisati

on (

min

)

0 100 2000

100

200

300

400

500

600

H2O2 (mM)

+IL-1b

Oxidative stress prolongs NF-B activity by suppressing its nuclear export

HeLa cells

Non-stim IL-1b + H2O2IL-1b

60 mins

IL-1bH2O2

TSA

--

-

-+

-

+-

-

++

-

--

+

+-

+

Acetylated H4 IP

NF-kB p65 IP

INPUT

ROS stress does not enhance rate of p65 nuclear entry: stimulates histone acetylation

HDAC activity

Control CSM 0.150

2500

5000

7500 **

HD

AC

act

ivit

y(A

FU

/10

g)

Cigarette smoke reduces HDAC activity in macrophages

Y146 - activityY253 - expression