Identifying New Therapeutic Targets for Severe Asthma:The S100A8/A9-RAGE Axis

Andrew J. Halayko, PhDCanada Research Chair in

Airway Cell & Molecular Biology

ASM

*

Mucousgland

Epi

Smooth muscle

Mucous gland

Bloodvessels

(Myo)fibroblasts

Mast cellEosinophil NeutrophilLymphocyte

INFLAMMATION, TISSUE DAMAGE & REPAIR

TH-2 / Th-1 cytokines & chemokines

TGF1, EGF

eotaxin, IL-8,IL-6, collagen

Airway Remodeling in Asthma

Halayko & GhavamiCan J Physiol Pharmacol

87:743-755, 2009

Neutrophils & Airway Inflammation• Steroid refractory asthma and COPD (smoking):

neutrophilic inflammation (+/- eosinophils) & airway remodeling

• Need to identify neutrophil-released factors that circumvent steroid therapy

• Degranulation•Oxidative burst (ROS)

•Proteases (elastase)• Cytokines (IL-1β, 6, 8, TNF,

GMCSF)• Lipid mediators (LTB4)

• Other? (S100A8 & S100A9)

“priming”

“activation”

Ca2+

Ca2+

S100 Protein Family (Calgranulins)

• >20 acidic Ca2+-binding proteins (1q21 gene cluster)

• Cell, tissue & species-specific expression

Intracellular role:• Ca2+ signaling; microtubule-associated reg. of migration

Extracellular role (a.k.a. alarmins; endokines):• Pro-inflammatory Damage-Associated Molecular Pattern

proteins (eg. HMGB1, S100s, IL-33)• Receptors include TLRs & RAGE

S100A8

Ca2+

Ca2+

S100A8 (MRP8) & S100A9 (MRP9)

• Heterodimer secretion induced in phagocytes, endothelium, and epithelium (eg. foreign surface contact; LPS)

• Pro-inflammatory (leukocyterecruitment) & innate immunity regulation (inhibit dendriticcell differentiation via MDSC)

S100A8monomer

• >40% of soluble cytoplasmic proteins in neutrophils & macrophages

S100A8 corticosteroid refractory in LPS-induced neutrophilic lung inflammation in mice (Bozinovski et al. J Proteome Res 4: 136-45,

2005 )

(AGEs; β-amyloid protein; HMGB1 S100s incl. S100A12, S100A8/A9)

• Multiple variants & signaling (cell, tissue & environment specific profile)

• Low expression in “healthy” tissue EXCEPT lung

• RAGE KO mice resistant to bleomycin lung fibrosis

S100A8/A9 Receptor: RAGEReceptor for Advanced Glycation End Products

intracellularsR

AG

E

• Multi-ligand immunoglobulin family receptor:

S100A8/A9 in Human Asthma

250

500

2500

3000

S1

00

A8

/A9

(n

g/m

L)

Human BALF

Healthy SevereAsthma

P<0.05

Healthy

Asthma

S100-Receptor Axis in Human Airway Smooth Muscle Cells

S100A8

S100A9

HM

GB1

TLR

4

RAG

E

GAPD

H

100 bp

200 bp

300 bp

650 bp

RAGE: A Functional S100A8/A9 Receptor on Human ASM

Collagen (130kDa)

Total ERK

p-ERK

GAPDH

BSA (10μM) -

S100A8/A9 (2.5μg/mL) -AGE-BSA (10μM) -

+

--

-

-+

-

+-

+

+-

S100A8/A9 Induces Proliferation:Human ASM

PR

OLIF

ER

ATIO

N(B

rdU

% C

on

trol)

0

500

1000

1500

2000

0

200

400

600

800

1000

1200

1400

Eotaxin-1

IL-8

Control

TNF

(10ng/mL)

S100A8/A9 (g/mL)

Eota

xin

-1 (

ng

/mL)

IL-8

(n

g/m

L) *

*

**

*

*

2 10 20

Pro-Inflammatory Role of S100A8/A9: Cultured Human ASM

Pro-Fibrotic Role of S100A8/A9:Cultured Human ASM

GAPDH (37 kD)

Time (hrs) 0 24 48 72 24 48 72

S100A8/A9 (2 µg/ml) - + + + - - -

S100A8/A9 (10 µg/ml) - - - - + + +

Collagen 1 (140 kD)

RAGE (42 kD)

“Cell Associated”

Time (hrs) 0 24 48 72 24 48 72

S100A8/A9 (2 µg/ml) - + + + - - -

S100A8/A9 (10 µg/ml) - - - - + + +

Collagen 1 (140 kD)

Mature collagen 1 (70 kD)

“Secreted”

The existence of a link between S100 protein expression and asthma symptoms, pathogenesis and severity is unresolved.

Murine models of allergic airway inflammation that lead to asthma-like symptoms and pathology hold promise to asses this link.

S100A8/A9 is casually linked with airway inflammation, hyperresponsiveness & remodeling in

murine models of “asthma”

Current State of Knowledge

Hypothesis

S100A8

S100A9

HM

GB1

TLR

4RAG

EG

APD

H

100 bp

200 bp

300 bp

S100-Receptor Axis in Murine Lung

Week 2 Weeks 3 - 7Week 1

35L HDM (0.7mg/mL)

Intranasal HDM Challenge

D1

D2

D3

D4

D5

D1

D2

D3

D4

D5

D1

D3

D5

AcuteChronic

Respiratory Mechanics & BALF, Serum

35L HDM (0.7mg/mL)

35L HDM (0.7mg/mL)

Acute & Chronic HDM Exposure Mouse ModelD. pteronnyssinus

0

15000

30000

45000

60000

75000

Naïve mid week 8 hr IN 24 hr IN 48 hr IN 72 hr IN

0

5000

10000

15000

20000

25000

Naïve mid week 8 hr IN 24 hr IN 48 hr IN 72 hr IN

**

**

**

****

* *

*

Time after HDM (hrs)

Mid-wk

Naive

8 24 72

**

Eosinophils

Neutrophils

**

Cells/m

L

BA

LF

Cells/m

L

BA

LF

48

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

cmH

2O.s

/ml

Acute HDM

Naive

Airway Resistance

Raw

(c

mH

2O

.s/m

L)

Methacholine (mg/mL)

Acute HDM Challenge: Temporal Pattern of Neutrophil & Eosinophil Recruitment

0

500

1000

1500

2000

2500

3000

Naïve mid week 8hr IN 24hr IN 48hr IN 72hr IN

S1

00

A8

/A9

(n

g/m

l)

S100A8/A9 (ng/mL) BALF

**

Time after HDM (hrs)

Mid-wk

Naive

8 24 48 72

Eosinophils

Neutrophils

… & tracks with neutrophil infiltration

Acute HDM Challenge InducesS100A8/A9 Accumulation in Airways

Acute HDM Challenge InducesAirway S100A8/A9 Accumulation

Naïve Balb/c HDM Challenge (8hrs post)

Acute Chronic

Total-p38

phospho-p38

Total-ERK1/2

phospho-ERK1/2

β-Actin

RAGE

Control HDM HDMControl

HDM Challenge Induces RAGE and Associated Downstream Signaling

Whole lung lysate – Balb/c mice

Acute HDM Challenge InducesAirway RAGE Expression

HDM Challenge (8hrs post)Naïve Balb/c

ASM

Nai

ve

7 w

eeks

H

DM

H & E sm--actin collagen

Chronic HDM Challenge Induces Airway Remodeling & AHR

Airway Resistance

Raw

(c

mH

2O

.s/m

L)

Methacholine (mg/mL)Saline 3 6 12 25 50

0.6

0.9

1.2

1.5

1.8

0.3

0

Chronic HDM

Naive

Chronic HDM Challenge: Sustained Airway Eos and Neutrophils

05000

1000015000200002500030000350004000045000

TimeControl 5

wk

5 wksHDM IN

TimeControl 7

wk

7 wksHDM IN

TimeControl 9

wk

9 wksHDM IN

05000

1000015000200002500030000350004000045000

TimeControl 5

wk

5 wksHDM IN

TimeControl 7

wk

7 wksHDM IN

TimeControl 9

wk

9 wksHDM IN

**

**

*** *B

ALF

cells

/mL

**

Eosinophils Neutrophils

NaiveHDM

NaiveHDM

NaiveHDM

NaiveHDM

NaiveHDM

NaiveHDM

5 Wks 7 Wks 9 Wks 5 Wks 7 Wks 9 Wks

S100A8/A9 (ng/mL)

BALF

HDM Challenge

Naiv

e

Acut

e

Chro

nic

(5 W

KS)

Chro

nic

(9 w

ks)

0

500

1000

1500

2000

2500

3000

S100A8/A9 (ng/mL)

Serum

HDM Challenge

Naiv

e

Acut

e

Chro

nic

(5 W

KS)

Chro

nic

(9 w

ks)

0

300

600

900

Chronic HDM Challenge: Sustained BALF & SERUM S100A8/A9

Chronic HDM Challenge Sustains Elevated Lung S100A8/A9

NaïveBalb/c

5 WeeksHDM

epi

epi

Chronic HDM Challenge Induced Sustained RAGE Expression

Naïve Balb/c 5 Weeks HDM

Acute Chronic

RAGE

Control HDM HDMControl

Summary• S100A8/A9 increased in asthmatic airway (tissue and BALF) & RAGE induced by allergic inflammation

• S100A8/A9, via RAGE, promotes remodeling responses in airway mesenchymal cells

• Allergen airway challenge induces sustained increase in RAGE and S100A8/A9 in airways and serum

• S100A8/A9 accumulation in airways tracks with neutrophil recruitment and development of airway remodeling

ASM

Unanswered Questions• S100A8/A9 & RAGE causally linked to airway remodeling and AHR? (S100A9-/- & RAGE-/- mice)

• S100A8/A9 role in mucosal (innate) immunity in the airways

• Source of S100A8/A9 in allergic inflammation? Selective suppression?

• Is S100A8/A9 expression linked with asthma phenotype & severity? (biomarker; steroid response; exacerbation trigger [RTI])

ASM

Contributors

Sujata BasuKaren DetillieuxSaeid GhavamiAruni JhaHessam KashaniAndrea KroekerMark MutaweDedmer SchaafsmaPawan SharmaJacquie SchwartzGerald StelmackSoma TripathiBehzad Yaganeh

Jamila Chakir, LavalMichel Laviolette, Laval

Johannes Roth, MuensterThomas Vogl, Muenster

John Gordon, Saskatchewan

Abdel Soussi GounniHelmut Unruh

Canada Research Chairs ProgramCanadian Institutes of Child HealthCanada Foundation for Innovation

Man. Inst. Child Health