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Nov 23 at 8am
Sala Dios Candangos
Non-Eosinophilic Asthma:
Mechanisms and
Treatment
Prof Peter G Gibson
NHMRC Centre for Respiratory
and Sleep Medicine
Newcastle, Australia
2 people with asthma…
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FEV1%:
BDR%:
PC20 :
SABA :
70% pred
15%
0.13
6.5
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88%
13%
0.45 mg/ml
3.8 puffs/day
Turner MO etal Thorax 1995;50:1057
GINA guidelines
“…….a condition in which
many cells and cellular
elements play a role…….”
HETEROGENEITY
Inflammatory Phenotypes:
relevance
 diagnosis
 pathophysiology
 response to treatment
Treatment response depends
on inflammatory phenotype
Meijer Clin Exp Allergy 2002
Sputum strategy reduced
Eosinophilic exacerbations:
Jayaram, Pizzichini ERJ 2006
Eosinophilic
Exacerbations
Noneosinophilic
Exacerbations
sputum
Mechanisms
Allergens
Acquired
Immunity
IgE
Activated TH2 Cells
IL-4, 5, 9,13
Eosinophils
ECP
MBP
LTC-4
Eosinophilic
Asthma
Allergen avoidance
Allergens
Acquired
Immunity
IgE
Activated TH2 Cells
Omalizumab
Immunotherapy
Corticosteroids
IL-5
Eosinophils
ECP
MBP
LTC-4
Eosinophilic
Asthma
LTRA
Non-Eosinophilic asthma
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Symptoms
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Increased Airway responsiveness

Eosinophils within normal range
Allergens
Acquired
Immunity
Particulates, Pollutants, Virus,
Endotoxin, Bacteria
IgE
Activated TH2 Cells
TLR
Macrophages and Epithelial Cells
ICS X
Eosinophilic
Asthma
Innate
Immunity
X LABA
Neutrophilic
Asthma
Inflammatory cell activation
X LABA
hyperresponsiveness
Inflammatory Phenotypes
Eosinophilic
Neutrophilic
Paucigranulocytic
Inflammatory Phenotypes in
Stable Asthma
31%
41%
Eosinophilic
Neutrophilic
Paucigranulocytic
28%
Simpson J et al, Respirology 2006;11:54-61
Bronchial Biopsy
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EA n=12, HC n=10,
NEA n=11
Bronchial biopsy,
BAL, Ind sputum
Immunopathology
NON-response to
ICS confirmed in rct
Eosinophils/sq. mm
25
20
15
10
5
0
Berry M etal Thorax, 2008
Ctl
Eos A
NEA
Pathology of NEA
10
9
8
7
6
5
4
3
2
1
0
HC
EA
NEA
Mast cells in ASM
Cells / sq.mm
Subepith Thick'n um
Berry M etal, 2008
HRCT in Neutrophilic Asthma
Bronchial Wall Thickening Score 2 or more
Total Emphysema Score
% subjects
Bronchial Wall Thickening
**
60
50
40
30
20
10
0
Neutrophilic
Asthma
COPD
350
Emphysema Score
*
250
150
50
Healthy Smokers
Neutrophilic Asthma
COPD
Healthy Smokers
* p=0.005 versus neutrophilic asthma
Simpson J, Milne D, Gibson PG
submitted
Neutrophilic Asthma: how is it
different ?
Characteristics of Neutrophilic
Asthma

Stable asthma: n=259
Eosinophilic n=135, 52%
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Neutrophilic alone n=60, 23%
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B2 only n=35
Less atopy
Older
Later onset asthma
Green RH et al Thorax 2002;57:875-9
Neutrophil Elastase
40
*
% of samples postivie
35
30
25
20
107
mg/mL
15
10
5
56
mg/mL
0
Neutrophilic
Asthma
Eosinophilic
Asthma
Paucigranulocytic
Asthma
Healthy Controls
* p<0.04 versus Eosinophilic & Paucigranulocytic Asthma
Simpson J Am J Resp Crit Care Med 2005;172:559
Zymography: MMP-9
201kDa
118kDa
Pro MMP-9 ~ 92kDa
Active MMP-9~ 88kDa
83kDa
MMP-2 ~ 72kDa
48kDa
Ladder
Control
NA
HC
PGA
EA
Simpson J Am J Resp Crit Care Med 2005;172:559
% Active MMP-9
MMP-9% Active
100
75
50
*
25
0
NA
* p<0.001 versus all other groups
EA
PGA
HC
Simpson J Am J Resp Crit Care Med 2005
Enhanced IL-8 Release
Resting
*
* spont.
17500
17500
15000
15000
IL-8 (pg/mL)
IL-8 (pg/mL)
500
400
300
200
100
0
NEA
EA
12500
12500
10000
10000
IL-8 (pg/mL)
600
LPS
Stimulated
LPS
75007500
50005000
25002500
0
0
NEANEA
EA EA
Non-Eosinophilic Asthma
Eosinophilic Asthma
Katie Baines
Baines subm.
K etal
Resting Neutrophils:
Distinct Gene Profiles in Asthma Subtypes
Eosinophilic
Eosinophilic
Asthma
Asthma
Neutrophilic
Neutrophilic
Asthma
Asthma
Tues
27/3 ASIG
...
3.30pm Baines K
Eosinophilic Asthm
Tues
Baines
27/3
K, ASIG
submitte
3.30pm Baines K
Neutrophilic Asthma is
different…..
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In the airway
In the blood
Different genes
Different mediators
……….…different disease ?
Neutrophilic Asthma:
why is it different ?
Gene array: Classification of
biological function
+
6
Up
Down
5
+
+
4
3
2
1
0
Cell motility
Inhibition of
Apoptosis
NF-kB pathway
+ means more genes than expected
(category is overrepresented)
What do we know about the
NF-kB pathway ?
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Innate immune response
Input:
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Toll-Like Receptors
Oxidant stress
Output:
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I L-8
Neutrophils
What is innate immunity?
Acquired
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antigen
dependent
antigen specific
late
memory
cytokines
T/B/macrophage
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Innate
antigen independent
pattern recognition
early
immediate
antimicrobial
molecules
neutrophil
Hoebe K, etal. Adv Immunol 2006; 91:175
Innate immune responses
Non-allergic triggers
TLRs and SPs
Innate Immune
Activation
NF-kB
IL-8, IL-6
Noneosinophilic
asthma
Inflammation
Douwes, Gibson, Pearce etal Thorax; 2002:57:643
Innate Immune Activation in
Asthma Phenotypes
Asthma
N=49
Eos
Neuts
Paucigranulocytic
Bronchiectasis, n=9
Healthy Control n=12
Simpson JL, Thorax 2007;63:211
Inflammatory Cell Counts
Neutrophils, %
100
80
Neutrophilic
Eosinophilic
60
Paucigranulocytic
Bronchiectasis
40
20
0
6
Eosinophil, %
5
Simpson JL, Thorax
2007;63:211
4
3
2
1
0
Sputum IL-8 mRNA
Neutrophilic Asthma
Eosinophilic Asthma
*†º
IL-8 mRNA x10-1
60
Healthy
Bronchiectasis
50
40
Paucigranulocytic Asthma
*†º
30
20
10
0
* versus Healthy Controls
† versus Eosinophilic Asthma
º versus Paucigranulocytic Asthma
Subject Group
Simpson JL, Thorax 2007
TLR4 mRNA x10-2
6
TLR2 protein
5
4
3
2
1
0
TLR2 mRNA x10-2
30
*
20
*
10
*
Subject Group
Neutrophilic Asthma
Eosinophilic Asthma
Paucigranulocytic Asthma
Healthy
Bronchiectasis
0
Subject Group
Simpson JL, Thorax 2007;63:211
Non-allergic triggers
Clarithromycin
Reduces IL-8
TLRs and SPs
6
NF-kB
*
5
4
3
IL-8, IL-6
2
1
0
Pre
CAM
Off
Inflammation
Neutrophilic
asthma
Non-allergic triggers….
Pollution
Particulates
TLR2/4
Bacteria,
endotoxin
TLR 4
Virus
RSV
TLR4/SP-D
… also trigger innate immune activation
Stable Asthma, no ICS
nonsmokers
smokers
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
0
EA
NEA
EA
NEA
Chalmers, CHEST 2001
Allergens
Acquired
Immunity
IgE
Activated TH2 Cells
Particulates, Pollutants,
Virus, Endotoxin
TLR
Innate
Immunity
Macrophages and Epithelial Cells
NF-kB
IL-5
IL-8
Eosinophils
ECP
Neutrophils
MBP
MMP-9
Eosinophilic
Asthma
NE
Neutrophilic
Asthma
Oxidative
stress
10 days: low antioxidant diet
Effects of Antioxidant
withdrawal on Asthma
Asthma Control
Sputum Neuts
*
1.4
60
1.2
50
1
40
0.8
ACQ
0.6
0.4
neut
eos
30
20
0.2
0
*
10
day 0
day 10
Wood LG, Free Radic Biol 2008
0
day 0
day 10
% Neutrophils
mcg/
2.0
1.5
75
Antioxidant Supplement : lycopene
reduces
neutrophilic
inflammation
Lycopene
1.0
0.5
50
25
0.0
Placebo
Juice
Extract
0
Placebo
Neutrophil Elastase Activity
(ng/mL)
p<0.05
p<0.05
p<0.05
12500
8500
4500
75
%
Extract
Elastase
Neutrophils %
100
Juice
3000
50
2000
1000
25
0
Placebo
Juice
Extract
0
Placebo
Juice
Extract
Wood LG, Free Rad Biol
What about treatment ?
Allergens
Acquired
Immunity
Particulates, Pollutants, Virus,
Endotoxin, Bacteria
IgE
Activated TH2 Cells
TLR
Innate
Immunity
Macrophages and Epithelial Cells
ICS X
Eosinophilic
Asthma
Neutrophilic
Asthma
Inflammatory cell activation
X LABA
hyperresponsiveness
LABA reduces Neutrophilic
Inflammation
1600
1400
1200
*
1000
*
800
0 wks
4 wks
600
400
75
200
0
70
IL-8
PMN
Barnes PJ, Chest 2005
128:1936
65
*
60
55
IL-8
Reid DW, ERJ, 2003
Macrolides for Persistent
Asthma: Inclusion Criteria
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Symptomatic Asthma
AHR to hypertonic
saline
Severe Persistent
asthma according to the
2002 GINA guidelines
No sensitivity to
macrolide antibiotics
Clarithromycin
Simpson JL, AJRCCM, 208
Study Design
V1
V2
V3
V4
V5
CAM 1000mg
Screening
Follow Up
R
Placebo
t=-4
t=0
t=8
t=12
Consort Chart
Enrolment
n=79
Excluded (n=33)
Allocated
n=46
1 lost to follow up
Placebo =23
Active n=23
Completed
n=22 placebo, n=23 active
Participant Characteristics
n
Age mean (range)
45
58 (27-80)
Gender M|F
Atopy n (%)
FEV1% predicted mean (SD)
FEV1/FVC
22|23
32 (80)
70.6 (17.4)
65.7 (14.4)
Asthma control (Juniper)
ICS daily dose mcg, median
1.46 (0.8)
2000 (1000-2000)
Taken OCS in previous year
n(%)
20 (44)
Non-Eosinophilic Asthma
70

28 participants with
NEA
Similar age, gender,
ICS dose, lung
function and asthma
control
50
% participants

60
40
30
20
10
0
Eosinophilic
NonEosinophilic
CAM reduces NEA IL-8 mRNA
PLACEBO
70
70
60
60
50
50
*
40
30
20
IL-8 mRNA
IL-8 mRNA
ACTIVE
40
30
20
10
10
0
0
Before
* p= 0.023 versus before treatment
After
Before
After
CAM reduces NEA IL-8
Protein
Clarithromycin
Placebo
14
Sputum IL-8 ng/mL
12
10
8
6
*#
4
2
0
Baseline After Treatment Treatment Baseline After Treatment Treatment
Withdrawn
Withdrawn
Improved Quality of* Life
0.8
0.7
Quality of Life Score
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
Macrolide Treatment
Placebo
* p=0.016 versus placebo
QOL Domains
7
#
*
Baseline
After Treatment
Treatment Withdrawn
Median QOL Score
6
5
4
3
2
1
Activities
Symptoms
Emotions
Environmental
stimuli
Allergens
Acquired
Immunity
Particulates, Pollutants, Virus,
Endotoxin, Bacteria
IgE
Activated TH2 Cells
TLR
Macrophages and Epithelial Cells
X LABA Macrolides
ICS X
Eosinophilic
Asthma
Innate
Immunity
? diet
Neutrophilic
Asthma
Inflammatory cell activation
X LABA
hyperresponsiveness
Noneosinophilic Asthma
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‘new’ mechanism: innate immunity
Airway AND systemic disease
Triggers: diet, ETS
Treatment
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Explains exceptional synergy of ICS-LABA
New uses for old drugs: macrolides
Surfest Newcastle !
Treatment Prospects for
Neutrophilic Asthma
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Treatable exposure
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Infection
non-infection
Modify host response
Antagonise Neutrophils
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Chemokine/elastase inhibition
Reduce steroids
Asthma Control:2 components
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Current Control
Symptoms
Reliever use
‘bedside’ lung
function: PEF
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Future Risk
Exacerbations
Rapid decline in lung
function
Side-effects
‘pathological and physiological markers
provide information about the
underlying phenotype and the level of
residual disease activity on treatment,
and may serve as surrogate
markers for future risk’
ERS/ATS Task Force,
ERJ 2008
ERJ
ASTHMA CONTROL
Current clinical
control
Future risk
ASTHMA SEVERITY
TREATMENT
(“difficulty to treat”)
Genetic and
environmental
factors
DISEASE ACTIVITY
ASTHMA PHENOTYPES
Smoking
ASTHMA CONTROL
Current clinical
control
Future risk
Exacerbations
Loss of FEV1
80
70
ASTHMA SEVERITY
60
(“difficulty to treat”)
50
40
30
DISEASE ACTIVITY
20
10
0
EA
NEA
ASTHMA PHENOTYPES
Noneosinophilic
asthma
Smoking
ASTHMA CONTROL
Current clinical
control
Future risk
Exacerbations
Loss of FEV1
ASTHMA SEVERITY
(“difficulty to treat”)
DISEASE ACTIVITY
ASTHMA PHENOTYPES
Increased
Neutrophils, IL-8
Noneosinophilic
asthma
Smoking
ASTHMA CONTROL
Current clinical
control
0.3
0.2
0.1
0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
-0.7
-0.8
Future risk
ASTHMA SEVERITY
(“difficulty to treat”)
DISEASE ACTIVITY
FEV,l
Control
ASTHMA PHENOTYPES
Never
Ex
Smokers
Exacerbations
Loss of FEV1
Non-response to
steroid
Increased
Neutrophils, IL-8
Noneosinophilic
asthma
Smoking
ERJ
ASTHMA CONTROL
Current clinical
control
Future risk
ASTHMA SEVERITY
(“difficulty to treat”)
DISEASE ACTIVITY
ASTHMA PHENOTYPES
Exacerbations
Loss of FEV1
Non-response to
steroid
Increased
Neutrophils, IL-8
Noneosinophilic
asthma
Smoking
ERJ
ASTHMA CONTROL
Current clinical
control
Future risk
ASTHMA SEVERITY
TREATMENT
(“difficulty to treat”)
Genetic and
environmental
factors
DISEASE ACTIVITY
ASTHMA PHENOTYPES
GINA Guidelines
Assess control
Modify treatment
Asthma Control: future risk
Exacerbations
Loss of FEV1
 Trigger Management
 Trigger Management
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Avoidance
Vaccination
? Specific
antiviral/IFNb
Steroids
LABA
? Macrolides

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
Smoking cessation
Asthma control
Studies needed to
examine specific
therapies
Neutrophilic asthma




Asthma with neutrophilic
bronchitis
Nonallergic triggers
Different mechanisms..
? Different disease
Allergens
Acquired
Immunity
IgE
Particulates, Pollutants, Virus,
Endotoxin, Bacteria
TLR
Activated TH2 Cells
Macrophages and Epithelial Cells
IL-5
IL-8
Eosinophils
ECP
Neutrophils
MBP
MMP-9
Eosinophilic
Asthma
NE
Neutrophilic
Asthma
Innate
Immunity