The role of microfibrillar-associated
protein 4 (MFAP4) in asthma
Bartosz Pilecki
PhD student
Institute of Molecular Medicine
University of Southern Denmark, Odense
Asthma
• Common chronic airway disease
• Decrease in lung function due to persistent
inflammation, airway remodeling and airway
hyperresponsiveness (AHR)
• Current treatments effective only in selected
subsets of patients
MFAP4
• Extracellular matrix (ECM) protein that binds to elastin and
collagen
• Abundant in heart, lung, skin etc.
• Promotes proliferation and migration of vascular SMC in an
integrin-dependent manner (Schlosser et al, submitted)
Wulf-Johansson et al, 2013
Hypothesis:
MFAP4 contributes to asthmatic airway disease,
mainly due to its interaction with airway smooth
muscle cells
this
In
In
study
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investigate
we
we
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investigate
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the
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role
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of
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In
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the
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his study we investigate the possible role In
of In
MFAP4
in in asthma.
inmay
inin
asthma.
WeWeWe
hypothesize
We
hypothesize
hypothesize
that
that
MFAP4
that
MFAP4
MFAP4
may
asthma.
We
hypothesize
that
MFAP
asthma.
inasthma.
asthma.
hypothesize
We
hypothesize
that
MFAP4
that
MFA
m
asthma. We hypothesize that MFAP4
contribute
contribute
contribute
to to
the
to
development
to
the
the
development
development
ofof
allergic
of
of
allergic
allergic
asthma.
asthma.
asthm
contribute
to
the
development
of
allergic
asth
contribute
contribute
the
development
to
the
development
allergic
of
allergic
asthma.
asth
ntribute to the development of allergic asthma.
C
In vivo allergy models
METHODS
METHODS
METHODS
METHODS
METHODS
METHODS
METHODS
Neutrophils / ml BAL
40000
*
30000
BALB/c
BALB/c
BALB/c
wild
wild
wild
type
type
type
mice
mice
mice
and
and
MFAP4-deficient
and
MFAP4-defic
MFAP4-de
BALB/c
wild
type
mice
and
MFAP4BALB/c
BALB/c
wild
wild
type
type
mice
mice
and
MFAP4-deficie
and
MFAP4LB/c wild type mice and MFAP4-deficient
20000
model:
House
dust
mite
(HDM)
littermates
littermates
littermates
were
were
subjected
were
subjected
subjected
toto
acute
to
to
acute
ovalbumin
acute
ovalbumin
ovalbumin
(OVA)(O
littermates
were
subjected
to
acute
ovalbumi
littermates
littermates
were
subjected
were
subjected
acute
to
ovalbumin
acute
ovalbum
(OVA
ermates were OVA
subjected
to acute ovalbumin
(OVA)induced
induced
induced
asthma
asthma
asthma
model.
model.
model.
Inflammatory
Inflammatory
Inflammatory
cell
cell
cell
counts
coun
and
induced
asthma
model.
Inflammatory
cell
cou
induced
induced
asthma
asthma
model.
model.
Inflammatory
Inflammatory
cellcounts
counts
cell
co
a
uced asthma model. Inflammatory cell counts
and
10000
chronic
model:
cytokine
cytokine
cytokine
secretion
secretion
secretion
measurements
measurements
measurements
were
were
performed
were
performe
perform
on
secretion
measurements
were
perfo
cytokine
cytokine
secretion
secretion
measurements
measurements
were
performed
were
perfo
okine secretion measurements were performed
oncytokine
0
bronchoalveolar
bronchoalveolar
bronchoalveolar
lavage
lavage
lavage
(BAL).
(BAL).
(BAL).
Lungs
Lungs
Lungs
were
were
processed
were
proce
pro
bronchoalveolar
bronchoalveolar
lavage
lavage
(BAL).
(BAL).
Lungs
were
Lungs
process
were
bronchoalveolar
lavage
(BAL).
Lungs
were
pp
nchoalveolar lavage (BAL). Lungs were processed
KO PBS WT PBS KO OVA WT OVA
forfor
histology
for
for
histology
histology
and
assessed
and
and
assessed
assessed
forfor
pathological
for
for
pathological
pathological
changes.
change
chan
histology
for
histology
and
assessed
and
assessed
pathological
for
pathological
changes.
cha
for
histology
and
assessed
for
pathological
cha
histology and assessed for pathological changes.
Figure
3.
(A-C)
Cellular
infiltration
measured
Day: 0
7
14-16
deficient mice. (D) Alveolar macrophage activa
Day:
Day:
0Day:
0in 0KO
7 7compared
14-16
14-16
171717
1717
Day:
0Day:
Day:
7 14-16
14-16
17
to their
WT
litter
070 7mice
714-16
14-16
0-4
5-6
*P < 0.05, **P < 0.01, calculated by Student s t
17
KO
rest
2 mg alum +
20 ug OVA i.p.
20 ug OVA i.n.
Sacrifice
2 mg
2alum
mg
2alum
mg
alum
+20+20
ug
OVA
20
ug
i.n.
ug
OVA
OVA
i.n.
Sacrifice
i.n.
Sacrifice
Sacrifice
2 mg
2+mg
mg
+ HDM
alum
+20
ug
OVA
20
i.n.
ug
OVA
Sacrifice
i.n.
Sacrific
2alum
alum
+i.n.
20
ug
OVA
i.n.
Sacrifice
25
ug
20 20
ug20
OVA
20
ug
i.p.
ug
OVA
OVA
i.p.
i.p.i.p.
ug
OVA
20
i.p.
ug
OVA
i.p.
20
ug
OVA
Figure
Figure
1.Figure
Experimental
1. Experimental
1. 1.
Experimental
setup.
setup.
setup.
Figure
1.
Figure
Experimental
1.Experimental
Experimental
setup.
setup.
Figure
setup.
H-E
re 1. Experimental setup.
RESULTS
A
IL4
RESULTS
RESULTS
RESULTS
RESULTS
RESULTS
RESULTS
AAAA
A
A
B
IL5
7 weeks
IL4 IL4 IL4 IL4IL4
IL4
B
BBB
B
B
IL5 IL5 IL5 IL5IL5
IL
MFAP4 levels are increased in asthma
WT PBS
BAL
BAL
*
15
10
10
U/ml
U / ml
15
5
5
0
0
PBS
PBS
OVA
HDM
Serum
WT OVA
Serum
400
*
100
300
U/ml
80
U / ml
***
60
200
40
100
20
0
0
PBS
OVA
PBS
HDM
MFAP4 deficiency attenuates eosinophilic infiltration
3×1005
2×1005
1×1005
1.0×1006
5.0×1005
0.0
0
WT PBS
KO PBS
WT OVA
WT PBS
KO OVA
4×1005
KO PBS
WT HDM KO HDM
1.5×1006
*
Eosinophils/ml BAL
Eosinophils/ml BAL
p=0.0616
1.5×1006
*
Cells/ml BAL
Cells/ml BAL
4×1005
3×1005
2×1005
1×1005
0
**
1.0×1006
5.0×1005
0.0
WT PBS
KO PBS
WT OVA
KO OVA
WT PBS
KO PBS
WT HDM KO HDM
Eosinophil chemoattractants are downregulated
in MFAP4-deficient mice
CCL11
*
500
**
60
pg/mg protein
pg/mg protein
80
CCL24
40
20
400
300
200
100
0
0
WT PBS
KO PBS
WT OVA
KO OVA
WT PBS
KO PBS
WT OVA
KO OVA
Lack of MFAP4 attenuates mucus production
WT OVA
IL13
pg/mg protein
100
*
80
60
40
20
0
WT PBS
KO PBS
WT OVA
KO OVA
No. goblet cells / mm basement membrane
PBS
KO OVA
Mucus production
*
50
40
30
20
10
0
WT OVA
KO OVA
MFAP4 contributes to asthmatic
smooth muscle deposition
Smooth muscle thickness
20
Smooth muscle thickness
25
****
Thickness [um]
Thickness [um]
ns
15
10
5
**
20
15
10
5
0
0
WT PBS
KO PBS
WT OVA
KO OVA
WT PBS
KO PBS
WT HDM KO HDM
MFAP4 deficiency partially protects from AHR
Pulmonary resistance
3
2
1
0
0
1
3
MCh [mg/ml]
10
30
% of baseline
1500
KO PBS
WT PBS
KO OVA
WT OVA
****
cmH2O.s/ml
4
dose response resistance
KO PBS
WT PBS
KO HDM
WT HDM
1000
500
0
1
3
10
MCh [mg/ml]
30
50
Conclusions
MFAP4 is increased in circulation and BAL of asthmatic mice.
MFAP4 contributes to development of experimental asthma by:
1. Attraction of eosinophils through CCL11/CCL24
2. Goblet cell metaplasia
3. Smooth muscle deposition
4. AHR
It suggests that MFAP4 can be a potential therapeutic target for
allergic asthma.
Acknowledgements
Christopher Stevenson,
Novartis, UK
Jorgen Vestbo, OUH, DK
Niels Marcussen, OUH, DK
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