PBS HDM

A B C D

Annexin A1 Annexin A1

E Resistance F Resistance 100mg/ml Mch G Compliance 100mg/ml Mch

5 ++
5 0.03
PBS WT *
4 +
HDM WT
cmH2O.s/mL

4
*
PBS ANXA1-/- cmH2O.s/mL

mL/cmH2O
0.02
3 3 * *
HDM ANXA1-/-
2 2
0.01
1 1

0 0 0.00
PBS HDM PBS HDM PBS HDM PBS HDM
Baseline PBS 3 10 30 100
WT ANXA1-/-
WT ANXA1-/-

H Total BAL Leukocytes I BAL Eosinophils J BAL Th2 Lymphocytes

60 15 20
* + ++
x105cells/ml BAL

x105cells/ml BAL

x103cells/ml BAL
** * 15
40 10 **
10
20 PBS WT HDM WT
* PBS KO HDM KO
5
** 5

0 0 0
PBS HDM PBS HDM PBS HDM PBS HDM PBS HDM PBS HDM
WT ANXA1-/- WT ANXA1-/- WT ANXA1-/-

K IL-4 L Lung IL-5 M 50 Lung IL-13

100 ** 150
++
+ 40 *
80
pg/ml Lung
pg/ml Lung

*
pg/ml Lung

100
60 30
PBS WT HDM WT
PBS ANXA -/- ANXA-/-
HDM
40 20
50
20 10

0 0 0
PBS WT HDM WT PBS KO HDM KO PBS HDM PBS HDM PBS HDM PBS HDM
WT ANXA1-/- WT ANXA1-/-

Figure X. Annexin A1 is induced following HDM challenge and mice deficient in ANXA1 have exacerbated airway hyper-
reactivity. (A) Schematic of house dust mite allergen dosing protocol. (B,C) Immunofluorescent staining of paraffin embedded
lung tissue sections incubated with anti-Annexin A1, counterstained with DAPI (magnification?). (D) mRNA expression of annexin
A1 in lung tissue following HDM exposure, expression normalized to housekeeping gene hprt. Mice receiving HDM were analysed
for changes in airway hyper-reactivity following methacholine (MCh) challenge in tracheotomized restrained mice. (E) Dose
response curve with increasing concentrations of MCh (F) airway resistance at top MCh dose 100mg/ml (G) compliance at top
MCh dose 100mg/ml, (H) total BAL leukocytes (I) BAL eosinophils , (J) T1/ST2+ lymphocytes quantified in the BAL. IL-4 (K) IL-5
and (L) IL-13 quantified by ELISA. * p<0.05 and ** p<0.01 relative to PBS control group by Mann Whitney test. + p<0.05 and ++
p<0.01 comparing HDM AnnexinA1 KO mice relative to HDM WT group by Mann Whitney test. Representative of two
experiments.
 x105cells/ml BAL

0
5
10
15

PBS
WT
**

HDM
+
Exp SM26

PBS

Figure 2H
BAL Eosinophils

ANXA1-/-
HDM

PB x105cells/ml BAL
S
0
5
10
15

H +
D Ve
PB M h
S +
H + Veh
H DM WR
D
M + W W4
PB + A R
S nx W4
+ A
Exp SM33

A 2-
nx 50
A
Eosinophils

2-
50
Figure 4F, 5F
Figure X (Mimetic)

Resistance 100mg/ml Mch

Resistance
6 6 +

PBS + Veh
cmH2O.s/mL

cmH2O.s/mL
4 HDM + Veh 4 *
HDM + ANXA2-50

2 2

0 0
Baseline PBS 3 10 30 100

50
Ve

Ve

2-
XA
+

+
S

N
PB

A
H

+
M
D
H
Eosinophils Th2 Lymphocytes
30
* 80
x105cells/ml Lung

x103cells/ml Lung

* *
20 60 *

40
10
20

0
0
h

50

50
Ve

Ve

nx h
50

50
2-

2-

Ve

Ve

2-

2-
A

A
+

nx

A
+

+
S

nx
PB

M
D

PB

D
H

A
+

+
S

M
PB

PB

D
H

IL-5
IL-4
250
60
++
200 *
*
pg/ml Lung

*
pg/ml Lung

40 * 150

100
20
50

0 0
PBS HDM PBS HDM
h

50

50
Ve

Ve

2-

2-

WT AnxA (2-50)
A

A
+

nx

nx
S

M
PB

A
H

+
S

M
PB

D
H