Maintenance of vascular integrity via ARF6-GTP inhibition ... · Maintenance of vascular integrity...
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Maintenance of vascular integrity via ARF6-GTP inhibition protects mice from MDR Acinetobacter infection Lin Lin, 1 Teclegiorgis Gebremariam, 1 Lina Zhang, 1,2 Samuel French, 1 Alan L. Mueller, 3 Dean Li, 4 and Ashraf S. Ibrahim 1 Grant Support: NIAID ARF6-GTP Inhibitors: Navigen Pharmaceuticals, Salt Lake City 1 LA Biomed. Res. Inst. at Harbor-UCLA Med Ctr, Torrance, California, U.S.A; 2 College of Wildlife Resources, Northeast Forestry University, Harbin, China; 3 Navigen Pharmaceuticals, Salt Lake City, Utah, U.S.A; 4 University of Utah, Salt Lake City, Utah, U.S.A
Transcript of Maintenance of vascular integrity via ARF6-GTP inhibition ... · Maintenance of vascular integrity...
Maintenance of vascular integrity via ARF6-GTP inhibition protects mice from MDR Acinetobacter
infection
Lin Lin,1 Teclegiorgis Gebremariam,1 Lina Zhang,1,2 Samuel French,1 Alan L. Mueller,3
Dean Li,4 and Ashraf S. Ibrahim1
Grant Support: NIAID
ARF6-GTP Inhibitors: Navigen Pharmaceuticals, Salt Lake City
1 LA Biomed. Res. Inst. at Harbor-UCLA Med Ctr, Torrance, California, U.S.A;2
College of Wildlife Resources, Northeast Forestry University, Harbin, China;3
Navigen Pharmaceuticals, Salt Lake City, Utah, U.S.A; 4 University of Utah, Salt
Lake City, Utah, U.S.A
Introduction
• Septicemia due to Multidrug resistant (MDR) Gram
negative bacteria (GNB) such as Acinetobacter
baumannii (AB) is a predominant cause of healthcare-
associated infections world-wide with high mortality
rates (Boucher et al., CID 2009).
• GNB septicemia is treated with few highly toxic
antibiotics and in many cases are untreatable (Boucher
et al., CID 2009).
• Hence, novel approaches of treatment are urgently
needed which can be facilitated by understanding the
pathogenesis of the infection.
Introduction
• LPS acute lung injury triggers a TLR4-mediated
activation of the MyD88/NF-kB cascade leading to
robust inflammatory immune response (Zhu…Li.,
Nature 2012; Davis….Li, J Immunol. 2014).
• LPS also triggers a MyD88/ARF6 activation pathway
that leads to increased vascular leak via internalization
of VE-cadherin intracellularly (London…Li Sci Transl
Med 2010).
• The increased vascular leak results in tissue edema,
organ failure, and ultimate death which is a common
feature of septicemia.
• LPS plays a major role in pathogenesis of many GNB
including AB (Lin et al. mBio 2012)
Hypothesis
Aims
•We sought to determine the role of GNB LPS,
using AB as a prototype bacterium, in activation
of MyD88/ARF6-GTP pathway and its
consequence of vascular permeability in vitro
(using umbilical vein endothelial cells [HUVEC])
and in mice.
•Given its convergence point in destabilizing
vascular integrity, we wanted to investigate the
role of novel ARF6-GTP inhibitors in protecting
against AB-induced infections in murine models
Methods
• AB-mediated ARF6-GTP formation in HUVEC and theeffect of ARF6-GTP inhibitors was studied byimmunoprecipitation (IP) and trans-well permeabilityassays.
• HUVEC VE-Cadherin expression was tracked byimmunofluorescence.
• Contribution of ARF6-GTP to AB virulence in vitro and invivo was studied by reduction of ARF6-GTP expression(siRNA) and by using ARF6-/- mice, respectively.
• ARF6-GTP Inhibitors were evaluated for their protectiveeffect in neutropenic mice with AB pneumonia.
Results(AB LPS induces HUVECs permeability via TLR-4 signaling)
ARF6
EC
alone
EC +
AB
EC + AB +
NAV2729
EC +
NAV2729
ARF6-GTP
GAPDH
D E
EC alone EC + AB EC + AB + NAV2729
ATCC17978HUMC1
cells sup
En
do
the
liu
mP
erm
ea
bilit
y
(rela
tive
to
no
tre
atm
en
t)
cells sup En
do
the
liu
mP
erm
ea
bilit
y
(rela
tive
to
no
tre
atm
en
t)
HUMC1+
Isotype Ab
cells sup
HUMC1+
anti-TLR4 Ab
cells sup
B
HUMC1
cells sup
HUMC1+
SLIT2
cells sup
C
HUMC1 cells
**
*
*
*
*
* *
*
En
do
the
liu
mP
erm
ea
bilit
y
(rela
tive
to
no
tre
atm
en
t)A
-10
10
0
20
30
40
50
-10
10
0
20
30
40
50
-10
10
0
20
30
40
50
-20*
ARF6
EC
alone
EC +
AB
EC + AB +
NAV2729
EC +
NAV2729
ARF6-GTP
GAPDH
D E
EC alone EC + AB EC + AB + NAV2729
ATCC17978HUMC1
cells sup
En
do
the
liu
mP
erm
ea
bil
ity
(re
lati
ve
to
no
tre
atm
en
t)
cells sup En
do
the
liu
mP
erm
ea
bil
ity
(re
lati
ve
to
no
tre
atm
en
t)
HUMC1+
Isotype Ab
cells sup
HUMC1+
anti-TLR4 Ab
cells sup
B
HUMC1
cells sup
HUMC1+
SLIT2
cells sup
C
HUMC1 cells
**
*
*
*
*
* *
*
En
do
the
liu
mP
erm
ea
bil
ity
(re
lati
ve
to
no
tre
atm
en
t)A
-10
10
0
20
30
40
50
-10
10
0
20
30
40
50
-10
10
0
20
30
40
50
-20*
ARF6
EC
alone
EC +
AB
EC + AB +
NAV2729
EC +
NAV2729
ARF6-GTP
GAPDH
D E
EC alone EC + AB EC + AB + NAV2729
ATCC17978HUMC1
cells sup
En
do
the
liu
mP
erm
ea
bil
ity
(re
lati
ve
to
no
tre
atm
en
t)
cells sup En
do
the
liu
mP
erm
ea
bil
ity
(re
lati
ve
to
no
tre
atm
en
t)
HUMC1+
Isotype Ab
cells sup
HUMC1+
anti-TLR4 Ab
cells sup
B
HUMC1
cells sup
HUMC1+
SLIT2
cells sup
C
HUMC1 cells
**
*
*
*
*
* *
*
En
do
the
liu
mP
erm
ea
bil
ity
(re
lati
ve
to
no
tre
atm
en
t)A
-10
10
0
20
30
40
50
-10
10
0
20
30
40
50
-10
10
0
20
30
40
50
-20*
Results(AB activates ARF6-GTP formation-Pull Down Assay)
ARF6
HUVEC
alone
EUVEC
+AB
EUVEC+AB
+NAV2729
HUVEC
+NAV2729
ARF6-GTP
GAPDH
AB
0
0.2
0.4
0.6
0.8
1
1.2
AR
F6
-GT
P/A
RF
6 r
ati
o
(rel
ati
ve
to E
C +
AB
)
AR
F6
-GT
P/A
RF
6 r
ati
o
(re
lati
ve
to
HU
VE
C+
AB
)
Results(AB compromises vascular stability via ARF6-mediated intraceullar recruitment of VE-cadherin)
ARF6
EC
alone
EC +
AB
EC + AB +
NAV2729
EC +
NAV2729
ARF6-GTP
GAPDH
D E
EC alone EC + AB EC + AB + NAV2729
ATCC17978HUMC1
cells sup
En
do
theli
um
Pe
rme
ab
ilit
y
(re
lati
ve
to
no
tre
atm
en
t)
cells sup En
do
theli
um
Pe
rme
ab
ilit
y
(re
lati
ve
to
no
tre
atm
en
t)HUMC1+
Isotype Ab
cells sup
HUMC1+
anti-TLR4 Ab
cells sup
B
HUMC1
cells sup
HUMC1+
SLIT2
cells sup
C
HUMC1 cells
**
*
*
*
*
* *
*
En
do
theli
um
Pe
rme
ab
ilit
y
(re
lati
ve
to
no
tre
atm
en
t)A
-10
10
0
20
30
40
50
-10
10
0
20
30
40
50
-10
10
0
20
30
40
50
-20*
FL1-H
Count
100
101
102
103
104
0
2
4
6
7
FITC Anti-VE cadherin Fluorescence
HUVEC alone
HUVEC+AB
HUVEC+AB
+NAV2729
A
B
0
10
20
30
40
50
60
70
80
90
100
% V
E c
ad
her
in e
xp
ress
ion
C
Co
un
t
% V
E-c
ad
he
rin
exp
ress
ion
Results(Down regulation of MyD88/ARNO/ARF6 genes attenuate HUVEC permeability in response to AB in vitro)
A
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Scrambled
siRNA
ARF6
siRNA
ARNO
siRNA
MyD88
siRNA
ROBO4
siRNA
Fold
Ch
an
ge
(rela
tive t
o c
on
trol
scram
ble
siR
NA
)
* **
*
0
0.2
0.4
0.6
0.8
1
1.2B
AR
F6-G
TP
Fo
ld C
han
ge
(re
lati
ve
to
Sc
ram
ble
siR
NA
+A
B)
En
do
theliu
mp
erm
eab
ilit
y
(rela
tive t
o n
o t
rea
tmen
t)
***
*
scra
mb
led
siR
NA
+H
UM
C1
Arf
6 s
iRN
A+H
UM
C1
RO
BO
4 s
iRN
A+S
lit2
+H
UM
C1
scra
mb
led
siR
NA
+S
lit2
+H
UM
C1
AR
NO
siR
NA
+H
UM
C1
MyD
88 s
iRN
A+H
UM
C1
co
ntr
ol E
C +
HU
MC
1
-4 0
-2 0
0
2 0
4 0
6 0
8 0C
Results(ARF6-/- mice are resistant to AB pneumonia)
0%
20%
40%
60%
80%
100%
0 3 6 9 12 15 18 21
% S
urv
iva
l
Days post infection
Wild-type
ARF6 null
N=13 wild-type mice and 14 ARF6 null mutant mice
P=0.003
*
Wild-type
ARF6 null
325x
325x
ResultsARF6 inhibitors prolong survival of neutropenic mice with AB pneumonia without affecting the inflammatory
immune response.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 3 6 9 12 15 18 21
% S
urv
iva
l
Days post infection
UninfectedPlaceboNAV-2729NAV4424NAV-4543Colistin *
**
*
A
Placebo NAV2729 Colistin
Eu
/ml
0
5
10
15
‡
BloodD 80
Eu
/ml
0
40
60
20
Placebo NAV2729 Colistin
‡
Lungs
B
Placebo NAV2729 Colistin
# ***
Lo
g C
FU
/g o
f lu
ng
s
0
1
2
3
4
5
6 ¥¥
C
**
**
*
Placebo NAV2729 Colistin
* *
*
Lu
ng
sS
ple
en
E
¥
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 3 6 9 12 15 18 21
% S
urv
iva
l
Days post infection
UninfectedPlaceboNAV-2729NAV4424NAV-4543Colistin *
**
*
A
Placebo NAV2729 Colistin
Eu
/ml
0
5
10
15
‡
BloodD 80
Eu
/ml
0
40
60
20
Placebo NAV2729 Colistin
‡
Lungs
B
Placebo NAV2729 Colistin
# ***
Lo
g C
FU
/g o
f lu
ng
s
0
1
2
3
4
5
6 ¥¥
C
**
**
*
Placebo NAV2729 Colistin
* *
*
Lu
ng
sS
ple
en
E
¥
0
10
20
30
40
50
60
70
80
90
100
WT AB AB+NAV2729
ug E
BD
/ g T
issu
e
Uninfected
Mice
AB AB+
NAV2729
Lu
ng
pe
rme
ab
ilit
y
(µg
Eva
ns
Blu
e/g
tis
su
e)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 3 6 9 12 15 18 21
% S
urv
ival
Days post infection
UninfectedPlaceboNAV-2729NAV4424NAV-4543Colistin *
**
*
A
Placebo NAV2729 Colistin
Eu
/ml
0
5
10
15
‡
BloodD 80
Eu
/ml
0
40
60
20
Placebo NAV2729 Colistin
‡
Lungs
B
Placebo NAV2729 Colistin
# ***
Lo
g C
FU
/g o
f lu
ng
s
0
1
2
3
4
5
6 ¥¥
C
**
**
*
Placebo NAV2729 Colistin
* *
*
Lu
ng
sS
ple
en
E
¥
D
*
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 3 6 9 12 15 18 21
% S
urv
iva
l
Days post infection
Uninfected
Placebo
NAV-4424
NAV-5093
NAV-5093 is the prodrug of NAV-4424
*
* P<0.004 compared to placebo or NAV-4424 treated mice (n=10 mice per arm).
Results(Water soluble prodrug ARF6-GTP inhibitor is protective against AB pneumonia model)
Summary/Conclusions
• AB activates MyD88/ARNO/ARF6 pathway via TLR4 stimulation by LPS.
• Activation of ARF6-GTP formation results in enhanced endothelium vascular permeability through intracellular recruitment of VE-Cadherin.
• Down regulation of any of the MyD88/ARNO/ARF6 expression in HUVEC protect them from AB-induced vascular permeability in vitro.
• Conditional HUVEC ARF6 knockout mice are more resistant to AB pneumonia than wild-type mice.
• Treatment of wild-type neutropenic mice with ARF6-GTP inhibitors protect them from AB pneumonia via a mechanism that involves stabilizing vascular integrity.
• The ARF6-GTP inhibitors can potentially have an effect on other GNB infection and potentially any other organisms that activate the MyD88/ARF6 pathway (e.g. MRSA, Candida sepsis)
• Continued investigations of ARF6-GTP inhibitors as a novel treatment for MDR organisms are warranted.
The Model
Serum proteins
TLR4
VE-cadherin
Death
Organ failure
Tissue edema
Vascular leak
MyD88
ARF6
GDPARF6
GTPNF-κB
Inflammatory Cytokines
AR
NO
Leukocytes
AB
ABAB
Acknowledgments
Harbor-UCLA Medical Center• Lin Lin• Teklegiorgis Gebremariam• Lina Zhang• Francisco Bautista• Sondus Alkhazraji• Samuel French• UCLA CTSI
University of Utah• Dean Li• Shannon Odelberg
Navigen Pharmaceuticals• Alan Mueller• Brandi Simpson
Research support
NIAID (R21 AI119339)
