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The role of neutrophils in corneal wound healing in
HO-2 null mice
Giovanni Li Volti, MD, PhD
Department of Clinical and Molecular Medicine
University of Catania
HEME OXYGENASE
a potent and efficient
scavenger of reactive oxygen
species and is involved in
proliferation and neurons
survival
decreases LPS-induced proinflammatory
transcription factors (NF-kB) and cytokines (IL-6, TNF-α)
anti-inflammatory�suppresses pro-inflammatory cytokines
�increases anti-inflammatory IL-10
expression
Heme oxygenase 1 (HO-1)
(288 amino acids; 33 kDa)
contains no cysteine residues is an
inducible isoform in response to
stress such as oxidative stress,
hypoxia, heavy metals, cytokines, etc.
Heme oxygenase 2 (HO-2)
(316 amino acids; 36 kDa) contains
three cysteine residues is a
constitutive isoform which is
expressed under homeostatic
conditions.
HEME OXYGENASE
Today's presentation
FIRST PART: Evaluation of the role of heme oxygenase and its metabolites to the
would healing process using an in vitro model of epithelial scratch injury in
primary and immortalized HCE cells
SECOND PART: Examination of the possible relationship between HO-2 and the
recruitment of neutrophils following a corneal surface injury in wild type (WT)
and HO-2 knockout (HO-2-/-) mice treated with Gr-1 monoclonal antibody to
deplete peripheral neutrophils
THIRD PART: Evaluation of a possible correlation beetwen heme oxygenase and
phagocytic activity of the macrophages
A
control
1h
24h
HO-1 MergedDAPI HO-2 DAPI Merged
control
1h
24h
*p<0.05 to control; **p<0.05 to 8h wound edge
Distal to wound edgeWound edgeB
2
HO-2
control 1 4 8 240.0
0.5
1.0
1.5
hours post injury
(re
lati
ve
to
co
ntr
ol)
Flu
ore
sce
nce
in
ten
sity
HO-1
control 1 2 4 8 24hours post injury
*
**
*
*
*
**
(re
lati
ve
to
co
ntr
ol)
Flu
ore
sce
nce
in
ten
sity
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
150 µm
Halilovic A; Patil K; Bellner L; Marrazzo G et al, J Cell Physiol. 2011 Jul;226(7):1732-40. doi: 10.1002/jcp.22502
ControlBVDCORM A1
0
20
40
60
80
100
12 h 24 h
Pe
rce
nt
reg
row
th
*
* *B
Halilovic A; Patil K; Bellner L; Marrazzo G et al, J Cell Physiol. 2011 Jul;226(7):1732-40. doi: 10.1002/jcp.22502
Control
CrMP
BVD
CORM-A1
D
0
0.1
0.2
0.3
0.4
0.5
0 12 24
Time (h) after injury
Re
lati
ve
ab
sorb
an
ce
B
0
20
40
60
80
100
* *
Control CrMP BVD CORM A1CrMP
Pe
rce
nt
He
ali
ng
12h
‡
‡‡
CP
erc
en
t H
ea
lin
g
0
20
40
60
80
100
Control CrMP BVD CORM A1
* *24h
‡
CrMPHalilovi A; Patil K; Bellner L; Marrazzo G et al, J Cell Physiol. 2011 Jul;226(7):1732-40. doi: 10.1002/jcp.22502
Today's presentation
FIRST PART: Evaluation of the role of heme oxygenase and its metabolites to the
would healing process using an in vitro model of epithelial scratch injury in
primary and immortalized HCE cells
SECOND PART: Examination of the possible relationship between HO-2 and the
recruitment of neutrophils following a corneal surface injury in wild type (WT)
and HO-2 knockout (HO-2-/-) mice treated with Gr-1 monoclonal antibody to
deplete peripheral neutrophils
THIRD PART: Evaluation of a possible correlation beetwen heme oxygenase and
phagocytic activity of the macrophages
The HO-2 (-/-) mice vs. WT when injured showed:
• Impaired and delayed wound healing
• Exagerated inflammatory response
• Consistent increase of corneal neovascularization
• 4- Fold higher number of inflammatory cells that infiltrate the corneal
stroma
• Impaired induction on HO-1 under mechanical injury stimulus
BACKGROUND
Control B6129SF2/J
(WT)
HO-2 null
(HO-2-/-)
control
Gr-1
treated
Adhesion
assay
Isolation of neutrophils from
peripheral blood
Monitoring wound closure 48 hours
after injury
Corneal epithelium removal using
Algerbrush II cornal rust ring
remover
Corneas collected for assessment of
mRNA levels and histology
Confluent mouse aortic endothelial cells (mAEC)
isolated from WT and HO-2 null mice
EXPERIMENTAL PROTOCOL:
A
B
WT
(injured)
HO-2-/-
(injured)
Control Neutrophil-
depletedControl Neutrophil-
depleted
Day 0 Day 2
†
WT Control WT N-depleted HO-2-/- Control HO-2-/- N-depleted0
10
20
30
40
50
60
% R
e-e
pit
he
lia
liza
tio
n
*
**‡
Marrazzo G, Bellner L, Halilovic A, Li Volti G, Drago F, et al. PLoS ONE 6(6): e21180, doi:10.1371/journal.pone.0021180
A WT
(20X)
Control
(injured)Neutrophil-depleted
HO-2-/-
(20X)
WT (63X)
HO-2-/-
(63X)
Control
(uninjured)
Marrazzo G, Bellner L, Halilovic A, Li Volti G, Drago F, et al. PLoS ONE 6(6): e21180, doi:10.1371/journal.pone.0021180
A WT
Control
WT
Neutrophil-
depleted
HO-2-/-
Control
HO-2-/-
Neutrophil-
depleted
Gr- 1 DAPI Merged
BWT
Control
WT
Neutrophil-
depleted
HO-2-/-
Control
HO-2-/-
Neutrophil-
depleted
CD 68 DAPI Merged
Marrazzo G, Bellner L, Halilovic A, Li Volti G, Drago F, et al. PLoS ONE 6(6): e21180, doi:10.1371/journal.pone.0021180
*
WT
Control
WT
N-depleted
HO-2-/-
Control
HO-2-/-
N-depleted
0
1
2
3
HO
-1 m
RN
A (
Re
lati
ve
Ex
pre
ssio
n)
Marrazzo G, Bellner L, Halilovic A, Li Volti G, Drago F, et al. PLoS ONE 6(6): e21180, doi:10.1371/journal.pone.0021180
** ** †
Control TNF- α α α α treated
0
20
40
60
80
100
120
140
160WT mAEC
HO-2-/- mAECN
eu
tro
ph
il A
dh
esi
on
(R
FU
)
Marrazzo G, Bellner L, Halilovic A, Li Volti G, Drago F, et al. PLoS ONE 6(6): e21180, doi:10.1371/journal.pone.0021180
WT mAEC HO-2-/- mAEC0
1
2
3
4
Md
k m
RN
A
(Re
lati
ve
Ex
pre
ssio
n)
*
*
WT mAEC HO-2-/- mAEC0
1
2
3
VE
-Ca
dh
eri
n m
RN
A
(Re
lati
ve
Ex
pre
ssio
n)
Marrazzo G, Bellner L, Halilovic A, Li Volti G, Drago F, et al. PLoS ONE 6(6): e21180, doi:10.1371/journal.pone.0021180
Today's presentation…
FIRST PART: Evaluation of the role of heme oxygenase and its metabolites to the
would healing process using an in vitro model of epithelial scratch injury in
primary and immortalized HCE cells
SECOND PART: Examination of the possible relationship between HO-2 and the
recruitment of neutrophils following a corneal surface injury in wild type (WT)
and HO-2 knockout (HO-2-/-) mice treated with Gr-1 monoclonal antibody to
deplete peripheral neutrophils
THIRD PART: Evaluation of a possible correlation beetwen heme oxygenase and
phagocytic activity of the macrophages
EXPERIMENTAL PROTOCOL:
RAW 264.7 CELLS
MURINE MACROPHAGES
INCUBATED 30
min WITH TEXAS
RED-LABELED
ZYMOSAN
�INDUCED BY SnCl2
�TREATED WITH shRNA
AGAINST HO-2
�EVALUATION OF THE
PHAGOCITIC ACTIVITY
�Q-PCR
Marrazzo G et al. In preparation for
Current Pharmaceutical Designs
0
10
20
30
40
50
60
no plasmid Scramble ShRNA HO-2
**
0,00
0,20
0,40
0,60
0,80
1,00
1,20
1,40
1,60
no plasmid Scramble shRNA HO-2
HO
-2 m
RN
A
(Re
lati
ve
Ex
pre
ssio
n)
A
B
% o
f p
ha
go
cyto
sis
Transiently transfected RAW cells
**
0
10
20
30
40
50
60
70
80
control induced (1uM) induced (5uM) induced (10 uM)
*
% o
f p
ha
go
cyto
sis
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
control 1 uM 5uM 10 uM
HO-1
HO-2
RAW cells treated with different concentration of
SnCl2A
B
HO
-1;
HO
-2 m
RN
A
(Re
lati
ve
Ex
pre
ssio
n)
*
CONCLUSIONS
�HO-2 is critical for a self-resolving inflammatory and repair response in the
cornea.
�Epithelial injury in HO-2 null mice leads to impaired wound closure and chronic
inflammation in the cornea.
�Systemic and corneal neutrophil depletion worsened rather than improved the
wound healing process in both WT and HO-2-/- mice
�The absence of HO-2 gene within corneal cells contributed to the impaired
corneal healing in the HO-2 null mice.
ACKNOWLEDGMENT
Prof. Filippo Drago
Prof. Claudio Bucolo
Dr Letizia Marrazzo
Department of Clinical and Molecular Medicine
University of Catania Prof. Francesco Cappello
Prof. Michal L. Schwartzman