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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