116/11/2018 Sophie Lanone

Deficient macrophage autophagyprotects mice from CeO2 nanoparticle-induced alveolar remodeling

B. Annangi, Z. Lu, A. Ridoux, J. Boczkowski, S. Lanone

Sophie.lanone@inserm.fr

216/11/2018 Sophie Lanone

Introduction - Cerium oxide nanoparticles (CeO2 NP)

CeO2 are used as diesel fuel catalyst to reduce emissions of particulate matter emission of CeO2 nanoparticles (NP)

Pulmonary exposure to CeO2 NP

Granuloma formation

Lung remodeling (fibrosis)

Exact underlying mechanism?

Epithelio-mesenchymal transition

Autophagy?

Ma et al. Toxicol Appl. Pharmacol. 2012Ma et al. Toxicol Appl. Pharmacol. 2014

Hussain et al. ACS Nano 2012Ma et al. Toxicol Appl. Pharmacol. 2017

Aalapati et al. Nanotoxicology 2017

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Introduction - Autophagy

A self-eating physiological process

Adapted from Cohignac et al. Nanomaterials 2015

Lanone et al. Med&Sci. 2017

Cohignac et al. Autophagy 2018

Role in health and diseases

Modulated, in macrophages, in response to NP, depending on their physico-chemical characteristics

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Hypothesis - Objectives

Hypothesis:

Lung remodeling induced by pulmonary exposure to CeO2 NP could be linked to modifications of autophagy

Objectives:

– Characterize lung remodeling in response to CeO2 NP

– Evaluate the contribution of macrophagic autophagy in this response

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Pulmonary exposure to CeO2 NP: induction of lung remodeling?

Saline CeO2 Saline CeO2 Saline CeO2

1 day 7 days 28 days

HES

Progressive perialveolar and peribronchial thickening after single oropharyngeal administration of CeO2 NP

Oropharyngeal aspiration of saline of 50µg CeO2 NP Sacrifice 24h, 1 week or 28 days later

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Pulmonary exposure to CeO2 NP: induction of lung remodeling?

Saline

CeO2

HES Sirius red

5 µg 50 µg Saline

CeO2

5 µg 50 µg

Dose-dependent perialveolar and peribronchial thickening after single oropharyngeal administration of CeO2 NP

Oropharyngeal aspiration of saline of 5 or 50µg CeO2 NP Sacrifice 28 days later

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Pulmonary exposure to CeO2 NP: induction of autophagy?

Activation of (macrophagic) autophagy after single oropharyngealadministration of CeO2 NP

Saline CeO2 Saline CeO2 Saline CeO2

1 day 7 days 28 days

Oropharyngeal aspiration of saline of 50µg CeO2 NPSacrifice 24h, 1 week or 28 days laterGFP-LC3 mice

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Macrophage exposure to CeO2 NP: activation of autophagy?

Macrophage exposure to CeO2 NP induces a functional autophagy

Control

CeO2

10 µg/mL

MergedLAMP1 (Red )LC3 (green)

Peritoneal macrophages (GFP-LC3 mice) exposed to 10µg/ml CeO2 NPLC3 (green): Autophagosome; LAMP1 (red): lysosome; merged (orange): autolysosome

916/11/2018 Sophie Lanone

Deficient macrophagic autophagy: protection from remodeling in response to CeO2 NP?

Oropharyngeal aspiration of saline of 50µg CeO2 NP - Sacrifice 28 days laterAtg5floxLysMCre mice inactivation of macrophagic autophagy

Saline CeO2

Atg5+/-

WT

Atg5+/-WT

Saline CeO2 Saline CeO2

WT 0 WT 50 Atg5 0 Atg 5 50

0

10

20

30

40

50

Bro

nch

iola

r w

all t

hic

kn

ess (µ

M)

* *

WT 0 WT 50 Atg5 0 Atg 5 50

0

10

20

30

40

50

Bro

nch

iola

r w

all t

hic

kn

ess (µ

M)

Bronchiolar wall thickness

Saline CeO2

Atg5+/-

WT

Atg5+/-WTWT WT 50 Atg Atg 50

0

10

20

30

40

Alv

eo

lar

wall t

hic

kn

ess (

A.U

.)

CeO2 Saline CeO2Saline

*

Alveolar wall thickness

Deletion of autophagy in macrophagesprotection againstalveolar but not bronchiolar remodelingafter single oropharyngealadministration of CeO2 NP

1016/11/2018 Sophie Lanone

Deficient macrophagic autophagy: protection from remodeling in response to CeO2 NP?

Oropharyngeal aspiration of saline of 50µg CeO2 NP - Sacrifice 28 days laterAtg5floxLysMCre mice inactivation of macrophagic autophagy

Atg5+/-WT

Saline CeO2 Saline CeO2

*Collagen III

Atg5+/-WT

CeO2 Saline CeO2Saline

*

Collagen I

Deletion of autophagy in macrophages protection againstalveolar remodeling after single oropharyngeal administration of CeO2 NP

0

5

10

15

20

rela

tive %

are

a o

f C

ola

gen

I

0.0

0.5

1.0

rela

tive %

are

a o

f C

ola

gen

III

0

1

2

3

4

rela

tive %

are

a o

f a

SM

A

-1

0

1

2

3

4

% a

rea o

f T

GF

b

Atg5+/-WT

Saline CeO2 Saline CeO2

*

SMA

Atg5+/-WT

Saline CeO2 Saline CeO2

*

TGFß

0

1

2

3

% a

rea o

f To

tal co

llag

en

Atg5+/-WT

CeO2 Saline CeO2Saline

*

Total Collagen

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Mechanism of protection: modification of M1/M2 balance?

Saline CeO2

Atg5+/-

WT

Oropharyngeal aspiration of saline of 50µg CeO2 NP - Sacrifice 28 days laterQuantification/qualification of macrophages

Mac3

Saline CeO2 Saline CeO2

Atg5+/-WT

* *

Total number of macrophages

0

200

400

600

800

# o

f m

ac3+

cells/1

0 f

ield

s

Deletion of autophagy in macrophages

Modification of M1/M2 phenotype in favor of M2

0

1

2

3

4

5

rela

tive %

are

a o

f iN

OS

Saline CeO2 Saline CeO2

Atg5+/-WT

Saline CeO2 Saline CeO2

Atg5+/-WT

Saline CeO2 Saline CeO2

Atg5+/-WT

0

2

4

6

8

rela

tive %

are

a o

f A

rgin

ase I

Saline CeO2 Saline CeO2

Atg5+/-WT

Saline CeO2 Saline CeO2

Atg5+/-WT

Saline CeO2 Saline CeO2

Atg5+/-WT

* **

0

2

4

6

8

10

rela

tive %

are

a o

f C

D68

0

2

4

6

rela

tive %

are

a o

f C

D80

0

1

2

3

4

5

rela

tive %

are

a o

f iN

OS

CD 86 CD 68 iNOS

CD 206CD 163 Arginase 1

0

2

4

6

8

rela

tive %

are

a o

f C

D163

0

1

2

3

4

rela

tive %

are

a o

f C

D206

0

2

4

6

8

rela

tive %

are

a o

f A

rgin

ase I *

* *

1216/11/2018 Sophie Lanone

Mechanism of protection: modification of M1/M2 balance?

WT

Control CeO2 NP

Atg5+/-

iNOS

WT

Control CeO2 NP

Atg5+/-

Arginase 1

WT

Control CeO2 NP

Atg5+/-

CD68

WT

Control CeO2 NP

Atg5+/-

CD206

Deletion of autophagy in macrophages

Modification of M1/M2 phenotype in favor of M2

1316/11/2018 Sophie Lanone

Mechanism of protection: macrophage autophagy?

Oropharyngeal aspiration of saline of 50µg CeO2 NP - Sacrifice 28 days laterRepeated administration of clodronate Depletion of macrophages

Depletion of macrophages no more protection against alveolarremodeling in Atg5+/- mice.

WT

Liposome Clodronate/CeO2

NP

Atg5+/-

HES

1416/11/2018 Sophie Lanone

Conclusions

Exposure to CeO2 NP induces

– lung remodeling in mice

– M1 macrophage differenciation

Deletion of macrophagic autophagy protects from alveolar remodeling and

M1 differenciation

Macrophagic autophagy: a new target to consider against lung remodeling?

1516/11/2018 Sophie Lanone

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