deficient macrophage autophagy protects mice from ceo ... nanosafe... · 2017 aalapati et al....
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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
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
316/11/2018 Sophie Lanone
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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