Rozen Le Panse CNRS UMR 8162 Centre Chirurgical Marie lannelongue Le Plessis Robinson - France
16
Rozen Le Panse CNRS UMR 8162 Centre Chirurgical Marie lannelongue Le Plessis Robinson - France Thymic changes in Myasthenia Gravis: Involvement of chemokines in thymic hyperplasia associated with an inflammatory environment International conference on Myasthenia December 1-2, 2009 - Paris - France
description
Thymic changes in Myasthenia Gravis:. Involvement of chemokines in thymic hyperplasia associated with an inflammatory environment. Rozen Le Panse CNRS UMR 8162 Centre Chirurgical Marie lannelongue Le Plessis Robinson - France. International conference on Myasthenia - PowerPoint PPT Presentation
Transcript of Rozen Le Panse CNRS UMR 8162 Centre Chirurgical Marie lannelongue Le Plessis Robinson - France
Rozen Le PanseCNRS UMR 8162
Centre Chirurgical Marie lannelongueLe Plessis Robinson - France
Thymic changes in Myasthenia Gravis:Involvement of chemokines in thymic hyperplasia
associated with an inflammatory environment
International conference on MyastheniaDecember 1-2, 2009 - Paris - France
Implication of the thymus in MG
Cellules épithéliales corticales
Cellules épithéliales médullaires
Thymocytes
Cellules épithéliales corticales
Cellules épithéliales médullaires
Thymocytes
AChR+ Myasthenia Gravis (MG) AChR expression (Wakkach et al., 1996) Anti-AChR autoreactive T cells (Melms et al., 1988)B cells producing anti-AChR antibodies (Leprince et al., 1990
AChR
Thymus
Muscle
!
Méraouna et al., Blood 2006
Ectopic germinal centers (GCs)
Germinal centers (CD21 labeling)Epithelial cells (keratin labeling)
Adult control MG + corticoïdsMG
Thymic hyperplasia in AChR+ MG
% o
f CD
21+
area
s
0.01
0.1
1
10
p<0.003p<0.001
MG + Cortico MG Controls
Neoangiogenesis is associated with thymic hyperplasia
Adult
MH
HEVs
KeratinGCs
Number of HEVs
0
50
100
150
200
p=0.0714p=0.0357
HEV
s/th
ymic
sec
tion
MG
HEVsGCs
Ectopic high endothelial venules (HEVs)
Berrih-Aknin et al., Ann. of Neurol. 2009
Lymphangiogenesis
Adults MH0
50
100
150
200
250
300
p<0.05
Adults MH0
25
50
75
100
125
150
175
p<0.01
VEG
FR3
mR
NA
(AU
)
PRO
X1 m
RN
A (A
U)
Adult control
HEVs
StromaGCs
Role of chemokine in thymic hyperplasia
Expression of chemokines involved in GC formation
Thymic transcriptome analysis revealed a chemotactic signature in the hyperplastic thymus of MG patients.
Chemotactic assays
Le Panse et al., J. Immunol 2006
CXCL13 CCL21 BB Ad MH Cortico
0
2
4
6
8
10 p<0.001 p<0.001
CXC
L13
(ng/
mg
of p
rote
in)
BB Ad MH Cortico0
1
2
3
4p<0.001 p<0.001
CC
L21
(ng/
mg
of p
rote
in)
Ad ML MH SN-100
0
100
200
300
400
500
p<0.05
p<0.05
Cel
l mig
ratio
n(fl
uore
scen
ce m
ean)
ELISA
Analysis of CXCL13 overexpression in MG
Méraouna et al., Blood 2006
CXCL13 overexpression by thymic epithelial cells
Ad BB MG
100
200
Ad BB MG
100
200
CXC
L13
mR
NA
(AU
)
Q-PCR on thymicepithelial cells
Laser-capturemicrodissection
Q-PCRon CXCL13
Generation of transgenic mice for CXCL13
WT1 WT2 WT3 Tg-A1 Tg-A2 Tg-B10
50
100
150
CXC
L13
mR
NA
norm
aliz
ed o
n G
APD
H (A
U)
Increased expression of CXCL13 and B-cell recruitment
Weiss et al., in progress
polyAKeratin 5 promoter CXCL13
Kpn I
5,2 kb EGFP0,8 kb 1,2 kb
Not I Not I0,5 kb
Promoteur K5 Intron β-Globine
Poly-A5,2 kb EGFP
0,8 kbEGFP0,8 kb 1,2 kb
Not I Not I0,5 kb
Promoteur K5 Intron β-Globine
Poly-A
Kpn I
Kpn IKpn I
5,2 kb EGFP0,8 kb 1,2 kb
Not I Not I0,5 kb
Promoteur K5 Intron β-Globine
Poly-A5,2 kb EGFP
0,8 kbEGFP0,8 kb 1,2 kb
Not I Not I0,5 kb
Promoteur K5 Intron β-Globine
Poly-A
Kpn I
Kpn IKpn I
5,2 kb EGFP0,8 kb 1,2 kb
Not I Not I0,5 kb
Promoteur K5 Intron β-Globine
Poly-A5,2 kb EGFP
0,8 kbEGFP0,8 kb 1,2 kb
Not I Not I0,5 kb
Promoteur K5 Intron β-Globine
Poly-A
Kpn IKpn I
Kpn I
Not1 Not1
pEYFP-1
Heterozygous…
B220Keratin
Q-PCR on CXCL13 Immunofluorescence
Analysis of CCL21 overexpression in MG
VEGFR3CCL21
0 0.5 5 50 125 250 5000
100
200
300350550750950
1150 T cellsB cells
p<0.0001ng/ml
0 100 200 300 400 500 600 700 800
CD27-IgD+
CD27-IgD-
% of migration towards CCL21compared to passive migration (100%)
Naive B cells
DifferentiatedB cells
p=0.
0317
CD
19+
B c
ells0
500
10005000
7500
10000
WS
_
LEC
CCL2
1+
vess
els
HUVEC
WS
_
CCL2
1+
vess
els
Non-MGadults
MG (MH)patients
PROX1
mRNA e
xpre
ssio
n (A
U)
norm
alised
with
28S
p=0.076 p=0.05
0
500
10005000
7500
10000
D6 m
RNA e
xpre
ssio
n (A
U)
norm
alised
with
28S
Non-MGadults
MG (MH)patients
WS
_
LEC
CCL2
1+
vess
els
HUVEC
WS
_
CCL2
1+
vess
els
p=0.076
D6 m
RNA
(AU)
WS
_
LEC
CC
L21
+ve
ssel
s
HU
VE
C
WS
_
CCL2
1+
vess
els
PRO
X1 m
RNA
(AU)
0
500
10005000
7500
10000
WS
_
LEC
CCL2
1+
vess
els
HUVEC
WS
_
CCL2
1+
vess
els
Non-MGadults
MG (MH)patients
PROX1 mRNA e
xpress
ion (A
U)
norm
alised
with
28S
p=0.076 p=0.05
0
500
10005000
7500
10000
D6 mRNA e
xpress
ion (A
U)
norm
alised
with
28S
Non-MGadults
MG (MH)patients
WS
_
LEC
CCL2
1+
vess
els
HUVEC
WS
_
CCL2
1+
vess
els
p=0.076
% o
f cel
l mig
ratio
nCCL21 overexpression by lymphatic vessels
HEVsCCL21
Berrih-Aknin et al., Ann. of Neurol. 2009
Immunofluorescence Transwell assaysQ-PCR for PROX1 and D6
Julia Weiss : Poster 16
Expression of SDF1 on thymic HEVs
Search for chemokines on HEVs
HEVsSDF1
MG
Transcytosis ?
Immunofluorescence
Roles of chemokines in thymic hyperplasia
Signals triggering these thymic dysregulations ?
Thymus
Epithelial cells
CXCL13
Lymphaticendothelial cells
CCL21
GC
T
TnDC
BB
BB
BB
B
B
B
B
BB
BFDC
B
T
T
T
BT
B
Tn
Tn
TT
TT
TnTn
BB
BB
BB
B
B
B
B
BB
FDC
B
BB
BB
BB
B
B
B
BBBB
BBB
BBBB
BB
BB
BB
BB
BBBB
B
BB
T
T
BTT
B
TnTn
HEV
SDF1-α
CXCL10 (Feferman et al., 2003)
CXCL9-CXCL11 (Transcriptomic analysis)
RANTES (Colombarra et al., 2005)
Peripheral cell recruitment
Microarray analysis revealed a role for IFN-I in MG
Adults MH Cortico0.0
0.5
1.0
1.5
2.0 p=0.007 p=0.008
Mic
roar
ray
ratio
s of
type
Iin
terf
eron
indu
ced
gene
s
Ad MG0
20
40
60
80
100p<0.05
-IF
N (p
g/m
g of
pro
tein
)
Ad MG0
1
2
3
4
-IF
N s
ubty
pes
(pg/
mg
of p
rote
in)
Ad MG0
50
100
150
200p<0.01
IFN-
m
RNA
expr
essi
on
Ad MG0
200
400
600
IFN-
2
mRN
A ex
pres
sion
Increased expression of IFN- in MG thymuses
Cufi et al., in progress
-IFN
-IFN
Microarray data Q-PCR and ELISA
Roles of IFN-I on chemokine expression
IFN-I IFN-IIU/ml
CCL21
Lymphatic endothelial cells(Dermal LECs)
Perrine Cufi : Poster 14
IFN-I induces the expression of :• CXCL13 and CCL21• -AChR
Thymic epithelial cells
IFN-I IFN-IIU/ml
CXCL13
IFN-I
-AChR
Potential involvement of viral infection in MG
Cufi et al., in progress
Specific effect of Poly(I:C)
Selective effect on the -AChR subunit
0
400
800
1200
0 25 50 100
AC
hR s
ubun
it m
RN
A (A
U)
Poly(I:C) (µg/ml)
0
600
1200
1800
100
p<0.0001
p<0.002
Auto
antig
en m
RN
A ex
pres
sion
(AU
)
-AChR-AChr-AChR
-AChR-AChr-AChR
0
1000
2000
3000p<0.001
-A
ChR
mR
NA
(AU
)
TLR1/2
TLR3
TLR4 TLR6/2 TLR7 TLR7/8 TLR9TLR5
In vivo confirmation of Poly(I:C) effects on -AChR
Poly(I:C) Control
-AChR -AChR
Cufi et al., in progress
Thymic upregulation of -AChR expression by Poly(I:C)
Control Poly(I:C)0
50
100
150
200
250 p<0.009
-A
ChR
mR
NA
(AU
)
Control Poly(I:C)0
50
100
150
200
-A
CH
R m
RN
A (A
U)
Q-PCR on AChR subunits
Could a viral infection, under particular conditions, trigger thymic changes involved in MG?
Conclusion
Thymic Hyperplasia
MDA5
RIG-IPKR
TLR3
dsRNA/Poly(I:C)
Thymic Epithelial Cells
Endosome
Nucleus IFN-β α-ACHR
IFNAR1
IFNAR2
α-ACHR
CCL21
Lymphatic endothelial cells
CXCL13
Julia Weiss
Perrine Cufi
Nathalie Ruhlman
Géraldine Cizeron-Clairac
Jacky Bismuth
Frédérique Truffault
Amel Méraouna
Sonia Berrih-Aknin
Nicole Kerlero de Rosbo
WEIZMANN INSTITUTE
Idit ShacharFrida Lantner
UMR 8162
Acknowledgements
