EP2 receptor signaling supresses TLR3/TLR4-mediated inflammation in microglia and organotypic hippocampal slice cultures via MAPKs Nizar M. Yousif, Antonio Carlos Pinheiro de Oliveira, Simone Brioschi, Michael Huell, Knut Biber and Bernd L. Fiebich Laboratory of Translational Psychiatry, Department of Psychiatry and Psychotherapy, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
Introduction Brain inflammation is a critical factor involved in neurodegeneration. In this regard, increased levels of prostaglandin E2 have been found in patients and animal models of AD, PD and ALS, supporting the phenomena that inflammation via the PGE2 pathway is playing a role in these neuropathologies. However, clinical trials with nonsteroidal anti- inflammatory drugs (NSAIDS) that act at the COX-1 and COX-2 level or COX-2 selective inhibitors have failed to hold promises as potential therapy for CNS diseases. In the search for new alternatives, the PGE2 EP receptors (designated EP subtypes 1-4) have gained much of attention. The advantage of targeting these receptors is that their modulation, unlike inhibition of COX-2, would not affect other prostanoids with beneficial physiological functions.
Results
LP S +
LP S
ro l Co nt
Bu ta Bu ta
LP S +
LP S
Co nt ro l
0
-
+ -
+
+ +
+ +
h
l
+ -
24
h
Microglia
600
Microglia
***
**
p38 MAPK, ERK1/2 and JNK contribute to the anti-inflammatory effects mediated by EP2
150 100
200
50
) )
C)
+ ta u B
P
C) (I: y ol
PGE2 (pg/ml)
0
0
Bu ta
+
Po
I: ly(
)
ta Bu
Po ly (I: C
Co nt ro l
20
100
Control
0
Control
Poly(I:C)
l
Control
Poly(I:C)
Poly(I:C)
***
200
p-ERK (44/42 kDa)
150
pp38-MAPK (43 kDa) p38-MAPK (43 kDa) 150
l
50
)
LPS (10ng/ml) Poly(I:C) (25µg/ml Butaprost
-
+ -
+ -
-
+ - + + +
-
+ -
+ -
-
+ - + + +
Bu ta
+
Po ly (I: C
Po ly (I: C)
co nt ro l
0
LPS (10ng/ml) Poly(I:C) (25µg/ml Butaprost
150
150
*** 100
50
0
*** 100
50
0 Control
PPJNK/ -actin (% of PolyI:C)
150
Poly(I:C)
Control
Poly(I:C)
CCL2 Relative expression (% Poly(I:C) control)
ERK (44/42 kDa)
TNF Relative expression (% of Poly(I:C) control)
100
IL-6 Relative expression (% of Poly(I:C) control)
CCL2/GAPDH Fold induction (Relative to control)
Po ly (I: C)
Co nt ro l
Microglia
100
200
0
l tro n Co Bu ta
0
40
150
**
50
0
20
Po ly Bu (I: C) ta + Po ly (I: C)
50
300
**
***
200
40
0
60
250
(I: C
100
60
100
Po ly
TNF pg/ml
200
50
**
TNF- pg/ml
80
300
***
100
MG MG-Depleted
+
*
Bu ta
150
IL-6 pg/ml
p.ERK/ERK (% of PolyI:C)
*
100
400
IL-6 pg/ml
150
+ Bu ta
OHSCs
OHSCs
Clodronate
Po ly (I: C
Po ly (I: C)
Bu ta
Co nt ro l
) Po ly (I: C
Bu ta
Control
+
Po ly (I: C)
0 Bu ta
Co nt ro l
0
Microglial depletion reduces Poly(I:C)induced inflammatory markers in OHSCs
Bu ta
Merge
Po ly (I: C)
NeuN
TNFa pg/ml
GFAP
IL-6 pg/ml
400
Bu ta
200
Co nt ro l
Clodronate liposomes treatment does not deplete neurons or astrocytes
EP2 signaling suppresses proinflammatory cytokine and chemokine expression stimulated by TLR3 agonist
pp38/p38 (% of PolyI:C)
12
h
0 6
h
h 24
12
h 6
h 3
h 1
h 0
50
COX-2 (70 kDa)
LPS Poly(I:C) Butaprost
h
l
0
l
1
h
5
***
*** 100
+
LP S
)
Bu ta
+
Po l
y( I:C )
Bu ta
0
Microglia
Actin (42 kDa)
3
***
COX-2/ -actin (%of Poly(I:C)/LPS controls)
50
150
2
h
10
1
***
S
Bu ta
***
*
0
*** 100
50
0 Control
Poly(I:C)
** 100
50
Bu ta
+
Po ly
(I: C
)
Po ly (I: C)
Co nt ro l
0 Bu ta
TLR3/GAPDH Relative expression
***
3 TLR4/GAPDH Relative expression
MG MG-Depleted
15
+
LP
(I: ly Po
+ Bu ta
*** 100
Po ly (I: C
PGE2 (% of Poly(I:C)/LPS controls)
Clodronate
Microglia
150
0
LP
S
C)
C) (I: ly
Po
Co nt
ro l
Bu ta
0
50
LP S
Control
200
Bu ta
PGE2 (pg/ml)
400
**
100
Bu ta
Merge
NeuN
OHSCs
Po ly (I: C) +P ol y( I:C )
**
150
Bu ta
600
COX-2/GAPDH (% of Poly(I:C)/LPS controls)
OHSCs
Iba1
Butraprost affects microglial Iba1 immunoreactivity and morphology in Poly(I:C) challenged OHSCs, without affecting the overall number of microglial cells
Activation of EP2 reduces PGE2 release in microglia by modulating COX-2
Bu ta Po l y( Bu I:C ta ) + Po ly (I: C)
TLR3 and TLR4 receptor expression elevated in challenged OHSCs is microglia dependent
Conclusion
Acknowledgements
p-JNK (54/46 kDa)
β-Actin (42 kDa)
Our data identify an anti-inflammatory function for EP2 signaling in diverse innate immune
LPS (10ng/ml) Poly(I:C) (25µg/ml Butaprost
-
+ -
+ -
-
+ +
+ +
Nizar M. Yousif is supported by a doctoral
responses, through a mechanism that involves the mitogen-activated protein kinases pathway. l Therefore, we suggest that targeting EP2 receptor pathway may serve as a therapeutic target for
scholarship from Landesgraduiertenförderung l
neurodegenerative pathologies driven by neuroinflammation.
Freiburg.
(LGFG-IGA), Albert-Ludwigs-Universität
[email protected]