Beruflich Dokumente
Kultur Dokumente
Background
Biliary atresia is a life-threatening condition in
infants in which the bile ducts inside or outside
the liver become inflamed and obstructed.
Complement-dependent inflammation is
required for duct obstruction in biliary atresia.
Complement receptor C5aR is increased in the
mouse model of biliary atresia
Loss of the complement receptor, C5aR in mice
prevents bile duct atresia and promotes long
term survival.
Background cont.
C5aR-expressing cells populate the livers of
mice with experimental biliary atresia.
Myeloid cells and more specifically
Macrophages express high levels of C5aR.
Previous studies have implicated macrophages
in the pathogenesis of biliary atresia.
Microarray analysis of EHBDs from RRVchallenged mice showed increased M1 and
decreased M2 markers of macrophage
activation above control mice.
Overall Objectives
1. To quantify preserved epithelium in WT
and C5aR-KO mice.
2. To link C5aR to macrophage activity and
identify M1/M2 marker expression in WT
and C5aR-KO mice.
Antigen
M1
Macrophage
ProInflammatory
Antigen
Presenting
Cell
Macrophages
M2
Macrophage
AntiInflammatory
Response
Th1
Respon
se
IFNg, IL-12,
IL-6, IL-1b
TNFa
Th2
Respons
e
IL-4, IL-10, IL13, IL-1,
Stablin1
Methods
Quantify the total surface area of preserved
epithelium and peribiliary glands at D14 of bile
ducts using Olympus Cellsens and Adobe
Photoshop software.
Isolate RNA from livers and perform cDNA
synthesis for the following cohorts of mice:
WT-Saline and WT-RRV
C5aR-KO Saline & C5aR-KO RRV
Analyze microarray data of control subjects and
patients with BA for macrophage M1/M2
markers .
WT
Saline
WT
RRV
C5aR-KO
RRV
Quantification of Epithelium:
duct
100
***
P<0.0001
80
60
40
20
0
C5aR-KO EHBDs
WT EHBDs
Experimental Groups
60
WT
RRV
C5aR-KO
RRV
***
P<0.0001
40
20
C5aR-KO EHBDs
WT EHBDs
Experimental Groups
Macrophages
Saline
*
15
10
800
RRV
Relative CD68 expression
% Intrahepatic Macrophages
20
600
400
200
0
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
Day 3
Day 7
Day 14
Days after Saline or RRV injections
Genes of Interest
M1 Markers
M2 Markers
Microarray of EHBD
The genes that possess an increased signature
in the EHBD, which show a clear up-regulation
of M1 genes(IFNg, TNFa, IL-6), which induce a
pro-inflammatory environment and enhance
the polarization of the M1 phenotype and Th1
response.
There is a significant down-regulation of M2
genes(Ym1, Fizz1, Mrc2), which potentially
fails to contribute to the change in polarization
of a pro-inflammatory microenvironment.
10
15 30
40
50
Day 7
CD14
Nos2
Il12b
Dectin1
Ccr7
Ccr2
Ccl5
Ccl4
Ccl3
Ccl2
Cxcl11
Cxcl10
Cxcl9
Il6
Il1b
TNF-a
M1 Macrophage markers
Day 3
CD14
Nos2
Il12b
Dectin1
Ccr7
Ccr2
Ccl5
Ccl4
Ccl3
Ccl2
Cxcl11
Cxcl10
Cxcl9
Il6
Il1b
TNF-a
M1 Macrophage markers
M1 Macrophage markers
10
20
50
55
60
Day 14
CD14
Nos2
Il12b
Dectin1
Ccr7
Ccr2
Ccl5
Ccl4
Ccl3
Ccl2
Cxcl11
Cxcl10
Cxcl9
Il6
Il1b
TNF-a
0
Day 3
Pdgfc
Stab1
Mrc2
Cd302
Mrc1
Mgl2
Mgl1
Ccl17
Stab1
M2 Macrophage markers
Cd209a
Cd209a
Mrc2
Cd302
Mrc1
Mgl2
Mgl1
Ccl17
Cd209a
Mrc2
Cd302
Mrc1
Mgl2
Mgl1
Ccl17
Fizz1
Fizz1
Fizz1
Arg1
Arg1
Arg1
-1
Day 14
Pdgfc
Stab1
M2 Macrophage markers
M2 Macrophage markers
Day 7
Pdgfc
-1
-1
40
WT Saline
WT RRV
10
Day 3
Day 7
Day 14
Days after Saline or RRV injections
2.5
WT Saline
WT RRV
20
10
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
2.0
1.5
1.0
0.5
0.0
Day 3
Day 7
Day 14
Days after Saline or RRV injections
30
0
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
50
15
300
100
WT Saline
WT RRV
150
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
200
100
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
150
100
50
0
Day 3
Day 7
Day 14
Days after Saline or RRV injections
Day 3
Day 7
Day 14
Days after Saline or RRV injections
200
-1
1500
1000
500
0
WT Saline
WT RRV
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
2000
Day 3
Day 7
Day 14
Days after Saline or RRV injections
2500
15
Relative CD209 expression
10
WT Saline
WT RRV
15
250
Relative Ym1 expression
20
WT Saline
WT RRV
25
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
10
Day 3
Day 7
Day 14
Days after Saline or RRV injections
Results
There is a similar macrophage signature,
in the livers as found in the EHBD
M1
& M2
Since there is a distinguishing imbalance
in the M1/M2 macrophage expression
level, I want to further investigate
macrophages due to the subsequent
phenotypes and the differential
microenvironments that are exhibited.
C5aR Macrophages
+
% C5aR+ Macrophages
100
80
Saline
RRV
60
40
20
Day 3
Day 7
Day 14
Days after Saline or RRV injections
Day 3
Day 7
Day 14
Days after Saline or RRV injections
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
C5aR-KO RRV
10
40
WT Saline
WT RRV
C5aR-KO RRV
20
10
Day 3
Day 7
Day 14
Days after Saline or RRV injections
2.0
WT Saline
WT RRV
C5aR-KO RRV
1.5
1.0
0.5
0.0
Day 3
Day 7
Day 14
Days after Saline or RRV injections
30
2.5
50
WT Saline
WT RRV
C5aR-KO RRV
300
100
15
WT Saline
WT RRV
C5aR-KO RRV
150
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
C5aR-KO RRV
200
100
Day 3
Day 7
Day 14
Days after Saline or RRV injections
100
50
0
WT Saline
WT RRV
C5aR-KO RRV
600
400
200
Day 3
Day 7
Day 14
Days after Saline or RRV injections
4
Relative Fizz1 expression
800
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
C5aR-KO RRV
WT Saline
WT RRV
C5aR-KO RRV
-1
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
C5aR-KO RRV
2000
1000
Day 3
Day 7
Day 14
Days after Saline or RRV injections
3000
150
15
Relative CD209 expression
200
WT Saline
WT RRV
C5aR-KO RRV
250
Day 3
Day 7
Day 14
Days after Saline or RRV injections
WT Saline
WT RRV
C5aR-KO RRV
10
Day 3
Day 7
Day 14
Days after Saline or RRV injections
Results
There is a significant up regulation of M2 genes
in livers from C5aR-KO with a concomitant
decrease of M1 markers, reflecting a blunted
pro-inflammatory response.
Thus, absence of C5aR attenuates the
commitment of macrophages to an M1
phenotype and regulates cholangiocyte injury.
MFAP4
MPEG1
CD68
Normal
BA
500
1000
1500
Relative expression
2000
M1 Macrophage markers
IL7R
Macrophage M1 markers
Dectin-1
CCR7
CCR2
CXCL1
CCL20
CCL4
CCL3
CCL2
CXCL10
IL23A
Normal
BA
IL6
500
1000
Relative expression
1500
M2
M1macrophage
Macrophagemarkers
markers
IGF1
Macrophage M2 markers
CCL14
SRA1
CD302
STAB1
CD209
ARG1
IL13
Normal
BA
IL4
1000
2000
3000
Relative expression
4000
Conclusions
Absence of C5aR preserves cholangiocyte
cell architecture and prevents
inflammatory duct obstruction.
Polarization of macrophages to an M1
phenotype identifies a new role for C5aR in
maintaining intrahepatic M1/M2 balance in
biliary atresia.
Livers of children with BA show a dominant
M1 macrophage signature and decreased
M2 profile.
Acknowledgements
Thank you all for
taking the time to
listen to my work
this past summer
I would like to
specially thank...