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Complement C5aR-derived

signals reciprocally regulate


M1/M2 macrophage balance in
disease pathogenesis of biliary
atresia
Lakmal Ekanayake, Reena Mourya, Stephanie
Walters, Jorge Bezerra, Pranavkumar
Shivakumar
April 15th, 2016

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

Hypothesis and Aims


Hypothesis 1:
EHBDs show differential preservation of duct
epithelium in virus-infected WT and C5aR-KO mice.
Aim: To determine cholangiocyte cell surface area of
EHBDs from Day 14 RRV-challenged WT and C5aR-KO
mice.

Hypothesis and Aims


Hypothesis 2:
C5aR is an integral driver of macrophages as central
effectors of epithelial injury and duct obstruction.
Aim 1: To determine if EHBDs show differential
expression of M1/M2 macrophage markers.
Aim 2: To determine intrahepatic markers of M1 and M2
macrophages in nave and RRV-infected mice.

Hypothesis and Aims


Hypothesis 3:
Impaired M1/M2 macrophage balance regulates the
phenotype of human biliary atresia.
Aim: To analyze the microarray data of M1 and M2
macrophages.

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 .

Hypothesis and Aims


Hypothesis 1:
EHBDs show differential preservation of duct
epithelium in virus-infected WT and C5aR-KO mice.
Aim: To determine cholangiocyte cell surface area of
EHBDs from Day 14 RRV-challenged WT and C5aR-KO
mice.

WT
Saline

WT
RRV

C5aR-KO
RRV

Surface area of duct epithelium (mm2)

Quantification of Epithelium:
duct
100

***

P<0.0001

80
60
40
20
0

C5aR-KO EHBDs

WT EHBDs

Experimental Groups

Quantification of Epithelium: PBGs

Surface area of PBGs (mm2)

60

WT
RRV

C5aR-KO
RRV

***

P<0.0001

40

20

C5aR-KO EHBDs

WT EHBDs

Experimental Groups

Hypothesis and Aims


Hypothesis 2:
C5aR is an integral driver of macrophages as central
effectors of epithelial injury and duct obstruction.
Aim 1: To determine if EHBDs show differential
expression of M1/M2 macrophage markers.
Aim 2: To determine intrahepatic markers of M1 and M2
macrophages in nave and RRV-infected mice.

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.

M1 markers are increased in RRV-infected


EHBDs

10

15 30

40

Fold change above controls

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

EHBD Microarray data

10

20

50

55

Fold change above controls

60

Day 14

CD14
Nos2
Il12b
Dectin1
Ccr7
Ccr2
Ccl5
Ccl4
Ccl3
Ccl2
Cxcl11
Cxcl10
Cxcl9
Il6
Il1b
TNF-a
0

Fold change above controls

M2 markers are decreased in RRV-infected


EHBDs
EHBD Microarray data

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

Fold change above controls

Day 14

Pdgfc

Stab1

M2 Macrophage markers

M2 Macrophage markers

Day 7

Pdgfc

-1

Fold change above controls

-1

Fold change above controls

M1 markers are increased in livers of RRV


mice

Relative iNOS expression

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

Relative IL-6 expression

50

15

300

Relative Dectin-1 expression

100

Relative IL-1b expression

WT Saline
WT RRV

Relative TNFa expression

Relative IFNg expression

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

M2 markers are decreased in livers of RRV


mice

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

Relative Stabilin-1 expression

15

250
Relative Ym1 expression

Relative IL-13 expression

20

WT Saline
WT RRV

Relative Fizz1 expression

Relative IL-10 expression

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

Relative IL-1b expression

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

Relative IL-6 expression

50

WT Saline
WT RRV
C5aR-KO RRV

300

Relative Dectin-1 expression

100

15

Relative TNFa expression

WT Saline
WT RRV
C5aR-KO RRV

Relative iNOS expression

Relative IFN gexpression

150

M1 markers (WT and C5aRKO)

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

M2 markers (WT and C5aRKO)

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

Relative Mrc2 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

Relative Stabilin-1 expression

150

15
Relative CD209 expression

200

WT Saline
WT RRV
C5aR-KO RRV

Relative IL-13 expression

Relative Ym1 expression

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.

Human Biliary Atresia


Macrophage markers
Macrophage markers

MFAP4

MPEG1

CD68

Normal
BA

500

1000

1500

Relative expression

2000

Human Biliary Atresia


IL1R1

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

Human Biliary Atresia


CCL23

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...

All of the Bezerra


Lab
Dr. Bezerra
Dr. Shivakumar
Reena Mourya
Tatsuki Mizuochi
Aki
Stephanie Walters