El-Karef Amro 303D006 三重大学医学部病理 Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic hepatitis in mice

El-Karef Amro 303D006

三重大学医学部病理

Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic

hepatitis in mice

Chronic liver diseases Viral Hepatitis, Autoimmune Hepatitis etc

Progression from Hepatitis to Fibrosis

Activation and Recruitment of HSCs (Hepatic Stellate Cells) &MFBs (Portal Myofibroblasts)

Liver Fibrosis(Cirrhosis)

T LymphocytesHepatocyte Injury

Kupffer Cells

Inflammatory Response

Collagen Synthesis

Tenascin-C (TN-C) in Chronic Liver Diseases

• highly expressed in chronic viral hepatitis, autoimmune hepatitis and bile obstruction

• highly deposited in interfaces of fibrotic areas and in peri-sinusoidal spaces

• synthesized by HSCs

Possible involvement in liver fibrosis

1 2 3 4 5 A1 A2 A3 A4 B AD2 AD1 C D 6 7 8

FNIII Repeat TA EGFL Repeats Alternatively spliced region FG

876DCAD1AD2BA4A3A2A154321 876DCAD1AD2BA4A3A2A154321

8 7 6 D C AD1 AD2 B A4 A3 A2 A1 5 4 3 2 18 7 6 D C AD1 AD2 B A4 A3 A2 A1 5 4 3 2 1

Tenascin-C (TNC) is an ECM glycoprotein upregulated in remodeling tissues

Hexameric Structure

Molecular model of the subunit

Mice develop normally without Tenascin-C

(Saga Y et al. Gene Dev 1992)

Tenascin-C Knockout (TNKO) Mice

Abnormal repair in diseased tissues of adult mice

• Neuromuscular junction• Venom-induced glomerulonephritis• Chemically induced dermatitis• Corneal injury• Granulation tissue after cardiac injury

Aim of Study

Immune-mediated chronic hepatitis model[ Concanavalin A (ConA)-induced ]

×WT mice and TNKO mice

To obtain direct evidence for promotive roles of TNC in live fibrosis

Experimental Protocol of ConA Challenge

sacrifice

ExperimentalGroups

8-week-old female BALB/c mice

0w 13w

ConA challenges (20 mg/Kg/ week, i.v.)

12w

9w

6w

3wSaline alone

CTL2

CTL1

x

x

x

x

xx

Controls

Histological & immunohitochemical staining:

- H&E: Histological analyses for necrosis and inflammation.

- Picrosirus red: Deposited collagen fibrils

- TNC: TNC protein

- smooth muscle actin (α-SMA ): Activated HSCs/MFBs

Liver RNA extraction & RT-PCR:

- Quantitative real-time PCR (qPCR)

ECM proteins: TNC – Collagen I & III

Inflammatory cytokines: INF-γ, IL-4 & TNF-α

Fibrogenic cytokine: TGF-β

Histological and Biochemical Analyses

CTL1

6w

12w CTL2

3w

9w

Immunohistochemical staining of TNC

TNC deposition is gradually increased

after ConA administration.

Liver tissues of WT mice

0

1

2

3

4

5

6

CTL1 3w 6w 9w 12w CTL2T

NC

mR

NA

（fo

lds)

p<0.05

RT- PCR and qPCR for TNC mRNA

TNC mRNA levels sequentially increase in WT livers.

Quantitative real time PCR

-actin

TNC

- 540

- 344

CTL1 3w 6w 9w 12w CTL2

Conventional PCR

p<0.01

p<0.01 p<0.001

CTL2

12w

WT TNKO

Collagen deposition of WT & TNKO mice livers

Picrosirius red staining

Collagen deposition was less intense in TNKO mice after 12th ConA injection (12w) than WT mice.

0

5

10

15

20

25


Fib

rotic

are

a (%

)

0

1

2

3

4

5

6

7


ns

Fib

rotic

are

a (%

)

Image analysis of fibrosis

Total liver fibrosis Parenchymal fibrosis

Fibrosis areas in WT liver were more than in TNKO.

Quantification was performed by NIH image using picrosirius red-stained sections

WT TNKO

p<0.01p<0.001 p<0.001p<0.001p<0.05 p<0.05 p<0.05

Procollagen I Procollagen III

qPCR for procollagen I & III mRNA

WildKO Wild

KO

0

1

2

3

4

CO

L3A

1 m

RN

A (

fold

s)


1

2

3

4

5

6


CO

L1A

2 m

RN

A (

fold

s)

Procollagen I & III expression was upregulated in WT mice livers than in TNKO.

WT TNKO

p<0.001 p<0.05 p<0.05 p<0.01 p<0.01 p<0.05 p<0.001 p<0.05

TNC expressed in WT mice can

contribute to liver fibrosis.

How could TNC promote liver fibrosis?

TNKO

PT

PT

CV

CV

WT

Inflammatory cell infiltrate in WT & TNKO mice

PT: portal tract CV: central vein

Inflammatory infiltrate was more severe in WT mice livers than in TNKO.

Semi-quantification of the inflammatory infiltrate

Lymphocytic infiltrate scores were significantly higher in WT than in TNKO after Con A administration

modified Knodell’s HAI scoring system

Lym

phoc

ytic

Infil

trat

ion

3w 6w 9w 12w

3

2

1

0

3

1

2 2

1 3

3

2 4

43 24

2 1 3

* ns* ns

WT TNKO

* p<0.05

qPCR of inflammatory cytokines mRNA

Cytokines were significantly upregulated in WT than in

TNKO.

WT TNKO

02468

1012

02468

1012


INF-

γm

RN

A

(fo

ld v

alu

e) *** *** *

0

2

4

6

8


IL-4

mR

NA

(f

old

val

ue

) * * **

02468

1012


TN

F-

mR

NA

(f

old

val

ue

) ****** **

* p<0.05

** p<0.01

*** p<0.001

α-SMA immunostaining of WT & TNKO liver

α-SMA positive cells were more common in WT than in TNKO.

PT: portal tract CV: central vein

Arrows:Positive cells(Activated HSCs/MFBs)

α-SMA positive cell counts

100

200

-S

MA

-po

siti

ve c

ells

( /

mm

2 )

0

300


p<0.05

α-SMA positive cells significantly increased to a greater extent in WT than in TNKO.

WT TNKO

p<0.01 p<0.001

0

1

2

3

4

5


TG

F-β

1 m

RN

A (

fold

s)

qPCR for TGF-β mRNA

TGF-β1 mRNA was significantly increased in WT than in TNKO.

WT TNKO

p<0.001 p<0.001p<0.01

Con A

Kupffer cell

Liver cell injury

Activated T lymphocyte

TNF-α

INF-γIL- 4

TNC promotes inflammatory response & cytokine upregulation

LiverTNC

INF-γ

Rolling ↑Migration ↑

Cytokines ↑

(IL-1)

Kupffer cell

Liver cell injury

Liver

TNC Inflammatory Response ↑

TNF-α

(IL-1, NO, O*)

Quiescent HSC

(Lipid metabolites)

TNF-α

Activated HSC-SMA +)

Phenotypicchange ↑

TNC promotes activation of HSC

TNC promotes recruitment of HSCs/MFBs & TGF- upregulation

TNC

Quiescent HSC

Activated HSC-SMA +)TGF-

Expression/Signaling ↑

Portal MFBs

Activation & Migration ↑

Activated MFBs(-SMA +)

TGF-HSC proliferation

Liver

HSCs/MFBs provides TNC-rich matrix

TNC

Quiescent HSC



Portal MFBs




Liver

TNC-rich Matrix

TNC promotes liver fibrosis

TNC

Quiescent HSC



Portal MFBs




Liver

Procollagen ↑ Fibrosis

Tenascin-C actively contributes to hepatic fibrosis through possible mechanisms as follows:

1. Augments the inflammatory response & upregulates inflammatory cytokines as INF-γ, IL-4 & TNF-α.

2. Recruits & activates HSCs & MFBs.

3. Up-regulates TGF-β.

Conclusion

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El-Karef Amro 303D006 三重大学医学部病理 Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic hepatitis in mice