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

Progression from Hepatitis to Fibrosis Chronic liver diseases Viral Hepatitis, Autoimmune Hepatitis etc Inflammatory Response Hepatocyte Injury T Lymphocytes Kupffer Cells Activation and Recruitment of HSCs (Hepatic Stellate Cells) & MFBs (Portal Myofibroblasts) Collagen Synthesis Liver Fibrosis (Cirrhosis)

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

Molecular model of the subunit Tenascin-C (TNC) is an ECM glycoprotein upregulated in remodeling tissues Hexameric Structure FNIII Repeat TA EGFL Repeats Alternatively spliced region FG 1 2 3 4 5 A1 A2 A3 A4 B AD2 AD1 C D 6 7 8 Molecular model of the subunit

Mice develop normally without Tenascin-C (Saga Y et al. Gene Dev 1992) Tenascin-C Knockout (TNKO) Mice Mice develop normally without Tenascin-C (Saga Y et al. Gene Dev 1992) 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 8-week-old female BALB/c mice Experimental Groups ConA challenges (20 mg/Kg/ week, i.v.) 12w 0w 13w x 9w sacrifice x 6w x 3w x Saline alone Controls CTL2 x CTL1 x

Histological and Biochemical Analyses Histological & immunohitochemical staining: - H&E: Histological analyses for necrosis and inflammation. - Picrosirus red: Deposited collagen fibrils - TNC: TNC protein - a 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

Immunohistochemical staining of TNC Liver tissues of WT mice CTL1 3w 6w 9w TNC deposition is gradually increased after ConA administration. 12w CTL2

RT- PCR and qPCR for TNC mRNA 6 p<0.01 p<0.001 5 b-actin TNC - 540 - 344 4 p<0.01 p<0.05 TNC mRNA （folds) 3 2 CTL1 3w 6w 9w 12w CTL2 1 Conventional PCR CTL1 3w 6w 9w 12w CTL2 Quantitative real time PCR TNC mRNA levels sequentially increase in WT livers.

Collagen deposition of WT & TNKO mice livers CTL2 12w Picrosirius red staining Collagen deposition was less intense in TNKO mice after 12th ConA injection (12w) than WT mice.

Image analysis of fibrosis WT TNKO Total liver fibrosis Parenchymal fibrosis 25 7 p<0.001 p<0.001 p<0.05 p<0.001 p<0.05 ns p<0.05 p<0.01 20 6 5 15 Fibrotic area (%) 4 Fibrotic area (%) 10 3 2 5 1 CTL1 3w 6w 9w 12w CTL2 CTL1 3w 6w 9w 12w CTL2 Quantification was performed by NIH image using picrosirius red-stained sections Fibrosis areas in WT liver were more than in TNKO.

qPCR for procollagen I & III mRNA WT TNKO Procollagen I Procollagen III 6 4 p<0.05 p<0.05 p<0.01 p<0.001 p<0.01 p<0.05 p<0.001 p<0.05 5 Wild 3 KO Wild 4 KO COL1A2 mRNA (folds) COL3A1 mRNA (folds) 3 2 2 1 1 CTL1 3w 6w 9w 12w CTL2 CTL1 3w 6w 9w 12w CTL2 Procollagen I & III expression was upregulated in WT mice livers than in TNKO.

How could TNC promote liver fibrosis? TNC expressed in WT mice can contribute to liver fibrosis. How could TNC promote liver fibrosis?

Inflammatory cell infiltrate in WT & TNKO mice CV PT PT: portal tract CV: central vein TNKO PT CV Inflammatory infiltrate was more severe in WT mice livers than in TNKO.

Semi-quantification of the inflammatory infiltrate WT TNKO * ns ns * * p<0.05 3 3 2 2 2 4 2 Lymphocytic Infiltration 1 3 1 4 3 3 4 2 3 1 2 1 3w 6w 9w 12w modified Knodell’s HAI scoring system Lymphocytic infiltrate scores were significantly higher in WT than in TNKO after Con A administration

qPCR of inflammatory cytokines mRNA 2 4 6 8 10 12 CTL1 3w 6w 9w 12w CTL2 INF - γ mRNA (fold value) ** * *** IL 4 mRNA TNF a WT TNKO * p<0.05 ** p<0.01 *** p<0.001 Cytokines were significantly upregulated in WT than in TNKO.

α-SMA immunostaining of WT & TNKO liver PT: portal tract CV: central vein Arrows: Positive cells (Activated HSCs/MFBs) α-SMA positive cells were more common in WT than in TNKO.

a-SMA-positive cells ( /mm2 ) α-SMA positive cell counts WT TNKO p<0.001 p<0.05 p<0.01 300 200 a-SMA-positive cells ( /mm2 ) 100 CTL1 3w 6w 9w 12w CTL2 α-SMA positive cells significantly increased to a greater extent in WT than in TNKO.

qPCR for TGF-β mRNA WT TNKO 5 p<0.01 p<0.001 p<0.001 4 3 TGF-β1 mRNA (folds) 2 1 CTL1 3w 6w 9w 12w CTL2 TGF-β1 mRNA was significantly increased in WT than in TNKO.

TNC TNC promotes inflammatory response & cytokine upregulation Con A Rolling ↑ Migration ↑ TNC Liver Activated T lymphocyte Cytokines ↑ INF-γ INF-γ IL- 4 Kupffer cell Liver cell injury TNF-α (IL-1)

TNC TNC promotes activation of HSC Liver cell injury Liver Inflammatory Response ↑ Liver cell injury TNF-α Kupffer cell TNF-α (Lipid metabolites) Phenotypic change ↑ (IL-1, NO, O*) Quiescent HSC Activated HSC (a-SMA +)

TNC TNC promotes recruitment of HSCs/MFBs & TGF-b upregulation Liver Activated HSC (a-SMA +) Quiescent HSC TGF-b Expression/ Signaling ↑ TNC HSC proliferation TGF-b Activation & Migration ↑ Activated MFBs (a-SMA +) Portal MFBs

TNC-rich Matrix TNC HSCs/MFBs provides TNC-rich matrix Liver TGF-b Activated HSC (a-SMA +) Quiescent HSC TGF-b Expression/ Signaling ↑ TNC-rich Matrix TNC HSC proliferation TGF-b Activation & Migration ↑ Activated MFBs (a-SMA +) Portal MFBs

Procollagen ↑ Fibrosis TNC TNC promotes liver fibrosis Liver TGF-b Activated HSC (a-SMA +) Quiescent HSC TGF-b Expression/ Signaling ↑ Procollagen ↑ Fibrosis TNC HSC proliferation TGF-b Activation & Migration ↑ Activated MFBs (a-SMA +) Portal MFBs

Conclusion Tenascin-C actively contributes to hepatic fibrosis through possible mechanisms as follows: Augments the inflammatory response & upregulates inflammatory cytokines as INF-γ, IL-4 & TNF-α. Recruits & activates HSCs & MFBs. Up-regulates TGF-β.