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The title is: "Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic hepatitis in mice

This study investigates the role of tenascin-C (TN-C), an extracellular matrix glycoprotein, in liver fibrosis in immune-mediated chronic hepatitis. The findings show that TN-C is highly expressed and deposited in fibrotic areas of the liver, and its deficiency leads to reduced collagen deposition and fibrosis. The study provides direct evidence for the promotive role of TN-C in liver fibrosis.

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The title is: "Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic hepatitis in mice

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  1. 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

  2. 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)

  3. 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

  4. Tenascin-C (TNC) is an ECM glycoprotein upregulated in remodeling tissues Hexameric Structure FNIII Repeat TA EGFL Repeats Alternatively spliced region FG Molecular model of the subunit

  5. 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

  6. 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

  7. Experimental Protocol of ConA Challenge 8-week-old female BALB/c mice ConA challenges (20 mg/Kg/ week, i.v.) Experimental Groups 12w 0w 13w x 9w sacrifice x 6w x 3w x Saline alone Controls CTL2 x x CTL1

  8. 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

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

  10. - 344 TNC b-actin - 540 RT- PCR and qPCR for TNC mRNA 6 p<0.01 p<0.001 5 p<0.01 4 p<0.05 TNC mRNA （folds) 3 2 CTL1 3w 6w 9w 12w CTL2 1 Conventional PCR 0 CTL1 3w 6w 9w 12w CTL2 Quantitative real time PCR TNC mRNA levels sequentially increase in WT livers.

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

  12. WT TNKO Image analysis of fibrosis Total liver fibrosis Parenchymal fibrosis 25 7 p<0.05 p<0.05 p<0.01 ns p<0.001 p<0.001 p<0.05 p<0.001 6 20 5 15 4 Fibrotic area (%) Fibrotic area (%) 10 3 2 5 1 0 0 CTL1 3w 6w 9w 12w CTL1 3w 6w 9w 12w CTL2 CTL2 Quantification was performed by NIH image using picrosirius red-stained sections Fibrosis areas in WT liver were more than in TNKO.

  13. WT TNKO qPCR for procollagen I & III mRNA Procollagen I Procollagen III 6 4 p<0.05 p<0.05 p<0.01 p<0.001 p<0.05 p<0.001 p<0.05 p<0.01 5 3 Wild Wild KO KO 4 COL1A2 mRNA (folds) COL3A1 mRNA (folds) 2 3 2 1 1 0 0 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.

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

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

  16. WT TNKO Semi-quantification of the inflammatory infiltrate ns ns *p<0.05 * * 3 3 2 4 2 2 2 Infiltration Lymphocytic 3 3 3 4 4 2 1 1 3 0 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

  17. 12 12 * *** * ** 10 10 WT TNKO mRNA 8 8 (fold value) 6 6 γ - INF 4 4 2 2 0 0 12w CTL1 3w 6w CTL2 9w 8 * * ** 6 4 mRNA (fold value) 4 - IL 2 0 3w CTL1 6w 9w 12w CTL2 12 *** ** *** 10 mRNA 8 (fold value) 6 a - 4 TNF 2 0 12w CTL1 3w 6w CTL2 9w qPCR of inflammatory cytokines mRNA * p<0.05 ** p<0.01 *** p<0.001 Cytokines were significantly upregulated in WT than in TNKO.

  18. 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.

  19. WT TNKO α-SMA positive cell counts p<0.001 p<0.05 p<0.01 300 200 a-SMA-positive cells ( /mm2 ) 100 0 CTL1 3w 6w 9w 12w CTL2 α-SMA positive cells significantly increased to a greater extent in WT than in TNKO.

  20. WT TNKO qPCR for TGF-βmRNA 5 p<0.001 p<0.01 p<0.001 4 3 TGF-β1 mRNA (folds) 2 1 0 CTL1 3w 6w 9w 12w CTL2 TGF-β1mRNA was significantly increased in WT than in TNKO.

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

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

  23. 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 ↑ Portal MFBs Activated MFBs (a-SMA +)

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

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

  26. 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.

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