Cancer Immunology, Immunotherapy

, Volume 63, Issue 3, pp 235–245 | Cite as

TLR4-dependant immune response, but not hepatitis B virus reactivation, is important in radiation-induced liver disease of liver cancer radiotherapy

  • Zhi-Feng Wu
  • Xiao-Hui Zhou
  • Yun-Wen Hu
  • Le-Yuan Zhou
  • Ya-Bo Gao
  • Xiu-Hua Peng
  • Xiao-Hua Yang
  • Jian-Ying Zhang
  • Yong Hu
  • Zhao-Chong ZengEmail author
Original Article


Toll-like receptor 4 (TLR4) is an important trigger of the immune response against hepatitis B virus (HBV) infection and liver injuries. The roles of HBV reactivation versus TLR4-dependant immune response may be critical factors in preventing radiation-induced liver diseases (RILDs) after liver cancer radiotherapy. This study consists of three phases. In the primary phase, livers of mutant TLR4 (TLR4) mice were irradiated with 30 Gy in either the absence or presence of HBV infection. The latter was done by introduction of plasmid pAAV/HBV 1.2. In the advanced phase, RILDs were compared in normal TLR4 (TLR4+) versus TLR4 mice. In the validation phase, 28 liver cancer patients who had undergone radiotherapy before hepatectomy were enrolled. Liver biopsies near tumors, irradiated with 35–48 Gy, were used to construct tissue microarrays. HBV reactivation, TLR4 expression, and severity of RILDs were studied in both mouse and human. More HBV reactivation, without significant RILD, was observed in irradiated versus unirradiated TLR4 mice. RILD scores of TLR4+ mice were higher than TLR4 mice. In humans, serious RILDs tended to develop in patients with high TLR4 expression, but not in patients with low TLR4 or high HBV surface antigen expression. High TLR4 expression was seen in only 2 of 12 HBV-reactive patients, but in HBV-nonreactive patients, it was seen in 6 of 9 (P < 0.03). In summary, RILDs correlated with high TLR4 expression, but not with HBV reactivation, which is inhibited in liver with high TLR4 expression after liver cancer radiotherapy.


Hepatitis B virus Liver cancer Toll-like receptor TLR4 mutation Radiotherapy 



Hepatitis B virus


Radiation-induced liver disease


Toll-like receptor 4




Hepatocellular carcinoma


Hepatitis B surface antigen


Polymerase chain reaction


Tissue microarray







We thank Prof. Pei-Jer Chen (National Taiwan University) for kindly providing plasmid pAAV/HBV 1.2. This work was supported by the National Natural Science Foundation of China (Grant 81241012 to Z–C Zeng).

Conflict of interest



  1. 1.
    Ohishi W, Fujiwara S, Cologne JB, Suzuki G, Akahoshi M, Nishi N, Tsuge M, Chayama K (2011) Impact of radiation and hepatitis virus infection on risk of hepatocellular carcinoma. Hepatology 53(4):1237–1245PubMedCrossRefGoogle Scholar
  2. 2.
    Chou CH, Chen PJ, Lee PH, Cheng AL, Hsu HC, Cheng JC (2007) Radiation-induced hepatitis B virus reactivation in liver mediated by the bystander effect from irradiated endothelial cells. Clin Cancer Res 13(3):851–857PubMedCrossRefGoogle Scholar
  3. 3.
    Chang WW, Su IJ, Lai MD, Chang WT, Huang W, Lei HY (2005) Toll-like receptor 4 plays an anti-HBV role in a murine model of acute hepatitis B virus expression. World J Gastroenterol 11(42):6631–6637PubMedGoogle Scholar
  4. 4.
    Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M, Takeda K, Akira S (2003) Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301(5633):640–643PubMedCrossRefGoogle Scholar
  5. 5.
    Schmidt M, Raghavan B, Muller V, Vogl T, Fejer G, Tchaptchet S, Keck S, Kalis C, Nielsen PJ, Galanos C, Roth J, Skerra A, Martin SF, Freudenberg MA, Goebeler M (2010) Crucial role for human Toll-like receptor 4 in the development of contact allergy to nickel. Nat Immunol 11(9):814–819PubMedCrossRefGoogle Scholar
  6. 6.
    Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in TLR4 gene. Science 282(5396):2085–2088PubMedCrossRefGoogle Scholar
  7. 7.
    Chang WW, Su IJ, Lai MD, Chang WT, Huang W, Lei HY (2003) The role of inducible nitric oxide synthase in a murine acute hepatitis B virus (HBV) infection model induced by hydrodynamics-based in vivo transfection of HBV-DNA. J Hepatol 39(5):834–842PubMedCrossRefGoogle Scholar
  8. 8.
    Huang LR, Wu HL, Chen PJ, Chen DS (2006) An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proc Natl Acad Sci USA 103(47):17862–17867PubMedCrossRefGoogle Scholar
  9. 9.
    Batts KP, Ludwig J (1995) Chronic hepatitis. An update on terminology and reporting. Am J Surg Pathol 19(12):1409–1417PubMedCrossRefGoogle Scholar
  10. 10.
    Goodman ZD (2007) Grading and staging systems for inflammation and fibrosis in chronic liver diseases. J Hepatol 47(4):598–607PubMedCrossRefGoogle Scholar
  11. 11.
    Xu H, Wu Q, Dang S, Jin M, Xu J, Cheng Y, Pan M, Wu Y, Zhang C, Zhang Y (2011) Alteration of CXCR7 expression mediated by TLR4 promotes tumor cell proliferation and migration in human colorectal carcinoma. PLoS ONE 6(12):e27399PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Wang S, Astsaturov IA, Bingham CA, McCarthy KM, von Mehren M, Xu W, Alpaugh RK, Tang Y, Littlefield BA, Hawkins LD, Ishizaka ST, Weiner LM (2012) Effective antibody therapy induces host-protective antitumor immunity that is augmented by TLR4 against treatment. Cancer Immunol Immunother 61(1):49–61PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, Mignot G, Maiuri MC, Ullrich E, Saulnier P, Yang H, Amigorena S, Ryffel B, Barrat FJ, Saftig P, Levi F, Lidereau R, Nogues C, Mira JP, Chompret A, Joulin V, Clavel-Chapelon F, Bourhis J, Andre F, Delaloge S, Tursz T, Kroemer G, Zitvogel L (2007) Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 13(9):1050–1059PubMedCrossRefGoogle Scholar
  14. 14.
    Oblak A, Jerala R (2011) Toll-like receptor 4 activation in cancer progression and therapy. Clin Dev Immunol 2011:609579PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Manzano-Alonso ML, Castellano-Tortajada G (2011) Reactivation of hepatitis B virus infection after cytotoxic chemotherapy or immunosuppressive therapy. World J Gastroenterol 17(12):1531–1537PubMedCrossRefGoogle Scholar
  16. 16.
    Apetoh L, Ghiringhelli F, Tesniere A, Criollo A, Ortiz C, Lidereau R, Mariette C, Chaput N, Mira JP, Delaloge S, Andre F, Tursz T, Kroemer G, Zitvogel L (2007) The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy. Immunol Rev 220:47–59PubMedCrossRefGoogle Scholar
  17. 17.
    O’Neill LA (2008) When signaling pathways collide positive and negative regulation of toll-like receptor signal transduction. Immunity 29(1):12–20PubMedCrossRefGoogle Scholar
  18. 18.
    Seki E, Brenner DA (2008) Toll-like receptors and adaptor molecules in liver disease: update. Hepatology 48(1):322–335PubMedCrossRefGoogle Scholar
  19. 19.
    Liu C, Gao F, Li B, Mitchel RE, Liu X, Lin J, Zhao L, Cai J (2011) TLR4 knockout protects mice from radiation-induced thymic lymphoma by downregulation of IL6 and miR-21. Leukemia 25(9):1516–1519PubMedCrossRefGoogle Scholar
  20. 20.
    Eiro N, Altadill A, Juarez LM, Rodriguez M, Gonzalez LO, Atienza S, Bermudez S, Fernandez-Garcia B, Fresno-Forcelledo MF, Rodrigo L, and Vizoso FJ (2013) Toll-like receptors 3, 4 and 9 in hepatocellular carcinoma: Relationship with clinicopathological characteristics and prognosis. Hepatol ResGoogle Scholar
  21. 21.
    Wu J, Lu M, Meng Z, Trippler M, Broering R, Szczeponek A, Krux F, Dittmer U, Roggendorf M, Gerken G, Schlaak JF (2007) Toll-like receptor-mediated control of HBV replication by nonparenchymal liver cells in mice. Hepatology 46(6):1769–1778PubMedCrossRefGoogle Scholar
  22. 22.
    Soares JB, Pimentel-Nunes P, Roncon-Albuquerque R, Leite-Moreira A (2010) The role of lipopolysaccharide/toll-like receptor 4 signaling in chronic liver diseases. Hepatol Int 4(4):659–672PubMedCrossRefGoogle Scholar
  23. 23.
    Chen Z, Cheng Y, Xu Y, Liao J, Zhang X, Hu Y, Zhang Q, Wang J, Zhang Z, Shen F, Yuan Z (2008) Expression profiles and function of Toll-like receptors 2 and 4 in peripheral blood mononuclear cells of chronic hepatitis B patients. Clin Immunol 128(3):400–408PubMedCrossRefGoogle Scholar
  24. 24.
    Wu JF, Chen CH, Ni YH, Lin YT, Chen HL, Hsu HY, Chang MH (2012) Toll-like receptor and hepatitis B virus clearance in chronic infected patients: a long-term prospective cohort study in Taiwan. J Infect Dis 206(5):662–668PubMedCrossRefGoogle Scholar
  25. 25.
    Gao B, Seki E, Brenner DA, Friedman S, Cohen JI, Nagy L, Szabo G, Zakhari S (2011) Innate immunity in alcoholic liver disease. Am J Physiol Gastrointest Liver Physiol 300(4):G516–G525PubMedCrossRefGoogle Scholar
  26. 26.
    Testro AG, Visvanathan K (2009) Toll-like receptors and their role in gastrointestinal disease. J Gastroenterol Hepatol 24(6):943–954PubMedCrossRefGoogle Scholar
  27. 27.
    Du SS, Qiang M, Zeng ZC, Ke AW, Ji Y, Zhang ZY, Zeng HY, Liu Z (2010) Inactivation of kupffer cells by gadolinium chloride protects murine liver from radiation-induced apoptosis. Int J Radiat Oncol Biol Phys 76(4):1225–1234PubMedCrossRefGoogle Scholar
  28. 28.
    Zhou LY, Wang ZM, Gao YB, Wang LY, Zeng ZC (2012) Stimulation of hepatoma cell invasiveness and metastatic potential by proteins secreted from irradiated nonparenchymal cells. Int J Radiat Oncol Biol Phys 84(3):822–828PubMedCrossRefGoogle Scholar
  29. 29.
    Sun W, Ma J, Wu S, Yang D, Yan Y, Liu K, Wang J, Sun L, Chen N, Wei H, Zhu Y, Xing B, Zhao X, Qian X, Jiang Y, He F (2010) Characterization of the liver tissue interstitial fluid (TIF) proteome indicates potential for application in liver disease biomarker discovery. J Proteome Res 9(2):1020–1031PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Zhi-Feng Wu
    • 1
  • Xiao-Hui Zhou
    • 2
  • Yun-Wen Hu
    • 3
  • Le-Yuan Zhou
    • 1
  • Ya-Bo Gao
    • 1
  • Xiu-Hua Peng
    • 2
  • Xiao-Hua Yang
    • 4
  • Jian-Ying Zhang
    • 1
  • Yong Hu
    • 1
  • Zhao-Chong Zeng
    • 1
    Email author
  1. 1.Department of Radiation Oncology, Zhongshan HospitalFudan UniversityShanghaiChina
  2. 2.Shanghai Public Health Clinical CenterFudan UniversityShanghaiChina
  3. 3.Key Laboratory of Medical Molecular Virology of the Ministries of EducationFudan UniversityShanghaiChina
  4. 4.Shanghai Chest HospitalShanghai Jiaotong UniversityShanghaiChina

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