Skip to main content


Log in

Necroptosis in biliary atresia of the liver

  • Original Paper
  • Published:
Medical Molecular Morphology Aims and scope Submit manuscript


Biliary atresia (BA) is characterized by the occlusion of extrahepatic bile ducts due to sclerosing inflammation. Necroptosis is a recently characterized form of programmed cell death but has not been examined in BA. We, therefore, explored the potential involvement of necroptosis in the pathogenesis of BA by evaluating the correlation between necroptosis-related factors and clinicopathological features of BA patients. We studied liver biopsy specimens of 59 patients with BA and 30 with congenital biliary dilatation (CBD). We also evaluated 14 surgical BA cases, who eventually underwent liver transplantation and 9 normal liver from neonates and infants obtained at autopsy. Necroptosis-related factors including toll-like receptor 3 (TLR3), receptor-interacting protein kinase1 (RIP1), receptor-interacting protein kinase3 (RIP3), mixed lineage kinase domain-like (MLKL), and phosphorylated mixed lineage kinase domain-like (pMLKL) in these liver specimens were immunolocalized. TLR3, RIP1, MLKL in the intrahepatic cholangiocytes was significantly higher in BA than CBD. pMLKL immunoreactivity was significantly greater at an earlier age of BA patients. The native liver survival period was significantly prolonged in the high RIP3 group. The low RIP3 status could serve as an adverse clinical prognostic factor for the native liver survival among the necroptosis-related factors examined in this study.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.



Biliary atresia


Congenital biliary dilation


Toll-like receptor3


Receptor-interacting protein kinase1


Receptor-interacting protein kinase3


Mixed lineage kinase domain-like


Phosphorylated mixed lineage kinase domain-like


Danger-associated molecular patterns


TIR domain-containing adapter protein interferon-β


Phosphate-buffered saline

H score:

Histological score


Labeling index


  1. Nio M (2017) Japanese biliary atresia registry. Pediatr Surg Int 33:1319–1325

    Article  Google Scholar 

  2. Sasaki H, Tanaka H, Wada M, Kazama T, Nishi K, Nakamura M, Kudo H, Kawagishi N, Nio M (2014) Liver transplantation following the Kasai procedure in treatment of biliary atresia: a single institution analysis. Pediatr Surg Int 30:871–875

    Article  Google Scholar 

  3. Funaki N, Sasano H, Shizawa S, Nio M, Iwami D, Ohi R, Nagura H (1998) Apoptosis and cell proliferation in biliary atresia. J Pathol 186:429–433

    Article  CAS  Google Scholar 

  4. Galluzzi L, Bravo-San Pedro JM, Kroemer G (2014) Organelle-specific initiation of cell death. Nat Cell Biol 16:728–736

    Article  CAS  Google Scholar 

  5. Vercammen D, Brouckaert G, Denecker G, Van de Craen M, Declercq W, Fiers W, Vandenabeele P (1998) Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways. J Exp Med 188:919–930

    Article  CAS  Google Scholar 

  6. Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B, Tschopp J (2000) Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1:489–495

    Article  CAS  Google Scholar 

  7. Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, Chan FK (2009) Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137:1112–1123

    Article  CAS  Google Scholar 

  8. Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X, Wang X (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148:213–227

    Article  CAS  Google Scholar 

  9. Galluzzi L, Kepp O, Chan FK, Kroemer G (2017) Necroptosis: mechanisms and relevance to disease. Annu Rev Pathol 12:103–130

    Article  CAS  Google Scholar 

  10. Desmet VJ (1992) Congenital diseases of intrahepatic bile ducts: variations on the theme “ductal plate malformation.” Hepatology (Baltimore, MD) 16:1069–1083

    Article  CAS  Google Scholar 

  11. Morecki R, Glaser JH, Cho S, Balistreri WF, Horwitz MS (1982) Biliary atresia and reovirus type 3 infection. N Engl J Med 307:481–484

    Article  CAS  Google Scholar 

  12. Riepenhoff-Talty M, Gouvea V, Evans MJ, Svensson L, Hoffenberg E, Sokol RJ, Uhnoo I, Greenberg SJ, Schakel K, Zhaori G, Fitzgerald J, Chong S, Yousef M, Nemeth A, Brown M, Piccoli D, Hyams J, Ruffin D, Rossi T (1996) Detection of group C rotavirus in infants with extrahepatic biliary atresia. J Infect Dis 174:8–15

    Article  CAS  Google Scholar 

  13. Harada K, Sato Y, Itatsu K, Isse K, Ikeda H, Yasoshima M, Zen Y, Matsui A, Nakanuma Y (2007) Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia. Hepatology (Baltimore, MD) 46:1146–1154

    Article  CAS  Google Scholar 

  14. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA (2001) Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413:732–738

    Article  CAS  Google Scholar 

  15. Tatematsu M, Nishikawa F, Seya T, Matsumoto M (2013) Toll-like receptor 3 recognizes incomplete stem structures in single-stranded viral RNA. Nat Commun 4:1833

    Article  Google Scholar 

  16. Kariko K, Ni H, Capodici J, Lamphier M, Weissman D (2004) mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem 279:12542–12550

    Article  CAS  Google Scholar 

  17. Polykratis A, Hermance N, Zelic M, Roderick J, Kim C, Van TM, Lee TH, Chan FKM, Pasparakis M, Kelliher MA (2014) Cutting edge: RIPK1 Kinase inactive mice are viable and protected from TNF-induced necroptosis in vivo. J Immunol 193:1539–1543

    Article  CAS  Google Scholar 

  18. Kaiser WJ, Sridharan H, Huang C, Mandal P, Upton JW, Gough PJ, Sehon CA, Marquis RW, Bertin J, Mocarski ES (2013) Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J Biol Chem 288:31268–31279

    Article  CAS  Google Scholar 

  19. Wang H, Sun L, Su L, Rizo J, Liu L, Wang LF, Wang FS, Wang X (2014) Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell 54:133–146

    Article  CAS  Google Scholar 

  20. Nio M, Wada M, Sasaki H, Kazama T, Tanaka H, Kudo H (2016) Technical standardization of Kasai portoenterostomy for biliary atresia. J Pediatr Surg 51:2105–2108

    Article  Google Scholar 

  21. Zhao J, Jitkaew S, Cai Z, Choksi S, Li Q, Luo J, Liu ZG (2012) Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci USA 109:5322–5327

    Article  CAS  Google Scholar 

  22. Yamazaki Y, Nakamura Y, Shibahara Y, Konosu-Fukaya S, Sato N, Kubota-Nakayama F, Oki Y, Baba S, Midorikawa S, Morimoto R, Satoh F, Sasano H (2016) Comparison of the methods for measuring the Ki-67 labeling index in adrenocortical carcinoma: manual versus digital image analysis. Hum Pathol 53:41–50

    Article  CAS  Google Scholar 

  23. Ueki S, Fujishima F, Kumagai T, Ishida H, Okamoto H, Takaya K, Sato C, Taniyma Y, Kamei T, Sasano H (2020) GR, Sgk1, and NDRG1 in esophageal squamous cell carcinoma: their correlation with therapeutic outcome of neoadjuvant chemotherapy. BMC Cancer 20:161

    Article  CAS  Google Scholar 

  24. Liu W, Chen B, Wang Y, Meng C, Huang H, Huang XR, Qin J, Mulay SR, Anders HJ, Qiu A, Yang B, Freeman GJ, Lu HJ, Lin HY, Zheng ZH, Lan HY, Huang Y, Xia Y (2018) RGMb protects against acute kidney injury by inhibiting tubular cell necroptosis via an MLKL-dependent mechanism. Proc Natl Acad Sci USA 115:E1475-e1484

    Article  CAS  Google Scholar 

  25. Afonso MB, Rodrigues PM, Simao AL, Ofengeim D, Carvalho T, Amaral JD, Gaspar MM, Cortez-Pinto H, Castro RE, Yuan J, Rodrigues CM (2016) Activation of necroptosis in human and experimental cholestasis. Cell Death Dis 7:e2390

    Article  CAS  Google Scholar 

  26. Roychowdhury S, McCullough RL, Sanz-Garcia C, Saikia P, Alkhouri N, Matloob A, Pollard KA, McMullen MR, Croniger CM, Nagy LE (2016) Receptor interacting protein 3 protects mice from high-fat diet-induced liver injury. Hepatology (Baltimore, MD) 64:1518–1533

    Article  CAS  Google Scholar 

  27. Gautheron J, Vucur M, Reisinger F, Cardenas DV, Roderburg C, Koppe C, Kreggenwinkel K, Schneider AT, Bartneck M, Neumann UP, Canbay A, Reeves HL, Luedde M, Tacke F, Trautwein C, Heikenwalder M, Luedde T (2014) A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis. EMBO Mol Med 6:1062–1074

    Article  CAS  Google Scholar 

  28. Lu C, Xu W, Zhang F, Shao J, Zheng S (2016) Nrf2 knockdown disrupts the protective effect of curcumin on alcohol-induced hepatocyte necroptosis. Mol Pharm 13:4043–4053

    Article  CAS  Google Scholar 

  29. Gunther C, He GW, Kremer AE, Murphy JM, Petrie EJ, Amann K, Vandenabeele P, Linkermann A, Poremba C, Schleicher U, Dewitz C, Krautwald S, Neurath MF, Becker C, Wirtz S (2016) The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis. J Clin Investig 126:4346–4360

    Article  Google Scholar 

  30. Moriwaki K, Balaji S, McQuade T, Malhotra N, Kang J, Chan FK (2014) The necroptosis adaptor RIPK3 promotes injury-induced cytokine expression and tissue repair. Immunity 41:567–578

    Article  CAS  Google Scholar 

Download references


We appreciate the skillful technical assistance of Ms. Yayoi Aoyama (Department of Pathology, Tohoku University Hospital, Sendai, Japan). We would like to thank Enago ( for the English language review.


Not applicable.

Author information

Authors and Affiliations



MH carried out an immunohistological examination. MH performed the statistical analysis. MH, FF, TL, SJ, MN, and HS conceived the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Hironobu Sasano.

Ethics declarations

Conflicts of interest

The authors have no conflicts of interest.

Ethics approval and consent to participate

The study protocol was approved by the Ethics Committee of the Tohoku University School of Medicine (Accession No. 2019-1-470). Written informed consent was obtained from all patients prior to surgery.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.


Supplementary file1 (DOCX 2164 KB) The receiver operating characteristics (ROC) curve against the native liver survival of the H-score and LI

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hashimoto, M., Fujishima, F., Lomphithak, T. et al. Necroptosis in biliary atresia of the liver. Med Mol Morphol 54, 305–315 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: