Recapitulating Cholangiopathy-Associated Necroptotic Cell Death In Vitro Using Human Cholangiocyte Organoids

Abstract

Background & Aims Liver and bile duct diseases often are associated with extensive cell death of cholangiocytes. Necroptosis represents a common mode of programmed cell death in cholangiopathy, however, detailed mechanistic knowledge is limited owing to the lack of appropriate in vitro models. To address this void, we investigated whether human intrahepatic cholangiocyte organoids (ICOs) can recapitulate cholangiopathy-associated necroptosis and whether this model can be used for drug screening. Methods We evaluated the clinical relevance of necroptosis in end-stage liver diseases and liver transplantation by immunohistochemistry. Cholangiopathy-associated programmed cell death was evoked in ICOs derived from healthy donors or patients with primary sclerosing cholangitis or alcoholic liver diseases by the various stimuli. Results The expression of key necroptosis mediators, receptor-interacting protein 3 and phosphorylated mixed lineage kinase domain-like, in cholangiocytes during end-stage liver diseases was confirmed. The phosphorylated mixed lineage kinase domain-like expression was etiology-dependent. Gene expression analysis confirmed that primary cholangiocytes are more prone to necroptosis compared with primary hepatocytes. Both apoptosis and necroptosis could be specifically evoked using tumor necrosis factor α and second mitochondrial-derived activator of caspases mimetic, with or without caspase inhibition in healthy and patient-derived ICOs. Necroptosis also was induced by ethanol metabolites or human bile in ICOs from donors and patients. The organoid cultures further uncovered interdonor variable and species-specific drug responses. Dabrafenib was identified as a potent necroptosis inhibitor and showed a protective effect against ethanol metabolite toxicity. Conclusions Human ICOs recapitulate cholangiopathy-associated necroptosis and represent a useful in vitro platform for the study of biliary cytotoxicity and preclinical drug evaluation.

Liver Ischemia and Reperfusion Induce Periportal Expression of Necroptosis Executor pMLKL Which Is Associated With Early Allograft Dysfunction After Transplantation

“…To determine whether the activation of pMLKL is involved in rat hepatic IRI, rat liver biopsies were collected after 22 hours of cold storage and 24 hours of reperfusion or sham operation ( Figure 1A ), followed by immunohistochemistry staining of pMLKL using a validated antibody ( 27 , 40 , 41 ). Compared to sham-operated rats, a significant increase of serum ALT (1153.0 (441.5-1652.0) vs 10.0 (10.0-17.5), p =0.029) and AST (1294.0 (654.5-1497.0) vs 66.5 (47.0-94.0), p =0.029) levels was observed in rats after LT ( Figure 1B ).…”

“…In previous studies, apoptosis has been one of the key targets, but the translation into clinical practice has been merely lacking. Except for apoptosis, the emerging role of programmed cell death has been validated in hepatic IRI, expanding our knowledge of the different cell death programs during LT. We previously reported that most pMLKL positive cells were found in the portal triads in LT biopsies, but were absent in ischemia-free liver biopsies collected from living donors grafts, suggesting the involvement of pMLKL-mediated cell death in hepatic IRI ( 27 ). Myofibroblasts are the main effectors of liver fibrosis ( 31 ) and we concluded in this study that myofibroblasts represent a major cell type with pMLKL activation.…”

“…Myofibroblasts are the main effectors of liver fibrosis ( 31 ) and we concluded in this study that myofibroblasts represent a major cell type with pMLKL activation. In fact, the periportal expression pattern of pMLKL and its activation in myofibroblast was barely found in liver biopsies obtained from patients with end-stage liver diseases, which were generally featured by severe liver fibrosis and extensively activated fibroblast ( 27 ). This discrepancy may imply that the activation of pMLKL during liver transplantation represents an event with a unique mechanism that is different from other chronic etiologies.…”

“…Sections were stained with hematoxylin and eosin (H&E) according to standard procedures. Immunostaining was performed as previously described ( 27 ). In short, sections were dewaxed and rehydrated via gradient ethanol washes.…”

“…We have previously shown that necroptosis is involved in various human liver diseases in an etiology-dependent manner ( 27 ). Interestingly, although based on only a few cases, we found extensive expression of pMLKL in human liver biopsies during LT, implying the potential existence of pMLKL-mediated cell death in human liver IRI.…”