Injury to the biliary epithelium triggers inflammation and fibrosis, which can result in severe liver diseases and may progress to malignancy. Development of a type 1 immune response has been linked to biliary injury pathogenesis; however, a subset of patients with biliary atresia, the most common childhood cholangiopathy, exhibit increased levels of Th2-promoting cytokines. The relationship among different inflammatory drivers, epithelial repair, and carcinogenesis remains unclear. Here, we determined that the Th2-activating cytokine IL-33 is elevated in biliary atresia patient serum and in the livers and bile ducts of mice with experimental biliary atresia. Administration of IL-33 to WT mice markedly increased cholangiocyte proliferation and promoted sustained cell growth, resulting in dramatic and rapid enlargement of extrahepatic bile ducts. The IL-33-dependent proliferative response was mediated by an increase in the number of type 2 innate lymphoid cells (ILC2s), which released high levels of IL-13 that in turn promoted cholangiocyte hyperplasia. Induction of the IL-33/ILC2/IL-13 circuit in a murine biliary injury model promoted epithelial repair; however, induction of this circuit in mice with constitutive activation of AKT and YAP in bile ducts induced cholangiocarcinoma with liver metastases. These findings reveal that IL-33 mediates epithelial proliferation and suggest that activation of IL-33/ILC2/IL-13 may improve biliary repair and disruption of the circuit may block progression of carcinogenesis.

tion-mass spectrometry (LC-ESI-MS) analysis. We hereby report the exact identity of 16 oxysterols and downstream metabolites, including cholestenoic acids, found in human CSF (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI68506DS1). The most abundant of these metabolites (19.48-0.40 ng/ml; Supplemental Figure 1) were 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7αH,3O-CA), 3β-hydroxycholest-5-en-26-oic acid (3β-HCA), and 2 newly identified metabolites in CSF, 3β,7α-diHCA and 3β,7β-dihydroxycholest-5-en-26-oic acid (3β,7β-diHCA). Precursors of these acids, including 26-HC and newly identified 7α,26-dihydroxycholesterol (7α,26-diHC; cholest-5-ene-3β,7α,26-triol) and 7α,26-dihydroxycholest-4-en-3-one (7α,26-diHCO), were also found, but at lower levels (0.15-0.03 ng/ml). Our results thus identified 4 novel oxysterol metabolites in human CSF that were downstream of 26-HC ( Figure 1A). 26-HC is metabolized via 7α,26-diHC and 7α,26-diHCO, or via 3β-HCA and 3β,7α-diHCA, to 7αH,3O-CA. While 26-HC can cross the blood-brain barrier (BBB) and enter the brain from the circulation (25), 7αH,3O-CA traverses the BBB and is exported from the brain (26). Very low levels of 24S-hydroxycholesterol (24S-HC; cholest-5-ene-3β,24S-diol), 25-hydroxycholesterol (25-HC; cholest-5-ene-3β,25-diol), and newly identified 7α,25-dihydroxycholesterol (7α,25-diHC; cholest-5-ene-3β,7α,25-triol) and 7α,25-dihydroxycholest-4-en-3-one (7α,25-diHCO) were also found in CSF (0.08-0.03 ng/ml).Reduced levels of 7α-hydroxylated cholestenoic acids in CSF and plasma/serum of human patients with SPG5. SPG5 presents with upper motor neuron signs and results from mutations in CYP7B1, encoding the oxysterol 7α-hydroxylase responsible for 7α-hydroxylation of side-chain oxidized sterols that is required for extrahepatic synthesis of 7αH,3O-CA and its precursor, 3β,7α-diHCA ( Figure 1A and ref. 18). In order to examine the pathogenic role of such mutations, we sought to identify alterations in oxysterol and cholestenoic acid profiles in CSF and plasma from these patients and then examine the biological activities of the altered metabolites. We first studied the CSF from 3 patients with SPG5

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A self-organizing organoid model provides a new approach to study the mechanism of human liver organogenesis. Previous animal models documented that simultaneous paracrine signaling and cell-to-cell surface contact regulate hepatocyte differentiation. To dissect the relative contributions of the paracrine effects, we first established a liver organoid using human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as previously reported. Time-lapse imaging showed that hepatic-specified endoderm iPSCs (HE-iPSCs) self-assembled into three-dimensional organoids, resulting in hepatic gene induction. Progressive differentiation was demonstrated by hepatic protein production after organoid transplantation. To assess the paracrine contributions, we employed a Transwell system in which HE-iPSCs were separately co-cultured with MSCs and/or HUVECs. Although the three-dimensional structure did not form, their soluble factors induced a hepatocyte-like phenotype in HE-iPSCs, resulting in the expression of bile salt export pump. In conclusion, the mesoderm-derived paracrine signals promote hepatocyte maturation in liver organoids, but organoid self-organization requires cell-to-cell surface contact. Our model demonstrates a novel approach to identify developmental paracrine signals regulating the differentiation of human hepatocytes.

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