Biliary repair and carcinogenesis are mediated by IL-33–dependent cholangiocyte proliferation (original) (raw)
IL-33 increases in human and experimental biliary atresia. Based on the previous report of increased expression of IL1RL1 mRNA encoding the ST2 receptor in the liver at the time of diagnosis of biliary atresia (3), we first quantified the concentration of serum IL-33 in affected subjects. We found an increased serum concentration of this cytokine in 20 patients with biliary atresia (mean ± SD: 218.3 ± 887.0 pg/ml) above undetectable levels of age-matched healthy controls (Figure 1A). In a model of rhesus rotavirus type A–induced (RRV-induced) biliary injury in newborn mice (experimental biliary atresia), the expression of Il33 mRNA correlated with an increased profile of intrahepatic bile ducts and with the abundance of cholangiocytes in the epithelium of extrahepatic bile ducts (EHBDs) during progression of biliary injury (Figure 1B). In a similar fashion, immunostaining for the St2 receptor showed expression in cholangiocytes along the epithelium of EHBDs in neonatal mice, with a number of St2+ cholangiocytes decreasing during progression of epithelial injury after rotavirus (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI73742DS1). St2 was also expressed in intrahepatic bile ducts (in addition to surrounding hematopoietic cells) in neonatal mice, but the expression decreased after rotavirus challenge and was undetectable in cholangiocytes (Supplemental Figure 1B). In adult mice, St2 was also detected in cholangiocytes of extra- and intrahepatic bile ducts (Supplemental Figure 1C).
Expression of IL-33 is increased in humans and mice with biliary atresia. (A) Serum concentration of IL-33 in infants with biliary atresia (BA) at the time of diagnosis (n = 20, < 4 months of age) and in age-matched normal controls (NC) (n = 6). (B) Il33 mRNA expression (as a ratio to Hprt; graphs) and PanCK staining of liver and EHBDs at 3, 7, and 14 days after injection with RRV or normal saline (NS) in the first 24 hours of life. Each time point had n = 4–5 mice for normal saline and RRV groups. Representative immunostaining experiments included tissue sections from 3 mice for each group and time point. Mean ± SD. *P < 0.05; ***P < 0.001. Scale bars: 50 μm.
To investigate whether IL-33–St2 signaling plays a role in the response of the bile duct to an injury, we injected 0.5 × 106 fluorescent focus units (ffu) of rotavirus into newborn mice followed by daily i.p. administration of anti-St2 blocking antibody or IgG isotype (as control) beginning 24 hours later (12). This lower dose of rotavirus induces mild epithelial injury and atresia phenotype in less than 40% of mice. Mice receiving anti-St2 antibody had higher levels of serum alanine aminotransferase and bilirubin after rotavirus challenge than IgG control mice (Figure 2, A and B). Microscopically, mice in the anti-St2 antibody group showed a diffuse loss of the epithelial layer and duct lumen, while controls had a much greater residual epithelial surface area (Figure 2, C–E). The soluble form of St2, which serves as a decoy to neutralize IL-33–induced signaling, increased in the plasma at days 7 and 14 after virus challenge (Supplemental Figure 1D). These data suggested that loss of St2 signaling rendered mice more susceptible to biliary injury and functionally linked the high expression of IL-33–St2 signaling to the abundance of cholangiocytes in the duct epithelium.
Blocking of St2 by antibodies worsens experimental biliary atresia. Serum levels of bilirubin (A) and alanine aminotransferase (ALT) (B) increase in mice receiving anti-St2 antibody 7 days after RRV challenge when compared with IgG isotype controls. (C) Quantification of the epithelial surface area of EHBDs is significantly smaller in mice receiving anti-St2 than in control mice 11 days after RRV. (D and E) Representative histological sections show segments of intact epithelium of EHBDs in control mice, but not in anti-St2 mice. n = 8 mice for RRV + IgG and n = 8 mice for RRV + St2 Ab. Mean ± SD. **P < 0.01; ***P < 0.001. Scale bars: 200 μm.
IL-33 is a potent epithelial mitogen. To directly determine whether IL-33 induces proliferation of the duct epithelium, we administered 0.1 μg of IL-33 i.p. into newborn BALB/c mice. Measuring proliferation by BrdU incorporation, we found that BrdU+ cholangiocytes of EHBDs increased by approximately 2-fold above the levels of age-matched controls, which had high baseline levels of BrdU incorporation, within 24 hours of injection (Figure 3A). When injected into adult mice, when the baseline cholangiocyte proliferation was less than 1%, 1 μg of IL-33 i.p. increased BrdU uptake by 76-fold in the bile duct epithelium, neighboring peribiliary glands (a site of putative progenitor cells), and cytokeratin-19–negative (CK19–) submucosal cells (Figure 3, A, B, and D). The cholangiocyte proliferation in the epithelium was lower after 4 daily doses, although it remained well above the baseline level (Figure 3B), and was associated with a rise in the plasma concentration of the soluble St2 (Figure 3C). Similar proliferation was achieved with as little as 0.1 μg of IL-33 given to adult mice (Supplemental Figure 2A), and daily administration increased the diameter of EHBDs from 0.3 mm to approximately 3 mm in 1 week, without an obvious effect on the gallbladder (Figure 3, E–G, and Supplemental Figure 2B). In the liver, IL-33 induced cholangiocyte proliferation in hilar bile ducts, but not in small intrahepatic bile ducts in the periphery or in neighboring hepatocytes (Figure 4, A and B). Interestingly, while St2 was expressed in cholangiocytes of intrahepatic bile ducts, the expression was lost after one dose of IL-33 (Figure 4, C and D). In vitro, incubation of IL-33 with human cholangiocyte cell lines induced proliferation if the cells derived from EHBDs (Witt cell line), but not if they derived from intrahepatic bile ducts (H69 cell line) (Figure 4, E–G). These data identified IL-33 as a potent inducer of extrahepatic cholangiocyte proliferation in the absence of preexisting injury. However, the combination of a mild proliferative response in vitro and the inflammatory infiltration in the submucosal compartment suggested that other cellular and molecular signals may be involved in IL-33–induced proliferation.
IL-33 induces cholangiocyte proliferation. (A) Percentage of CK19+ epithelial cells in neonatal and adult EHBDs that stain for BrdU 1 day after PBS or IL-33 (0.1 μg in neonatal and 1 μg for adult mice) i.p. (n = 4 for each group). (B) BrdU uptake in CK19+ epithelial and CK19– submucosal cells of adult EHBDs after 1 and 4 daily doses of PBS or IL-33. (C) Plasma concentration of soluble St2 (sSt2) at the same time points. (D) Representative fluorescence images show BrdU uptake by CK19+ in epithelium (arrows) and peribiliary glands (PBGs) (arrowheads). (E) Representative macroscopic view of EHBDs from adult BALB/c mice after daily doses of IL-33; a segment of normal jejunum is included as a size control. (F and G) H&E staining of EHBD longitudinal sections after 4 daily doses of PBS or IL-33 shows normal epithelium after PBS (arrows; F) and irregular epithelial lining (arrows; G) after 1 μg IL-33 due to hyperplasia of duct mucosa, expansion of PBGs, and accumulation of inflammatory cells in the submucosal compartment. Mean ± SD (A–C) (n ≥ 3 animals). Experiments shown in E were repeated 4 times. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 50 μm.
IL-33 induces proliferation of cholangiocytes from hilar bile ducts and a cell line from EHBDs. BrdU staining (brown, arrows) of liver sections after 4 daily doses of IL-33 shows cholangiocyte proliferation in hilar bile duct (A), but not in the peripheral small duct (B, arrow, bile duct). PV, portal vein. (C) Detection of St2 in intrahepatic cholangiocytes by immunohistochemical staining with anti-St2 antibody (brown staining, arrow), which is lost 1 day after 1 dose of 1 μg IL-33 (D, arrow). (E) BrdU uptake in Witt cells (human extrahepatic cholangiocarcinoma cell line) and (F) H69 cells (human intrahepatic duct cell line) after culture with 10 ng/ml IL-33 for 48 hours. (G) Witt cell proliferation by the MTS assay after 48 hours of culture with different concentrations of IL-33. Mean ± SD, 4–5 replicates, repeated 3 times. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 50 μm (A and B); 20 μm (C and D).
ILC2s are required for IL-33–induced proliferation of cholangiocytes. The expansion of CK19– submucosal cells after IL-33 injection provided clues as to their potential role as an amplifier of proliferation. Using flow cytometry, quantification of mononuclear cells showed an increase in basophils and dendritic cells early (1 day) and neutrophils and eosinophils late (4 and 7 days) after IL-33 injection, while a decrease or no change in the number of B, T, and NK lymphocytes was noted after IL-33 injection (Supplemental Figure 3, A and B). Consistent with the ability of IL-33 to promote a Th2 response by activating ILC2s, a type of lineage-negative innate effector leukocyte that expresses the receptor St2 and mediates type-2 immunity (5, 6, 13), liver populations of Lin–St2+ cells increased, constituting nearly 50% of the hepatic mononuclear cells by 7 days after IL-33 injection (Figure 5, A–C). Lin–St2+ cells (defined by the lack of expression of CD3ε, CD4, CD8α, B220, CD11b, CD11c, CD49b, NK1.1, Gr-1, F4/80, Ter119, and FcεRIα, and expression of St2) expressed surface markers of ILC2s (CD45+Sca1+Icos+CD127+CD25+CD44+cKit–Flt3–; Figure 5D) and produced progressively increased amounts of IL-13 but not IFN-γ after restimulation with the lymphocyte-activating agents phorbol-12-myristate13-acetate and ionomycin in culture (Figure 5, E and F). These findings formed the basis for the hypothesis that hepatic ILC2 cells and the release of IL-13 mediate the proliferative effects of IL-33.
Hepatic ILC2s increase after IL-33 treatment. Representative dot plots (A) and quantification from 3 independent experiments (B and C) of flow cytometric assays show that hepatic ILC2s increase after 4 and 7 days of IL-33 injection. (D) By gating on Lin–ST2+ cells, plots show that IL-33 upregulates the expression of Sca1, ICOS, CD127, CD25, and CD44, but not that of CD45, cKit, or Flt3 (Flt3–). (E and F) Intracellular staining shows ILC2 cells harvested from livers at the specified time points after daily injections of IL-33 produce high levels of IL-13 and minimal amounts of IFN-γ after restimulation with phorbol 12-myristate 13-acetate and ionomycin in vitro for 4 hours. Data in C are absolute cell count at specified time points, E contains representative dot plots, and F shows percent of ILC2s expressing IL-13 from 3 independent experiments. Mean ± SD, 4–5 replicates. *P < 0.05; **P < 0.01; ***P < 0.001.
To directly determine whether ILC2s are required for IL-33–induced proliferation, we injected the cytokine into mice carrying the simultaneous inactivation of the Rag2 and Il2rgc genes (Rag2–/–gc–/– mice); these mice lack ILC2 as well as B, T, and NK cells (Figure 6, A and B, and ref. 14). While IL-33 induced the expected surge of proliferation in epithelial and peribiliary cholangiocytes in C57BL/6 (B6) control mice, bile ducts from Rag2–/–gc–/– mice were completely unresponsive to IL-33 (Figure 6C). In view of the simultaneous loss of other lineage-positive cells in these mice, we adoptively transferred purified ILC2s expressing CD45.1+ from B6/SJL/CD45.1+ mice into Rag2–/–gc–/– mice (expressing CD45.2+) to determine whether ILC2s are responsible for the loss of proliferation (Supplemental Figure 4). Adoptive transfer restored the proliferation of cholangiocytes in the epithelium and peribiliary glands after IL-33 administration to Rag2–/–gc–/– mice (Figure 6, D and E). We applied the same strategy to Rorasg/sg mice, which have a developmental defect in ILC2s, without affecting other immune cell types (15, 16). Cholangiocytes of the EHBDs from Rorasg/sg mice were unresponsive to daily doses of IL-33, while WT controls had the typical surge in proliferation (Supplemental Figure 5, A and B) accompanied by an increase in the number of ILC2s in the liver (Supplemental Figure 5, C and D). Adoptive transfer of bone marrow cells from Rora+/+ control mice into Rag2–/–gc–/– mice restored the proliferative response of cholangiocytes to IL-33 administration accompanied by an increase in the number of ILC2s (Supplemental Figure 5, E and F), which did not occur when bone marrow donors were isolated from Rorasg/sg mice (Figure 6F). These data identify ILC2s as key targets of IL-33 and raise the possibility that soluble factor or factors released by these cells may provide proliferative signals to cholangiocytes.
ILC2s mediate IL-33–induced cholangiocyte proliferation. (A) Representative dot plots show that Rag2–/–gc–/– mice lack Lin–ST2+ population after 4 days of IL-33 injections (B6 strain used as control). (B) Quantification of Lin–ST2+ cells from total hepatic mononuclear cells from 3 independent experiments. (C) Quantification of BrdU uptake by CK19+ and CK19– cells in EHBDs by immunofluorescence staining 1 day after administration of IL-33 into Rag2–/–gc–/– mice compared with B6 controls. (D) BrdU staining showing the recovery of proliferation in EHBDs of Rag2–/–gc–/– mice following adoptive transfer of B6/SJL/CD45.1+ Lin–ST2+ cells after IL-33, with (E) quantification of BrdU uptake. (F) BrdU uptake in EHBDs of Rag2–/–gc–/– mice after i.p. administration of PBS or 5 × 106 bone marrow cells from Rora+/+ or Rorasg/sg mice into Rag2–/–gc–/– mice, followed by IL-33 treatment i.p. for 6 weeks. Mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 50 μm.
IL-13 mediates IL-33 induced–proliferation of cholangiocytes. Based on the high expression of IL-13 by hepatic ILC2s after IL-33 administration, we quantified IL-13 expression in livers (mRNA) and hepatic mononuclear cells (protein) after IL-33 injection in Rag2–/–gc–/– and Rorasg/sg mice and found it to be increased only after reconstitution with ILC2s in Rag2–/–gc–/– mice (Supplemental Figure 6A) or in the presence of ILC2s in Rorasg/sg mice (Supplemental Figure 6, B and C). Direct i.p. administration of IL-33 to Il13–/– mice failed to induce the high BrdU uptake in epithelial and peribiliary cholangiocytes typical of WT mice injected with IL-33, while the proliferative effect on CK19– submucosal cells was similar between Il13–/– and WT controls; i.p. administration of IL-13 in a separate group of Il13–/– mice restored the increased proliferation of cholangiocytes after injection of 1 μg IL-33 (Figure 7, A and B). Examining whether IL-13 is able to induce cholangiocyte proliferation, we injected increasing amounts of IL-13 i.p. into BALB/c mice. BrdU+ cholangiocytes increased with as little as 1 μg, but reached the levels of proliferation induced by IL-33 when the dose reached 20 μg (Figure 7, C and D), a dose previously reported to induce expulsion of Nippostrongylus brasiliensis from murine small intestine (17). In vitro, we were unable to detect IL-13 in the conditioned medium from wells containing Witt cells cultured in the presence of IL-33. Additional experiments incubating Witt cells with 10 to 100 ng/ml IL-13 neither induced proliferation nor modified the proliferative response due to IL-33 (Supplemental Figure 7). These data suggest that IL-13 is a secondary signal required for the full proliferative effect of IL-33 in normal cholangiocytes in vivo. However, in experiments with a cholangiocarcinoma cell line, we observed that the mitogenic effect of IL-33 is independent of IL-13.
IL-13 as a molecular effector of IL-33–induced proliferation. Fluorescence images (A) and percentage of BrdU uptake from 3 to 5 animals (B) in representative sections of EHBDs 1 day after injection of IL-33 and/or IL-13 into WT or Il13–/– mice. (C and D) Percentage of BrdU cells after different doses of IL-13 in representative EHBDs (C) or quantification from 3 animals (D) 1 day after injection of 0.1, 1, 10, and 20 μg of IL-13 into WT mice. Mean ± SD. ***P < 0.001. Scale bars: 50 μm.
IL-33 improves the bile duct epithelium in experimental biliary atresia. To investigate the relevance of the proliferative properties of IL-33 to biliary repair and carcinogenesis, we first examined whether the cytokine promotes repair following rotavirus-induced epithelial injury in experimental biliary atresia. The full phenotype was induced by the i.p. administration of 1.5 × 106 plaque-forming units of RRV into BALB/c mice soon after birth (3). Then we injected 0.02 μg IL-33 i.p. daily for 7 days; we used this lower dose because neonatal mice did not tolerate the higher dose, as evidenced by decreased activity in the cage, poor feeding, and a lethargic appearance after repeated administration of 0.1 μg IL-33. On examination of the EHBDs 7 days later, when the bile duct lumen of RRV-infected mice showed inflammatory obstruction and diffuse epithelial loss (which is typical of the disease model; Figure 8, A and C), IL-33 treatment was associated with a patent duct lumen and the epithelium was largely intact, with a much greater surface area than in mice that did not receive IL-33 (Figure 8, B, D, and E).
IL-33 promotes epithelial repair in experimental biliary atresia. The epithelial injury and lumen obstruction of EHBD 7 days after rotavirus (RRV) infection (A; dashed lines depicts obstructed duct lumen) are prevented in mice that also receive daily doses of 0.02 μg IL-33 (B; arrows show epithelium; arrowheads show peribiliary glands). Quantification of the surface area of the mucosal lining of EHBDs from RRV-infected mice followed by daily doses of PBS (C, as control) or IL-33 (D) for 7 days shows a greater abundance of epithelium in the IL-33 group (E). Mean ± SD. ***P < 0.001. Scale bars: 50 μm (A and B); 100 μm (C and D).
IL-33 facilitates biliary carcinogenesis. Next, exploring a potential link between IL-33 and biliary carcinogenesis, we injected IL-33 daily into adult BALB/c mice for 10 weeks. Extrahepatic bile ducts increased in size and thickness, largely due to a substantial expansion of peribiliary glands, with remarkable features of glandular metaplasia (Figure 9), raising the possibility that IL-33 requires the activation of other growth-promoting circuits for neoplastic transformation.
Long-term administration of IL-33 promotes epithelial metaplasia. Cross sections of a representative adult mouse EHBD before (A) and after (B) 10 weeks of daily injections of IL-33 show glandular metaplasia. (C and D) Cross sections stained with Alcian blue show intense staining in abundant peribiliary glands. Arrows show bile duct epithelium, and arrowheads point to peribiiary glands. Scale bars: 50 μm (A and C); 100 μm (B and D).
Based on the proposed oncogenic roles of Akt and Hippo pathways in the biliary epithelium (18, 19), we investigated whether IL-33 promotes carcinogenesis in mice whose biliary tract is primed to cholangiocarcinoma, the neoplasm originating from cholangiocytes. Genetic priming was induced by the intrabiliary injection of a transposon-transposase complex containing constitutively active Akt (myr-Akt) and Yap (YapS127A) coupled with lobar bile duct ligation to retain the transposon-transposase complex within the bile ducts of B6 mice. Animals received 1 μg of IL-33 or vehicle i.p. for 3 consecutive days and were examined for tumor burden and tumor characteristics 8 weeks later. In the group of animals that received both the transposons and IL-33, 10 of 17 (or 58.8%) mice developed advanced tumors with intrahepatic metastases (Figure 10, A and B) as compared with none of the animals in the group injected with transposons without IL-33 (n = 4, P < 0.05). The livers containing cancer had an average of 38 ± 9.4 macroscopic nodules, with the nodule size averaging 1.3 ± 0.07 mm. Microscopically, tumor nodules were easily identifiable by H&E staining in a background of normal liver (Figure 10C). Intrahepatic tumors exhibited neoplastic glands that strongly expressed pAkt and Yap (the activated forms of the oncogenes), and the 2 markers of biliary differentiation pancytokeratin (PanCK) and Sox9, without expression of HepPar1, a marker of hepatocellular cells (Figure 10C). Thus, exogenous administration of IL-33 to genetically susceptible mice potentiates oncogene-associated biliary tract carcinogenesis.
IL-33 facilitates biliary carcinogenesis. (A) liver appearance of mice after intrahepatic injection of myr-Akt and YapS127A Sleeping Beauty transposon-transposase complexes coupled with lobar bile duct ligation, and without (upper photo) or with (lower photo) daily injections of IL-33 (1 μg i.p. for 3 days). The lower photo shows liver nodules (arrows) representing neoplasms in mice with constitutively active Akt and Yap and injection of IL-33. (B) Percentage of animals with liver tumors (*P < 0.05). (C) Intrahepatic neoplastic nodules stained by H&E; expression of p-AKT; Yap; PanCK, and Sox 9 (nuclear staining), markers of biliary differentiation; and negative staining for HepPar1, a hepatocyte marker. Scale bars: 50 μm.