Biliary repair and carcinogenesis are mediated by IL-33-dependent cholangiocyte proliferation

Abstract

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.

Figure 1. 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.

Figure 2. 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.

Figure 3. 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.

Figure 4. 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).

Figure 5. 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.

Figure 6. 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 Rora^sg/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.

Figure 7. 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.

Figure 8. 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).

Figure 9. 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).

Figure 10. 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.