Hedgehog Signaling Modulates Interleukin‐33‐Dependent Extrahepatic Bile Duct Cell Proliferation in Mice

Abstract

Hedgehog (HH) signaling participates in hepatobiliary repair after injury and is activated in patients with cholangiopathies. Cholangiopathies are associated with bile duct (BD) hyperplasia, including expansion of peribiliary glands, the niche for biliary progenitor cells. The inflammation‐associated cytokine interleukin (IL)‐33 is also up‐regulated in cholangiopathies, including cholangiocarcinoma. We hypothesized that HH signaling synergizes with IL‐33 in acute inflammation‐induced BD hyperplasia. We measured extrahepatic BD (EHBD) thickness and cell proliferation with and without an IL‐33 challenge in wild‐type mice, mice overexpressing Sonic HH (pCMV‐Shh), and mice with loss of the HH pathway effector glioma‐associated oncogene 1 (Gli1lacZ/lacZ). LacZ reporter mice were used to map the expression of HH effector genes in mouse EHBDs. An EHBD organoid (BDO) system was developed to study biliary progenitor cells in vitro. EHBDs from the HH overexpressing pCMV‐Shh mice showed increased epithelial cell proliferation and hyperplasia when challenged with IL‐33. In Gli1lacZ/lacZ mice, we observed a decreased proliferative response to IL‐33 and decreased expression of Il6. The HH ligands Shh and Indian HH (Ihh) were expressed in epithelial cells, whereas the transcriptional effectors Gli1, Gli2, and Gli3 and the HH receptor Patched1 (Ptch1) were expressed in stromal cells, as assessed by in situ hybridization and lacZ reporter mice. Although BDO cells lacked canonical HH signaling, they expressed the IL‐33 receptor suppression of tumorigenicity 2. Accordingly, IL‐33 treatment directly induced BDO cell proliferation in a nuclear factor κB‐dependent manner. Conclusion: HH ligand overexpression enhances EHBD epithelial cell proliferation induced by IL‐33. This proproliferative synergism of HH and IL‐33 involves crosstalk between HH ligand‐producing epithelial cells and HH‐responding stromal cells.

C‐X‐C motif chemokine ligand 1 induced by Hedgehog signaling promotes mouse extrahepatic bile duct repair after acute injury

“…MouseEHBDandlivertissuewasisolated12,24,or 48hafteroperationandfixedin10%neutralbuffered formalin(ThermoFisherScientific)at4°Covernight, followed by processing for immunofluorescence as described. [8] Antibodies for immunofluorescence are listed in Table S1.Hematoxylinandeosin(H&E) staining was done following manufacturer's protocol (Vector Labs). X-gal staining was performed as described.…”

“…[8] Notably, the EHBDHHsignalingaxisinvolvedepithelial-stromalcell crosstalk,whereIndianHedgehog(IHH)ligandexpres-sioninEHBDcholangiocytessignaledtostromalcells expressing transcriptional response genes Gli1, Gli2, and Gli3andtheHHreceptorPatched1(Ptch1). [8] The identitiesoftheHH-dependentstromalcellsignalsthat provide feedback to promote epithelial cell proliferationareunknown.HHsignalingup-regulationwasalso reported in human PSC and mouse models of chronic cholestatic injury. [13][14][15][16][17] Inflammatory cells are recruited to sites of tissue injury,wheretheyorchestratecomplextissueresponses toresolveorexacerbatetheinjury.Neutrophilsinparticular play a major role after tissue injury, serving as first responders of the innate immune system to initiate tissue repair.…”

“…[ 8 ] Notably, the EHBD HH signaling axis involved epithelial‐stromal cell crosstalk, where Indian Hedgehog (IHH) ligand expression in EHBD cholangiocytes signaled to stromal cells expressing transcriptional response genes Gli1 , Gli2 , and Gli3 and the HH receptor Patched1 ( Ptch1 ). [ 8 ] The identities of the HH‐dependent stromal cell signals that provide feedback to promote epithelial cell proliferation are unknown. HH signaling up‐regulation was also reported in human PSC and mouse models of chronic cholestatic injury.…”

“…Cytokines are commonly up‐regulated in cholangiopathies, including IL‐6, IL‐8, IL‐17, IL‐33, C‐X‐C motif chemokine ligand (CXCL) 8, TNF‐α, and TGF‐β2. [ 2 , 8 , 9 , 10 , 11 ] Our group and others recently demonstrated in mouse models that acute inflammatory challenge with IL‐33 stimulates biliary proliferation in EHBDs. [ 8 , 9 , 12 ] Further, we showed an important role for HH signaling in the IL‐33 proliferative response.…”

“…[ 2 , 8 , 9 , 10 , 11 ] Our group and others recently demonstrated in mouse models that acute inflammatory challenge with IL‐33 stimulates biliary proliferation in EHBDs. [ 8 , 9 , 12 ] Further, we showed an important role for HH signaling in the IL‐33 proliferative response. [ 8 ] Thus, transgenic overexpression of HH enhanced the IL‐33 proliferative response, and HH inhibition through deletion of the gene encoding the key transcriptional effector GLI family zinc finger 1 ( Gli1 ) blunted the response.…”