Mechanisms regulating cellular crosstalk in extrahepatic bile duct responses to injury - Cholangiopathies are incurable bile duct disorders often affecting patients younger than 40 years old and characterized by biliary obstruction, inflammation, and hyperproliferation leading to cirrhosis and deadly cancer development in more than 10% of patients. This application aims to answer fundamental questions about the poorly understood mechanisms promoting inflammation and hyperproliferation during extrahepatic bile duct (EHBD) injury. The rationale for these studies is that unveiling mechanisms of cellular crosstalk in EHBD responses to injury can improve understanding of cholangiopathy pathogenesis. Our preliminary data shows profound cholangiocyte hyperproliferation 24 hours after acute EHBD injury with bile duct ligation (BDL). This is associated with an upregulation of the Indian Hedgehog (IHH) ligand in cholangiocytes, which targets GLI1+ fibroblasts. IHH upregulation is linked with increased chemokine CXCL1 expression and neutrophil recruitment to injured EHBDs. In turn, neutrophils promote biliary proliferation. Our data also shows that post-BDL, biliary hyperproliferation is associated with increased expression of pro-proliferative IL1β in neutrophils and AP-1 transcription factor complex in cholangiocytes. Our project will uncover molecular signaling in the complex crosstalk between cholangiocytes, fibroblasts and neutrophils. The overarching hypothesis for this proposal is that IHH orchestrates EHBD response to injury by inducing CXCL1 in GLI1+ fibroblasts to recruit neutrophils, which in turn upregulates the IL1β/AP-1 axis to promote cholangiocyte proliferation. We will use state-of-the art in vivo mouse approaches combined with in vitro human and mouse organoid, fibroblast, and neutrophil culture models. BDL will model obstructive cholangiopathy. Aim 1 will focus on mechanisms of GLI1+ fibroblast-mediated neutrophil recruitment into injured EHBDs and test the effects of HH-activated fibroblasts and CXCL1 on neutrophil migration. We will use primary fibroblast and neutrophil cultures for in vitro analyses. Mouse loss-of-function models will be used to test the effects of CXCL1 inhibition on neutrophil influx. Aim 2 will test if neutrophils promote biliary proliferation via an IL1β/AP-1-dependent mechanism. We will use genetic/pharmacologic mouse models to test the effects of cell-specific Il1β and AP-1 deletion on biliary proliferation post-BDL. RNAseq and ATACseq approaches will define transcriptional changes to IL1β/AP-1 signaling/targets and accessibility of AP-1 binding sites post-BDL. Aim 3 will define GLI1 target genes relevant to EHBD injury response. We will map GLI1 DNA targets using epitope-tagged mouse fibroblasts and determine HH/GLI1-mediated gene expression changes with RNAseq from EHBDs of ligated mice treated with a HH inhibitor. In addition, in Aims 1-3 we will analyze archived biliary tissues from patients with/without cholangiopathies for HH, CXCL1, IL1β and AP-1 expression. Ultimately, defining the mechanisms that regulate cellular crosstalk in EHBD injury responses may inform novel therapeutic strategies to improve outcomes in patients with cholangiopathies.
