TWEAK/Fn14 Signalling Regulates the Tissue Microenvironment in Chronic Pancreatitis.

Chronic pancreatitis increases the risk of developing pancreatic cancer through the upregulation of pathways favouring proliferation, fibrosis, and sustained inflammation. We established in previous studies that the ligand tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) signals through its cognate receptor fibroblast growth factor-inducible 14 (Fn14) to regulate these underlying cellular processes in the chronic liver injury niche. However, the role of the TWEAK/Fn14 signalling pathway in pancreatic disease is entirely unknown. An analysis of publicly available datasets demonstrated that the TWEAK receptor Fn14 is upregulated in pancreatitis and pancreatic adenocarcinoma, with single cell RNA sequencing revealing pancreatic ductal cells as the main Fn14 producers. We then used choline-deficient, ethionine-supplemented (CDE) diet feeding of wildtype C57BL/6J and Fn14 knockout littermates to (a) confirm CDE treatment as a suitable model of chronic pancreatitis and (b) to investigate the role of the TWEAK/Fn14 signalling pathway in pancreatic ductal proliferation, as well as fibrotic and inflammatory cell dynamics. Our time course data obtained at three days, three months, and six months of CDE treatment reveal that a lack of TWEAK/Fn14 signalling significantly inhibits the establishment and progression of the tissue microenvironment in CDE-induced chronic pancreatitis, thus proposing the TWEAK/Fn14 pathway as a novel therapeutic target.

Single-nucleus RNA sequencing of pre-malignant liver reveals disease-associated hepatocyte state with HCC prognostic potential.

Current approaches to staging chronic liver diseases have limited utility for predicting liver cancer risk. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte (daHep) transcriptional state. These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep-enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled, and risk stratified.

Previous liver regeneration induces fibro-protective mechanisms during thioacetamide-induced chronic liver injury.

Chronic liver injury is characterised by continuous or repeated epithelial cell loss and inflammation. Hepatic wound healing involves matrix deposition through activated hepatic stellate cells (HSCs) and the expansion of closely associated Ductular Reactions and liver progenitor cells (LPCs), which are thought to give rise to new epithelial cells. In this study, we used the murine thioacetamide (TAA) model to reliably mimic these injury and regeneration dynamics and assess the impact of a recovery phase on subsequent liver injury and fibrosis. Age-matched naïve or 6-week TAA-treated/4-week recovered mice (C57BL/6 J, n = 5-9) were administered TAA for six weeks (C57BL/6 J, n = 5-9). Sera and liver tissues were harvested at key time points to assess liver injury biochemically, by real-time PCR for fibrotic mediators, Sirius Red staining and hydroxyproline assessment for collagen deposition as well as immunofluorescence for inflammatory, HSC and LPC markers. In addition, primary HSCs and the HSC cell line LX-2 were co-cultured with the well-characterised LPC line BMOL and analysed for potential changes in expression of fibrogenic mediators. Our data demonstrate that recovery from a previous TAA insult, with LPCs still present on day 0 of the second treatment, led to a reduced TAA-induced disease progression with less severe fibrosis than in naïve TAA-treated animals. Importantly, primary activated HSCs significantly reduced pro-fibrogenic gene expression when co-cultured with LPCs. Taken together, previous TAA injury established a fibro-protective molecular and cellular microenvironment. Our proof-of principle HSC/LPC co-culture data demonstrate that LPCs communicate with HSCs to regulate fibrogenesis, highlighting a key role for LPCs as regulatory cells during chronic liver disease.