Hepatocyte dedifferentiation profiling in alcohol-related liver disease identifies CXCR4 as a driver of cell reprogramming

Highlights

• Hepatobiliary cells are associated with poor outcomes in patients with ArLD.

• Hepatobiliary cells express pathways related to inflammation and stemness.

• Cxcr4 drives hepatocyte dedifferentiation in chronic liver injury.

• Cxcr4 overexpression in hepatocytes promotes hepatocyte dedifferentiation and injury progression.

• Genetic deletion or pharmacological inhibition of Cxcr4 reverts hepatocyte dedifferentiation.

Background & Aims

Loss of hepatocyte identity is associated with impaired liver function in alcohol-related hepatitis (AH). In this context, hepatocyte dedifferentiation gives rise to cells with a hepatobiliary (HB) phenotype expressing biliary and hepatocyte markers and showing immature features. However, the mechanisms and impact of hepatocyte dedifferentiation in liver disease are poorly understood.

Methods

HB cells and ductular reaction (DR) cells were quantified and microdissected from liver biopsies from patients with alcohol-related liver disease (ArLD). Hepatocyte-specific overexpression or deletion of C-X-C motif chemokine receptor 4 (CXCR4), and CXCR4 pharmacological inhibition were assessed in mouse liver injury. Patient-derived and mouse organoids were generated to assess plasticity.

Results

Here, we show that HB and DR cells are increased in patients with decompensated cirrhosis and AH, but only HB cells correlate with poor liver function and patients’ outcome. Transcriptomic profiling of HB cells revealed the expression of biliary-specific genes and a mild reduction of hepatocyte metabolism. Functional analysis identified pathways involved in hepatocyte reprogramming, inflammation, stemness, and cancer gene programs. The CXCR4 pathway was highly enriched in HB cells and correlated with disease severity and hepatocyte dedifferentiation. In vitro, CXCR4 was associated with a biliary phenotype and loss of hepatocyte features. Liver overexpression of CXCR4 in chronic liver injury decreased the hepatocyte-specific gene expression profile and promoted liver injury. CXCR4 deletion or its pharmacological inhibition ameliorated hepatocyte dedifferentiation and reduced DR and fibrosis progression.

Conclusions

This study shows the association of hepatocyte dedifferentiation with disease progression and poor outcome in AH. Moreover, the transcriptomic profiling of HB cells revealed CXCR4 as a new driver of hepatocyte-to-biliary reprogramming and as a potential therapeutic target to halt hepatocyte dedifferentiation in AH.

Impact and implications

Here, we show that hepatocyte dedifferentiation is associated with disease severity and a reduced synthetic capacity of the liver. Moreover, we identify the CXCR4 pathway as a driver of hepatocyte dedifferentiation and as a therapeutic target in alcohol-related hepatitis. Therefore, this study reveals the importance of preserving strict control over hepatocyte plasticity in order to preserve liver function and promote tissue repair.

Introduction

Alcohol-related hepatitis (AH) is an acute-on-chronic liver injury condition developed in patients with underlying alcohol-related liver disease (ArLD) and recent alcohol intake. AH is characterised by cholestasis, cell damage, inflammatory cell infiltration, hepatocellular failure, and loss of hepatocyte identity.[1], [2], [3] Moreover, patients with AH are characterised by the expansion of the ductular reaction (DR), which is associated with liver failure and high short-term mortality.^1,4

Both hepatocytes and biliary cells present a high plasticity potential, which is essential for their response to stress and injury. In liver injury, hepatocyte plasticity plays a key role in cell response to stress and is critical for cell proliferation and liver regeneration.[5], [6], [7] In this context, the biliary epithelium gives rise to immature cells and the expansion of DR, which emerges as a regenerative response of the liver to sustain the biliary compartment.^8,9 However, in the context of advanced liver disease, DR expansion and hepatocyte reprogramming become a maladaptive regenerative response of the liver and are associated with disease progression.^10,11

Hepatocyte-to-biliary reprogramming involves a progressive acquisition of biliary features by hepatocytes while reducing hepatocyte characteristics and functional competence, giving rise to hepatobiliary (HB) cells. Several histological studies have shown the presence of HB expressing both hepatocyte and biliary markers in cirrhosis, acute liver failure (ALF) and cholestatic liver diseases.^10,12 In AH, we have shown that the transforming growth factor beta (TGFβ) pathway plays a key role in promoting hepatocyte reprogramming.² In the same line, the activation of the Hippo/YAP pathway is involved in adult hepatocyte loss of identity in patients with ALF and AH.[13], [14], [15] In mice, lineage tracing studies have provided evidence for hepatocyte plasticity in chronic injury models.^16,17 In this context, Notch, YAP, and Wnt pathways promote hepatocyte dedifferentiation and hepatocyte-to-biliary transdifferentiation.^7,9,16,18 In addition, hepatocytes can be the source of both intrahepatic cholangiocarcinoma,¹⁹ thus suggesting the association of hepatocyte-to-biliary reprogramming with tumorigenesis. Although hepatocyte reprogramming has been extensively studied in animal models, its impact in human liver disease and the molecular mechanisms driving this event are not well understood.

These studies revealed the importance of preserving tight control of hepatocyte reprogramming in response to injury to maintain liver function and tissue repair, and underline cell reprogramming as a potential therapeutic target in chronic liver disease. In this study, we aimed to dissect the molecular mechanisms regulating hepatocyte-to-biliary reprogramming, inducing the appearance of HB cells in ArLD. We reveal that HB cells constitute a hallmark of AH, correlating with disease severity and reduced synthetic capacity of the liver. Moreover, we describe the transcriptomic and functional profile of human HB cells, identifying C-X-C motif chemokine receptor 4 (CXCR4) as a signalling pathway underlying hepatocyte-to-biliary reprogramming and as a potential therapeutic target to revert hepatocyte dedifferentiation in AH.