Hepatic stellate cell heterogeneity and function during the hepatic regenerative response

INTRODUCTION: Acetaminophen (APAP) poisoning is the commonest cause of acute liver failure (ALF) in the Western world1 and results in centrilobular hepatic necrosis. In non-severe cases the liver regenerates to normal microarchitecture and function. However, in severe APAP-induced ALF, transplantation remains the only curative treatment. Hepatic stellate cells (HSCs) play a central role during liver injury and regeneration, driving matrix remodelling and impacting proliferation and differentiation of other hepatic cell types2,3. Traditionally HSCs were considered a homogenous population, with global inhibition reducing hepatic regeneration4,5, but recent single cell RNA sequencing (scRNA-seq) studies of hepatic fibrosis have identified functional heterogeneity within HSCs6. However, little is known of the role of different HSC subpopulations during the hepatic regenerative response. AIMS AND METHODS: Combining an APAP-induced liver injury (AILI) mouse model with scRNA-seq, heterogeneity within HSCs was investigated during liver regeneration. 6–12-week-old male mice were used for all experiments. Mice were fasted for 12 hours prior to intraperitoneal (IP) injection of APAP (300mg/kg). PDGFRb-bac-eGFP mice were used to isolate eGFP+(PDGFRb+) mesenchymal cells by flow cytometry for scRNA-seq using 10X Chromium. To investigate the function of a p75NTRexpressing HSC subpopulation, C57BL/6J mice received Tat-Pep57 (TP5, cell-permeable peptide inhibitor which specifically blocks the activation of Rho signalling through p75NTR) 2 hours post- AILI. Further experiments were undertaken using P75NTR+/- mice. To investigate the function of a TNC-expressing HSC subpopulation, transgenic TNC-/- mice were used. Hepatotoxicity was assessed by measuring plasma levels of hepatic enzymes (including alanine aminotransferase, ALT), and histological examination of Periodic Acid-Schiff-stained tissue sections. RESULTS: (1) scRNA-seq analyses of PDGFRb+ cells from healthy and APAP-injured mouse livers identified HSC heterogeneity, with two distinct subpopulations (defined by expression of HSC specific markers, p75NTR or TNC). Immunofluorescence (IF) staining demonstrated restriction of the p75NTR+ HSC subpopulation to the peri-portal region and the TNC+ population to the peri-central region in murine and human liver. p75NTR and TNC were not expressed in other hepatic cell lineages. (2) AILI resulted in upregulation of both p75NTR and TNC expression (quantified by IF staining) in both human and murine liver. (3) Pharmacological inhibition of p75NTR/Rho signalling in vivo reduced hepatic injury in a mouse model of AILI, with reduced ALT (mean ALT 6605 U/L vs. 1869 U/L, p=0.0006) and necrosis (35.25% vs. 14.07%, p=0.0078) in TP5-treated mice compared with vehicle control at 24 hours post-AILI. TP5 was as effective as N-acetylcysteine (NAC) in reducing liver injury following AILI at 24 hours. Furthermore, genetic depletion of p75NTR using P75NTR+/- mice reduced injury in a mouse model of AILI, with reduced ALT (8481U/L vs. 6517U/L, p=0.020) and necrosis (39.03% vs. 22.07%, p=0.0022) at 24 hours post-AILI. (4) Transgenic depletion of TNC resulted in delayed repair (72 hours mean necrosis 3.27% (WT) vs. 20.74% (TNC- /-), p=0.0079) and persistent matrix deposition in a mouse model of AILI. CONCLUSIONS: This work has characterised, both transcriptionally and spatially, two distinct HSC subpopulations in both murine and human tissue following AILI; identified by the HSC-specific markers - p75NTR and Tnc. These HSC subpopulations have opposing roles in the regenerative response. Further studies will determine if manipulating Tnc and p75NTR signalling in HSC could represent an effective novel therapeutic strategy for patients with AILI.

Grantor dc:publisher

The University of Edinburgh

Year dc:date.issued

2023

Author dc:creator

• Kitto, Laura

Advisors dc:contributor.advisor

• Henderson, Neil
• Ponting, Chris

• paracetamol poisoning
• liver injury
• hepatic stellate cells
• scRNA-seq
• mouse model

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.7488/era/3729

OAI identifier oai:identifier

oai:era.ed.ac.uk:1842/40978