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Nonalcoholic Steatohepatitis Linked to Inflammation-Related Liver Cell Signature

NEW YORK – Using single-nucleus transcriptome sequencing and epigenomic profiling, a University of Pennsylvania-led team has teased out liver cell features linked to an advanced form of nonalcoholic fatty liver disease (NAFLD) called nonalcoholic steatohepatitis (NASH), identifying a group of hepatocyte liver cells marked by a cell adhesion- and migration-related expression signature.

"NAFLD represents a spectrum of diseases that range from nonalcoholic fatty liver (NAFL) with simple lipid accumulation to advanced stage nonalcoholic steatohepatitis (NASH) characterized by progressive inflammation and fibrosis," senior author Mitchell Lazar, a diabetes, obesity, and metabolism researcher at UPenn's Perelman School of Medicine, and his colleagues wrote, noting that liver fibrosis "poses the greatest risk of liver-related morbidity and mortality in patients with NASH, but approved therapy for treating fibrosis in NASH is still lacking."

As they reported in Science Translational Medicine on Wednesday, the researchers relied on single-nucleus RNA sequencing (snRNA-seq) and single-nucleus ATAC-seq (snATAC-seq) to profile gene expression patterns in liver samples from mouse models of NASH, which were fed an "American lifestyle-induced obesity syndrome" (ALIOS) diet linked to liver inflammation, fibrosis, weight gain, and moderate glucose intolerance in the mice.

They noted that the ALIOS diet led to NAFLD in six of seven mice at the three-month point, with the remaining mouse progressing to NASH. On the other hand, NASH appeared in all the mice after nine months on the diet.

With single-nucleus profiles generated on more than 28,300 liver cells from mice fed a normal or ALIOS diet for three or nine months, the team tracked down a range of NAFL- and NASH-related transcriptome, transcription factor, and open chromatin changes in hepatocyte and non-parenchymal cells.

In particular, the researchers flagged a cluster of hepatocytes expressing a receptor tyrosine kinase enzyme known as the Ephrin type B receptor 2 (EphB2), which is not active in normal hepatocyte cells.

From there, they incorporated snATAC-seq profiles to demonstrate that EphB2 gets activated by Notch signaling in the NASH-related subset of hepatocytes, kicking off inflammation-related processes — NASH-associated changes that were subsequently verified in 54,847 nuclei from NASH-affected or healthy human liver samples.

"We identified one subset of murine hepatocytes unexpectedly expressing receptor tyrosine kinase Ephrin type B receptor 2 (EphB2) at the NASH stage, which was corroborated in human NASH livers," the authors reported, noting that EphB2 activation "was sufficient to generate cell-autonomous inflammation in hepatocytes."

In a series of knockdown experiments in a NASH mouse model, meanwhile, the team showed that it was possible to reverse inflammation and fibrosis by dialing down EphB2 with AAV-based CasRx-mediated RNA targeting, for example, pointing to the possibility of developing NASH treatments aimed at staunching EphB2 activity in hepatocytes.

"Our comprehensive characterization of transcriptomic and epigenomic changes in all major liver cell types during NASH progression revealed two distinct hepatocyte trajectory branches and the implication of TFs potentially responsible for the development of NAFL as well as the transition to NASH in mouse models and human patients," the authors wrote.