Transcriptomic profiling across the nonalcoholic fatty liver disease spectrum reveals gene signatures for steatohepatitis and fibrosis

Abstract

The mechanisms that drive nonalcoholic fatty liver disease (NAFLD) remain incompletely understood. This large multicenter study characterized the transcriptional changes that occur in liver tissue across the NAFLD spectrum as disease progresses to cirrhosis to identify potential circulating markers. We performed high-throughput RNA sequencing on a discovery cohort comprising histologically characterized NAFLD samples from 206 patients. Unsupervised clustering stratified NAFLD on the basis of disease activity and fibrosis stage with differences in age, aspartate aminotransferase (AST), type 2 diabetes mellitus, and carriage of PNPLA3 rs738409, a genetic variant associated with NAFLD. Relative to early disease, we consistently identified 25 differentially expressed genes as fibrosing steatohepatitis progressed through stages F2 to F4. This 25-gene signature was independently validated by logistic modeling in a separate replication cohort (n = 175), and an integrative analysis with publicly available single-cell RNA sequencing data elucidated the likely relative contribution of specific intrahepatic cell populations. Translating these findings to the protein level, SomaScan analysis in more than 300 NAFLD serum samples confirmed that circulating concentrations of proteins AKR1B10 and GDF15 were strongly associated with disease activity and fibrosis stage. Supporting the biological plausibility of these data, in vitro functional studies determined that endoplasmic reticulum stress up-regulated expression of AKR1B10, GDF15, and PDGFA, whereas GDF15 supplementation tempered the inflammatory response in macrophages upon lipid loading and lipopolysaccharide stimulation. This study provides insights into the pathophysiology of progressive fibrosing steatohepatitis, and proof of principle that transcriptomic changes represent potentially tractable and clinically relevant markers of disease progression.

Clinical implications of hepatic progenitor cell activation in non-alcoholic fatty liver disease

“…Over recent years, high-through transcriptomic analyses have been used in efforts to examine changes that occur as NAFLD progresses [ Table 1]. Though some studies are limited to dichotomous comparisons between mild and advanced disease, the expression of HPC markers has been found to be enriched in more advanced stage of the disease [78][79][80][81][82][83][84] . In a small cohort of 22 patients with NAFLD or alcoholic liver disease, Starmann et al [78] reported the expression of KRT7, KRT23, and p62 to be enriched in steatohepatitis when compared to steatosis.…”

“…K23 has been described as a stress-related DR marker associated with prominent inflammation and fibrosis in chronic liver disease, that can be induced in vitro by treatment with TWEAK and the type I acute phase inducer interleukin (IL)-1b [85] . Interestingly, other transcriptomics studies using larger NAFLD cohorts, have reported that the expression of TNF receptor superfamily member 12A (TNFRSF12A), the receptor for TWEAK, is increasingly expressed in advanced NAFLD [83,84] . Immunopositivity for TNFRSF12A in NASH has been described to be found in DR, endothelial cells and myofibroblasts [86] .…”

“…This would explain the association between DR and the histological severity in NAFLD. Recently, our group showed that the expression of HPC markers was associated with a high NAFLD Activity Score using RNA sequencing performed on 206 NAFLD biopsy samples [84] . Unsupervised clustering stratified the 206 NAFLD patients based on disease activity, fibrosis stage, age, serum aspartate aminotransferase, type 2 diabetes mellitus, and carriage of the PNPLA3 rs738409 variant [84] .…”

“…Recently, our group showed that the expression of HPC markers was associated with a high NAFLD Activity Score using RNA sequencing performed on 206 NAFLD biopsy samples [84] . Unsupervised clustering stratified the 206 NAFLD patients based on disease activity, fibrosis stage, age, serum aspartate aminotransferase, type 2 diabetes mellitus, and carriage of the PNPLA3 rs738409 variant [84] . Intriguingly, this was correlated with a high gene expression of HPC markers including CD24, EPCAM, JAG1, KRT7, KRT19, LAMC2, SOX9, TACSTD2, THY1, and TNFRSF12A [ Table 1].…”

“…Hoang et al [83] 2019 NAFL or NASH F0/1 vs. NASH F2 or F3 or F4 n = 206 RNAseq 25 THY1, TNFRSF12A Govaere et al [84] 2020 NAS ≥ 4 vs. NAS < 4 n = 206 RNAseq 369 CD24, EPCAM, GLI2, SPP1, TNFRSF12A Govaere et al [84] Govaere et al [84] 2020 HPC: hepatic progenitor cell; NAFLD: Non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis…”