Profibrotic cytokines in nonalcoholic steatohepatitis (NASH) liver biopsies visualized and quantified by chromogenic in situ hybridization

Background Non-alcoholic steatohepatitis (NASH) leads to liver fibrosis and subsequently to liver cirrhosis and increases the risk for hepatocellular carcinoma (HCC). Chronic inflammation of liver tissue in NASH represents a key role in the development of liver fibrosis. The aim of the study is to identify pro-inflammatory pathways for potential new therapeutical interventions.

Method Formalin-fixed paraffin-embedded (FFPE) human liver biopsies from patients with NASH (n=10) and control samples (n=4) were analysed. Chromogenic in situ hybridization (cISH) was used to detect proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin-1-beta (IL-1B), interleukin-6 (IL-6) and transforming growth factor beta (TGF-beta) in Semi-quantitative histological scoring based on ACD criteria was used for analysis.

Results Liver biopsies were evaluated for the level of fibrosis and steatosis. NASH biopsies were scored between 1–6 using the hepatitis activity score. Steatosis was graded semiquantitatively with a score of 5%-50%. The control group had no or low-grade fibrosis and no steatosis. NASH samples showed in comparison to the control group significantly up-regulation for the pro-inflammatory cytokines TNF-alpha (p = 0.04) and IL-1b (p = 0.02). IL-6 (p = 0.1059) showed no difference compared to the control group. The downstream pro-fibrotic marker TGF-beta showed a significant up-regulation in the NASH group (p = 0.04). Both pro-inflammatory and pro-fibrotic cytokines were mainly expressed by immune cells in the periportal region.

Conclusion cISH allows visualization of cytokine expression on mRNA level in FFPE tissue and revealed up-regulation of pro-inflammatory and pro-fibrotic cytokines in NASH samples, mostly located in the periportal region.

Publication History

Article published online:
26 January 2022