Characterization of two types of intranuclear hepatocellular inclusions in NAFLD

Suzan Schwertheim,Holger Jastrow,Sarah Theurer,Ali Canbay,Heiner Wedemeyer,Hideo Andreas Baba,Kurt Werner Schmid,Christoph Matthias Schaefer,Saskia Ting,Julia Kälsch

doi:10.1038/s41598-020-71646-y

Abstract

Nuclear inclusions (NI) are a common finding in hepatocytes from patients with liver disease especially in diabetes mellitus and non-alcoholic fatty liver disease (NAFLD) but studies examining the shape and content of these inclusions in detail are lacking. In this study we define two distinct types of NI in NAFLD: inclusions bounded by the nuclear membrane, containing degenerative cell organelles and heterolysosomes (type1) and inclusions with deposits of glycogen but without any kind of organelles and delimiting membrane (type2). NI in 77 paraffin-embedded patients of NAFLD including NAFL and non-alcoholic steatohepatitis (NASH) were analyzed. In 4–12% of type1 NI immunopositivity for the autophagy-associated proteins LC3B, ubiquitin, p62/sequestosome1, cathepsin D and cathepsin B were detected with co-localizations of ubiquitin and p62; type2 NI showed no immunoreactivity. Three-dimensional reconstructions of isolated nuclei revealed that NI type1 are completely enclosed within the nucleus, suggesting that NI, although probably derived from cytoplasmic invaginations, are not just simple invaginations. Our study demonstrates two morphologically different types of inclusions in NAFLD, whereby both gained significantly in number in advanced stages. We suggest that the presence of autophagy-associated proteins and degenerated organelles within type1 NI plays a role in disease progression.

Highlights

Nuclear inclusions (NI) are a common finding in hepatocytes from patients with liver disease especially in diabetes mellitus and non-alcoholic fatty liver disease (NAFLD) but studies examining the shape and content of these inclusions in detail are lacking
We examined whether there is a relationship between the manifestation of NI and autophagy. p62, ubiquitin, LC3B, cathepsin B and cathepsin D immunohistochemistry was performed in all 77 cases (Fig. 5)
As the prevalence of NAFLD is rapidly rising in industrialized countries and because of its association with diabetes and progression to advanced forms such as non-alcoholic steatohepatitis (NASH), it becomes more important to clarify the factors driving its progression

Summary

Introduction

Nuclear inclusions (NI) are a common finding in hepatocytes from patients with liver disease especially in diabetes mellitus and non-alcoholic fatty liver disease (NAFLD) but studies examining the shape and content of these inclusions in detail are lacking. Nuclear vacuolation in hepatocytes was first reported over a century ago by Paul E hrlich[5] He showed glycogen filled vacuoles in nuclei with eccentrically displaced chromatin in liver tissue obtained from autopsies of diabetic patients. Nuclear vacuolation was described in different liver diseases, such as non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis or Wilson’s disease[8,9,10,11,12]. Intranuclear inclusions (NI) have been studied for over 100 years[13] They have been considered to have a homogenous morphology until Cowdry et al were the first to divide these NI in patients with viral infections into two groups depending on alterations of cell morphology as a reaction to a virus, the cowdry b odies[14,15]. Jaskolski et al described that autophagy is involved in the pathogenesis of intranuclear vacuolation in m eningeomas[22]; their electron microscopic studies revealed lysosomal bodies and autophagic vacuoles within these NI, suggesting an active macroautophagy p rocess[22]

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