The presence of a BH is an important finding in the histological diagnosis of human NASH. BHs are considered a special form of cellular degeneration that is caused by the accumulation of fatty acids and glycogen, abnormal protein accumulation due to unfolded protein response dysfunction, and autophagy suppression. However, the mechanism underlying BH formation is not well understood (5, 14–17). Histological features of BHs include hepatocyte ballooning (1.5- to 2-fold increase in size compared to normal hepatocytes), accumulation of fat droplets, abnormal arrangement of CK, abnormalities in organelles such as those in the mitochondria and endoplasmic reticulum, and the presence of MDBs (5, 18–20). In this study, we found that the exposure of PHH/HSC sheets to a glucolipotoxicity environment resulted in the production of BHs with features similar to those observed in human NASH. Functional analysis showed the involvement of fibrosis factors such as SHH and TGF-β1 but not the inflammatory cytokines such as TNFα and IL-8. Interestingly, BH was produced without the involvement of Kupffer cell (KC) or inflammatory cytokines. These results suggest that fibrosis is the major factor whereas inflammation is a supporting factor in BH formation. This observation is supported by the pathological correlation between fibrosis and BH formation (21, 22), and both correlate with clinical outcomes (10).
Although fibrosis is the most important factor in BH formation, it is not well understood why exposure of PHH/HSC sheets to a glucolipotoxicity environment results in BH formation. In a previous study, it was shown that ballooning did not occur in co-culture sheets with 3T3-J2 cells (12), which have been used as a feeder for maintaining PHH function (23), but ballooning did occur in co-culture with NHDF, a fibroblast cell line(12). These results suggest that fibrosis, hyperglycemia, and insulin resistance are important for ballooning. One of the factors that could produce BH was the change of co-culture cells to HSCs. HSCs are non-parenchymal cells of the liver and have a variety of functions (24) One pathway of HSCs is activated by SHH, which functions as a protection against apoptosis when the liver is damaged, and are transformed into myofibroblasts (25), to produce extracellular matrix (ECM) by increasing desmin and αSMA (24). After ECM production, activated HSCs regress to inactive forms or undergo apoptosis (26). However, HSCs continue to be activated under chronic damage such as fatty acid and oxidative stress and produce excessive ECM while further producing fibrosis-inducing factors such as TGF-β, causing liver fibrosis (24). It has also been reported that hyperglycemia and insulin resistance indirectly promote fibrosis (27). The action of HSCs as fibroblasts, chronic stimulation by fatty acids, and indirect effects of hyperglycemia and insulin resistance may have been factors involved in BH formation. Since ballooning does not occur in simple co-cultures (28, 29), it is assumed that there are some factors that lead to the production of BH in PHH/HSC sheets. We hypothesized that cells in the sheet will become three-dimensional (3D). This is supported by our observation that cells in the glucotoxicity, lipotoxicity, and glucolipotoxicity groups were layered compared to the normal group. Moreover, the 3D PHH/HSC sheets better represent the complex human liver due to enhanced cell-to-cell interaction (11, 30, 31). Recently, the sinusoidal pressure hypothesis has been proposed, and a correlation between pressure and fibrosis has been shown (32). We suspect that aggregation of PHH/HSC sheets during detachment exerts a strong force on cells (Fig. 2A), which in turn triggers fibrosis and HSC activation. In support of this, we observed that αSMA staining on day 0 of sheet fabrication showed strong activation of HSCs (Fig. 3A). During PHH/HSC sheet fabrication in the absence of FFA, αSMA was mainly localized at the basal side, while in the presence of FFA, αSMA showed no polarity and was localized to all regions including the periphery of hepatocytes (Fig. 3A). Thus, during sheet fabrication, stress due to external forces and glucolipotoxicity environment may have contributed to BHs formation.
MDBs are not specific to NASH but are also observed in various other diseases such as alcoholic steatohepatitis, liver cell carcinoma, and Wilson's disease (33). MDBs are thought to be formed as a result of the complex interaction between several factors, such as fibrosis, inflammation, suppression of autophagy, and cellular stress. All these factors lead to an imbalance in protein homeostasis within the cell and accumulation of abnormal intracellular proteins such as ubiquitin and p62 in addition to CK (33–35). However, the detailed underlying mechanisms remain unclear. Autophagy, a mechanism through which homeostasis is maintained by the degradation of unwanted cytoplasmic components and abnormal proteins, is thought to be important for MDB formation (36). In general, damaged organelles and abnormal proteins are modified with p62 and degraded. Once autophagy is inhibited, degradation cannot be performed, and p62 accumulates in the cell, which leads to MDB formation (37). Histological and functional analyses in our study showed that Ballooning, fibrosis, and fat accumulation are mainly induced by FFA exposure. The presence of MDBs suggest the involvement of hyperglycemia and hyperinsulinemia because these bodies appeared in the glucotoxicity and glucolipotoxicity groups. Interestingly, MDBs appeared in the glucotoxicity group, even though autophagy was only mildly suppressed as evidenced by a slight increase in p62 expression. It might be possible that mechanisms involved in autophagy suppression are different in PHHs and PHH/HSC sheets. It has been reported that autophagy is suppressed in hepatocytes with fat accumulation in human NASH (38), which is consistent with the results of this study. We investigated the presence of MDBs in PHHs from three different donors and found that MDBs were present in PHHs from only one donor (HU8317), even though all donors showed BH phenotypes such as ballooning and fatty degeneration. Clinically, MDBs have been reported only in 10–70% of patients with NASH (33, 39), which is consistent with the results of our in vitro experiment, and it is thought that there may be individual differences among donors.
Although various molecules are involved in NASH development and progression, the key factors are thought to be metabolic abnormalities due to hepatocyte steatosis, progression of fibrosis due to HSC activation, and inflammatory stimulation by KCs (2). This study has some limitations. Our PHH/HSC model could partially reproduce hepatocyte steatosis and fibrosis but could not reproduce inflammation. Since no inflammation was observed in our in vitro model, oxidative stress and endoplasmic reticulum stress could not be detected as well. It has also been shown that KCs and inflammatory cytokines can cause fibrosis and cellular damage (40, 41). Therefore, a tri-culture model with KCs is essential to better reproduce human NASH. Moreover, our model can produce BHs and fibrosis in approximately 2 weeks; therefore, it could also be used as a fibrosis model with BHs (13). Furthermore, patients with MDBs have been reported to have a worse prognosis, and thus, the presence of MDBs can be a clinically important indicator (42). Hence, the use of healthy hepatocytes from the donors in this model and screening for the appearance of MDBs may be applied to predict the prognosis of the donor after future development of NASH.
In summary, in vitro BH can be produced by exposing PHH/HSC sheets to a glucolipotoxicity environment. BH formation in this model is not due to inflammatory cytokines production but due to transformation of cells to a 3D structure and fibrosis. Our model will further help in understanding the mechanisms underlying BH formation. In the future, this model could be further developed by incorporating inflammatory cells in the PHH/HSC sheets and creating a new type of in vitro NASH model with BHs.