*1.2. Roles of Di*ff*erent Hepatic Cell Types in Liver Fibrosis*

The process of fibrosis development, fibrogenesis, can be analyzed from a cellular perspective. The regeneration of hepatocytes works in a streaming fashion, as shown in a rat model by Zajicek and colleagues in 1985. Hepatocytes located at the portal space gradually stream towards the hepatic vein where they are eliminated by apoptosis. This cellular traveling was estimated to last 201 days in rats [2]. However, during liver fibrosis, the empty spaces left by the missing hepatocytes are frequently replenished with ECM by HSC, rather than with fresh hepatocytes. During the course of cell death, certain molecules are released by hepatocytes, which function as danger signals for other cell types, for instance for HSC [12]. These "alarm bells" also attract immune cells which themselves secrete pathogenic factors that can induce apoptosis of hepatocytes. These processes result in an amplification of the fibrogenic response. Innate immune cells are well known to initiate liver inflammation in NAFLD and they express pattern recognition receptors (PRR) which can sense danger-associated and pathogen-associated molecular pattern molecules (DAMP and PAMP), as well as inflammatory mediators [12].

In recent years, the roles of cholangiocytes, particularly in cholestatic liver injury, started to be explored. For instance, cholangiocyte proliferation is a substantial driver of liver fibrosis in biliary atresia. Researchers demonstrated that a long non-coding RNA has a major impact on the proliferation of cholangiocytes and thus represents a therapeutic target in this regard [13]. Damage of cholangiocytes in toxic liver injury leads to a hampered production of bile acids. Sato and colleagues further revealed that extracellular vesicles and microRNAs might be critical factors which regulate cyclic adenosine monophosphate (cAMP) metabolism in cholangiocytes [14].

Mast cells are another cell type reported to further amplify hepatic fibrosis and injury. Particularly, mast cell-deficient mice exhibited less pronounced fibrosis. It was reported that mast cells regulate the proliferation of cholangiocytes and contribute to the activation of HSC [15]. A means to specific deactivation of mast cells might therefore represent a novel therapeutic strategy. However, the involvement of mast cells in fibrosis remains controversial since they have been reported to be both harmful and protective [16].

The hepatic macrophages form a complex mixture of cells of various activation stages and cellular origin. They can be distinguished as resident macrophages, which were originally defined as Kupffer cells, as well as monocyte-derived macrophages (MoMF). These cells can be separated using cell sorting applications [17]. Kupffer cells were first discovered by Karl Wilhelm von Kupffer in 1876 [18], even before the relevance of phagocytic cells was first published by Metchnikoff in 1888 in Tuberculosis [19]. While cell sorting has unraveled hepatic macrophage subpopulations that were characterized in RNA bulk sequencing where thousands of cells are analyzed in a single RNA isolate [17], single cell RNA sequencing has begun to start unraveling the real complexity of hepatic macrophage subtypes [20]. Single cell RNA sequencing has also enabled identification of subpopulations of HSC [21]. The so-called resting or quiescent HSC (qHSC) form a homogenous population characterized by high platelet-derived growth factor receptor β (PDGFRβ) expression. However, the activated HSC, which are also called myofibroblasts, can further be sub-divided into populations expressing α-smooth muscle actin (α-SMA), collagens, or immunological markers. The S100 calcium binding protein A6 (S100A6) was identified as a universal marker of activated HSC, myofibroblasts (MFB), for both mRNA and protein expression [21]. The so-called transdifferentiation of the resting and vitamin A storing HSC into MFB, which are proliferative and which express huge amounts of collagen, is central for fibrogenesis [22]. The activation of HSC can be induced through a variety of extracellular signals from other liver cell types like hepatocytes and macrophages. Further, intracellular processes like oxidative stress, autophagy, endoplasmatic reticulum stress, or metabolic dysregulations, have been studied in great detail and are regarded as causative for HSC activation [23]. The fact that fibrosis is a bidirectional path is evidenced by reports on fibrosis regression [24]. HSC thus represent a valuable target for fibrosis therapy since they are the source of excessive matrix production. Inhibiting the construction of different collagens, which in part takes place in the extracellular space by specific inhibitors, for example, Lysyl oxidase-like 2 (LOXL2), has recently been proposed to be done by small molecules [25].

It was shown that, if a circumvention of the inflammatory insult is achieved, among other factors, the anti-inflammatory and restorative activities of macrophages persist and impact the hepatic microenvironment [26]. During fibrosis regression, MFB are eliminated through cell death induction, become senescent, or may even revert into cells which resemble quiescent HSC [27]. The excessively deposited ECM was shown to be degraded, i.e., by matrix metalloproteinases (MMP) that are released by certain subtypes of macrophages [24] (Figure 1).

**Figure 1.** Cells, their roles, and potential targets in liver fibrosis. Liver disease is in most cases initiated by a noxa that leads to hepatocyte cell death. Cytokines secreted by immune and other cell types promote hepatocyte cell injury, i.e., the tumor necrosis factor (TNF) triggers apoptosis of hepatocytes. Hepatic collagen deposition by activated hepatic stellate cells is a hallmark of fibrosis and in part is facilitated by extracellular enzymes.
