*3.3. BDL-Induced Cholestasis in Mast Cell-Depleted KitW-sh*/*W-sh Mice*

In order to delineate pro-fibrogenic effects of MCs in more detail, the crosstalk of MCs with other liver resident and infiltrating cells has been analyzed in murine models of cholestatic liver injury. Due to the localization of MCs in the portal tracts of the liver, different animal models and human biopsies have been analyzed (BDL, *Mdr2*−/<sup>−</sup> mice, porcine serum injection, PBC and PSC samples) [97,136]. The animal models develop portal fibrosis and a bile duct reaction encompassing bile duct proliferation (cholangiocyte proliferation), bile duct hyperplasia, and angiogenesis as critical features [137].

In order to delineate the effects of MC absence in vivo on processes induced by cholestasis, wild-type and MC-depleted mice (*KitW-sh*/*W-sh*) underwent BDL surgery. Wild-type mice subjected to BDL displayed increased focal necrosis (infarct) and portal inflammatory changes that are consistent with obstruction. In BDL *KitW-sh*/*W-sh* mice, the degree of focal necrosis (infarct) was reduced. Liver enzymes were significantly upregulated in wild-type mice after BDL, but this was completely ablated in BDL *KitW-sh*/*W-sh* mice, suggesting diminished hepatocyte damage in the absence of MCs. Biliary and cholangiocyte proliferation was reduced in BDL *KitW-sh*/*W-sh* mice. With respect to HSC function, the collagen deposition was dramatically reduced in BDL *KitW-sh*/*W-sh* mice, as monitored by Fast Green/Sirius Red staining. In agreement, the expression of α-SMA, fibronectin-1, collagen I type 1α, and SYP-9 as activation markers of HSCs were decreased in BDL *KitW-sh*/*W-sh* mice. In addition, there was a significant reduction in both TGF-β1 expression and secretion in BDL *KitW-sh*/*W-sh* mice, while TGF-β1 levels were increased in *KitW-sh*/*W-sh* mice injected with cultured MCs, supporting the concept that MCs are an important source of TGF-β1 during fibrosis progression [138]. TGF-β1 expression and secretion by antigen-stimulated MCs has been shown to induce proliferation, collagen Iα1, and monocyte chemoattractant protein-1 (MCP-1) expression in fibroblasts [139–141].

As a confirmation of the aforementioned findings, HSCs were cultured in vitro with conditioned supernatants derived from cholangiocytes isolated from BDL treated *KitW-sh*/*W-sh* animals. Only when the cholangiocytes were isolated from *KitW-sh*/*W-sh* animals supplemented with MCs, the corresponding supernatants have the potential to activate HSCs, as displayed by higher α-SMA expression. Those results imply that the presence of MCs triggers cholangiocytes to secrete factors that are able to cause HSC activation in vitro [138].

### *3.4. Tryptase*/*Protease-Activated Receptor-2 Axis*

One of the prominent effectors secreted by MCs upon degranulation is the protease tryptase. In contrast to chymase, tryptase is found in both human MCs (MCTC and MCT) [142]. On the other hand, in the mouse, tryptase is only expressed in CTMCs but not in MMCs [143]. Tryptase, in addition to trypsin and coagulation factors, activates the protease-activated receptor (PAR)-2 via limited proteolysis of its N-terminus. Protease-activator receptor-2 (PAR-2) is expressed in hepatic endothelial cells, Kupffer cells, hepatocytes, bile duct epithelial cells, and HSCs. The expression of

PAR-2 in HSCs is increased during liver fibrosis [144]. In agreement, the analysis of PAR-1 and PAR-2 expression in isolated rat HSCs showed that mRNA expression increases during the transdifferentiation of HSCs to MFBs in culture [145].

In vivo, a PAR-2 knockout had no impact on initial liver fibrosis after 5 weeks of CCl4 treatment but prevented the progression of fibrosis with sustained CCl4 treatment after 8 weeks, as reflected by lower hydroxyproline content. A similar behavior was seen in the expression of α-SMA, TGF-β1, matrix metalloproteinase (MMP)-2 (gelatinase A), and tissue inhibitor of metalloproteinase (TIMP)-1. The expression of PAR-1 compensates for PAR-2 deficiency, but only in the early phase (5 weeks) and not in the late phase (8 weeks). Since activation of PAR-2 leads to the recruitment and activation of macrophages, their presence at the different time points were quantified. F4/80<sup>+</sup> macrophages are reduced after 5 and 8 weeks of treatment, whereas CD68<sup>+</sup> macrophages were only reduced at 8 weeks. These observations are consistent with a role for PAR-2 in the recruitment and later activation of macrophages in CCl4-induced hepatic fibrosis. Therefore, these in vivo data implicate that PAR-2 is involved in the activation of HSCs, TGF-β1 synthesis, and ECM deposition, as well as macrophage recruitment.

To substantiate these findings with in vitro data, human HSC LX2 cells were induced for 48 h with the peptidic PAR-2 agonist SLIGKV. This stimulation induced proliferation of HSCs similar to that of PDGF, representing the most potent inducer of HSC proliferation. The PAR-2 agonist also led to an increase in collagen I and TGF-β1 production. Those effects were also observed when using a PAR-1 agonist, but the induction achieved with the PAR-1 and PAR-2 agonists on collagen I and TGF-β1 protein content were not additive [144]. Agonists for PAR-1, such as thrombin and SFFLRN, or agonists for PAR-2, such as tryptase and SLIGRL, induced a proliferative response in rat HSCs. Due to the sensitivity of this response to PD98059, it is most likely that the observed effects are mediated by ERK1/2 activation. In addition, tryptase and SLIGRL increased the collagen I secretion by HSCs [145]. In a more recent study, MC tryptase induced the activity of PAR-2 and increased HSC activation and proliferation, thus promoting hepatic fibrosis and implying that MCs interact directly with HSCs to drive fibrosis. The application of the MC tryptase inhibitor APC366 in BDL-induced hepatic fibrosis in rats showed that APC366 reduced hepatic fibrosis scores, collagen content, and serum biochemical parameters. Moreover, the reduced fibrosis was associated with a decreased expression of PAR-2 and α-SMA. Therefore, it was suggested that MC tryptase induces PAR-2 activation to augment HSC proliferation and promote hepatic fibrosis in rats [146].
