*5.2. Signaling Pathways as Targets for Potential Future Therapies in Liver Fibrosis*

Another critical inflammatory mediator within the fibrotic process in the liver is the cytokine IL-1β [203]. IL-1β is a peculiar cytokine in that it is not translated by ER membrane-bound ribosomes and not secreted via the classical ER–Golgi route [204]. In contrast, pro-IL-1β is synthesized by cytoplasmic ribosomes, and for maturation and secretion, the inflammasome, a multiprotein complex (e.g., the NLRP3 inflammasome), is required for caspase-1 activation and the subsequent cleavage of pro-IL-1β to yield mature IL-1β, which then is released by the process of pyroptosis [204]. Human chymase has been demonstrated to convert the 31 kDa pro-form of IL-1β to an 18 kDa biologically active molecule [205]. This cleavage product differs from the caspase-1-processed molecule in three amino acids; however, the activity of both cytokines is comparable. Since pro-IL-1β can be released, amongst others, from necrotic cells in an alarmin-like fashion, MC chymase could contribute to the generation of IL-1β and hence to the promotion of fibrosis.

So far, the described functions of the MC proteases, tryptase and chymase, for the development of liver fibrosis and associated hepatic alterations indicated a promoting role for these diseases. However, chymase was also reported to degrade two alarmins, IL-33 and HMGB1 [206], which have been shown to play detrimental pro-fibrotic roles in liver disease. An overproduction of the mRNAs for IL-33 and its receptor ST2 was observed in mouse and human fibrotic livers, and activated HSCs were identified as the major source of IL-33 [207]. IL-33 expression in liver was sufficient for severe hepatic fibrosis in vivo by the activation and expansion of liver-resident innate lymphoid cells (ILC2), which then contribute to fibrosis by producing IL-13 [208–210]. With respect to the second alarmin, HMGB1, which is produced and released by hepatocytes and Kupffer cells, and signals via the receptor for advanced glycation end-products (RAGE) in HSCs, activation of MAPK pathways and the induction of the increased deposition of collagen has been described [211,212]. In conclusion, chymase is a double-edged sword with regard to the development of liver fibrosis; on the one hand, it cleaves/activates pro-fibrotic proteins, such as angiotensin I, MMP-9, TGF-β, and type I pro-collagen, and on the other hand, it can degrade pro-fibrotic IL-33 and HMGB1. Thus, a thorough analysis of the respective disease development and regulators involved has to be performed before chymase inhibition can be recommended.

Another central MC mediator is the biogenic amine, histamine, which also has been reported to be involved in the regulation of fibrotic diseases. Histamine is synthesized by decarboxylation of the amino acid histidine via the enzyme L-histidine decarboxylase (HDC) [213]. Degradation of histamine is catalyzed by the enzyme monoamine oxidase B (MAOB) [213]. As with tryptase and chymase, histamine as a preformed mediator is stored in secretory lysosomes and is released by the process of degranulation. The effect(s) of histamine on target cells is dependent on the type of histamine receptor(s) expressed by these cells. Histamine receptors (H1R, H2R, H3R, and H4R) belong to the family of G-protein-coupled receptors and exert their differential functions by coupling to different G proteins, which again regulate differential signaling enzymes/pathways. Whereas H1R via Gq proteins activates PLC-γ, and thus induces Ca2<sup>+</sup>- and PKC-regulated signaling enzymes and transcription factors, H2R via Gs proteins positively couples to adenylyl cyclase, and thus regulates cellular processes by means of cAMP/PKA-dependent signal transduction and gene transcription. In an opposite fashion, H3R and H4R via Gi proteins inhibit adenylyl cyclase [214]. Thus, although histamine is only a single substance, it can trigger multiple cellular reactions and affect diseases in various ways.

In the livers of infants suffering from biliary atresia, histamine levels were significantly increased and positively correlated with the severity of fibrosis. Infants with severe fibrosis showed an elevated and reduced expression of HDC and DIO, respectively [213]. The *Abcb4* gene (aka *Mdr2*) encodes for the multidrug-resistant protein MDR2, and *Mdr2*−/<sup>−</sup> mice spontaneously develop severe biliary fibrosis via gross dysregulation of pro- and anti-fibrotic genes [215]. Interestingly, using H1R and H2R antagonists (mepyramine and ranitidine, respectively) alone or in combination, liver and biliary damage, as well as fibrosis in *Mdr2*−/<sup>−</sup> mice, was attenuated [160]. Moreover, these H1R and H2R antagonists also decreased the growth of CCA, angiogenesis, and epithelial–mesenchymal transition [160], clearly demonstrating the detrimental role for histamine in liver pathology.

Though so far not studied in liver, histamine was shown to affect the biology of fibroblasts in several ways. Stimulation of normal adult human lung fibroblasts with histamine enhanced the proliferation of these cells in a dose-dependent manner, mediated through the histamine receptor H2R and not H1R, which indicated a role for cAMP/PKA signaling [216]. These data were obtained using the H2R antagonist cimetidine and the H1R antagonist pyrilamine maleate. In normal human dermal fibroblasts, histamine treatment caused a clear enhancement in α-SMA expression, suggesting a role for histamine in fibroblast–myofibroblast progression [217]. Moreover, in human skin fibroblasts, histamine was found to increase proliferation and collagen production [218]. The action of histamine; however, is not only dependent on the histamine receptor(s) expressed, but also on the microenvironment of the respective cells. Hence, Lin et al. demonstrated that TGF-β1-induced expression of α-SMA in human skin fibroblasts was suppressed by histamine. In this situation, H1R activation, but not H2R or H4R activation, was responsible for the suppressive effect by histamine [219]. These few examples already clearly indicate that thorough knowledge about histamine receptor expressing cells in tissues of interest, and in addition, about the type(s) of histamine receptors expressed in these cells, must be obtained to take advantage of the substantial tool-box of various histamine receptor antagonists.
