Macrophages have been found to both promote liver fibrosis and contribute to its resolution by acquiring different phenotypes based on signals from the micro-environment. The best-characterized phenotypes in the macrophage spectrum are labeled M1 (classically activated) and M2 (alternatively activated). Until now the in situ localization of these phenotypes in diseased livers is poorly described. In this study, we therefore aimed to localize and quantify M1- and M2-dominant macrophages in diseased mouse and human livers. The scarred collagen-rich areas in cirrhotic human livers and in CCl4-damaged mouse livers contained many macrophages. Though total numbers of macrophages were higher in fibrotic livers, the number of parenchymal CD68-positive macrophages was significantly lower as compared to normal. Scar-associated macrophages were further characterized as either M1-dominant (IRF5 and IL-12) or M2-dominant (CD206, TGM2 and YM-1) and significantly higher numbers of both of these were detected in diseased livers as compared to healthy human and mouse livers. Interestingly, in mouse livers undergoing resolution of fibrosis, the total number of CD68+ macrophages was significantly lower compared to their fibrotic counterparts. M2-dominant (YM-1) macrophages were almost completely gone in livers undergoing resolution, while numbers of M1-dominant (IRF5) macrophages were unchanged and the proteolytic activity (MMP9) increased. In conclusion, this study shows the distribution of macrophage subsets in livers of both human and murine origin. The presence of M1-and M2-dominant macrophages side by side in fibrotic lesions suggests that both are involved in fibrotic responses, while the persistence of M1-dominant macrophages during resolution may indicate their importance in regression of fibrosis. This study emphasizes that immunohistochemical detection of M1/M2-dominant macrophages provides valuable information in addition to widely used flow cytometry and gene analysis.

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic.

Cytokines, growth factors and other locally produced mediators play key roles in the regulation of disease progression. During liver fibrosis, these mediators orchestrate the balance between pro- and antifibrotic activities as exerted by the hepatic cells. Two important players in this respect are the profibrotic mediator platelet derived growth factor BB (PDGF-BB) and the antifibrotic cytokine interferon gamma (IFNγ). PDGF-BB, produced by many resident and infiltrating cells, causes extensive proliferation, migration and contraction of hepatic stellate cells (HSCs) and myofibroblasts. These cells are the extracellular matrix producing hepatic cells and they highly express the PDGFβ-receptor. On the other hand, IFNγ is produced by natural killer cells in fibrotic livers and is endowed with pro-inflammatory, antiviral and antifibrotic activities. This cytokine attracted much attention as a possible therapeutic compound in fibrosis. However, clinical trials yielded disappointing results because of low efficacy and adverse effects, most likely related to the dual role of IFNγ in fibrosis. In our studies, we targeted the antifibrotic IFNγ to the liver myofibroblasts. For that, we altered the cell binding properties of IFNγ, by delivery of the IFNγ-nuclear localization sequence to the highly expressed PDGFβ-receptor using a PDGFβ-receptor recognizing peptide, thereby creating a construct referred to as “Fibroferon” (i.e. fibroblast-targeted interferon γ). In recent years, we demonstrated that HSC-specific delivery of IFNγ increased its antifibrotic potency and improved its general safety profile in vivo, making Fibroferon highly suitable for the treatment of (fibrotic) diseases associated with elevated PDGFβ-receptor expression. The present review summarizes the knowledge on these two key mediators, PDGF-BB and IFNγ, and outlines how we used this knowledge to create the cell-specific antifibrotic compound Fibroferon containing parts of both of these mediators.

Purpose Delivery of apoptosis-inducing compounds to hepatic stellate cells (HSC) may be an effective strategy to reverse liver fibrosis. The aim of this study was therefore to examine the selective targeting of the apoptosis-inducing drug 15-deoxy-Δ12,14-prostaglandin J₂ (15dPGJ₂) with two different HSC-carriers: human serum albumin modified with the sugar mannose-6-phosphate (M6PHSA) or albumin modified with PDGF-receptor recognizing peptides (pPBHSA). Methods and Results After chemical conjugation of 15dPGJ₂ to the carriers, the constructs displayed pharmacological activity and specific receptor-mediated binding to HSC in vitro. Unlike 15dPGJ₂-pPBHSA, the cellular binding of 15dPGJ₂-M6PHSA was reduced by a scavenger receptor antagonist. In vivo, both conjugates rapidly accumulated in fibrotic livers. Intrahepatic analysis revealed that 15dPGJ₂-M6PHSA mainly accumulated in HSC, and to a lesser extent in Kupffer cells. 15dPGJ₂-pPBHSA also predominantly accumulated in HSC with additional uptake in hepatocytes. Assessment of target receptors in human cirrhotic livers revealed that M6P/IGFII-receptor expression was present in fibrotic areas. PDGF-β receptor expression was abundantly expressed on human fibroblasts. Conclusions These studies show that 15dPGJ₂ coupled to either M6PHSA or pPBHSA is specifically taken up by HSC and is highly effective within these cells. Both carriers differ with respect to receptor specificity, leading to differences in intrahepatic distribution. Nevertheless, both carriers can be used to deliver the apoptosis-inducing drug 15dPGJ₂ to HSC in vivo.

Osteoprotegerin (OPG) serum levels are associated with liver fibrogenesis and have been proposed as a biomarker for diagnosis. However, the source and role of OPG in liver fibrosis are unknown, as is the question of whether OPG expression responds to treatment. Therefore, we aimed to elucidate the fibrotic regulation of OPG production and its possible function in human and mouse livers. OPG levels were significantly higher in lysates of human and mouse fibrotic livers compared to healthy livers. Hepatic OPG expression localized in cirrhotic collagenous bands in and around myofibroblasts. Single cell sequencing of murine liver cells showed hepatic stellate cells (HSC) to be the main producers of OPG in healthy livers. Using mouse precision-cut liver slices, we found OPG production induced by transforming growth factor β1 (TGFβ1) stimulation. Moreover, OPG itself stimulated expression of genes associated with fibrogenesis in liver slices through TGFβ1, suggesting profibrotic activity of OPG. Resolution of fibrosis in mice was associated with decreased production of OPG compared to ongoing fibrosis. OPG may stimulate fibrogenesis through TGFβ1 and is associated with the degree of fibrogenesis. It should therefore be investigated further as a possible drug target for liver fibrosis or biomarker for treatment success of novel antifibrotics.

Alkaline phosphatase (AP) activity is highly upregulated in plasma during liver diseases. Previously, we demonstrated that AP is able to detoxify lipopolysaccharide (LPS) by dephosphorylating its lipid A moiety. Because a role of gut-derived LPS in liver fibrogenesis has become evident, we now examined the relevance of phosphate groups in the lipid A moiety in this process. The effects of mono-phosphoryl and di-phosphoryl lipid A (MPLA and DPLA, respectively) were studied in vitro and LPS-dephosphorylating activity was studied in normal and fibrotic mouse and human livers. The effects of intestinal AP were studied in mice with CCL4-induced liver fibrosis. DPLA strongly stimulated fibrogenic and inflammatory activities in primary rat hepatic stellate cells (rHSCs) and RAW264.7 macrophages with similar potency as full length LPS. However, MPLA did not affect any of the parameters. LPS-dephosphorylating activity was found in mouse and human livers and was strongly increased during fibrogenesis. Treatment of fibrotic mice with intravenous intestinal-AP significantly attenuated intrahepatic desmin⁺− and αSMA⁺ −HSC and CD68⁺− macrophage accumulation. In conclusion, the lack of biological activity of MPLA, contrasting with the profound activities of DPLA, shows the relevance of LPS-dephosphorylating activity. The upregulation of LPS-dephosphorylating activity in fibrotic livers and the protective effects of exogenous AP during fibrogenesis indicate an important physiological role of intestinal-derived AP during liver fibrosis.