BACKGROUND & AIMS: Macrophages are key regulators of inflammation and cancer promotion in the liver, and their recruitment and activation is linked to chemokine receptor signaling. However, the exact roles of the chemokine receptors CCR2 and CCRS for macrophage functions in the liver is obscure. METHODS: To study CCR2 and CCRS in inflammatory liver injury, we used mice with a hepatocyte-specific knock-out of the nuclear factor kappa B (NF-kappa B) essential modulator (NEMO), termed NEMOLPC-KO mice, and generated NEMO(LPC-KO )Ccr2(-/-) and NEMO(LPC-KO)Ccr5(-/-) mice. NEMOLPC-KO mice develop hepatitis and fibrosis after two and liver tumors after six months. RESULTS: We found that both CCR2 and CCRS deficiency led to reduced fibrosis, while CCRS deficiency increased steatosis and tumor burden in NEMOLPC-KO mice. CCR2 was required for recruitment of hepatic macrophages, whereas CCRS promoted stellate cell activation. The reduction of monocytes and macrophages by either anti-Gr1 antibody or clodronate-loaded liposomes (CLL), but not of CD8(+) T cells or NK cells, significantly aggravated liver injury in NEMOLPC-KO mice and was further increased in NEMO(LPC-KO )Ccr5(-/-) mice. CLL-induced liver injury was dampened by the adoptive transfer of ex vivo generated macrophages, whereas the adoptive transfer of control CD115(+) immature monocytes or B cells did not reduce liver injury. CONCLUSIONS: Although CCR2 and CCRS principally promote liver fibrosis, they exert differential functions on hepatic macrophages during liver disease progression in NEMOLPC-KO mice. While CCR2 controls the recruitment of monocytes to injured livers, CCRS-dependent functions of liver macrophages limit hepatic injury, thereby reducing steatosis and hepatocarcinogenesis.

Einleitung: Makrophagen erfüllen essentielle Funktionen in chronischen Lebererkrankungen, können aber sowohl zu einer Progression als auch Regression beitragen. Die verschiedenen molekularen Faktoren, die die funktionelle Differenzierung der Lebermakrophagen bestimmen, sind derzeit unklar. Aktuelle Arbeiten mit Tumormodellen zeigten, dass das in der Leber generierte Histidin-reiche Glykoprotein (HRG) zu einer Polarisierung der anti-inflammatorischen (M2) zu pro-inflammatorischen (M1) Makrophagen führt. Daher vermuteten wir, dass HRG einen wichtigen Modulator der hepatischen Makrophagenfunktion darstellt und folglich eine Auswirkung auf die Leberentzündung und -fibrose hat.

Pathogen-and injury-related danger signals as well as cytokines released by immune cells influence the functional differentiation of macrophages in chronic inflammation. Recently, the liver-derived plasma protein, histidine-rich glycoprotein (HRG), was demonstrated, in mouse tumor models, to mediate the transition of alternatively activated (M2) to proinflammatory (M1) macrophages, which limit tumor growth and metastasis. We hypothesized that liver-derived HRG is a critical endogenous modulator of hepatic macrophage functionality and investigated its implications for liver inflammation and fibrosis by comparing C57BL/6N wild-type (WT) and Hrg(-/-) mice. In homeostatic conditions, hepatic macrophages were overall reduced and preferentially polarized toward the anti-inflammatory M2 subtype in Hrg(-/-) mice. Upon chronic liver damage induced by CCl4 or methionine-choline-deficient (MCD) diet, liver injury and fibrosis were attenuated in Hrg(-/-), compared to WT, mice. Macrophage populations were reduced and skewed toward M2 polarization in injured livers of Hrg(-/-) mice. Moreover, HRG-deficient mice showed significantly enhanced hepatic vascularization by micro-computed tomography and histology, corroborating proangiogenic activities of M2-polarized liver macrophages. Purified HRG protein induced, but HRG-deficient serum prevented, M1 macrophage differentiation in vitro. Accordingly, Hrg(-/-) mice transplanted with Hrg(+/+) bone marrow, but not Hrg(-/-)-transplanted Hrg(+/+) mice, remained protected from experimental steatohepatitis. Consistent with these findings, patients with chronic hepatitis C and nonalcoholic steatohepatitis significantly up-regulated hepatocytic HRG expression, which was associated with M1 polarization of adjacent macrophages. Conclusions: Liver-derived HRG, similar to alarmins, appears to be an endogenous molecular factor promoting polarization of hepatic macrophages toward the M1 phenotype, thereby promoting chronic liver injury and fibrosis progression, but limiting angiogenesis. Therefore, controlling tissue levels of HRG or PGF might be a promising strategy in chronic inflammatory liver diseases.

Objectives In chronic liver injury, angiogenesis, the formation of new blood vessels from pre-existing ones, may contribute to progressive hepatic fibrosis and to development of hepatocellular carcinoma. Although hypoxia-induced expression of vascular endothelial growth factor (VEGF) occurs in advanced fibrosis, we hypothesised that inflammation may endorse hepatic angiogenesis already at early stages of fibrosis. Design Angiogenesis in livers of c57BL/6 mice upon carbon tetrachloride- or bile duct ligation-induced chronic hepatic injury was non-invasively monitored using in vivo contrast-enhanced micro computed tomography (µCT) and ex vivo anatomical µCT after hepatic Microfil perfusion. Functional contributions of monocyte-derived macrophage subsets for angiogenesis were explored by pharmacological inhibition of CCL2 using the Spiegelmer mNOX-E36. Results Contrast-enhanced in vivo µCT imaging allowed non-invasive monitoring of the close correlation of angiogenesis, reflected by functional hepatic blood vessel expansion, with experimental fibrosis progression. On a cellular level, inflammatory monocyte-derived macrophages massively accumulated in injured livers, colocalised with newly formed vessels in portal tracts and exhibited pro-angiogenic gene profiles including upregulated VEGF and MMP9. Functional in vivo and anatomical ex vivo µCT analyses demonstrated that inhibition of monocyte infiltration by targeting the chemokine CCL2 prevented fibrosis-associated angiogenesis, but not fibrosis progression. Monocyte-derived macrophages primarily fostered sprouting angiogenesis within the portal vein tract. Portal vein diameter as a measure of portal hypertension depended on fibrosis, but not on angiogenesis. Conclusions Inflammation-associated angiogenesis is promoted by CCL2-dependent monocytes during fibrosis progression. Innovative in vivo µCT methodology can accurately monitor angiogenesis and antiangiogenic therapy effects in experimental liver fibrosis.

Macrophages constitute a major proinflammatory component during chronic liver diseases and are considered a key factor in promoting hepatic fibrosis. However, there is increasing evidence that distinct monocyte and macrophage subsets exert critical functions in regression from organ fibrosis as well. Experimental mouse models of fibrosis regression have identified “restorative” macrophages as Ly-6C (Ly6C, Gr1) low-expressing, monocyte-derived cells. We investigated molecular pathways balancing proinflammatory and restorative macrophages during fibrosis regression as well as pharmacologically augmenting beneficial macrophage functionality in fibrosis resolution. Therefore, we employed a Spiegelmer-based inhibitor of the chemokine, C-C motif chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1), termed mNOX-E36, in the regression phase of two murine models of toxic (CCl4) and metabolic (methionine-choline–deficient diet) liver fibrosis. Although inflammation rapidly declined after cessation of injury, we observed a transient influx of Ly-6C+ infiltrating monocytes (iMΦ), which are characterized by typical macrophage morphology, up-regulated expression of CCR2, and the pro-inflammatory cytokine, tumor necrosis factor (TNF), in injured liver. By inhibiting the early influx of Ly-6C+ iMΦ by the CCL2 inhibitor, mNOX-E36, the intrahepatic macrophage equilibration shifted toward the “restorative” Ly-6C- subset of iMΦ. Consequently, fibrosis resolution was significantly accelerated upon mNOX-E36 administration in both models. Blocking transient recruitment of infiltrating Ly-6C+ monocytes, but not direct effects of the inhibitor on the remaining macrophages, resulted in reduced intrahepatic levels of proinflammatory cytokines. Conclusion: Transient CCL2-dependent recruitment of infiltrating Ly-6C+ monocytes during fibrosis regression counteracts scar resolution by perpetuating inflammatory reactions through release of proinflammatory cytokines such as TNF. Pharmacological inhibition of Ly-6C+ monocyte recruitment using the CCL2-inhibitor, mNOX-E36, accelerates regression from toxic and metabolic liver fibrosis in two independent experimental models. (HEPATOLOGY 2014;59:1060–1072)

Background:In chronic hepatitis C (CHC), the transition from mild to moderate fibrosis is a major prognostic step.The aim of this study was to identify serum protein signature to differentiate mild from moderate fibrosis in CHC.Methods: Liver biopsies from 244 untreated CHC patients were studied.Among them, 66% had mild fibrosis (F1, Metavir) and 34% moderate fibrosis (F2).Patients were mainly infected with genotype 1 (55%), 2 (11%), 3 (11%), 4 (20%) and 5-6 (3%) respectively.Real-time quantitative RT-PCR assays were used to analyse the mRNA expression of 51 genes involved in fibrogenesis.The concentration of 6 proteins was assayed in duplicate by ELISA in the serum of 228 patients and reliable data were obtained for 216 (65% with mild fibrosis and 35% with moderate fibrosis).Results: 28 genes were found to be upregulated in F2 patients.These genes were mainly involved in extracellular matrix production and remodelling, in cell-cell and cell-extracellular matrix interactions, in cell cycle, or encode growth factors/cytokines families.We focused our interest on up-regulated genes that code for circulating proteins.The 6 genes with the highest statistical discriminating values were: A2M, CXCL10, SPP1, S100A4, IL8 and ENG.Therefore, we assayed the concentration of these 6 proteins in the serum of patients.The protein signature with the highest discriminating value was composed of A2M, CXCL10, IL8, and SPP1.This 4-protein signature was able to discriminate F2 patients from F1 patients with a sensitivity of 68%, and a specificity of 69% (AUC = 0.684).Addition of clinical parameters to this 4-protein signature allowed reaching 72% sensitivity and 75% specificity (AUC = 0.750). Conclusion:We demonstrated in a large independent cohort that mild and moderate fibrosis have different liver gene expression.The most notable changes occurred mainly in cell-matrix turn-over.Several genes that are up-regulated in the liver encode molecules detected in the serum and provide a logical functional approach for the development of serum markers of fibrosis progression.A 4-protein signature (A2M, CXCL10, IL8, and SPP1) was identified that demonstrates high value for the diagnosis of early fibrosis and particularly the discrimination of F1/F2 stages.

The deposition of extracellular matrix (ECM) proteins, such as collagen and elastin, is one of the hallmarks of liver fibrosis. In recent years it has become increasingly clear that tissue repair and remodeling are highly dynamic processes, resulting in continuous synthesis and turnover of ECM components during hepatofibrogenesis, and in disease state-specific changes in both the quantitative amount and qualitative composition of the ECM.1 In the June 2012 issue of HEPATOLOGY, Pellicoro et al.2 elegantly demonstrate that elastin accumulation represents a distinct feature of advanced-stage liver fibrosis, because of both increased synthesis and decreased macrophage metalloelastase (MMP12)-mediated degradation. Taking these findings, and the results recently reported by Polasek et al.3 on a collagen-specific magnetic resonance (MR) contrast agent into account, we reasoned that elastin might be a promising novel target for molecular MR monitoring of ECM-remodeling during hepatic fibrosis. We therefore evaluated the accumulation of the gadolinium-containing elastin-specific MR contrast agent ESMA, which has been shown to facilitate noninvasive assessment of atherosclerotic plaque burden4 in experimental murine liver fibrosis using a clinical 3.0T Philips Achieva MRI scanner. Two hours after intravenous administration of 0.2 mmol/kg ESMA into healthy and carbon tetrachloride (CCl4)-treated c57bl/6 mice (0.6 mL CCl4/kg body weight; thrice weekly for 4 weeks; n = 3 mice per group), a three-dimensional high-resolution inversion recovery gradient echo delayed-enhancement MRI (DE-MRI; see Makowski et al.4 for details on MR parameters and methodology) of liver tissue indicated clear differences between normal and diseased animals (Fig. 1): while healthy livers displayed no focal contrast enhancement upon ESMA administration (Fig. 1D,E), very distinct perivascular signals were observed in large and medium-sized vessels in fibrotic livers (Fig. 1A,B). This observation was in line with periportal ECM deposition visualized using Elastica-Van-Gieson staining (Fig. 1C,F). Although these findings require further investigation (with regard to fibrosis stage, ESMA dose, timing, specificity, and quantification), they demonstrate that elastin-based molecular MRI, like collagen-based molecular MRI,3 may be suitable for noninvasive monitoring of ECM remodeling during liver fibrosis. As the collagen-to-elastin-ratio changes during the progression and regression of liver fibrosis,2 the selective or combined use of different molecular MR probes might be a promising strategy for translating the differential regulation of ECM proteins during fibrosis pro- and regression into novel noninvasive imaging techniques for the clinic. Elastin-based molecular MRI of liver fibrosis. C57bl/6 mice were treated with CCl4 thrice weekly (0.6 mL/kg, intraperitoneally) for 4 weeks to induce liver fibrosis (A-C) or treated with the vehicle (corn oil) as controls (D-F) and subjected to MRI using a clinical 3 T MR scanner (Achieva 3.0T, Philips Healthcare, Best, Netherlands). Two hours after the intravenous injection of an elastin-specific gadolinium-containing MR contrast agent (ESMA, BMS753951), significant perivascular T1 contrast enhancement (see arrows) was observed in fibrotic (A,B), but not in healthy control livers (D,E). Periportal elastin accumulation in fibrotic (C) versus healthy livers (F) was confirmed by way of Elastica-Van-Gieson staining, clearly demarcating the presence of collagen and elastin fibers in light red and dark purple, respectively. Results are representative of n = 3 animals per group. For details on MR scanning methodology and ESMA specificity, see Makowski et al.4 Supported by the German Research Foundation (DFG SFB/TRR57; TA434/2-1; EH412/1-1; LA2937/1-1) and British Heart Foundation (RG/12/1/29262). ESMA was kindly provided by David Onthank (Lantheus Medical Imaging, North Billerica, MA). JOSEF EHLING, M.D.1 MATTHIAS BARTNECK, PH.D.2 VIKTOR FECH2 BRITTA BUTZBACH, M.D.3 RICHARD CESATI, PH.D.4 RENE BOTNAR, PH.D.3 TWAN LAMMERS, PH.D., D.SC.1 FRANK TACKE, M.D., PH.D.2 1Department of Experimental Molecular Imaging University Hospital RWTH Aachen Aachen, Germany 2Department of Medicine III University Hospital RWTH Aachen Aachen, Germany 3Division of Imaging Sciences and Biomedical Engineering King's College London London, UK 4Lantheus Medical Imaging North Billerica, MA, USA

cytokines secretion. In vitro study, DIM inhibited ConA-induced proliferation of splenic CD4+T cells, DIM enhanced the protein expression of CYP1A1 and CYP1B1, and decreased which of TLR4 on Con A-stimulated splenic CD4+T cells. Furthermore, blocking AhR with CH223191 significantly reversed the ability of DIM to promote the differentiation of Tregs, and caused marked inhibition of Th17 differentiation. On the other hand, blockage with single anti-TLR4 neutralizing antibody show the similar immune-regulatory effects of DIM to induc the production of Tregs. Conclusions: DIM may be an efficient theraprutic candidate of liver fibrosis, and high dietary intake of cruciferous vegetables may be an effective nutritional supplement for the patients with chronic liver disease.