The fractalkine receptor CX3CR1 protects against liver fibrosis by controlling differentiation and survival of infiltrating hepatic monocytes

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Chemokines modulate inflammatory responses that are prerequisites for organ fibrosis upon liver injury. Monocyte-derived hepatic macrophages are critical for the development, maintenance, and resolution of hepatic fibrosis. The specific role of monocyte-associated chemokine (C-X3-C motif) receptor 1 (CX3CR1) and its cognate ligand fractalkine [chemokine (C-X3-C motif) ligand 1)] in liver inflammation and fibrosis is currently unknown. We examined 169 patients with chronic liver diseases and 84 healthy controls; we found that CX3CL1 is significantly up-regulated in the circulation upon disease progression, whereas CX3CR1 is down-regulated intrahepatically in patients with advanced liver fibrosis or cirrhosis. To analyze the functional relevance of this pathway, two models of experimental liver fibrosis were applied to wild-type (WT) and CX3CR1-deficient mice. Fractalkine expression was induced upon liver injury in mice, primarily in hepatocytes and hepatic stellate cells. CX3CR1−/− animals developed greater hepatic fibrosis than WT animals with carbon tetrachloride–induced and bile duct ligation–induced fibrosis. CX3CR1−/− mice displayed significantly increased numbers of monocyte-derived macrophages within the injured liver. Chimeric animals that underwent bone marrow transplantation revealed that CX3CR1 restricts hepatic fibrosis progression and monocyte accumulation through mechanisms exerted by infiltrating immune cells. In the absence of CX3CR1, intrahepatic monocytes develop preferentially into proinflammatory tumor necrosis factor–producing and inducible nitric oxide synthase–producing macrophages. CX3CR1 represents an essential survival signal for hepatic monocyte–derived macrophages by activating antiapoptotic bcl2 expression. Monocytes/macrophages lacking CX3CR1 undergo increased cell death after liver injury, which then perpetuates inflammation, promotes prolonged inflammatory monocyte infiltration into the liver, and results in enhanced liver fibrosis. Conclusion: CX3CR1 limits liver fibrosis in vivo by controlling the differentiation and survival of intrahepatic monocytes. The opposing regulation of CX3CR1 and fractalkine in patients suggests that pharmacological augmentation of this pathway may represent a possible therapeutic antifibrotic strategy. (HEPATOLOGY 2010;52:1769-1782)

Sustained inflammation is a common characteristic of chronic liver injury in mice and men and induces the development of hepatic fibrosis.1 The intrahepatic inflammatory response following hepatic injury is a highly regulated process that involves the activation of distinct nonparenchymal cells in the liver, including hepatic stellate cells (HSCs), resident hepatic macrophages (Kupffer cells), and sinusoidal endothelial cells, and the attraction of specific immune cell populations that in turn exert critical proinflammatory or anti-inflammatory actions in the injured liver.1, 2 Chemokines (chemotactic cytokines) are essential mediators for attracting immune cells and for activating nonparenchymal liver cells.3, 4 As such, circulating Gr1-expressing monocytes are massively recruited after liver injury in mice by mechanisms dependent on chemokine (C-C motif) receptor 2 (CCR2) and its main ligand, monocyte chemoattractant protein 1 (MCP1).5-7 These monocytes differentiate into hepatic macrophages and promote the progression of liver fibrosis by releasing proinflammatory and profibrogenic cytokines such as tumor necrosis factor (TNF) and transforming growth factor β and by directly activating collagen-producing HSCs.5

The chemokine fractalkine [chemokine (C-X3-C motif) ligand 1 (CX3CL1)] differs from other chemokines in several respects. First, it is the only member of the CX3C chemokine family and lacks redundancy because there is only one known receptor, chemokine (C-X3-C motif) receptor 1 (CX3CR1), corresponding to this chemokine. Second, CX3CL1 is synthesized as a transmembrane protein with its chemokine domain presented on an extended mucin-like stalk; this allows tight, integrin-dependent adhesion of CX3CR1-expressing leukocytes.8 In addition, constitutive and inducible cleavage by metalloproteinases can result in the release of soluble CX3CL1 fragments from the cell membrane and thereby act as classic soluble chemoattractants.9 The fractalkine receptor, CX3CR1, is primarily expressed on circulating monocytes, tissue macrophages, and tissue dendritic cell populations but is also expressed on T cell and natural killer cell subsets.10 In the liver, CX3CR1 expression has been described on the biliary epithelium, infiltrating mononuclear cells, HSCs, and even hepatoma cell lines.11, 12

Preliminary observations have linked fractalkine and its receptor CX3CR1 to the pathogenesis of chronic liver diseases. Fractalkine and CX3CR1 were found to be up-regulated in biopsy samples of patients with acute and chronic liver injury11 and especially cholestatic diseases.13, 14 Furthermore, CX3CR1 gene polymorphisms have been associated with fibrosis progression in patients with chronic hepatitis C.12 Experimentally, the shedding of CX3CL1 by HSCs has promoted the chemoattraction of monocytes in vitro,15 and the adhesion of human CD16+ monocytes to liver sinusoidal endothelium is partially mediated by CX3CR1.16 Therefore, we conducted experiments to define the roles of fractalkine and CX3CR1 in liver inflammation and fibrosis.

Abbreviations

α-SMA, α-smooth muscle actin; ADAM, a disintegrin and metallopeptidase; ALT, alanine aminotransferase; APC, allophycocyanin; BCL2, B cell lymphoma 2; BDL, bile duct ligation; BM, bone marrow; BMT, bone marrow transplantation; CCL, chemokine (C-C motif) ligand; CCl4, carbon tetrachloride; CCR, chemokine (C-C motif) receptor; CX3CL1, chemokine (C-X3-C motif) ligand 1, alternative name: fractalkine; CX3CR1, chemokine (C-X3-C motif) receptor 1; FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; Gr1, myeloid differentiation marker; HSC, hepatic stellate cell; IL, interleukin; iNOS, inducible nitric oxide synthase; MCP1, monocyte chemoattractant protein 1; MELD, Model for End-Stage Liver Disease; MIP, macrophage inflammatory protein; mRNA, messenger RNA; NS, not significant; qPCR, quantitative polymerase chain reaction; RANTES, regulated upon activation, normal T cell expressed, and secreted; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; WT, wild type.

Materials and Methods

Human Patients and Controls.

The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki; this was reflected in a priori approval by the local ethics committee, and written, informed consent was obtained from each patient.17 As controls, healthy volunteers with normal aminotransferase activity, no history of liver disease or alcohol abuse, and negative serology for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus were recruited from the local blood transfusion institute. Samples from 30 liver biopsy procedures and 33 explanted cirrhotic livers (staged by a blinded pathologist) were subjected to messenger RNA (mRNA) expression analysis. Fractalkine serum concentrations were measured with a cytometric bead array (#560265, BD Biosciences).

Animals and Fibrosis Models.

C57BL/6 wild-type (WT), congenic Ly5.2 (CD45.1) C57BL/6, and CX3CR1−/− mice (backcrossed for more than eight generations to a C57BL/6 background) were maintained in a pathogen-free environment. The CX3CR1-deficient mice had green fluorescent protein (gfp) inserted into the CX3CR1 genetic locus.10 Experiments were performed with age-matched and sex-matched animals at 6 to 8 weeks of age. Mice were treated with carbon tetrachloride (CCl4; Merck; 0.6 mL/kg of body weight) mixed with corn oil or were treated with corn oil (for controls). Mice were sacrificed 48 hours after the last injection. Bile duct ligation (BDL) was performed according to standard procedures. All animals received humane care, and the experiments were approved by appropriate German authorities.

Flow Cytometry Analysis of Blood and Intrahepatic Leukocytes.

The isolation of blood cells and intrahepatic leukocytes was performed,5 and they were then subjected to fluorescence-activated cell sorting (FACS) with the following monoclonal antibodies: F4/80 (Serotec); CD45, Ly6G, natural killer 1.1, CD3, and CD8 (BD); and CD115, CD4, CD11b, CD45.1, CD45.2, and CD11c (eBiosciences). For the analysis of intrahepatic macrophage apoptosis, intrahepatic leukocytes were stained with CD45, CD11b, F4/80, and annexin V/allophycocyanin (APC; BD). CCR2 staining was performed with an unconjugated antibody (Epitomics) or with a corresponding isotype control (rabbit immunoglobulin G; BD), which was followed by secondary goat anti-rabbit APC (Invitrogen). Data analyses were performed with FlowJo (Tree Star), and intrahepatic leukocytes were identified by CD45 staining, by live cell staining (with Hoechst dye), by the exclusion of cell doublets (via forward scatter width), and by positive forward scatter/side scatter gates for live cells.18

Histology, Biochemical Assays, Western Blotting, and Real-Time Quantitative Polymerase Chain Reaction (qPCR).

Hematoxylin-eosin and Sirius red staining was performed as described.5 Immunofluorescence staining was performed on frozen sections with CD11b (BD), CD4 (eBioscience), B220 (Cedarlane), and appropriate isotype controls (BD).5 The terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay (Roche) was performed on frozen liver sections according to the manufacturer's instructions. Measurements of the hepatic hydroxyproline content, western blotting for α-smooth muscle actin (α-SMA)/glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and measurements of alanine aminotransferase (ALT) were conducted as described.5 RNA was extracted from the sorted cells or total liver, and qPCR was performed with the SYBR Green reagent (Invitrogen). All reactions were performed twice in triplicate, and β-actin expression was used to normalize gene expression. Primer sequences are available upon request.

Bone Marrow Transplantation (BMT).

Recipient mice were subjected to total body irradiation with a dose of 12 Gy for 20 minutes.19 Total bone marrow (BM) cells from WT (CD45.1) or CX3CR1gfp/gfp mice were injected via the tail vein. After BM transfer, recipient mice were maintained in a pathogen-free environment and given drinking water containing antibiotics (0.02% Borgal) for 2 weeks before the actual experiments were started.

Primary Cell Isolation and FACS Sorting.

Primary hepatocytes, Kupffer cells, and sinusoidal liver endothelial cells were isolated as described before.20 For the sorting of intrahepatic monocytes, CD45+CD11b+F4/80+CD4 live cells were sorted from intrahepatic leukocytes with the FACSAria II (BD). HSCs were sorted because of their negativity for CD45 and positive autofluorescent signals in the ultraviolet channel (355 nm).

Statistics.

Data from human patients are presented as medians and ranges because of the skewed distributions of most variables. Differences between two groups were assessed with the Mann-Whitney U test, and multiple comparisons were assessed with the Kruskal-Wallis analysis of variance and the Mann-Whitney U test for post hoc analysis (SPSS). Correlations between variables were assessed with the Spearman rank correlation test.17 Data from experimental studies are presented as means and standard errors of the mean. A two-tailed Student t test was used for comparisons between experimental groups with GraphPad Prism.

Results

Fractalkine and Its Receptor CX3CR1 Are Associated With the Progression of Liver Fibrosis in Patients With Chronic Liver Disease.

In order to evaluate the clinical relevance of the CX3CL1-CX3CR1 axis for liver fibrosis progression in humans, we first determined serum concentrations of fractalkine in a large cohort of patients with chronic liver diseases at different stages of fibrosis/cirrhosis (Table 1). Patients with chronic liver diseases showed significantly elevated serum fractalkine levels (n = 169, median = 41.3 pg/mL) in comparison with healthy controls (n = 84, median = 27.4 pg/mL, P < 0.001; Fig. 1A). Patients with cirrhosis had significantly higher circulating CX3CL1 levels than patients without cirrhosis (P < 0.001), whereas differences between early (Child A) and decompensated (Child C) cirrhosis did not reach statistical significance (Fig. 1A). The underlying disease etiology did not influence the serum fractalkine level (Fig. 1B). Moreover, the serum fractalkine level correlated with clinical scores of disease progression [r = 0.236 and P = 0.021 for Child-Pugh points and r = 0.336 and P = 0.001 for Model for End-Stage Liver Disease (MELD) scores; Fig. 1C], correlated inversely with liver function (e.g., r = −0.296 and P < 0.001 for albumin, r = 0.365 and P < 0.001 for bilirubin, r = −0.364 and P < 0.001 for cholinesterase, and r = 0.236 and P = 0.002 for the international normalized ratio), and correlated with noninvasive quantitative fibrosis markers (r = 0.388 and P < 0.001 for hyaluronic acid and r = 0.465 and P < 0.001 for procollagen III peptide; Fig. 1C).

Figure 1.

Regulation of CX3CL1 and CX3CR1 in patients with chronic liver disease and the association with fibrosis progression. (A) Serum levels of fractalkine (CX3CL1) were compared between healthy controls (n = 84) and patients with chronic liver disease (n = 169); patients were further subdivided into subjects without cirrhosis (n = 67) and subjects with cirrhosis (n = 102: Child A cirrhosis, n = 42; Child B cirrhosis, n = 30; and Child C cirrhosis, n = 30). Box plots are displayed: the bold, black lines indicate the median per group; the boxes represents 50% of the values; the horizontal lines show minimum and maximum values of the calculated nonoutlier values; and the open circles indicate outlier values. Significant differences (U test) are marked with asterisks. ***P < 0.001. (B) The underlying disease etiology (viral hepatitis, n = 65; biliary or autoimmune disease, n = 21; alcohol, n = 47; and other, n = 36) did not affect the serum CX3CL1 concentrations. (C) Serum CX3CL1 concentrations in patients were correlated with the MELD scores and procollagen III peptide serum levels (Spearman rank correlation test). (D) The gene expression of cx3cl1 and cx3cr1 was quantified by real-time polymerase chain reaction in livers without fibrosis (n = 13), with early fibrosis (F1/F2, n = 7), or with advanced fibrosis and cirrhosis (F3/F4, n = 43). The bars express the fold induction, and the error bars represent the standard errors of the mean. ***P < 0.001. Abbreviation: NS, not significant.

Table 1. Characteristics of the Patient Cohort and Serum Fractalkine Concentrations
 Healthy Controls (n = 84)All Patients (n = 169)Stages of Liver Cirrhosis
No Cirrhosis (n = 67)Child A (n = 42)Child B (n = 30)Child C (n = 30)
  1. For quantitative variables, medians are given with the ranges in parentheses.

Male/female, n49/35103/6640/2722/2017/1325/5
Age, years42 (18-68)54 (17-82)43 (17-65)62 (30-82)61 (28-77)53 (21-76)
Liver disease etiology, n  Viral hepatitis  Biliary or autoimmune  Alcohol or cryptogenic  OtherNot applicable65 21 47 3637 11 5 1417 6 12 78 3 10 93 1 20 6
Serum fractalkine (CX3CL1), pg/mL27.4 (<2-80.5)41.3 (<2-464.1)32.5 (<2-362.8)37.4 (<2-251.4)55.1 (<2-464.1)52.1 (<2-303.3)

We next assessed the intrahepatic gene expression of CX3CL1 and CX3CR1 in patients with different stages of fibrosis by real-time qPCR. The intrahepatic expression of cx3cl1 was down-regulated when we compared nonfibrotic or fibrotic livers with cirrhotic livers (Fig. 1D). Intrahepatic cx3cr1 expression was strongly reduced in cirrhotic livers versus fibrotic or nonfibrotic livers (Fig. 1D). This finding was in sharp contrast to the increased numbers of macrophages that were observed in cirrhotic livers,17 and this suggested that the down-regulation of CX3CR1 in the cirrhotic liver (not a lack of CX3CR1-expressing cells) was responsible for this finding. Collectively, these data demonstrate that progressive liver fibrosis in humans is associated with an increase in circulating fractalkine and a reduction of intrahepatic CX3CR1 expression.

Liver Injury and Intrahepatic Monocyte Infiltration Are Prolonged in CX3CR1−/− Mice After Acute CCl4-Induced Liver Damage.

In order to address the functional role of CX3CR1 in hepatic injury and fibrogenesis, WT and CX3CR1-deficient mice were subjected to CCl4-induced liver injury. After a single injection of CCl4 and during chronic liver injury induced by twice weekly CCl4 injections for 6 weeks, fractalkine gene expression was significantly up-regulated in the livers of WT and CX3CR1−/− mice (Supporting Fig. 1 and data not shown). At 24 and 48 hours after a single intraperitoneal administration of CCl4, WT and CX3CR1-deficient mice displayed massive hepatocyte necrosis and high ALT levels (Fig. 2A,B). However, CX3CR1−/− mice showed prolonged histological signs of injury and significantly elevated ALT levels at 72 and 120 hours (Fig. 2A,B), whereas WT animals fully recovered within 5 days after CCl4, as anticipated from previous studies.5

Figure 2.

Prolonged liver injury and monocyte infiltration in CX3CR1−/− mice after acute CCl4-induced liver damage. (A,B) Mice received a single injection of CCl4 (0.6 mL/kg of weight), which resulted in maximal damage at 48 hours as evidenced by a histological examination with hematoxylin-eosin (left; the arrows point to areas that are enlarged in the insets) or by the serum ALT activity (right; n = 6 per group and time point). Although WT mice fully recovered within 5 days after the injection, CX3CR1−/− mice display sustained signs of injury and persistent ALT elevations. (C,D) FACS analysis demonstrated increased numbers of intrahepatic leukocytes (CD45+ cells) and monocytes (CD45+CD11b+F4/80+ cells), respectively, in CX3CR1−/− mice after a single CCl4 injection (n = 6 per group and time point). Representative FACS plots, pregated on CD45 and live cells, are shown for the time points after the CCl4 injection. (E) Intrahepatic gene expression analyses of chemokines by qPCR 48 hours after CCl4 are displayed as the fold induction (the expression level of oil-treated WT mice was set to 1). *P < 0.05, **P < 0.01, and ***P < 0.001. Abbreviations: MIP, macrophage inflammatory protein; RANTES, regulated upon activation, normal T cell expressed, and secreted.

We next analyzed leukocyte infiltration into livers after CCl4-induced injury by FACS. In line with prolonged liver damage, CX3CR1−/− mice displayed a prolonged elevation of intrahepatic leukocytes at 72 and 120 hours, whereas intrahepatic leukocyte counts were almost normalized in WT mice at 120 hours after CCl4 treatment (Fig. 2C). The predominant intrahepatic leukocytes were CD11b+F4/80+ monocyte-derived macrophages, which remained high in CX3CR1−/− mice and decreased in WT mice at late time points after injury (Fig. 2D). Moreover, intrahepatic expression of MCP1, the specific ligand for the monocytic chemokine receptor CCR2, was more strongly induced in CX3CR1−/− mice versus WT mice; this contrasted with chemokines targeting other nonmonocytic immune cells such as chemokine (C-C motif) ligand 3 (CCL3), CCL5, and CCL20 (Fig. 2E). Notably, the expression of CCR2 by hepatic or circulating monocytes did not differ between WT and CX3CR1-deficient mice, although overall intrahepatic ccr2 expression was increased in CX3CR1−/− animals because of the higher numbers of infiltrating CCR2-expressing monocytes (Supporting Fig. 2). Furthermore, the increased accumulation of monocytes in the liver was mirrored by reduced levels of circulating total monocytes and a shift toward the inflammatory Gr1hi monocyte subpopulation in the peripheral blood of CX3CR1−/− mice versus WT animals (Supporting Fig. 3). These observations demonstrate that CX3CR1−/− mice have an impaired ability to limit the inflammatory response after injury, and this is associated with enhanced monocyte infiltration into the liver in the absence of CX3CR1.

Figure 3.

CX3CR1 promoted the survival and differentiation of intrahepatic monocyte–derived macrophages in the injured liver via bcl2. (A) The gene expression of bcl2 (normalized to β-actin) was quantified by real-time polymerase chain reaction from livers of WT or CX3CR1−/− mice at different time points after CCl4. The fold induction is displayed (the untreated WT liver was set to 1). The bar graph summarizes the findings for four to six animals per group. (B) TUNEL staining was performed to quantify apoptotic cells after the CCl4 injection. Representative pictures are shown in Supporting Fig. 4. The bar graph summarizes the findings for four to six animals per group. (C) Annexin V staining by FACS identified apoptotic hepatic monocytes in CX3CR1−/− mice; the left panel presents representative FACS plots 72 hours after CCl4, and the right panel presents a quantitative analysis based on four mice per group. (D) Hepatic monocytes were sorted by FACS from mice 48 hours after CCl4; the left panel presents representative FACS plots confirming the purity of the sorted cells. Bcl2 gene expression was quantified by real-time polymerase chain reaction in sorted hepatic monocytes and primary hepatocytes; both were isolated 48 hours after the CCl4 injection (n = 6 per group). The expression in untreated WT control cells (hepatic monocytes or hepatocytes) was set to 1. (E) The expression of tnf, il-1β, and il-10 was also tested in sorted hepatic monocytes 48 hours after the CCl4 injection (n = 3-6 per group). *P < 0.05, **P < 0.01, and ***P < 0.001. Abbreviation: NS, not significant.

CX3CR1 Regulates the Survival and Differentiation of Intrahepatic Monocyte–Derived Macrophages in the Injured Liver.

Besides mediating the chemotaxis of leukocytes and other mesenchymal cells as a classic chemoattractive cytokine,21 fractalkine has recently been found to also promote antiapoptotic effects on monocytes/macrophages in patients with inflammatory conditions such as atherosclerosis.22, 23 We thus assessed levels of several proapoptotic and antiapoptotic genes in the liver after CCl4 injury. Unlike bcl-XL (B cell lymphoma extra large) and other genes that we tested (Supporting Fig. 4A and data not shown), bcl2 (B cell lymphoma 2) was significantly down-regulated throughout the time course in the livers of CX3CR1−/− mice versus WT animals (Fig. 3A). Concordantly, CCl4-induced liver damage was associated with significantly higher numbers of TUNEL+ cells in CX3CR1−/− mice versus WT mice (Fig. 3B and Supporting Fig. 4B). Interestingly, there was a high association between TUNEL staining and gfp expression in CX3CR1−/− mice (with gfp insertion into the CX3CR1 gene locus), and this suggested apoptosis of gfp-expressing monocytes/macrophages within the injured liver of knockout mice (Supporting Fig. 4B). We therefore subjected intrahepatic leukocytes isolated from mice at different time points after the CCl4 injection to annexin V staining by FACS, and this revealed a distinct increase in annexin V+ hepatic monocytes in CX3CR1−/− mice 72 hours after CCl4 injury (Fig. 3C).

Figure 4.

CX3CR1−/− mice developed greater liver fibrosis with CCl4-induced injury. (A) Mice were injected intraperitoneally with CCl4 twice per week and were sacrificed at the time points indicated. Representative Sirius red stains revealed more progressive hepatic fibrosis in livers of CX3CR1−/− mice versus WT mice. The scale bars represent 200 μm. (B) Hydroxyproline levels (n = 6-8 per group) confirmed increased collagen deposition in CX3CR1−/− mice versus WT mice after chronic CCl4 treatment. (C) Western blotting for α-SMA (three representative samples per condition) confirmed increased HSC activation in CX3CR1−/− mice versus WT mice after chronic CCl4 treatment.

In line with our observations of whole liver tissue (Fig. 3A), studies of monocytes/macrophages from atherosclerotic mice and during lung inflammation have suggested that CX3CR1-CX3CL1 provides an essential survival signal for macrophages via Bcl2.22, 24 In order to dissect the molecular mechanism of increased hepatic monocyte/macrophage apoptosis after injury in CX3CR1−/− mice, hepatic CD11b+F4/80+ monocytes were purely isolated 48 hours after CCl4 injection by FACS sorting (Fig. 3D). Moreover, primary hepatocytes were isolated from untreated and CCl4-treated animals. Although bcl2 expression in either cell compartment did not differ between untreated WT and knockout mice (not shown), hepatic monocytes (but not hepatocytes) of CX3CR1−/− mice had significantly down-regulated bcl2 expression in comparison with WT mice. Moreover, intrahepatic monocytes of CX3CR1−/− after injury displayed higher tnf and lower interleukin-10 (il-10) expression, and this suggested that they were skewed toward a more proinflammatory macrophage phenotype than that in WT mice (Fig. 3E). These data demonstrate that CX3CR1 is a key signal regulating the survival and differentiation of intrahepatic monocyte–derived macrophages in the injured liver through the promotion of antiapoptotic pathways (i.e., bcl2 expression).

CX3CR1 Protects Against Liver Fibrosis In Vivo.

In order to address the functional role of CX3CR1 in hepatic fibrogenesis, two well-established experimental models of liver fibrosis were tested. After twice weekly intraperitoneal administrations of CCl4 for 6 weeks, CX3CR1−/− mice developed significantly more fibrosis than WT animals. This was evidenced by collagen deposition in the histological examination (Fig. 4A), the intrahepatic hydroxyproline content (Fig. 4B), and the expression of α-SMA protein (Fig. 4C) and by the increased expression of collagen and α-SMA according to qPCR (not shown). Interestingly, these differences were apparent throughout the whole duration of the experiment. These results suggest that CX3CR1-dependent mechanisms are relevant during the initiation and progression of fibrosis in the chronic CCl4 model.

Increased Monocyte Accumulation in CX3CR1−/− Mice During Liver Fibrosis.

We have previously demonstrated that inflammatory Gr1+ (Ly6C+) monocytes are massively recruited into the injured liver during chronic liver damage and that this is dependent on the chemokine receptor CCR2-mediated release of immature monocytes from BM.5 However, Gr1+ monocytes can also use CX3CR1 for immigration into chronically inflamed tissue, as exemplarily shown for their entry into atherosclerotic plaques.25 We therefore characterized intrahepatic immune cell populations in animals with CCl4-induced fibrosis by FACS analysis. In line with the acute injury model, CX3CR1−/− mice did not display reduced intrahepatic leukocytes, but there was a significant increase in the number of immune cells after chronic CCl4 injury (Fig. 5A,B). Specifically, CD11b+F4/80+ monocyte-derived macrophages were found in higher numbers during the course of CCl4-induced fibrosis in CX3CR1−/− mice versus WT mice (Fig. 5C,D). In contrast, the intrahepatic CD4+ or CD8+ T cell, B cell, and natural killer T cell compartments did not differ between WT and CX3CR1−/− mice (Fig. 5D and data not shown).

Figure 5.

Increased monocyte accumulation in CX3CR1−/− mice during liver fibrogenesis. (A) Mice were injected intraperitoneally with CCl4 twice per week and were sacrificed at the time points indicated. Representative hematoxylin-eosin stains revealed more pronounced inflammatory infiltrates surrounding necrotic areas in the livers of CX3CR1−/− mice versus WT mice. The inset shows a higher magnification of the area marked by the arrow. (B) The infiltration of intrahepatic leukocytes after chronic CCl4 administration was quantified by FACS and is depicted as the percentage of CD45+ cells among all liver cells. (C) Intrahepatic leukocytes were further characterized by FACS analysis, which confirmed a strong increase in the number of CD11b+F4/80+ macrophages upon CCl4 treatment (representative FACS plots are shown on the left; they were pregated on CD45+ and live cells). During the course of CCl4 injury, CX3CR1−/− mice showed significantly more intrahepatic monocytes (n = 6-8 animals per group and time-point). (D) The results were confirmed by immunofluorescent staining for CD11b (monocytes), CD4 (T cells), and B220 (B cells). Representative pictures taken 6 weeks after CCl4 are shown. The inset shows an area with CD11b+ cells (arrow) at a higher magnification. *P < 0.05, **P < 0.01, and ***P < 0.001.

In order to exclude model-specific confounding effects, mice were subjected to surgical BDL, which led to severe cholestatic fibrosis within 21 days. Similarly to the CCl4 model, CX3CR1−/− mice developed more severe hepatic fibrosis after BDL than WT animals, as demonstrated by periportal collagen deposition with Sirius red staining (Fig. 6A) and by the intrahepatic hydroxyproline content (Fig. 6B). Notably, CX3CR1-deficient mice also displayed an increased mortality rate in comparison with WT animals after BDL (Fig. 6C) as well as higher serum bilirubin and ALT levels, which indicated greater liver damage in knockout animals induced by this model (Supporting Fig. 5). These experiments confirm that CX3CR1 limits the development of liver fibrosis in vivo independently of the nature of the injury.

Figure 6.

CX3CR1−/− mice developed greater liver fibrosis and increased monocyte accumulation upon BDL. Mice underwent BDL or sham surgery and were sacrificed 21 days later (n = 6-8 per group). (A) Sirius red staining and (B) the intrahepatic hydroxyproline content demonstrated more progressive hepatic fibrosis in livers of CX3CR1−/− mice versus WT mice. The scale bars represent 200 μm. (C) CX3CR1−/− mice also displayed increased mortality (Kaplan-Meier curve) in comparison with WT mice after BDL. Increased liver injury in CX3CR1−/− mice after BDL was evident from serum bilirubin and ALT levels (Supporting Fig. 5). CX3CR1−/− mice showed (D) more hepatic leukocytes and (E) an accumulation of intrahepatic macrophages in comparison with WT animals after BDL. *P < 0.05, **P < 0.01, and ***P < 0.001.

We also analyzed immune cell infiltration in the BDL fibrosis model and found that the total number of leukocytes and the accumulation of monocytes/macrophages were also significantly higher in CX3CR1−/− mice versus WT mice after BDL (Fig. 6D). Compared with CCl4-induced fibrosis (Fig. 5B,C), BDL had an even stronger effect on the recruitment of monocytes/macrophages into the injured liver. These data indicate that during fibrogenesis, a lack of CX3CR1 promotes the infiltration of monocytes into the damaged liver independently of the injury model.

Infiltrating Immune Cells but Not Resident Nonparenchymal Hepatic Cells Require CX3CR1 to Limit Liver Fibrosis.

Although CX3CR1 is predominantly expressed in immune cells and especially circulating monocytes, CX3CR1 expression has been also described in (activated) HSCs, sinusoidal endothelial cells, biliary epithelium, and even hepatoma cell lines.11, 12 To functionally dissect the contribution of CX3CR1 to infiltrating immune cells of hematopoietic origin and liver-resident cell populations, we generated WT-CX3CR1−/− chimeric mice with irradiation and BMT. Successful BMT and reconstitution were demonstrated with staining for CD45.1 (WT BM donor) or gfp expression of CX3CR1-deficient BM by FACS (data not shown). Four weeks after BMT, liver fibrosis was induced by chronic CCl4 administration. WT or CX3CR1−/− mice that underwent transplantation with control BM (of their original genotype) developed hepatic fibrosis similar to that of their nontransplanted counterparts, as shown by Sirius red staining, hydroxyproline contents, and α-SMA blotting (Fig. 7A-C). In contrast, mice that were CX3CR1-deficient in resident hepatic cells but expressed (WT) CX3CR1 in BM displayed the same (low) level of fibrosis as WT mice (Fig. 7A-C). On the other hand, a lack of CX3CR1 only in hematopoietic cells was sufficient to significantly enhance fibrosis development in transplanted WT mice (Fig. 7A-C). Interestingly, the increased accumulation of total hepatic leukocytes and intrahepatic monocyte–derived CD11b+F4/80+ macrophages also depended on CX3CR1 deficiency in BM-derived cells (Fig. 7D). These experiments provide evidence that CX3CR1 restricts hepatic fibrosis progression through mechanisms exerted by hematopoietic cells and strongly suggest a specific function of CX3CR1 in infiltrating monocytes.

Figure 7.

Fibrosis progression and monocyte accumulation depended on CX3CR1 expression by infiltrating immune cells but not by resident hepatic cells. (A-C) BMT was performed to generate WT mice with WT BM, WT mice with CX3CR1−/− BM, CX3CR1−/− mice with CX3CR1−/− BM, and CX3CR1−/− mice with WT BM (n = 6 per group). BMT mice underwent CCl4 treatment for 6 weeks, and hepatic fibrosis was evaluated by (A) Sirius red staining, (B) hydroxyproline levels, and (C) α-SMA western blotting. This revealed that mice with CX3CR1−/− BM had more progressive liver fibrosis. In contrast, CX3CR1−/− mice that underwent transplantation with WT BM developed less severe fibrosis. (D) FACS analysis demonstrated increased numbers of intrahepatic leukocytes (CD45+ cells; left) and macrophages (CD45+CD11b+F4/80+ cells; right) in mice with CX3CR1−/− BM and CCl4-induced fibrosis. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 8.

CX3CR1 affected the differentiation and promoted the survival of infiltrating monocytes during hepatic fibrogenesis. (A) Different cell types were isolated from WT mice after 6 weeks of CCl4 by either standard cell isolation techniques with collagenase digestion and gradient centrifugation (hepatocytes, endothelium, and Kupffer cells) or FACS sorting (HSCs and monocytes). The fold induction is displayed, and the bar graph summarizes the findings for four to six mice per group. (B) The gene expression of bcl2 (normalized to β-actin) was quantified by real-time polymerase chain reaction in livers of WT or CX3CR1−/− mice 6 weeks after CCl4 and 21 days after BDL. The fold induction is displayed, and the untreated WT liver was set to 1. The bar graph summarizes four to six animals per group. (C) Annexin V staining by FACS identified apoptotic hepatic monocytes in CX3CR1−/− mice; the left panel presents representative FACS plots 4 weeks after CCl4, and the right panel presents a quantitative analysis of the findings from four mice per group. (D) Hepatic monocytes were sorted by FACS from mice 4 weeks after CCl4; CD45+CD11b+F4/80+ staining was used (cf. Fig. 2D). The gene expression of tnf, iNOS, and arginase-1 was quantified by real-time polymerase chain reaction (n = 3-6 per group). *P < 0.05, **P < 0.01, and ***P < 0.001.

CX3CR1 Affects the Differentiation and Promotes the Survival of Infiltrating Monocytes During Hepatic Fibrogenesis.

Monocyte-derived macrophages previously were found to enhance hepatic fibrosis by proinflammatory cytokines and HSC activation.5 Hence, the increased accumulation of infiltrating monocytes in CX3CR1−/− mice could causally link hepatic macrophages to the fibrosis phenotype of these animals. We isolated different primary cell types (hepatocytes, HSCs, endothelial cells, Kupffer cells, and infiltrating monocytes) from fibrotic livers after 6 weeks of CCl4 treatment and revealed that fractalkine is expressed primarily by (injured) hepatocytes and to a lesser extent by (activated) HSCs. These findings indicate that hepatocytes and HSCs provide essential signals via CX3CL1 to the CX3CR1+ infiltrating monocytes in the fibrotic liver (Fig. 8A). In analogy to the observations after acute injury, we determined whether CX3CL1 controls the survival of infiltrating monocytes. In fact, intrahepatic expression of antiapoptotic bcl2 was down-regulated in both fibrosis models in CX3CR1−/− mice versus WT animals (Fig. 8B). Annexin V staining revealed increased numbers of apoptotic cells among intrahepatic CD11b+F4/80+ monocytes in CX3CR1−/− mice after chronic CCl4 administration (Fig. 8C). Moreover, monocyte-derived macrophages in CX3CR1−/− mice displayed a more proinflammatory, M1-type differentiation because sorted CD11b+F4/80+ intrahepatic monocytes showed higher tnf and inducible nitric oxide synthase (iNOS) expression but unaffected arginase-1 expression with chronic CCl4 treatment (Fig. 8D). These data demonstrate that the CX3CL1-CX3CR1 pathway provides functionally important signals regulating the survival and differentiation of infiltrating intrahepatic monocytes and results in increased cell death, perpetuated inflammation, and preferential development of TNF/iNOS-producing macrophages in CX3CR1−/− mice upon chronic liver injury.

Discussion

Accumulating functional and genetic evidence demonstrates that chemokines, small chemotactic cytokines, play critical roles in acute and chronic liver diseases. The initial studies were mainly focused on the chemokine-directed infiltration of immune cells (monocytes and T cells) into the injured liver along a concentration gradient.3, 5, 26 Later, it became apparent that chemokines might also directly affect the biology of liver-resident cells, such as HSCs and hepatocytes, during inflammatory and fibrogenic tissue responses.4, 26 We have now identified fractalkine and CX3CR1 as a chemokine-chemokine receptor pathway that primarily modulates the differentiation and survival of infiltrating hepatic monocytes.

In this study, we first tested the potential clinical relevance of fractalkine (CX3CL1) and its specific receptor CX3CR1 in a large cohort of patients with chronic liver diseases at different stages of fibrosis progression. Interestingly, circulating fractalkine concentrations were significantly elevated in patients versus controls and especially in patients with cirrhosis. This finding is in agreement with a recent in vitro study demonstrating that HSCs shed CX3CL1 via the sheddases a disintegrin and metallopeptidase 10 (ADAM10) and ADAM17,15 and this results in increasing local and systemic CX3CL1 concentrations. Interestingly, we observed a correlation between quantitative serum fibrosis markers (i.e., hyaluronan or procollagen III peptide) and serum CX3CL1 in patients, and this indicates that the number of activated HSCs may influence fractalkine serum levels. Surprisingly, despite high systemic levels of fractalkine, the hepatic expression of cx3cr1 was low in patients with liver cirrhosis, and this suggests that disease progression is associated with the down-regulation of cx3cr1 by hepatic cells in vivo. In fact, we have observed that human monocytes that are cocultured with primary human HSCs down-regulate CX3CR1 surface expression ex vivo (H.W.Z. and F.T., unpublished data, 2010). Furthermore, advanced fibrosis in patients has been associated with decreased hepatic CX3CL1 expression. Collectively, our clinical data reveal that patients with liver fibrosis/cirrhosis have up-regulated serum fractalkine levels but down-regulated hepatic CX3CR1 and CX3CL1 expression, and this suggests the functional involvement of this pathway during liver fibrogenesis in humans.

We therefore analyzed the functional role of CX3CL1/CX3CR1 in experimental fibrosis in mice. Strikingly, CX3CR1−/− mice developed more progressive fibrosis than WT animals in two independent models. These results contrast with findings from other organ injury models in which CX3CR1−/− mice were partially protected from renal interstitial fibrosis after ischemia/reperfusion injury27 and in which atherosclerosis-prone ApoE−/− animals developed less progressive atherosclerotic lesions.25 Interestingly, in CX3CR1−/− livers, persistently more intrahepatic inflammatory cells and pronounced and specific intrahepatic macrophage accumulation were evident in experimental liver damage.

At this point, it is important to determine whether increased monocyte accumulation in CX3CR1−/− mice after liver injury is an epiphenomenon of CX3CR1-mediated actions on other cells or is directly linked to CX3CR1 effects on monocytes/macrophages. By using BM chimeric mice, we have demonstrated that CX3CR1 expression by infiltrating immune cells and not by resident parenchymal or nonparenchymal liver cells is required to limit liver inflammation and fibrosis. Our data provide experimental evidence that the main mechanisms of CX3CR1-mediated actions in the injured liver promote the survival of infiltrating monocytes and guide the differentiation of monocyte-derived macrophages. Although CX3CL1 was originally defined as a chemoattractant for monocytes,21, 25 a growing body of evidence indicates that CX3CR1 is involved in controlling cell survival. This was initially unraveled in the central nervous system. There, CX3CR1 controls the neurotoxicity of brain macrophages (microglia) and promotes neuronal survival.28, 29 This was later expanded to intestinal epithelial cells.30 Our present data demonstrate that CX3CL1 acts as a crucial survival signal for infiltrating CX3CR1+ monocytes that develop into monocyte-derived hepatic macrophages after liver injury because increased monocyte/macrophage apoptosis in the livers of CX3CR1−/− mice is associated with increased monocyte infiltration and inflammation (a condition likely promoting fibrosis progression). It has recently been shown in a model of atherosclerosis that sustained induction of apoptosis in lesional macrophages results in significant increases in inflammation and lesion size.31 In this model, the defective clearance of apoptotic macrophages in advanced lesions favors enhanced recruitment of monocytes and thus leads to enhanced atherogenesis.31 It is thus very likely that in our model increased hepatic macrophage apoptosis provides a strong signal for the infiltration of additional monocytes and thereby perpetuates the inflammatory response.

By dissecting the expression of proapoptotic and antiapoptotic genes in different hepatic cell populations, our results suggest that bcl2 is the specific molecular target for CX3CR1-mediated survival signals in hepatic monocytes/macrophages. In agreement, bcl2 down-regulation has also been reported in circulating CX3CR1−/− monocytes and inflamed tissue macrophages by other investigators.22, 24 Moreover, overexpression of bcl2 in CX3CR1-deficient monocytes/macrophages could restore their survival,22 and enforced cell survival by the transduction of CX3CR1-deficient BM with bcl2-overexpressing constructs restored the phenotype of CX3CR1−/− mice in an atherosclerosis model.22

Moreover, in the absence of CX3CR1, hepatic monocytes/macrophage displayed a more proinflammatory TNF/iNOS-producing phenotype. Interestingly, this skewing toward the proinflammatory M1-type macrophage subtype1 was apparent already after acute injury, and this suggests that CX3CL1 limits the activation of macrophages in vivo. This conclusion is strongly supported by recent in vitro experiments using murine liver macrophages, which demonstrated increased TNF expression and reduced arginase 1 expression by CX3CR1-deficient macrophages upon CCl4 stimulation.32 Furthermore, CX3CL1 induced preferential arginase 1 expression in WT liver macrophages.32 Similarly, the pretreatment of (BM-derived) macrophages with fractalkine suppressed the release of TNF upon lipopolysaccharide stimulation.33 Collectively, the in vitro data and our in vivo models provide evidence that CX3CR1-deficient macrophages deviate toward a proinflammatory M1 phenotype upon activation and that in turn CX3CL1 inhibits skewing toward an M1 phenotype in WT macrophages.

By activating antiapoptotic and anti-inflammatory signals in hepatic macrophages, the fractalkine-CX3CR1 pathway represents a protective mechanism that limits liver inflammation and fibrosis in vivo. Thus, pharmacological augmentation of this pathway may represent a possible therapeutic antifibrotic strategy for patients with chronic liver inflammation. However, it is important to keep in mind that fractalkine can act in its membrane-bound form or, after shedding, as a soluble ligand with its free chemokine domain.9 The expression of fractalkine in the membrane-bound form on hepatocytes12 and the shedding of the soluble ligand by HSCs have been described.15 Future studies are warranted to determine which of the two forms is functionally more relevant in the liver and during fibrogenesis before fractalkine is tested as a potential therapeutic agent in hepatic fibrosis.

Acknowledgements

The authors thank Aline Müller, Carmen Tag, and Sibille Sauer-Lehnen for their excellent technical assistance.

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