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Division of Gastroenterology, Department of Medicine, University of California San Diego School of Medicine, La Jolla, CA
Division of Gastroenterology, Department of Medicine, University of California San Diego School of Medicine, 9500 Gilman Drive, MC 0702, Leichtag Biomedical Research Building, Room 332MM, La Jolla, CA 92093-0702
Potential conflict of interest: Nothing to report.
Chronic liver disease is associated with hepatocyte injury, inflammation, and fibrosis. Chemokines and chemokine receptors are key factors for the migration of inflammatory cells such as macrophages and noninflammatory cells such as hepatic stellate cells (HSCs). The expression of CX3CR1 and its ligand, CX3CL1, is up-regulated in chronic liver diseases such as chronic hepatitis C. However, the precise role of CX3CR1 in the liver is still unclear. Here we investigated the role of the CX3CL1-CX3CR1 interaction in a carbon tetrachloride (CCl4)–induced liver inflammation and fibrosis model. CX3CR1 was dominantly expressed in Kupffer cells in the liver. In contrast, the main source of CX3CL1 was HSCs. Mice deficient in CX3CR1 showed significant increases in inflammatory cell recruitment and cytokine production [including tumor necrosis factor α (TNF-α); monocyte chemoattractant protein 1; macrophage inflammatory protein 1β; and regulated upon activation, normal T cell expressed, and secreted (RANTES)] after CCl4 treatment versus wild-type (WT) mice. This suggested that CX3CR1 signaling prevented liver inflammation. Kupffer cells in CX3CR1-deficient mice after CCl4 treatment showed increased expression of TNF-α and transforming growth factor β and reduced expression of the anti-inflammatory markers interleukin-10 (IL-10) and arginase-1. Coculture experiments showed that HSCs experienced significantly greater activation by Kupffer cells from CCl4-treated CX3CR1-deficient mice versus WT mice. Indeed, augmented fibrosis was observed in CX3CR1-deficient mice versus WT mice after CCl4 treatment. Finally, CX3CL1 treatment induced the expression of IL-10 and arginase-1 in WT cultured Kupffer cells through CX3CR1, which in turn suppressed HSC activation. Conclusion: The CX3CL1-CX3CR1 interaction inhibits inflammatory properties in Kupffer cells/macrophages and results in decreased liver inflammation and fibrosis. (Hepatology 2010)
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Liver inflammation is caused by hepatocyte damage associated with acute and chronic liver diseases, including alcoholic liver diseases, hepatitis B and C, and nonalcoholic steatohepatitis. In liver inflammation, various types of cells, including natural killer cells, natural killer T cells, T cells, dendritic cells, and macrophages, are recruited and activated.1, 2 In particular, the hepatic resident macrophage, the Kupffer cell, is a key player in producing inflammatory cytokines and reactive oxygen species to provoke liver inflammation upon liver injury.1 Both inflammatory cytokines [e.g., tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6] and anti-inflammatory cytokines (e.g., IL-10) are produced by Kupffer cells.1 It has been suggested that the balance between proinflammatory and anti-inflammatory responses is strictly regulated. Sustained and chronic liver injury and inflammation activate hepatic stellate cells (HSCs) and cause liver fibrosis. After liver injury, activated Kupffer cells produce various inflammatory and fibrogenic cytokines, such as monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor β (TGF-β), which lead to transdifferentiation of quiescent HSCs into myofibroblasts. Myofibroblasts express α-smooth muscle actin (α-SMA) and produce extracellular matrix proteins, such as collagen types I, III, and IV, in the liver.3, 4 The excessive production and deposition of extracellular matrix proteins result in liver fibrosis. Kupffer cell–derived TGF-β is essential for HSC activation, and Kupffer cell–depleted animals show significant reductions of liver fibrosis; this indicates that Kupffer cells are required for HSC activation and fibrogenic responses.5 Thus, the interaction of Kupffer cells with HSCs is crucial for HSC activation in chronic liver disease.
Chemokines are small, secreted proteins that promote cell migration, chemotaxis, and cell homing, which is a key event in development under normal conditions and in inflammation and disease under pathological conditions, including neovascularization, fibrosis, cancer cell growth, and metastasis.6, 7 Chemokines are classified into four subfamilies: C, CC, CXC, and CX3C. Chemokine receptors are the 7-transmembrane G protein–coupled receptors. Monocytes and macrophages express various chemokine receptors. Kupffer cells express CCR (CC-chemokine receptor) 1 CCR2, and CCR5, which contribute to the recruitment of inflammatory cells into inflammatory sites in the liver.8-10 CX3CL1 also known as fractalkine, is a membrane-bound type of chemokine. The soluble form of CX3CL1 is released after proteolysis by a disintegrin and metalloprotease 10 (ADAM10) and ADAM17.11-13 CX3CL1 is involved in cell recruitment and cell survival through binding to CX3CR1.14 The membrane-bound type of CX3CL1 binds to CX3CR1, which functions as an adhesion molecule independently of integrins.15 In addition, CX3CR1-expressing monocytes circulate in the steady state and differentiate into alternatively activated macrophages.16
CCR2 promotes inflammation such as atherosclerosis and lung, kidney, and liver diseases through the trafficking of monocytes to inflammatory sites.8, 17-19 In contrast, the role of CX3CR1 in inflammation is still controversial. CX3CR1-deficient mice showed reduced inflammation and injury after kidney ischemia/reperfusion and in atherosclerosis.20-23 On the other hand, loss of CX3CR1 exaggerates lipopolysaccharide (LPS)-induced neuronal damage, corneal neovascularization after alkali injury, and autoimmune uveitis and encephalomyelitis.24-27 CX3CL1 inhibits the production of nitric oxide, IL-6, and TNF-α in activated microglia, the resident macrophages in the central nervous system.28 Although increased levels of CX3CL1 and CX3CR1 have been observed in the livers of patients with chronic hepatitis C29, 30 and primary biliary cirrhosis,31 the precise role of the CX3CL1-CX3CR1 interaction in liver inflammation and fibrosis is unclear. Here we demonstrate that the disruption of CX3CR1 exacerbates liver inflammation and fibrosis by increased production of inflammatory and fibrogenic cytokines and reduced expression of IL-10 and arginase-1 in Kupffer cells. In addition, CX3CL1 treatment increases the expression of IL-10 and arginase-1 in Kupffer cells. These results suggest that the anti-inflammatory effects of CX3CR1 on Kupffer cells inhibit liver inflammation and fibrosis.
α-SMA, α-smooth muscle actin; ADAM, a disintegrin and metalloprotease; ALT, alanine aminotransferase; CCl4, carbon tetrachloride; CCR, chemokine (C-C motif) receptor; CYP2E1, cytochrome P450 2E1; ERK, extracellular signal-regulated kinase; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; GFP, green fluorescent protein; HSC, hepatic stellate cell; IL, interleukin; KC, Kupffer cell; KO, knockout; LPS, lipopolysaccharide; Ly6, lymphocyte antigen 6 complex; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; mRNA, messenger RNA; pan-CK, pan-cytokeratin; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RANTES, regulated upon activation, normal T cell expressed, and secreted; RPMI-1640, Roswell Park Memorial Institute 1640; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TNF, tumor necrosis factor; WT, wild type.
Materials and Methods
Animal Model of Chronic Liver Inflammation.
Specific pathogen-free, wild-type (WT) C57BL/6J mice and C57BL/6-background CX3CR1GFP/GFP mice, in which the CX3CR1 allele was replaced with a GFP-expressing cassette (Jackson Laboratories), were used.32 We used CX3CR1GFP/GFP mice as CX3CR1−/− mice.32 For the chronic liver inflammation model, mice were injected intraperitoneally with carbon tetrachloride (CCl4), which was diluted 1:3 in corn oil (Sigma), or with vehicle (corn oil) at a dose of 0.5 μL/g of body weight twice a week for a total of 12 injections. Mice were sacrificed 48 hours after the last injection. Serum levels of alanine aminotransferase (ALT) were measured with a commercial kit (Thermo Scientific). The mice received humane care according to the National Institutes of Health recommendations outlined in Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the institutional animal care and use committees of the University of California San Diego.
For immunohistochemical analysis, liver specimens were fixed in 10% buffered formalin and were incubated with monoclonal antibody against α-SMA (Sigma) with an M.O.M. kit (Vector Laboratories), rat anti-mouse F4/80 (eBioscience), or rat anti-mouse CD68 (AbD Serotec). For immunofluorescent staining, frozen sections were incubated with antibody to CX3CR1 (Novus Biologicals), F4/80, desmin (Neomarkers), pan-cytokeratin (pan-CK; Biolegend), or 4-hydroxynonenal (Alpha Diagnostic), and this was followed by imaging with fluorescent microscopy.8
Isolation of Kupffer Cells and HSCs.
WT or CX3CR1-deficient mice were treated with intraperitoneal injections of CCl4 or vehicle twice a week four times. Then, liver cells were fractionated into four major cell populations (hepatocytes, Kupffer cells, endothelial cells, and HSCs) as previously described.33 Briefly, mouse livers were digested by a two-step collagenase-pronase perfusion followed by three-layer, discontinuous density gradient centrifugation with 8.2% and 14.5% Nycodenz (Accurate Chemical and Scientific Corp.) to obtain the hepatocyte fraction, Kupffer cell/endothelial cell fraction, and HSC fraction. The Kupffer cell fraction and endothelial cell fraction were selected from the Kupffer cell/endothelial cell fraction by magnetic cell sorting (Miltenyi Biotec) with an anti-CD11b antibody and an anti–liver sinusoidal endothelial cell antibody, respectively. HSCs were isolated by digestion with collagenase and pronase, and this was followed by gradient centrifugation with 8.2% Nycodenz; then, CD11b+ Kupffer cells were removed by magnetic cell sorting. The >95% purity of the isolated HSCs was confirmed via immunostaining with anti-desmin antibody.
Total RNA was prepared from cells or frozen liver tissues with the TRIzol regent (Invitrogen), and it was cleaned with an RNeasy kit and then DNase treatment (Qiagen). RNA was reverse-transcribed with a high-capacity complementary DNA reverse-transcription kit (Applied Biosystems). Quantitative real-time PCR was performed with an ABI-Prism 7000 sequence detector (Applied Biosystems).9 PCR primer sequences are listed in Supporting Table 1. The expression of respective genes was normalized to 18S RNA as an internal control.
Liver mononuclear cells were prepared as previously described.10 Briefly, the liver was perfused and homogenated. The cells were resuspended in 36% Percoll and were centrifuged at 700g for 10 minutes. After Fc receptor blockade, cells were stained with anti-F4/80, CD11b, and lymphocyte antigen 6 complex C (Ly6C; eBioscience). In some experiments, cells were isolated from CX3CR1+/GFP mice as CX3CR1-GFP reporter mice, in which both GFP and CX3CR1 were expressed under the control of the endogenous CX3CR1 promoter, so that GFP-expressing cells represented CX3CR1-expressing cells. Samples were analyzed on a BD FACSAria flow cytometer and analyzed with Flow-Jo software (Tree Star).
Treatment of Primary Kupffer Cells.
Kupffer cells isolated from WT mice were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Invitrogen) containing 10% fetal bovine serum (FBS). After 24 hours of incubation, the medium was changed into an RPMI-1640 medium containing 1% FBS, and they were then incubated with 100 ng/mL recombinant CX3CL1 (ProSpec) or vehicle [phosphate-buffered saline (PBS)] for 6 hours more. In some experiments, Kupffer cells were pretreated with 2 μM LY294002 (AKT inhibitor; Sigma) or 20 μM U0125 (an extracellular signal-regulated kinase [ERK] inhibitor; Sigma) for 30 minutes before treatment with CX3CL1.5, 9
Measurement of Collagen-Driven GFP Expression in HSCs.
To measure collagen promoter activity, HSCs (1 × 105 cells/well) were isolated from collagen promoter–driven GFP transgenic mice (pCol9GFP-HS4,5 transgene).34 HSCs were cocultured with Kupffer cells (5 × 105 cells/well) for 48 hours in the presence or absence of 20 ng/mL soluble TGF-β receptor type II or 100 ng/mL recombinant CX3CL1 (ProSpec). Kupffer cells were isolated from WT or CX3CR1-deficient mice treated with or without CCl4. The number of GFP-positive cells was determined via the counting of GFP-positive cells and total cells in 10 randomly chosen high-power fields.5
Cell Migration Assay.
A cell migration assay was performed with a modified Boyden chamber as described previously.5 Briefly, WT Kupffer cells were placed into the upper chamber (5 × 104 cells/well) in RPMI-1640 medium without serum and were exposed to the vehicle or recombinant CX3CL1 (100 ng/mL) in the lower chamber. After 16 hours of incubation at 37°C, the cells that migrated to the lower side of the chamber were counted in eight randomly chosen fields (×100).
The preparation of whole cell protein extracts from frozen livers, electrophoresis, and subsequent blotting were performed as previously described.35 We incubated blots with mouse antibody to α-SMA (Sigma), cytochrome P450 2E1 (CYP2E1; Millipore), and β-actin (Sigma) and then visualized them with the enhanced chemiluminescence light method (Thermo Scientific).
All data are expressed as means and standard errors of the mean. Data between groups were analyzed with Student t tests. Differences between multiple groups were compared with one-way analysis of variance (GraphPad Prism 4.02, GraphPad Software); P values less than 0.05 were considered statistically significant.
CX3CR1 Is Expressed in Kupffer Cells in CCl4-Induced Liver Inflammation.
To investigate whether CX3CR1 and its ligand CX3CL1 were involved in liver inflammation and fibrosis, hepatic messenger RNA (mRNA) expression of CX3CR1 and CX3CL1 was measured after 12 injections of CCl4 in WT mice. Both CX3CR1 and CX3CL1 mRNA levels were significantly increased in the liver after CCl4 treatment (Fig. 1A). Next, we investigated the cellular source of CX3CR1 in the liver. CX3CR1 was expressed in F4/80-positive Kupffer cells/macrophages, but less CX3CR1 was detected in desmin-positive HSCs (Fig. 1B,C). CX3CR1 expression was not observed in hepatocytes, as determined by pan-CK immunostaining (Fig. 1D). Subsequently, we measured mRNA expression of CX3CR1 and CX3CL1 in hepatocytes, Kupffer cells/macrophages, and HSCs isolated from vehicle- or CCl4-treated mice. CX3CR1 mRNA was preferentially expressed in Kupffer cells/macrophages, and its level further increased after CCl4 treatment (Fig. 1E). Fluorescence-activated cell sorting (FACS) analysis determined that approximately 15% of F4/80-positive cells expressed CX3CR1; this was further increased to approximately 45% after CCl4 treatment. Ly6C is mainly expressed in bone marrow–derived infiltrated macrophages,10, 36 and CX3CR1 is largely expressed in Ly6C-negative monocytes in other organs, such as intestines.16, 37 Similarly, CX3CR1 was more highly expressed in Ly6C-negative macrophages in the liver (Fig. 1F).
Increased Liver Inflammation in CX3CR1-Deficient Mice After Chronic Treatment With CCl4.
To investigate the role of CX3CR1 in liver inflammation, WT and CX3CR1-deficient mice were treated with 12 injections of CCl4. Hepatic mRNA expression of inflammatory cytokines was measured with quantitative real-time PCR. After CCl4 treatment, hepatic inflammatory gene expression [including TNF-α; MCP-1; macrophage inflammatory protein 1α (MIP-1α); MIP-1β; and regulated upon activation, normal T cell expressed, and secreted (RANTES)] was significantly increased in CX3CR1-deficient mice versus WT mice (Fig. 2A). Inflammatory cell infiltration was enhanced in CX3CR1-deficient mice treated with CCl4, as determined by hematoxylin and eosin staining (Fig. 2B). Serum ALT levels were markedly elevated in CX3CR1-deficient mice versus WT mice after CCl4 injections (Fig. 2C). Because CYP2E1-mediated CCl4 metabolism plays an essential role in CCl4-induced liver damage,38 we examined CYP2E1 expression and generation of oxidative stress. We did not observe any differences in CYP2E1 levels or oxidative stress (Supporting Fig. 1A,B) between WT and CX3CR1-deficient mice after CCl4 treatment. These results demonstrate that a loss of CX3CR1 exacerbates liver inflammation and injury induced by CCl4 treatment.
Macrophage Infiltration Is Increased in CX3CR1-Deficient Mice After CCl4 Treatment.
To further investigate liver inflammation in CX3CR1-deficient mice, macrophage infiltration and activation were assessed. After CCl4 treatment, the expression of F4/80 and CD68, an activation marker for macrophages, was significantly increased in CX3CR1-deficient livers versus WT livers, as determined by immunohistochemistry and quantitative real-time PCR (Fig. 3A-E). FACS analysis demonstrated that the number of F4/80+ Kupffer cells was decreased in CX3CR1-deficient mice versus WT mice treated with vehicle (25.94% for WT mice versus 16.98% for CX3CR1−/− mice; Fig. 3F). CCl4 treatment increased the CD11b+F4/80+ population in WT mice (6.04% for vehicle versus 8.57% for CCl4). CD11b+F4/80+ cells were more increased in CX3CR1-deficient livers after CCl4 treatment (2.58% for vehicle versus 12.4% for CCl4; Fig. 3F).
Enhanced Inflammatory Features in CX3CR1-Deficient Kupffer Cells.
To characterize CX3CR1-deficient macrophages in the liver, liver Kupffer cells/macrophages were isolated from the control and fibrotic livers of both WT and CX3CR1-deficient mice. Compared with the levels in macrophages from WT livers, TNF-α and TGF-β1 mRNA levels were markedly increased in macrophages of CCl4-treated CX3CR1-deficient mice (Fig. 4A). Moreover, mRNA expression of the anti-inflammatory markers IL-10 and arginase-1 was significantly reduced in macrophages of CCl4-treated CX3CR1-deficient mice versus CCl4-treated WT mice or control CX3CR1-deficient mice (Fig. 4B). These results demonstrate that proinflammatory properties are augmented and anti-inflammatory properties are diminished in CX3CR1-deficient macrophages.
CX3CL1 Induces Anti-Inflammatory Features and Migration of Kupffer Cells.
The enhanced inflammatory features of CX3CR1-deficient Kupffer cells/macrophages prompted us to study the interaction of CX3CL1 and CX3CR1 in Kupffer cells. WT Kupffer cells were treated with CX3CL1 for 6 hours, and this was followed by measurement of the expression of anti-inflammatory markers. mRNA levels of IL-10 and arginase-1 were significantly increased in CX3CL1-treated WT Kupffer cells but not in CX3CR1-deficient Kupffer cells (Fig. 5A). Because the CX3CL1-CX3CR1 interaction induces AKT and ERK activation,39 we tested the requirement of AKT and ERK in CX3CL1-CX3CR1 signaling. Inhibition of AKT or ERK blocked IL-10 and arginase-1 expression in CX3CL1-treated Kupffer cells (Supporting Fig. 2). These results demonstrate that CX3CL1 induces anti-inflammatory properties through CX3CR1, AKT, and ERK. Moreover, CX3CL1 increased migration of CD11b+ Kupffer cells/macrophages (Fig. 5B). As shown in Fig. 1, CX3CR1 expression in HSCs is very low. To exclude the possibility that low levels of CX3CR1 contribute to HSC activation, we examined HSC activation treated with CX3CL1 by assessing collagen α1(I) promoter activity with HSCs isolated from collagen promoter–driven GFP transgenic (Coll-GFP) mice (Fig. 5C). CX3CL1 treatment did not have any effect on the enhancement of GFP expression or collagen α1(I) mRNA expression in HSCs (Fig. 5C-E). These results suggest that the CX3CL1-CX3CR1 interaction induces Kupffer cell migration and anti-inflammatory features but does not induce HSC activation.
CX3CR1-Deficient Kupffer Cells Enhance HSC Activation via TGF-β.
Inflammatory and fibrogenic cytokine production was augmented in CX3CR1-deficient Kupffer cells (Fig. 4). To investigate whether Kupffer cells of CX3CR1-deficient mice enhance HSC activation, HSCs of Coll-GFP mice were cocultured with Kupffer cells from control and fibrotic livers of either WT or CX3CR1-deficient mice. HSCs expressed higher GFP fluorescence in cocultures with Kupffer cells of CCl4-treated CX3CR1-deficient mice versus those of other mouse groups (Fig. 6A,B). Next, we treated cocultures of HSCs and Kupffer cells with the TGF-β receptor inhibitor, the soluble form of the type II TGF-β receptor. GFP expression of HSCs was significantly suppressed by treatment with the soluble type II TGF-β receptor (Fig. 6A,B), and this suggests that enhanced HSC activation by CX3CR1-deficient Kupffer cells requires TGF-β. Moreover, we examined whether exogenous CX3CL1 treatment suppresses the activation of HSCs cocultured with Kupffer cells. CX3CL1 treatment inhibited Coll-GFP HSCs cocultured with Kupffer cells from CCl4-treated WT mice but not those from CX3CR1-deficient mice (Fig. 6). These results suggest that CX3CL1 suppresses HSC activation through CX3CR1 in Kupffer cells. Further therapeutic potential of CX3CL1 was investigated in LPS-treated Kupffer cells. CX3CL1 treatment suppressed TNF-α and TGF-β induction in Kupffer cells in response to LPS (Supporting Fig. 3). Thus, the CX3CL1-CX3CR1 interaction inhibits inflammatory properties but induces anti-inflammatory properties in Kupffer cells.
Liver Fibrosis Is Exacerbated in CX3CR1-Deficient Mice After CCl4 Treatment.
Finally, we examined liver fibrosis in CX3CR1-deficient mice. Fibrogenic markers, including collagen α1(I), tissue inhibitor of metalloproteinase 1 (TIMP-1), and TGF-β1 mRNA levels, were significantly increased in CX3CR1-deficient mice versus WT mice after CCl4 treatment (Fig. 7A). Fibrillar collagen deposition was markedly increased in CX3CR1-deficient mice, as assessed by Sirius red staining and its quantification (Fig. 7B,C). Hepatic α-SMA expression, a marker for HSC activation, was enhanced in CX3CR1-deficient mice versus WT mice, as assessed by immunohistochemistry and immunoblotting (Fig. 7D,E). These results demonstrate that enhanced activation of Kupffer cells increases HSC activation and fibrosis in CX3CR1-deficient mice after CCl4 treatment.
A key function of chemokine–chemokine receptor interaction is chemoattractant activity. Recent studies have shown a role not only in cell migration but also in inflammation, fibrogenesis, and cell survival.6, 23 The present study demonstrates that CX3CR1 deficiency exacerbates liver inflammation and injury. Kupffer cells from CX3CR1-deficient mice lose their anti-inflammatory features, including IL-10 and arginase-1 expression, and enhance the expression of inflammatory cytokines and chemokines. Increased fibrogenic cytokines, including TGF-β, induce strong activation of HSCs, which leads to more liver fibrosis. Recombinant soluble CX3CL1 increases the expression of IL-10 and arginase-1 in Kupffer cells, which inhibit fibrogenic responses.40-43 These findings suggest that the CX3CL1-CX3CR1 interaction induces alternative activated macrophages that have anti-inflammatory properties and prevent excessive liver inflammation and fibrosis.
Chemokines are generally believed to act by recruiting immune cells that induce inflammation.7 Our previous studies have shown that CCR1, CCR2, and CCR5 signaling promotes liver inflammation and fibrosis.8, 9 Kupffer cells express CCR1, CCR2, and CCR5. The activation of these chemokine receptors results in the migration of macrophages to the injured site, which leads to inflammation and HSC activation and results in fibrosis.3 These classical inflammatory macrophages preferentially express inducible nitric oxide synthase, IL-12, and TNF-α and also express Ly6C. Interestingly, the functions of the CX3CL1-CX3CR1 axis are organ- and disease-specific. In atherosclerosis, inactivation of CX3CL1 or CX3CR1 reduces the severity.21, 23 Therefore, CX3CR1 antagonists are being developed for the therapy of atherosclerosis.44 On the other hand, the CX3CL1-CX3CR1 interaction exerts anti-inflammatory effects on microglia and neurons to protect the central nervous system from injury,28 and this corroborates our finding that CX3CR1 negatively regulates liver inflammation and fibrosis. Kupffer cells/macrophages express CX3CR1, which is further increased after CCl4 treatment (Fig. 1F). Hepatic natural killer cells and T cells also express CX3CR1,45 but the expression was not increased after CCl4 treatment (Supporting Fig. 4). Thus, we suggest that CX3CR1 in Kupffer cells/macrophages for the induction of alternative activated macrophages is critical in modulating liver inflammation.
Previous studies have demonstrated that disruption of CX3CR1 reduces Ly6Clow monocytes because of apoptosis.14 Overexpressed human Bcl-2 in CX3CR1-deficient mice restores the reduced number of Ly6Clow monocytes to a normal level.14 Furthermore, recombinant CX3CL1 treatment increases the survival of monocytes in the presence of serum deprivation and oxysterol-induced death.14 These findings suggest that the CX3CL1-CX3CR1 interaction prevents monocyte apoptosis, and this could explain the decreased number of hepatic macrophages in control CX3CR1-deficient mice (Fig. 3F). On the other hand, the number of monocytes/macrophages was increased in the livers of CX3CR1-deficient mice versus those of WT mice after CCl4 treatment. An increased number of CC chemokines in CCl4-treated CX3CR1-deficient mice (Fig. 2A) could recruit inflammatory cells through CCR1, CCR2, and CCR5 independently of CX3CR1.8-10
CX3CL1 is an exclusive ligand for CX3CR1 and is produced in a membrane-bound form.13, 15 Therefore, the CX3CL1-CX3CR1 interaction not only induces cell migration but also acts as an adhesion molecule for the capture and firm adhesion of CX3CL1-expressing cells and/or CX3CR1-expressing cells during cell recruitment and trafficking.15 As shown in Fig. 1, HSCs express much higher levels of CX3CL1 than Kupffer cells (Fig. 1E). CX3CR1 in Kupffer cells and the membrane-bound type of CX3CL1 in HSCs might interact directly and be important for Kupffer cell–HSC interaction, which is important for HSC activation. A previous study has shown that HSCs suppress immune cell activation.46 Our data demonstrate that HSC activation is increased in cocultures with CX3CR1-deficient Kupffer cells and is inhibited in cocultures with WT Kupffer cells treated with CX3CL1 (Fig. 6). CX3CL1 treatment also suppressed LPS-induced TNF-α and TGF-β production in Kupffer cells, and this is consistent with a previous study using microglia.28 Thus, Kupffer cell–HSC interaction activates HSCs and produces the inflammatory chemokines MCP-1 and RANTES but also prevents excessive activation of Kupffer cells/macrophages through the binding of HSC-derived CX3CL1 to CX3CR1 in Kupffer cells and inhibits HSC activation.
Clinical observations have demonstrated a correlation between the hepatic mRNA levels of CX3CR1 and the severity of histological fibrosis scores in patients with chronic hepatitis C.29, 30 CX3CL1 and CX3CR1 levels are also increased in the livers of patients with primary biliary cirrhosis, primary sclerosing cholangitis, and extrahepatic biliary obstructions.31 In these patients, CX3CR1 is expressed not only in leukocytes but also in pathological biliary epithelial cells. Moreover, patients with chronic hepatitis C with the CX3CR1 V249I polymorphism have more severe liver fibrosis and hepatic TIMP-1 mRNA levels.29 Leukocytes that express the CX3CR1 V249I variant have reduced binding activity with CX3CL1,47 and this corroborates our finding that CX3CR1 signaling negatively regulates liver inflammation. We suggest that increased CX3CR1 expression in severe liver fibrosis in humans and mice might inhibit liver fibrosis.29-31 Thus, modulation of the CX3CL1-CX3CR1 interaction may become a useful therapeutic target for chronic liver inflammation and fibrosis.