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. 2010 Oct;52(4):1390-400.
doi: 10.1002/hep.23795.

CX3CL1-CX3CR1 interaction prevents carbon tetrachloride-induced liver inflammation and fibrosis in mice

Tomonori Aoyama  1 , Sayaka Inokuchi, David A Brenner, Ekihiro Seki
Affiliations

CX3CL1-CX3CR1 interaction prevents carbon tetrachloride-induced liver inflammation and fibrosis in mice

Tomonori Aoyama et al. Hepatology. 2010 Oct.

Abstract

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 (CCl(4))-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 CCl(4) treatment versus wild-type (WT) mice. This suggested that CX3CR1 signaling prevented liver inflammation. Kupffer cells in CX3CR1-deficient mice after CCl(4) 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 CCl(4)-treated CX3CR1-deficient mice versus WT mice. Indeed, augmented fibrosis was observed in CX3CR1-deficient mice versus WT mice after CCl(4) 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.

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Conflict of interest statement

No conflicts of interest exist.

Figures

Fig 1
Fig 1. Kupffer cells/macrophages express CX3CR1 in the liver
Livers were analyzed from WT mice after 12 injections of CCl4 or vehicle (n=5). (A) Hepatic mRNA expression of CX3CR1 and CX3CL1 was measured by quantitative real time PCR. (B-D) CX3CR1 expression (in green) was detected by co-staining with (B) F4/80 (in red), (C) desmin (in red), and (D) pan-cytokeratin (in red) by fluorescence microscopy. Arrows indicate CX3CR1-positive and (B) F4/80 positive or (C) desmin positive cells. (E) mRNA levels of CX3CR1 and CX3CL1 in hepatocytes, Kupffer cells and HSCs isolated from vehicle- or CCl4-treated WT mice were detected by quantitative real time PCR (n=3). (F) Liver mononuclear cells isolated from control or CCl4-treated CX3CR1+/GFP mice, were stained with anti-F4/80, Ly6G, and Ly6C antibody followed by FACS analysis. F4/80 positive Ly6G negative cells were analyzed for Ly6C and GFP, as a measure of endogenous CX3CR1 expression. Representative FACS analysis is shown (n=3). * P<0.05.
Fig 2
Fig 2. Loss of CX3CR1 exacerbated liver inflammation after chronic treatment with CCl4
Chronic hepatitis was induced by intraperitoneal injection with CCl4 or vehicle twice a week for a total of 12 injections in WT (n=5) or CX3CR1-/- (KO) mice (n=5). (A) Hepatic mRNA expressions of TNF-α, MCP-1, MIP-1α, MIP-1β, and RANTES were measured by quantitative real time PCR. (B) Liver histology of CCl4-treated WT and CX3CR1-/- mice is assessed by H-E staining (original magnification: ×200). Arrows indicate inflammatory cell infiltration. (C) Serum ALT levels were measured. *P<0.05.
Fig 3
Fig 3. Increased macrophage infiltration in CCl4-treated CX3CR1-deficient mice
WT (n=5) or CX3CR1-/- (KO) mice (n=5) were intraperitoneally injected with CCl4 or vehicle 12 times. (A-D) Immmunohistochemistry for F4/80 (A) and CD68 (C), and (B, D) their quantification are shown (original magnification: ×200). (E) Hepatic mRNA expression of CD68 was measured by quantitative real time PCR. (F) Representative FACS analysis for F4/80 and CD11b expression in WT or CX3CR1-/- livers are shown (n=3). *P<0.05.
Fig 4
Fig 4. CX3CR1-deficient Kupffer cells have augmented inflammatory properties
Kupffer cells were isolated from WT or CX3CR1-/- (KO) mice treated with CCl4 or vehicle for 4 times (n=3). (A, B) mRNA levels of (A) TNF-α, TGF-β1, (B) IL-10 and arginase-1 were measured by quantitative real time PCR. *P<0.05, **P<0.01.
Fig 5
Fig 5. CX3CL1 induces alternative activation of Kupffer cells by expressing IL-10 and arginase-1 through CX3CR1
Kupffer cells isolated from WT and CX3CR1-deficient mice were incubated with 100 ng/ml CX3CL1 or vehicle (PBS). (A) mRNA levels of IL-10 and arginase-1 were measured by quantitative real time PCR. (B) Serum-free media containing recombinant CX3CL1 (100 ng/mL) was placed in the lower chamber and Kupffer cells of WT mice were placed in the upper chamber. Migration of Kupffer cells into the lower chamber was counted 16 hours after stimulation. (C, D) HSCs of collagen promoter driven GFP transgenic mice were incubated with 50, 100 or 300 ng/ml recombinant CX3CL1 or vehicle (PBS) for 48 hours. (C) Representative photomicrographs of HSCs and (D) their quantification are shown (original magnification: ×200). (E) Collagen α1(I) mRNA are shown. Similar results were obtained in three independent experiments. *P<0.05.
Fig 6
Fig 6. CX3CR1-deficient Kupffer cells enhanced and CX3CL1 treatment inhibited HSC activation
HSCs isolated from collagen promoter-driven GFP transgenic mice were co-cultured with WT or CX3CR1-/- Kupffer cells for 48 hours in the presence or absence of 20 ng/ml soluble TGF-β receptor type II, or 100ng/ml recombinant CX3CL1. (A) Representative photomicrographs of GFP positive HSCs and (B) their quantification are shown (original magnification: ×200). A representative result is shown. Similar results were obtained in three independent experiments. *P<0.05.
Fig 7
Fig 7. Increased Liver fibrosis in CX3CR1-deficient mice after CCl4 treatment
WT (n=5) or CX3CR1-/- (KO) mice (n=5) were treated with CCl4 or vehicle for 12 times. (A) Hepatic mRNA expressions of collagen α1(I), TIMP-1 and TGF-β1 mRNA were measured by quantitative real time PCR. (B) Fibrillar collagen deposition was evaluated by Sirius red staining (original magnification: ×200) and (C) its quantification. (D, E) Expression α-SMA in the liver was detected by immunohistochemistry (D) and western blotting (E). (original magnification: ×100). *P<0.05.
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