Hepatic macrophage migration and differentiation critical for liver fibrosis is mediated by the chemokine receptor C-C motif chemokine receptor 8 in mice

Abstract

Chemokines critically control the infiltration of immune cells upon liver injury, thereby promoting hepatic inflammation and fibrosis. The chemokine receptor CCR8 can affect trafficking of monocytes/macrophages, monocyte-derived dendritic cells (DCs) and T-helper cell (Th) subsets, but its role in liver diseases is currently unknown. To investigate the functional role of CCR8 in liver diseases, ccr8(-/-) and wild-type (WT) mice were subjected to chronic experimental injury models of carbon tetrachloride (CCl(4) ) administration and surgical bile duct ligation (BDL). CCR8 was strongly up-regulated in the injured liver. Ccr8(-/-) mice displayed attenuated liver damage (e.g., ALT, histology, and TUNEL) compared to WT mice and were also protected from liver fibrosis in two independent injury models. Flow cytometry revealed reduced infiltrates of liver macrophages, neutrophils and natural killer cells, whereas hepatic CD4(+) T cells increased. The main CCR8-expressing cells in the liver were hepatic macrophages, and CCR8 was functionally necessary for CCL1-directed migration of inflammatory but not for nonclassical monocytes into the liver. Moreover, the phenotype of liver macrophages from injured ccr8(-/-) animals was altered with increased expression of DC markers and enhanced expression of T-cell-attracting chemokine macrophage inflammatory protein 1-alpha (MIP-1α/CCL3). Correspondingly, hepatic CD4(+) T cells showed increased Th1 polarization and reduced Th2 cells in CCR8-deficient animals. Liver fibrosis progression, but also subsequent T-cell alterations, could be restored by adoptively transferring CCR8-expressing monocytes/macrophages into ccr8(-/-) mice during experimental injury.

Conclusions: CCR8 critically mediates hepatic macrophage recruitment upon injury, which subsequently shapes the inflammatory response in the injured liver, affecting macrophage/DC and Th differentiation. CCR8 deficiency protects the liver against injury, ameliorating initial inflammatory responses and hepatic fibrogenesis. Inhibition of CCR8 or its ligand, CCL1, might represent a successful therapeutic target to limit liver inflammation and fibrosis progression.

Fig. 1

CCR8 deficiency attenuates chronic liver injury and fibrosis. (A) H&E staining of WT and ccr8^−/− mice challenged with 0.6 mL/kg of CCl₄ IP repetitively three times per week. Magnification 10×. (B) Serum ALT levels (U/L) from WT and ccr8^−/− animals after repetitive CCl₄ challenge over 8 weeks (36 hours after last CCl₄ injection). Experiments were performed in groups of 3 animals, and results were confirmed in two independent experiments. (C) TUNEL assay of liver cryosections from WT and ccr8^−/− animals after 3 and 8 weeks of CCl₄ treatment. Apoptotic cells were stained green with FITC-dUTP, and nuclei were stained blue with DAPI. Magnification 10×. (D) Quantitative analysis of C. Five to seven independent view fields with 10× magnification were counted per animal in a blinded fashion. This experiment was performed in groups of 3 mice. (E) Sirius Red staining and IHC staining of alpha smooth muscle actin were performed on liver paraffin sections derived from WT and ccr8^−/− mice after 8 weeks of CCl₄ treatment. Magnification ×10. (F) Collagen deposition was quantified from Sirius Red stains using polarization microscopy (10 view fields per animal were counted from at least 6 animals per group and quantified by in silico morphometric analyis), as well as by measurement of hepatic hydroxyproline content. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SEM. All results are representative of two independent experiments with 3 animals per group. FITC-dUTP, fluorescein isothiocyanate/deoxyuridine triphosphate; DAPI, 4′,6-diamidino-2-phenylindole; α-SMA, alpha smooth muscle actin.

Fig. 2

CCR8 deficiency ameliorates infiltration of innate immune cells. (A) Pan-leukocyte marker CD45 was stained by IHC on 4-μm-thick paraffin-embedded sections. Magnification 10×. CD45⁺ cells were quantified manually by a pathologist who was blinded to experimental data. (B) Leukocytes were isolated from liver tissue of WT and ccr8^−/− animals after repetitive CCl₄ challenge over 8 weeks. Cells were stained for CD45 to discriminate between liver parenchymal cells and leukocytes. Dead cells were excluded by Hoechst 33258. Cells were stained for Ly6G to identify neutrophils, CD11b, and F4/80 for liver macrophages, NK1.1 for NK cells, and CD4⁺ and CD8⁺ for helper and cytotoxic T lymphocytes. (C) Statistical analysis of (B). Percentage values for total CD45 cells. This experiment was performed in groups of 3 animals, and results were confirmed in two independent experiments. Absolute cell counts of these hepatic leukocyte subsets are presented in Supporting Fig. 2. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± standard deviation.

Fig. 3

CCR8 is specifically expressed on macrophages and promotes macrophage migration. (A) Quantitative RT-PCR analysis of ccr8 expression from whole liver of WT animals after repetitive CCl₄ challenge over 8 weeks or controls. Expression levels were normalized against β-actin and calculated against unspecific background amplification in ccr8^−/− animals. (B) Quantitative RT-PCR analysis of leukocyte subpopulations after FACS sorting. Cells were sorted for CD45⁺ and further divided into Ly6G⁻CD3⁻NK1.1⁻CD11b⁺F4/80⁺ cells (macrophages, MF), CD3 NK1.1⁺ cells (NK cells, NK), and NK1.1⁻CD3⁺ cells (T cells). Expression levels were normalized against β-actin and calculated against unspecific background amplification in ccr8^−/− animals. (C) Transwell migration of liver leukocytes after CCl₄ challenge. Leukocytes were isolated 36 hours after the last CCl₄ injection and cultured in vitro in 5-μm Transwell chambers for 4 hours against a CCL1 gradient (100 ng/mL). Migrated cells were harvested from the bottom wells and characterized by FACS. Cells were stained for neutrophils, macrophages, NK cells, and CD4⁺ and CD8⁺ T-cells, and dead cells were excluded by Hoechst 33258. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SEM. All results are representative of two independent experiments with 3 animals per group.

Fig. 4

Phenotype of ccr8^−/− liver macrophages and DCs. (A) Phenotypic analysis of liver macrophages and DCs after CCl₄ challenge in WT and ccr8^−/− mice. Cells were isolated as described above and stained for CD45, Ly6G to gate out neutrophils, and CD11b, Gr-1/Ly6C, CD11c, and F4/80 for liver macrophage subtyping. (B) Statistical analysis of the macrophage/DC subtypes gated in (A). (C) Liver macrophages (CD11b⁺F4/80⁺) were isolated after 8 weeks of repetitive CCl₄ challenge by MACS purification against CD11b. Then, 2×10⁵ cells were seeded into 24-well plates and restimulated overnight, adding 100 ng/mL of LPS to the culture. For intracellular cytokine staining, cells were cultured in the the presence of GolgiPlug (BD Biosciences, Franklin Lakes, NJ) additionally 4 hours before analysis. Macrophages were stained extracellularly for I-A^b and CD86 (left, middle) and intracellularly for IL-12 (right). (D and E) Liver macrophages (CD11b⁺F4/80⁺) were isolated after repetitive CCl₄ challenge by MACS purification against F4/80, plated at 10⁵ cells/well, and cultured overnight without further stimulation. The release of cytokines and chemokines typical for classical macrophages (D) as well as of the T-cell-attracting chemokines (E), MIP1a (CCL3), MIP1β (CCL4), and RANTES (CCL5), was measured from the supernatant by multiplex assay. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SEM. All results are representative of two independent experiments with 3 animals per group. RANTES, regulated upon activation normal T cell expressed and secreted.

Fig. 5

Preferential differentiation of Th1-type effector cells in livers of ccr8^−/− mice. Liver-infiltrating helper T cells were analyzed regarding their activation and functional subtype after chronic CCl₄ challenge. (A) Intracellular cytokine staining of T cells isolated from inflamed liver tissue. CD4⁺ T cells were phenotyped as described after restimulation with PMA/ionomycin for 4 hours in the presence of GolgiPlug (BD Biosciences, Franklin Lakes, NJ) and stained intracellularly after fixation and permeabilization to detect IFN-γ, IL-13, and IL-10. (B) Analysis of Tregs in WT and ccr8^−/−. T cells were stained extracellularly for CD4 and CD25 and after fixation and permeabilization intracellularly for Foxp3 to identify Tregs. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SEM. All results are representative of two independent experiments with 3 animals per group.

Fig. 6

WT monocytes restore liver fibrosis in ccr8^−/− mice after adoptive transfer during CCl4-mediated liver damage. WT and ccr8^−/− mice were treated with CCl4 over 6 weeks, as described above, and were either injected weekly with 1×10⁶ monocytes isolated from CD45.1 donor mice using CD115 MACS purification or left further untreated. (A) Flow cytometric analysis of CD45.1⁺ cells isolated from the liver. Mice were sacrificed after 6 weeks of treatment with CCl₄ with or without additional monocyte transfer (72 hours before analysis). CD45.1⁺ cells were further subcharacterized using Ly6G to identify neutrophils and CD11b do detect monocytes/macropages. (B) Sirius Red stainings from liver paraffin sections of WT and ccr8^−/− mice challenged with CCl₄ and adoptively transferred WT monocytes. Collagen was visualized using polarization microscopy; pictures displayed are inverted and depicted as grayscale images. Magnification ×10. (C) Statistical analysis of (B). Four to six view fields per animal were counted and subsequently quantified by in silico morphometric analyis. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SEM. All results are representative of two independent experiments with 3 animals per group.